SYMMETRIC PROTEINS

Abstract

The invention relates to protein building block named the Self-Assembling Kelch (SAKe) protein. The protein has a stable, symmetric design with readily accessible loops that can be varied in both sequence and length to later bind larger molecules or scaffold a catalytic site.

Description

FIELD OF THE INVENTION

The invention relates to the design and expression of proteins with a symmetrical structures.

The invention relates to synthetic proteins with functional properties such as metal binding and enzymatic activity.

BACKGROUND OF THE INVENTION

The functional and structural diversity of proteins has inspired researchers to engineer them for various applications. Recent examples have demonstrated the engineering of proteins as enhanced catalysts, vaccines, biosensors, and building blocks for 2D/3D frameworks [McConnell et al. ACS Synth. Biol. 2020, 9, 381-391; Brouwer et al. Cell 2021, 184, 1188-1200; Schuster Biosensors 2018, 8, 40; Chen et al. J. Am. Chem. Soc. 2019, 141, 8891-8895; Pyles et al. Nature 2019, 571, 251-256; Zhang et al. Nat. Commun. 2020, 11, 1-12; Ben-Sasson et al. Nature 2021, 589, 468-473]. In many cases, proteins with new functions are obtained via the redesign of existing proteins. Advances in computational protein design have stimulated the development of unique proteins with various conformations and functionality [Kuhlman & Bradley Nat. Rev. Mol. Cell Bio. 2019, 20, 681-697]. Therefore, protein engineers are not limited to re-purposing natural proteins and are able to expand their toolkit with new molecules with improved properties. Symmetric proteins are highly desirable due to their stability and versatility as building blocks for the development of 2D/3D assemblies [Yeates Annu. Rev. Biophys. 2017, 46, 23-42]. An exceptionally stable, symmetric β-propeller protein called Pizza is described in [Voet et al. Proc. Natl. Acad. Sci. U.S.A. 2014, 111, 15102-15107]. To showcase its functional potential, Pizza was redesigned to obtain protein assemblies, artificial enzymes, and high affinity scaffolds for various metals and metal-oxo clusters [Vrancken et al. J. Struct. Biol.: 4 2020, 100027; Clarke et al. Chem. Commun. 2019, 55, 8880-8883; Voet et al. Angew. Chem. Int. Ed. 2015, 127, 9995-9998; Vandebroek et al. Chem. Commun. 2020]. However, Pizza lacks an extensively modifiable interface which limits its capacity to bind more complex molecules.

SUMMARY OF THE INVENTION

Advances in computational protein design have allowed for the development of new proteins with unique properties. Symmetric designer proteins have remarkable stability and can serve as versatile building blocks for the creation of macromolecular assemblies. The present invention describes the development of SAKe: A new symmetric, stable protein building block with modifiable loops. In the present invention polypeptides as claimed will be referred to as SAKE proteins. Following the observation of pH induced 3D self-assembly, metal binding sites were engineered along the protein's internal rotational axis to fabricate 2D surface arrays. Using atomic force microscopy, Cu(II) dependent on-surface 2D self-assembly is demonstrated. The present invention discloses a stable and highly modifiable SAKe protein scaffold, which for use as a building block for the creation of multi-functional macromolecular materials.

In the conserved sequence motif, the amino acids Xn are not numbered. As the sequence listing does number the Xn aminoacids, the numbering of amino acids cterminal of Xn will differ.

Thus X₁₄, X₁₆ and X₁₈ of the sequence motif become respectively positions 29, 31 and 33 in SEQ ID NO 34 of the sequence listing. Throughout the specification the numbering of the conserved sequence motif is used.

Throughout the specification, when wording is used such as “Ile4 has Leu or Phe as alternative” this means that at position 4 of the sequence Ile, Leu or Phe can be present.

The invention is further summarised in the following statements:

1. A polypeptide comprising a sequence having at least 60 or 70% identity with NGRIY₅AVG₈G₉-X_n-LNSVE₁₄AY₁₆DP₁₈ETDEW₂₃SFVAPM₂₉TTPR₃₃SGVG₃₇VAV₄₀L [SEQ ID NO:32] or comprising 2 to 9 repeats of said sequence, wherein Xn are between 1 and 15 amino acids, wherein x can be any amino acid,

• • and wherein the amino acids Tyr₅, Gly₈, Gly₉, Tyr₁₆, Trp₂₃, Arg₃₃, and Val₄₀ in said sequence are conserved.

2. A polypeptide comprising 2 to 9 repeats of a sequence, each of said sequences having at least 70% identity with [SEQ ID NO: 34] NGRIX₅AVG₈G₉-X_n-LNSVX₁₄AX₁₆DX₁₈ETDEW₂₃SFVAPM₂₉TTPR₃₃SGVG₃₇VAV₄₀L], wherein Xn are between 1 and 15 amino acids, wherein x can be any amino acid, and wherein repeats are separated from each other from between 0 and 15 amino acids, wherein the amino acids, Gly₈, Gly₉, and Trp₂₃ in each of said sequences are conserved,

• • and with the proviso that optionally for one of said sequences an aminoterminal part of said one sequence is located at the carboxyterminal end of said polypeptide and the remaining carboxyterminal part of said one sequence is located at the aminoterminal end of said polypeptide.

An example of this proviso is represented as follows:

A polypeptide with three repeats schematically represented as

• • ABCDEFGH-ABCDEFGH-ABCDEFGH

Can thus also occur as

• • BCDEFGH-ABCDEFGH-ABCDEFGH-A
  • CDEFGH-ABCDEFGH-ABCDEFGH AB
  • DEFGH-ABCDEFGH-ABCDEFGH ABC
  • EFGH-ABCDEFGH-ABCDEFGH-ABCD
  • FGH-ABCDEFGH-ABCDEFGH-ABCDE
  • GH-ABCDEFGH-ABCDEFGH-ABCDEF
  • H-ABCDEFGH-ABCDEFGH ABCDEFG

3. The polypeptide according to statement 2,

• • wherein, in one or more, or in all of said sequences,
  • X₅ is Tyrosine, Phenylalanine, Tryptophane, Histidine or Methionine, or wherein X₅ is Tyrosine Phenylalanine or Tryptophane, X₅ is Tyrosine or Phenylalanine,
  • X₁₆ is Tyrosine, Phenylalanine, or Tryptophane, or X₁₆ is Tyrosine or Phenylalanine, and
  • X₁₈ is Proline or Valine.

Embodiments for all above combinations of X₅, X₁₆ and X₁₈ are explicitly envisaged and disclosed herein.

In a specific embodiment X₅ is Tyrosine Phenylalanine or Tryptophane, and

• • X₁₆ is Tyrosine, Phenylalanine, or Tryptophane, and
  • X₁₈ is Proline or Valine.

4. The polypeptide according to statement 2 or 3, wherein, in one or more, or in all of said sequences X₁₄ is Glu, Asp, Cys, or Ser, or wherein X₁₄ is Glu or Asp.

5. The polypeptide according to any one of statements 1 to 4, wherein, in one or more, or in all of said sequences,

• • Arg₃₃, and/or Val₄₀ are conserved.
  • Val₄₀ is conserved.

5. The polypeptide according to any one of statements 2 to 4, wherein, in one or more, or in all of said sequences,

• • X₅ is Tyrosine,
  • X₁₄ is Glu,
  • X₁₆ is Tyrosine
  • X₁₈ is Proline,
  • Arg₃₃, and/or Val₄₀ is conserved.

6. The polypeptide according to any one of statements 2 to 5, wherein, in one or more, or in all of said sequences, the amino acids, Trp₂₃, Met₂₉, and Gly₃₇ are conserved.

Embodiments of all possible variations of X₅, X₁₄, X₁₆, X₁₈ are herewith envisaged and explicitly disclosed.

As recited above, apart from the absolute conserved Gly₈, Gly₉, and Trp₂₃ sequence variation is limited at position X₅, X₁₄, X₁₆, X₁₈, and sequence variation is less stringent for the remaining positions as long as the overall sequence identity is above the defined percentage.

Further specific embodiments of sequences falling under the definition of SEQ ID NO:34 are sequences wherein:

• • Ile4 has Leu or Phe as alternative; X₅ is Tyr, Met, Phe Ala or Val; Leu₁₀ has His as alternative; X₁₆ is Tyr, Phe or Trp; X₁₈ is Pro or Val; Met₂₉ has Leu as alternative; Arg₃₃ has as alternatives Ser or Met; Gly₃₅ has Ala as alternative; Gly₃₇ is conserved; Val₄₀ has Ser, His or Arg as alternative;
  • Sequences wherein Ile4 has Leu or Phe as alternative; X₅ is Tyr, Met, Ale or Val; Ans₁₁ has Asp as alternative; Ser₁₂ is has Lys as alternative; X₁₆ is Tyr or Phe; X₁₈ is Pro or Val; Thro has Arg as alternative; Met₂₉ has Leu as alternative; Arg₃₃ has as alternative Ser; Gly₃₅ has Ala as alternative; Gly 37 is conserved; Val₄₀ has His or Arg as alternative.

Sequences wherein Ile4 has Leu as alternative; Alas has Val as alternative; Leu₁₀ has His as alternative; Asn₁₁ has Asp as alternative; X₁₈ is Pro or Val; Met₂₉ has Leu as alternative; Gly₃₅ has Ala as alternative choice; Gly₃₇ is conserved

Sequences wherein Ile4 has Leu as alternative; Ala₆ has Val as alternative; Leu₁₀ has His as alternative; X₁₈ is Pro or Val; Met₂₉ has Leu as alternative; Gly₃₅ has Ala as alternative; Gly₃₇ is conserved.

For all the above sequences any sequence obtained by combination of the different possibilities for the recited amino acids is herewith explicitly disclosed.

The amino acids X of Xn wherein n=1 to 15, or the amino acids in between sequence in a repeat can be any of the 20 natural amino acids encountered in polypeptides as well as modified versions thereof as obtained by post-translation modification. Other side chain can be envisaged when synthetic peptides are produced as long as the amino acids can be incorporated via its NH₂ and COOH group in a polypeptide.

It is further envisaged that in the non-conserved parts of the sequence other amino acids occur which differ from the regular 20 naturally occurring amino acids as long as they are incorporated via its NH₂ and COOH group in a polypeptide.

7. The polypeptide according to any one of statements 1 to 6, wherein, in one or more, or in all of said sequences, the amino acids Tyr₅, Gly₈, Gly₉, Glu₁₄, Tyr₁₆, Pro₁₈, Trp₂₃, Met₂₉, Arg₃₃, and Gly₃₇ and Val₄₀ in said sequence are conserved.

8. The polypeptide according to any one of statements 1 to 7, comprising between 2 to 9 repeats of said sequence.

9. The polypeptide according to any one of statements 1 to 8, comprising 2, 3 or 6 repeats of said sequence.

10. The polypeptide according to any one of statements 1 to 9, wherein one or more, or all of said sequences have at least 80, 90 or 95% identity with SEQ ID NO: 32 or is identical to SEQ ID NO: 32.

As an example, for a polypeptide with 3 repeats of the sequence, one sequence can be for example 82% identical (>80%), a second 92% identical (>90%), and a third 97% identical (>95%).

11. The polypeptide according to any one of statements 1 to 10, wherein Xn are between 5 to 15 amino acids, or between 5 to 10 amino acids.

In these embodiments, for each of Xn the length can differ or can be identical, as long as the length falls within the defined range

12. The polypeptide according to any one statements 1 to 11, wherein one or more of said repeats of a sequence are separated from each other with 1 up to 5 amino acids.

In these embodiments, the length between two sequences can differ or can be identical, as long as the length falls within the defined range.

13. The polypeptide according to any one of statements 1 to 12, wherein one or more of said repeats of said sequence are immediately adjacent to each other.

14. The polypeptide according to any one of statements 1 to 13, wherein all of said repeats of said sequence are immediately adjacent to each other.

15. The polypeptide according to any one of statements 1 to 14, wherein two or more, or all repeats of said sequence are immediately adjacent to each other.

16. The polypeptide according to any one of statements 1 to 15, wherein two or more or all the repeats of said sequence are identical, with exception of the amino acids Xn.

17. The polypeptide according to any one of statements 1 to 16, comprising a first repeat and a second repeat of said sequence wherein said first and second repeat occur alternating in said polypeptide.

18. A multimeric polypeptide which of polypeptides as defined in any one of statements 1 to 17. In such multimer the polypeptides may be non-covalently bound to each other, and optionally via the presence of disulfide cystine bridges.

19. The multimeric polypeptide according to statement 18, which is a hexamer of 3 non non-covalently bound polypeptides as defined in any one of statements 1 to 9 having two repeats.

20. The multimeric polypeptide according to statement 19, which is a hexamer of 2 non non-covalently bound polypeptides as defined in any one of statements 1 to 9 having 3 repeats.

The invention further relates to nucleic acids encoding these polypeptides, as well as expressions vector comprising these nucleic acids, and bacterial yeast or eukaryotic cells comprising these vectors.

21. A method of producing a functional protein comprising the steps of:

• • a) providing a polypeptide according to any one of statements 1 to 17, wherein Xn is random selected or designed by an in silico method,
  • b) generating a multimeric protein of said polypeptides,
  • c) testing the multimeric protein for the function, and
  • d) selecting the multimer protein with the function.

22. The method according to statement 21, wherein the function is protein binding, an enzymatic activity, or the binding to an organic molecule.

23. The method according to statement 21 or 22, further comprising the step of determining whether the multimeric protein has rotational symmetry.

24. The method according to any one of statements 21 to 23, further comprising the step of determining whether the multimeric protein is stable at pH below 4, or wherein the multimeric protein is resistant against proteolytic degradation.

25. The method according to any one of statements 21 to 24, further comprising determining whether said multimeric protein assembles into quaternary structures, such as fibres, tubes, three dimensional cages or two-dimensional layers.

DETAILED DESCRIPTION

Figure Legends

FIG. 1. Overview of the RE₃Volutionary design strategy. The human keap1 β-propeller was used as a template (PDB: 1ZGK). A. The blades were separated. B. Using Rosetta SymDock, a perfect sixfold symmetric protein backbone was constructed from one blade. C. Simultaneously, a multiple sequence alignment (MSA) was constructed from the six unique blades. D. Putative ancestral sequences, derived from the MSA using the FastML server, are mapped on the perfect symmetric backbone. The resulting models were evaluated by their Rosetta Talaris2013 energy score and root mean square deviation (RMSD) from the ideal symmetric backbone. For each SAKe type, three designs were experimentally tested.

FIG. 2. A. APBS calculated surface electrostatics for the S6BE normal crystal structure at pH 8.0 and the self-assembled crystal structure at pH 4.0. B. Close-up of the axial hydrogen bonding networks found in the self-assembled S6BE crystal. C. Close-up of the lateral hydrogen bonding networks found in the self-assembled S6BE crystal. Additionally, there is a hydrophobic interaction between two prolines (5° A distance). The only structural difference between the normal and self-assembled S6BE crystals is found in the alternative conformations of the lateral glutamates, here shown in slate/wheat (pH 4) and white (pH 7-8).

FIG. 3. AFM imaging of S6BE-3HH:Cu(II) 2D arrays. A. Rectangular-dimeric lattices are formed at a ratio of 1:10 (protein:Cu(II)).(i) Topography map of 1 μM S6BE-3HH, 10 UM Cu(II) (100 nm scale bar) and (ii) selected line profiles. (iii) Topography map of 0.5 MM S6BE-3HH, 5 MM Cu(II) (20 nm scale bar). (iv) Dimensions calculated from the packing of a S6BE-3HH crystal (PDB code 7OPU)). B. Hexagonal lattices are generated at a ratio of 1:20 (1 μM S6BE-3HH:20 μM Cu(II)). (i) Topography map (100 nm scale bar) and (ii) selected line profiles. (iii) Topography map (20 nm scale bar), (iv) filtered zoom image of selected region (scale bar=10 nm) and (v) phase map (20 nm scale bar). (vi) Dimensions calculated from the packing of a S6BE-3HH crystal (PDB code 7OPV)). All AFM imaging was performed in 20 mM MES buffer (pH 5.6) and on a mica surface.

FIG. 4: Sequence logos generated with Consurf and WebLogo, 1,2 using 150 sequences from the NR proteins database and the default Consurf settings. (Top) Logo generated from the second blade of the human keap1 β-propeller (PDB code: 1ZGK).3 A clear region with low conservation corresponds to the protein's loops. (Bottom) Logo generated from the third blade of the Mycobacterium tuberculosis PknD β-propeller (PDB code: 1RWL).4 This protein was the template for the design of Pizza and lacks large variable regions.5

FIG. 5: (Left) Circular Dichroism (CD) spectra of SAKe proteins were similar to that of the parent human keap1 β-propeller. (Right) The CD signal at 233 nm was followed in function of temperature. Compared to keap1, all SAKe designs have an improved melting temperature. Between SAKe designs, loop length and composition seem to have the more pronounced impact on thermal stability. When loops are conserved (as is the case between S6A proteins), the scaffold sequence can still have a significant influence as well.

FIG. 6: Top and side views of crystal structures of Pizza6, A-type SAKe (S6A), B-type SAKe (S6B), S6BE-L1, S6BE-L2 and S6BE-L3. The modular loops are coloured in slate and their respective lengths are given. For S6BE-L2, the loops were too flexible and not visible in the electron density map. For visualization purposes, they have been modelled in this figure.

FIG. 7: Self assembled crystals of S6BE, grown in (A) 50 mM Na acetate-acetic acid pH 4.0 and (B) 50 mM Na citrate-citric acid pH 4.0. Both batches grew crystals after overnight dialysis of 20 mg/ml protein at 4° C. The bubbles have an approximate diameter of 5 mm.

FIG. 8: Various concentrations of S6BE were dialyzed in parallel to either 50 mM Na citrate-citric acid pH 4.0 or 4.5. At pH 4.5, crystals only grew after 144 h to 216 h. At pH 4.0, the proteins readily crystallized after 6 h to 72 h, depending on their concentration. The first pictures that show crystals are marked in green. The bubbles have a radius of approximately 5 mm.

FIG. 9: Various concentrations of S6BE-3HH were dialyzed in parallel to either 50 mM Na citrate-citric acid pH 4.0 or 4.5. At pH 4.5, crystals only grew after 144 h to 216 h. At pH 4.0, the proteins readily crystallized after 24 h to 216 h, depending on their concentration. The first pictures that show crystals are marked in green. The bubbles have a radius of approximately 5 mm.

FIG. 10: 5 mg/mL aliquots of S6BE and S6BE-3HH were dialyzed to 50 mM Na citrate citric acid at varied pH. S6BE yielded more crystals and they grew faster than S6BE-3HH. At pH 4.5, crystals remained stable. From pH 5.0 onward they dissolved again. The first pictures that show crystals were marked in green, while the first notices of disassembly were marked in red. The bubbles have a radius of approximately 5 mm.

FIG. 11: (A) The hexagonal crystal packings of normal (0.2 M K formate, 20% (w/v) PEG3350) and self-assembled (50 mM Na acetate-acetic acid pH 4.0) S6BE crystals are identical. The only obvious difference is found in the alternative rotamers for the lateral Glu. (B) In the normal crystal these Glu point outwards (0.48 occupancy of carboxy group, alternative rotamers were not visible). (C) The protonated Glu in the self-assembled crystal turn inward to form a putative hydrogen bridge with a serine hydroxyl group. Additionally, electrostatic repulsion in the normal crystal might direct the normal crystal's Glu outward.

FIG. 12: To enable metal-induced self-assembly, three double-histidine sites were added in S6BE (creating S6BE-3HH). A closely packed structure resembling the self-assembled crystal structure is obtained through a combination of metal-mediated and complementary interactions of adjacent proteins. The vacancies of the hexagonal lattice are likely to be occupied by S6BE-3HH proteins.

FIG. 13: DLS spectra of S6BE-3HH with different ratios of Cu(NO3)2 in MilliQ pH 7.0 (A) and 20 mM MES pH 5.6 (B). DLS spectrum of S6BE-3HH with different ratios of Zn(NO3)2 in 20 mM MES pH 5.6 (C).

FIG. 14: DLS spectra of S6BE with different ratios of Cu(NO3)2 in MilliQ pH 7 (A) and 20 mM MES PH 5.6 (B).

FIG. 15: AFM of 1 UM S6BE-3HH imaged in 20 mM MES pH 5.6 on mica. (A) Topography map, (B) phase map and (C) amplitude map. The scale bar on all images is 100 nm. (D) Height traces corresponding to the line profiles taken from the Topography map.

Particle analysis of the topography, (E) average radius and (F) maximum diameter.

FIG. 16: AFM of 0.5 UM S6BE-3HH, 5 UM Cu(NO3)2 imaged in 20 mM MES pH 5.6 on mica. Topography maps (A, D & G), phase maps (B, E & H) and amplitude maps (C, F & I). The scale bar is 100 nm for images A-C, 50 nm for D-F and 20 nm for G-I.

FIG. 17: AFM of 1.0 UM S6BE-3HH, 10 UM Cu(NO3)2 imaged in 20 mM MES pH 5.6 on mica. Topography map (A), phase map (B) and amplitude map (C). The scale bar is 100 nm. (D) Height traces corresponding to the line profiles taken from the topography map (A). (E) 2D-FFT map of the highlighted area selections in phase map (B), and schematic of the array's unit cell derived from the calculated distances of the 2D-FFT map and height traces (D).

FIG. 18: AFM of 1.0 UM S6BE-3HH, 20 UM Cu(NO3)2 imaged in 20 mM MES pH 5.6 on mica. Topography map (A), phase map (B) and amplitude map (C). The scale bar is 100 nm. (D) Height traces corresponding to the line profiles taken from the topography map (A). (E) 2D-FFT map of the highlighted area selection in phase map (B), and schematic of the array's unit cell derived from the calculated distances of the 2D-FFT map and height traces (D).

FIG. 19: AFM of 1.0 μM S6BE-3HH, 20 μM Cu(NO3)2 imaged in 20 mM MES pH 5.6 on mica. Topography map and 2D-FFT (A), phase map and 2D-FFT (B) and Amplitude map (C). The scale bar is 20 nm for A-C. (D) Projection and filtered image corresponding to the area selection in the topography map (A). The scale bar is 10 nm. (E) 2D-FFT map of the phase image (B), and schematic of the array's unit cell derived from the calculated distances of the 2D-FFT map.

FIG. 20: AFM of 1.0 μM S6BE-3HH, 30 μM Cu(NO3)2 imaged in 20 mM MES pH 5.6 on mica. Topography maps (A & D), phase maps (B & E) and amplitude maps (C & F). The scale bar is 100 nm for A-C and 50 nm for D-F. (G) Height traces corresponding to the line profiles taken from the topography map (A). (E) 2D-FFT map of the highlighted area selections in phase map (E), and schematic of the array's unit cell derived from the calculated distances of the 2D-FFT map and height traces (G).

FIG. 21: AFM of 1.0 μM S6BE-3HH, 20 μM Cu(NO3)2 imaged in 20 mM MES pH 5.6 on HOPG. Topography maps (A & D), phase maps (B & E) and amplitude maps (C & F). The scale bar is 100 nm for A-C and 20 nm for D-F. (G) Height traces corresponding to the line profiles taken from the topography maps (A & D). (E) 2D-FFT map of the highlighted area selections in topography map (D), and schematic of the array's unit cell derived from the calculated distances of the 2D-FFT map.

FIG. 22: AFM of 1.0 μM S6BE-3HH, 10 μM Zn(NO3)2 imaged in 20 mM MES pH 5.6 on Mica. Topography map (A), phase map (B) and Amplitude map (C). The scale bar is 100 nm. (D) Height traces corresponding to the line profiles taken from the topography map (A).

FIG. 23: AFM of 1.0 μM S6BE, 20 μM Cu(NO3)2 imaged in 20 mM MES pH 5.6 on mica. Topography maps (A & D), phase map (B) and amplitude map (C). The scale bar is 100 nm for A-C and 50 nm for D. (E) Height traces corresponding to the line profiles taken from the topography maps (A & D).

FIG. 24: Manual sequence alignment of all SAKe design sequences. Each entry contains only one blade. Proteins that have two blades per repeat span two entries. The amino acid sequences between the GG and LNS motifs are annotated as the protein's variable loop regions.

FIG. 25: SDS page from samples collected during preparative SEC for proteins S6BE-3CHR, S6BE-3 HR-L3, mEm-v22-S6BE-3 HR FIG. 26: SDS page from samples collected during nickel affinity chromatography expression testing of dS6AC. Clearly, the protein expresses as a genetic fusion of two S6AC units. F: Flow through. B: Wash B fraction. E: Elution fraction. I: Inclusion bodies.

FIG. 27: SDS page from samples collected during nickel affinity chromatography purification of S6BE-3 HR. BD: Before dialysis, with hexahistidine tag. AD: After dialysis, without hexahistidine tag. F: Flow through. A: Wash A fraction. B: Wash B fraction. C: Wash C fraction. Multiple thicker bands indicate presence of partially/non-denatured species. This has been reported for thermostable SAKe such as S6BE and S2BE.

FIG. 28: SDS page from samples collected during nickel affinity chromatography purification of S2BE-3 HR. BD: Before dialysis, with hexahistidine tag. AD: After dialysis, without hexahistidine tag. F: Flow through. A: Wash A fraction. B: Wash B fraction. C: Wash C fraction. Multiple thicker bands indicate presence of partially/non-denatured species. This has been reported for thermostable SAKe such as S6BE and S2BE.

FIG. 29: Size Exclusion Chromatograms for various SAKe mutants. A: S6BE-3 HR on a HiLoad Superdex 75 pg 16/600 column. B: S6BE-3CHR on a HiLoad Superdex 200 pg 16/600 column. C: S2BE-3 HR on a HiLoad Superdex 75 pg 16/600 column. D: S6BE-3 HR-L3 on a HiLoad Superdex 200 pg 16/600 column. E: mEm-v22S6BE-3 HR on a HiLoad Superdex 200 pg 16/600 column. F: dS6AC on a HiLoad Superdex 200 pg 16/600 column.

FIG. 30: Structures of Zn-induced SAKe cages, determined via xray diffraction. A to C: Upon addition of Zn(II), S6BE-3 HR and S6BE-3CHR assemble similar tetrameric cages in solution. D: In S6BE-3 HR the Zn-binding site is 2His-2Asp. E: In S6BE-3CHR, the Zn-binding site contains a 2His-2Cys Zinc finger motif.

FIG. 31: Crystal structure of S2BE-3 HR cages

The present invention discloses the design and engineering an improved protein building block named the Self-Assembling Kelch (SAKe) protein. SAKe has a stable, symmetric design with readily accessible loops that can be varied in both sequence and length to later bind larger molecules or scaffold a catalytic site. To demonstrate SAKe's versatility, its structure was modified to undergo metal-mediated self-assembly into 2D surface arrays. This highlights SAKe as a promising new protein scaffold which can be readily redesigned to target various applications.

Through an investigation into naturally occurring pseudosymmetric proteins, the human keap1 Kelch protein was identified as a template for the development of SAKe [Beamer et al. Acta. Crystallogr., Sect. D.: Biol. Crystallogr. 2005, 61, 1335-1342].

Kelch repeat proteins are β-propeller proteins composed of six nearly identical tandem sequence repeats that fold into 4-stranded anti-parallel sheets around a central cavity [Adams et al. Trends Cell Biol. 2000, 10, 17-24]. This structural family has well-conserved blades, with the first and second strands connected by loops that vary in length and sequence. Using a computational procedure that combines ancestral sequence reconstruction with computational protein backbone optimization and subsequent sequence scoring, a new family of proteins named SAKe were derived from the keap1 β-propeller (FIG. 1). Initially, six different SAKe proteins were designed and evaluated. They varied in their core amino acid sequence and length of surface loops. The variable “top-side” loops sit between the inner two β-strands of the propeller's blades; Atype SAKe (S6AE, S6AR and S6AC) loops have 10 amino acids while B-type SAKe (S6BE, S6BR and S6BC) loops have 6. Four of the six proteins were successfully expressed and readily crystallized: three A-type SAKes (S6AE, S6AR and S6AC) and one B-type SAKe (S6BE). Each protein has perfect 6-fold symmetry, with a central cavity surrounded by flexible loops on the “top” face. Circular Dichroism (CD) spectroscopy at elevated temperatures highlights their exceptional thermal stability, whereby S6BE exhibits a melting temperature (T_m) of over 95° C.

To investigate loop modification and further functionalisation, three loop variants of S6BE were created (FIG. 6). S6BE-L1 was created by conserving the intersection of S6Atype and S6B-type loops, while adding in 4 amino acids. For S6BE-L2, a larger part of the S6A-type loop was conserved with the addition of 4 amino acids. These 4 amino acid sequences were random Tyr-containing combinations of frequently occurring residues in nanobody CDR motifs [Zuo et al. BMC genomics 2017, 18, 797], and are not found in the natural Kelch proteins. S6BE-L3, was created, which is a 3-fold symmetric variant with two long loops of various length through grafting them from the Kelch domain of human KBTBD5 [Canning et al. Journal of Biological Chemistry 2013, 288, 7803-7814]. All 3 proteins were successfully purified, and their structures were confirmed via X-ray diffraction (XRD). However, the S6BE loop variants were found to be less stable than the original S6BE, with T_m dropping to a minimum of 51.7° C. as the loop lengths increased (FIG. 5). Nonetheless, all of these designs are significantly more stable than the template keap1 β-propeller, which unfolds at 44.1° C.

SAKe's symmetry and modifiable interfaces make it an ideal building block for construction of macromolecular assemblies. While dialyzing S6BE to low pH, a pH induced self-assembly was observed (FIG. 7. This pH responsive behaviour was further investigated through dialysis experiments and XRD. The tipping point for this self-assembly was found to be around pH 4.0 (FIGS. 8 and 9). At pH 4.0, large crystals were observed for S6BE within a few hours up to two days, depending on the concentration. In contrast, crystals only formed after 6 days at 5 mg/ml when dialyzing to pH 4.5. The self-assembled crystals grown at pH 4.0 remained stable at pH 4.5. However, they disassembled at pH 5.0 (FIG. 10), but readily re-assembled and formed crystals again when the pH was dropped to pH 4.0. To understand this reversible self-assembly mechanism, the crystals formed at different pH were investigated with XRD. These self-assembled structures revealed identical packing to the crystals grown via vapor diffusion at pH 7.0-8.5. At pH 8.0, His (pKa3 6.08-6.90) and Glu (pKa 4.3-4.4) are deprotonated and the net positive charge of S6BE likely causes mutual repulsion. However, at pH 4 these residues are protonated. Due to its symmetry and dipole-like character, S6BE shape and charge complementarity is then expected to allow for self-assembly into a tight hexagonal packing (FIGS. 2 and 11). A series of hydrogen bonds and hydrophobic interactions can then further stabilize the assembly. Similar mechanisms have been reported for the capsid proteins of cowpea chlorotic mosaic virus (CCMV) [Speir et al. Structure 1995, 3, 63-78; Lavelle et al. J. Phys. Chem. B 2009, 113, 3813-3819; Van Eldijk et al. J. Am. Chem. Soc. 2012, 134, 18506-18509].

The hexagonal packing and complementary interactions found in S6BE's self-assembled structures provide a promising starting point for its application as a supramolecular building block. Therefore, this protein was rationally reengineered to coordinate divalent metal-ions, and induce self-assembly into on-surface 2D arrays (FIG. 11). 6 residues in S6BE were selected to be mutated to His residues, creating the S6BE-3HH variant. These double His sites can be found on the bottom protrusions of the 1st, 3th and 5th blade, forming 3-fold rotational symmetry. Previous reports have combined a similar metal coordination strategy with naturally occurring symmetric protein oligomers to fabricate nano-particles and crystalline protein arrays [Salgado et al. Acc. Chem. Res. 2010, 43, 661-672; Huard et al. Nat. Chem. Biol. 2013, 9, 169-176; Suzuki et al. Nature 2016, 533, 369-373].

The metal-induced assembly of S6BE and S6BE-3HH proteins was first screened using Dynamic Light Scattering (DLS) in solution. Divalent metals (Cu(NO₃)₂ and Zn(NO₃)₂) were titrated into the protein solution in different buffers and pH, resulting in larger structures being formed as the ratio of metal:protein was increased (FIGS. 13 and 14). Interestingly, in both neutral and acidic conditions and at a ratio of 3:1 Cu(II): S6BE-3HH, an intermediate peak with a diameter of ≈10 nm can be recognized before the structures reached larger indistinguishable sizes at higher ratios.

To investigate the formation of on-surface 2D protein arrays, in solution amplitude-modulated atomic force microscopy (AFM) was utilized (FIG. 3). In the absence of metals and on a muscovite mica surface, it was recognized that the optimal conditions for imaging S6BE and S6BE-3HH was in a mildly acidic buffer (20 mM MES pH 5.6). In these conditions, both proteins adhered to the surface and adopted monomeric species with some small aggregates (FIG. 15). It is likely that in acidic conditions the proteins carry enough positively charged residues to form complementary interactions with the negatively charged mica surface. Organized 2D surface arrays were first observed for the S6BE-3HH protein at a ratio of 10:1 Cu(II): protein with an overall protein concentration of 0.5-1.0 μM, whilst imaging in 20 mM MES buffer (pH 5.6). The stable 2D surface crystals adopted high aspect ratio rectangular geometries with a preferred directional growth (FIG. 3A). Through analysing the dimensions derived from the 2D-FFT of selected surface crystals and their height profiles, the unit cell of these arrays could be obtained with c≈4.5 nm, a=7.23 nm, b=4.84 nm and γ=90.20 (FIG. 17). These unit cell dimensions correspond to arrays composed of S6BE-3HH dimers, and are similar to the 2D protein arrangements found in a S6BE-3HH crystal structure (FIG. 3A(iv)). Therefore, it is highly likely that these arrays are composed of protein dimers arranged bottom-bottom, which are bridged via Cu(II) ions and complementary interactions. At a ratio of ≥20:1 (Cu(II): S6BE-3HH), the surface arrays were found to transition from the rectangular-dimer species to hexagonal-honeycomb lattice. The 2D-FFT of these crystals confirmed their highly crystalline hexagonal packing with unit cell dimensions of a=4.62 nm, b=4.64 nm and γ=61.60 (FIGS. 3B and S18). This unit cell coupled with the height traces of individual domains (c˜ 3.2 nm) suggested that these 2D surface arrays were similar in geometry to the planar protein packing in a S6BE-3HH crystal (a=4.70 nm, b=4.72 nm, α=90.00, β=90.00 and γ=120.00). Similar crystalline structures were also observed on a highly oriented pyrolytic graphite (HOPG) surface (FIG. 21). The unit cell of the hexagonal array domains derived from the 2D-FFT (a=4.55 nm, b=4.51 nm and α=61.21) had similar dimensions to that obtained for the arrays on mica in the same conditions. However, the hexagonal arrays appear to have more vacancies and imperfections within the lattices. Through replacing Cu(NO₃)₂ with Zn(NO₃)₂, which is attributed with a similar atomic radius but a different tetrahedral coordination, no ordered arrays for S6BE-3HH could be obtained in the same imaging conditions (mica, 20 mM MES pH 5.6). Instead, aggregated proteins were observed with no obvious crystallinity (FIG. 22). This highlights the importance of Cu(II) and its square-planar coordination in the formation of these arrays.

The assembly of S6BE in the presence of Cu(II), which does not contain His mutations on its bottom loops was also investigated. At a ratio of 20:1 Cu(II): S6BE, amorphous aggregated proteins were observed with no obvious crystallinity (FIG. 23). This indicates the importance of the His mutations in S6BE-3HH and their role in facilitating Cu(II) coordination and subsequent assembly of 2D arrays. At pH 5.6, these His residues are close to their pka and are likely to adopt a mixture of protonated and deprotonated states. The pKa may also be lowered and mediated by nearby local bases such as Glu, further facilitating deprotonation and assembly in the presence of excess of Cu(II). In addition, pH 5.6 is close to S6BE-3HH pI (5.32), where the proteins will be predominantly neutralized and will no longer repel each other. A similar mechanism has been previously reported for Zn-mediated protein assemblies at pH 5.5 [Brodin et al. Nat. Chem. 2012, 4, 375-382]. Though demonstrating the Cu(II) dependent structural self-assembly, the square planar coordination of Cu(II) combined with the bi-chelate His mutations and shape complementarity of S6BE-3HH appears important for connecting adjacent proteins into close packed 2D arrays (FIG. 12).

Using a computational approach combining consensus design and Rosetta energy scoring, SAKe proteins were developed: a new symmetric, stable protein scaffold with modifiable loops. The loops can be varied in both length and sequence, highlighting their potential to be optimized for the binding of clinically relevant molecules or programming of catalytic activity. Following SAKe's modification with metal binding sites, Cu(II) induced self-assembly was observed of on-surface 2D arrays. The present invention discloses SAKe as a highly modifiable protein scaffold which can double as a building block for the fabrication of 2D protein assemblies. In summary, SAKe's versatility holds great promise for the creation of biotherapeutics and innovative on-surface materials.

The following crystal structures were submitted to RCSB PDB: SAKe6AE (7ON6), SAKe6AR (7ON8), SAKe6AC (7ONA), SAKe6BE (7ONC and 7ONE), SAKe6BE3HH (7OP4, 7OPU and 70PV), SAKe6BE-L1 (7ONG), SAKe6BE-L1 (7ON7) and SAKe6BE-L1 (7ONH).

Overview of DNA and Protein Sequences:

SEQ ID NO: 1 to SEQ ID NO: 19 (odd numbers): DNA sequences of designed SAKe proteins. The outermost emphasized 5′ and 3′sequences contain a start codon, NdeI restriction site, stop codon and XhoI restriction site.

SEQ ID NO: 1 to SEQ ID NO: 19 (even numbers): corresponding amino acid sequences.
S6AE [SEQ ID NO: 1]
GGGAATTCCATATGGATGGACGTATATACGCGGTTGGCGGGTATGATGGAAGTCCCGACGGTCATACGCA
CCTGAATTCTGTCGAAGCCTACGATCCCGAGACCGACACTTGGAGTCTGGTTGCCCCCATGAAAACTCGC
CGTTCTGGTGTAGGAGTTGCGGTCCTGGACGGCAGAATCTATGCCGTTGGCGGGTATGATGGATCCCCTG
ACGGCCACACTCACCTTAATTCAGTGGAGGCCTATGATCCCGAAACCGATACCTGGAGCTTGGTGGCCCC
CATGAAGACACGGCGGTCGGGCGTCGGCGTAGCGGTCTTGGATGGGAGAATATATGCAGTTGGTGGGTAC
GATGGGTCGCCTGATGGACACACTCATTTGAACTCCGTAGAAGCTTACGATCCGGAGACGGATACATGGT
CTCTTGTGGCACCTATGAAAACCCGGAGATCTGGCGTCGGAGTCGCCGTATTGGATGGCAGAATATACGC
TGTCGGGGGCTACGACGGCAGCCCTGACGGTCACACTCACCTTAATTCTGTAGAGGCATACGACCCCGAA
ACAGACACGTGGAGCCTGGTTGCTCCAATGAAAACACGCAGATCCGGCGTGGGGGTAGCGGTGCTGGATG
GCCGTATCTACGCGGTTGGAGGGTATGACGGCAGCCCCGATGGTCACACCCACCTGAATAGTGTGGAAGC
GTATGATCCTGAGACGGACACCTGGTCTTTGGTGGCTCCTATGAAAACGAGACGTTCAGGAGTAGGAGTT
GCAGTTTTGGACGGTCGTATTTATGCAGTTGGGGGATATGACGGATCCCCGGACGGCCACACGCACTTGA
ATTCAGTTGAAGCTTATGACCCAGAGACTGATACCTGGTCGCTGGTTGCTCCGATGAAAACGCGGCGGAA
CGGTGTTGGCGTGGCTGTCCTTTAACTCGAGCGG
S6AE [SEQ ID NO: 2]
MDGRIYAVGGYDGSPDGHTHLNSVEAYDPETDTWSLVAPMKTRRSGVGVAVL
DGRIYAVGGYDGSPDGHTHLNSVEAYDPETDTWSLVAPMKTRRSGVGVAVL
DGRIYAVGGYDGSPDGHTHLNSVEAYDPETDTWSLVAPMKTRRSGVGVAVL
DGRIYAVGGYDGSPDGHTHLNSVEAYDPETDTWSLVAPMKTRRSGVGVAVL
DGRIYAVGGYDGSPDGHTHLNSVEAYDPETDTWSLVAPMKTRRSGVGVAVL
DGRIYAVGGYDGSPDGHTHLNSVEAYDPETDTWSLVAPMKTRRNGVGVAVL
S6AR [SEQ ID NO: 3]
GGGAATTCCATATGAATAGATTGTTGTACGCTGTTGGGGGGTATGATGGTAGTCCAGATGGCCATACCCA
CCTGAACTCCGTTGAGTGCTACGATCCAGAGACCAACGAATGGTCGCTTGTAGCACCTATGAATACCCGC
CGTTCAGGCGTTGGGGTAGCTGTTTTAAATCGCCTGCTGTACGCGGTTGGCGGGTACGACGGATCCCCTG
ATGGGCACACCCACTTGAACTCTGTAGAATGCTATGACCCTGAAACGAATGAGTGGTCCCTTGTCGCTCC
CATGAATACGAGACGTTCAGGAGTTGGAGTTGCAGTTTTAAACCGCCTTCTTTATGCGGTTGGCGGTTAC
GACGGTAGCCCAGATGGACACACTCACTTGAATTCTGTGGAGTGCTACGACCCTGAGACGAACGAATGGA
GTTTAGTTGCACCGATGAATACGCGCCGGAGCGGTGTAGGTGTAGCCGTGCTTAATCGGTTACTTTACGC
AGTAGGGGGTTACGACGGATCTCCTGACGGTCACACCCACTTAAACTCTGTCGAATGTTACGATCCTGAG
ACTAACGAGTGGTCTTTAGTCGCTCCCATGAATACCCGCCGCAGCGGCGTAGGCGTAGCGGTACTTAACC
GGTTGTTGTATGCAGTTGGCGGGTACGACGGGTCACCGGACGGCCACACGCACCTGAACTCTGTCGAATG
TTACGACCCCGAAACAAATGAGTGGAGCCTGGTGGCCCCTATGAACACTCGCAGATCAGGAGTCGGGGTA
GCGGTGTTAAACAGACTTCTGTATGCTGTGGGAGGCTACGATGGATCCCCGGACGGGCATACCCATCTGA
ATTCAGTGGAGTGTTATGATCCCGAGACGAACGAATGGTCGCTTGTAGCGCCCATGAATACGCGGAGAAG
CGGTGTCGGCGTTGCGGTCCTTTAACTCGAGCGG
S6AR [SEQ ID NO: 4]
MNRLLYAVGGYDGSPDGHTHLNSVECYDPETNEWSLVAPMNTRRSGVGVAVL
NRLLYAVGGYDGSPDGHTHLNSVECYDPETNEWSLVAPMNTRRSGVGVAVL
NRLLYAVGGYDGSPDGHTHLNSVECYDPETNEWSLVAPMNTRRSGVGVAVL
NRLLYAVGGYDGSPDGHTHLNSVECYDPETNEWSLVAPMNTRRSGVGVAVL
NRLLYAVGGYDGSPDGHTHLNSVECYDPETNEWSLVAPMNTRRSGVGVAVL
NRLLYAVGGYDGSPDGHTHLNSVECYDPETNEWSLVAPMNTRRSGVGVAVL
S6AC [SEQ ID NO: 5]
GGGAATTCCATATGAATGGCCGCATTTATGCCGTAGGGGGGTATGACGGATCCCCAGACGGACATACACA
TTTAAATTCGGTAGAGGCATACGACCCGGAAACGGACGAGTGGTCGTTTGTAGCTCCCATGACAACGCCA
CGGTCCGGAGTCGGGGTTGCCGTGCTTAATGGCCGTATATATGCCGTCGGGGGTTACGACGGATCGCCAG
ATGGGCACACTCATTTGAATAGTGTTGAAGCTTACGATCCGGAGACGGATGAGTGGTCATTTGTTGCTCC
GATGACAACCCCGCGTAGTGGTGTTGGAGTTGCAGTCCTGAACGGCCGGATCTATGCCGTCGGCGGTTAT
GACGGTTCTCCTGATGGACACACGCACTTGAATTCAGTAGAGGCTTACGACCCTGAAACAGATGAATGGT
CCTTTGTTGCCCCGATGACTACACCAAGATCGGGAGTTGGGGTGGCTGTCCTGAATGGGAGAATTTATGC
GGTAGGCGGATACGATGGTAGTCCAGATGGCCACACACACTTAAACTCCGTCGAGGCATACGACCCGGAG
ACGGATGAATGGTCATTCGTTGCACCAATGACTACACCTCGGTCCGGGGTCGGCGTGGCAGTATTAAACG
GGCGTATCTATGCCGTTGGTGGCTACGACGGAAGTCCCGACGGCCATACGCATCTGAACTCGGTGGAAGC
CTATGACCCCGAAACTGATGAATGGAGTTTTGTGGCGCCCATGACGACGCCTCGCTCCGGCGTCGGGGTA
GCTGTGCTGAACGGGCGGATTTACGCGGTTGGTGGTTATGATGGATCCCCTGACGGACATACTCACTTGA
ATTCTGTGGAAGCTTATGACCCAGAGACCGACGAGTGGTCTTTCGTGGCGCCGATGACGACACCCCGCTC
AGGAGTCGGAGTGGCGGTATTGTAACTCGAGCGG
S6AC [SEQ ID NO: 6]
MNGRIYAVGGYDGSPDGHTHLNSVEAYDPETDEWSFVAPMTTPRSGVGVA
VLNGRIYAVGGYDGSPDGHTHLNSVEAYDPETDEWSFVAPMTTPRSGVGV
AVLNGRIYAVGGYDGSPDGHTHLNSVEAYDPETDEWSFVAPMTTPRSGVG
VAVLNGRIYAVGGYDGSPDGHTHLNSVEAYDPETDEWSFVAPMTTPRSGV
GVAVLNGRIYAVGGYDGSPDGHTHLNSVEAYDPETDEWSFVAPMTTPRSG
VGVAVLNGRIYAVGGYDGSPDGHTHLNSVEAYDPETDEWSFVAPMTTPRS
GVGVAVL
S6BE [SEQ ID NO: 7]
GGGAATTCCATATGAATGGACTGATATACGCGGTTGGCGGATATGATGGAAACACGCATTTAAACTCTGT
TGAAGCGTATGATCCCGAACGCAATGAGTGGTCCTTAGTTGCACCACTTTCGACTCGGCGGTCGGGGGTA
GGGGTTGCAGTATTAAATGGGCTTATTTATGCAGTTGGAGGCTATGATGGAAATACACATCTGAATTCTG
TGGAGGCCTATGATCCCGAGCGGAATGAGTGGTCTCTGGTGGCCCCGCTGAGCACTCGCCGTTCAGGGGT
CGGCGTGGCCGTTCTGAACGGGCTTATATATGCCGTGGGGGGTTACGACGGCAACACACACTTAAATTCT
GTAGAAGCTTACGATCCCGAGAGAAACGAGTGGTCCCTTGTCGCTCCTTTATCCACCCGCAGAAGCGGTG
TGGGTGTGGCTGTATTAAACGGTCTGATTTATGCCGTAGGGGGATACGACGGTAATACACACCTGAATAG
CGTTGAGGCCTATGATCCGGAGCGCAACGAATGGTCGTTGGTTGCGCCTTTGTCAACTCGCCGGTCTGGT
GTGGGGGTAGCCGTGTTGAACGGCCTGATATATGCGGTGGGTGGGTATGATGGAAACACTCACCTGAACT
CGGTAGAGGCGTACGATCCAGAGCGCAACGAGTGGTCTCTGGTCGCCCCACTGTCGACTCGCCGGTCAGG
CGTTGGGGTTGCCGTACTTAACGGCCTGATTTATGCGGTCGGTGGTTATGACGGGAACACTCACCTGAAT
TCCGTGGAAGCTTATGACCCAGAACGCAATGAATGGAGTCTGGTTGCACCCCTGTCTACTCGTAGATCGG
GGGTCGGGGTAGCTGTCTTGTAACTCGAGCGG
S6BE [SEQ ID NO: 8]
MNGLIYAVGGYDGNTHLNSVEAYDPERNEWSLVAPLSTRRSGVGVAVL
NGLIYAVGGYDGNTHLNSVEAYDPERNEWSLVAPLSTRRSGVGVAVL
NGLIYAVGGYDGNTHLNSVEAYDPERNEWSLVAPLSTRRSGVGVAVL
NGLIYAVGGYDGNTHLNSVEAYDPERNEWSLVAPLSTRRSGVGVAVL
NGLIYAVGGYDGNTHLNSVEAYDPERNEWSLVAPLSTRRSGVGVAVL
NGLIYAVGGYDGNTHLNSVEAYDPERNEWSLVAPLSTRRSGVGVAVL
S6BE-3HH [SEQ ID NO: 9]
CCAATTCATATGTTAATTTACGCTGTGGGCGGATACGACGGCAATACACATTTGAACAGTGTCGAGGCTT
ATGATCCTGAACGTAATGAGTGGTCCTTGGTGGCACCGTTGTCAACGCGCCGTTCCGGGGTAGGGGTAGC
AGTGTTAAATGGCTTGATTTATGCGGTGGGTGGTTATGACGGGAACACCCACTTAAATAGCGTTGAGGCC
TATGATCCGCATCACAACGAATGGAGTCTGGTCGCCCCTCTGAGTACCCGTCGCAGCGGAGTAGGTGTTG
CTGTCTTAAATGGCTTAATTTACGCGGTCGGCGGTTATGATGGAAATACGCACCTGAATTCCGTAGAAGC
TTACGATCCGGAACGCAATGAATGGTCATTAGTGGCGCCATTATCCACGCGTCGCAGCGGAGTAGGCGTG
GCGGTATTAAACGGTCTTATTTACGCAGTAGGGGGGTACGACGGAAATACTCACTTAAACTCCGTAGAGG
CGTATGACCCACATCACAATGAGTGGAGCCTGGTTGCGCCATTGTCTACACGCCGTAGTGGCGTCGGAGT
TGCTGTATTAAACGGGTTAATCTATGCCGTTGGTGGGTACGATGGAAACACACATCTGAATTCAGTGGAG
GCCTATGATCCAGAACGCAACGAATGGTCTCTGGTCGCACCGTTATCTACTCGCCGTAGCGGAGTGGGAG
TAGCAGTTTTGAATGGCTTGATCTACGCTGTGGGCGGTTACGACGGGAATACCCATCTTAACTCTGTCGA
GGCTTACGACCCTCATCACAACGAATGGTCACTGGTGGCACCCTTATCGACTCGTCGCTCTGGTGTGGGG
GTAGCAGTTCTTAATGGCTAACTCGAGTACACGAGG
S6BE-3HH [SEQ ID NO: 10]
MLIYAVGGYDGNTHLNSVEAYDPERNEWSLVAPLSTRRSGVGVAVLNG
LIYAVGGYDGNTHLNSVEAYDPHHNEWSLVAPLSTRRSGVGVAVLNG
LIYAVGGYDGNTHLNSVEAYDPERNEWSLVAPLSTRRSGVGVAVLNG
LIYAVGGYDGNTHLNSVEAYDPHHNEWSLVAPLSTRRSGVGVAVLNG
LIYAVGGYDGNTHLNSVEAYDPERNEWSLVAPLSTRRSGVGVAVLNG
LIYAVGGYDGNTHLNSVEAYDPHHNEWSLVAPLSTRRSGVGVAVLNG
S6BE-L1 [SEQ ID NO: 11]
GATATCCATATGAACGGGCTGATTTATGCCGTTGGAGGCTACGATGGGACAGGGTACAATACCCACCTGA
ATAGCGTAGAGGCTTATGATCCAGAACGTAATGAGTGGTCACTGGTCGCGCCTCTTTCCACGCGCCGCTC
AGGCGTTGGCGTGGCGGTTTTGAACGGATTGATCTACGCGGTTGGTGGTTACGACGGTACTGGCTACAAT
ACTCACTTGAATTCGGTGGAAGCTTATGACCCTGAACGCAATGAATGGAGTTTAGTAGCCCCGCTGAGCA
CGCGTCGCAGTGGCGTCGGCGTTGCGGTTCTTAACGGTTTGATCTATGCGGTCGGAGGATACGACGGGAC
AGGTTACAACACTCATTTAAACTCAGTCGAGGCTTACGATCCAGAGCGCAACGAGTGGTCCTTGGTTGCT
CCCCTTTCAACTCGTCGCTCAGGCGTCGGGGTTGCGGTTTTGAACGGCCTTATTTACGCTGTCGGAGGTT
ACGACGGAACGGGTTATAATACACATCTTAATAGCGTAGAGGCGTATGACCCGGAGCGCAATGAGTGGTC
TCTTGTAGCCCCTCTTAGCACTCGCCGCTCAGGGGTGGGTGTGGCTGTACTGAATGGCCTGATTTATGCA
GTTGGCGGATACGACGGCACGGGGTACAACACACACTTAAACAGTGTGGAAGCCTACGACCCTGAACGCA
ACGAGTGGTCTCTGGTCGCTCCGTTATCGACGCGCCGCTCGGGAGTAGGCGTAGCAGTGCTGAATGGTTT
AATCTACGCTGTGGGAGGATACGATGGCACTGGATACAATACGCATCTGAATTCCGTCGAAGCCTACGAC
CCCGAGCGTAATGAGTGGTCCTTAGTCGCCCCACTTTCGACTCGTCGTTCTGGAGTGGGGGTCGCAGTAT
TATAACTCGAGCGG
S6BE-L1 [SEQ ID NO: 12]
MNGLIYAVGGYDGTGYNTHLNSVEAYDPERNEWSLVAPLSTRRSGVGVAVL
NGLIYAVGGYDGTGYNTHLNSVEAYDPERNEWSLVAPLSTRRSGVGVAVL
NGLIYAVGGYDGTGYNTHLNSVEAYDPERNEWSLVAPLSTRRSGVGVAVL
NGLIYAVGGYDGTGYNTHLNSVEAYDPERNEWSLVAPLSTRRSGVGVAVL
NGLIYAVGGYDGTGYNTHLNSVEAYDPERNEWSLVAPLSTRRSGVGVAVL
NGLIYAVGGYDGTGYNTHLNSVEAYDPERNEWSLVAPLSTRRSGVGVAVL
S6BE-L2 [SEQ ID NO: 13]
GATATCCATATGAATGGATTGATTTACGCCGTAGGTGGTTATGATGGTAGTCCAGATTACTCGACAGGAA
CCCACTTGAATTCGGTAGAAGCCTACGATCCAGAACGTAATGAATGGTCGTTAGTGGCGCCGCTGTCCAC
ACGTCGTTCCGGGGTGGGAGTAGCAGTCTTGAACGGACTTATTTACGCCGTCGGGGGGTATGATGGATCC
CCGGATTACAGTACTGGAACCCACCTGAATAGTGTGGAAGCCTATGATCCCGAGCGCAATGAATGGAGTC
TGGTTGCACCCTTAAGTACCCGTCGTTCCGGCGTCGGAGTAGCAGTATTGAATGGCTTGATCTACGCGGT
AGGAGGGTACGACGGTTCTCCAGACTATAGTACTGGGACTCACCTTAATAGCGTCGAGGCCTACGATCCC
GAACGTAACGAGTGGTCCTTAGTTGCCCCCCTGTCCACACGCCGCTCTGGTGTAGGCGTAGCTGTCTTGA
ACGGGCTTATCTACGCTGTGGGGGGCTATGACGGATCGCCCGATTATTCCACCGGTACGCATTTGAATAG
CGTGGAAGCTTATGACCCAGAGCGTAACGAATGGAGTTTGGTTGCTCCTCTGAGCACCCGTCGCTCTGGG
GTAGGTGTCGCCGTATTGAATGGTTTGATTTATGCAGTTGGCGGCTACGACGGCTCTCCAGATTACAGCA
CTGGCACACACCTGAATTCTGTAGAGGCTTATGATCCGGAGCGCAACGAATGGTCCCTGGTGGCTCCACT
TAGCACACGCCGCAGTGGGGTTGGAGTTGCAGTGTTAAATGGACTGATCTATGCCGTAGGGGGTTACGAT
GGTAGCCCGGACTATTCTACAGGCACCCACCTTAACAGCGTGGAAGCGTACGACCCAGAACGCAATGAGT
GGTCTCTGGTAGCTCCGCTTTCCACACGTCGCTCAGGAGTAGGAGTTGCTGTATTGTAACTCGAGCGG
S6BE-L2 [SEQ ID NO: 14]
MNGLIYAVGGYDGSPDYSTGTHLNSVEAYDPERNEWSLVAPLSTRRSGVGVAVL
NGLIYAVGGYDGSPDYSTGTHLNSVEAYDPERNEWSLVAPLSTRRSGVGVAVL
NGLIYAVGGYDGSPDYSTGTHLNSVEAYDPERNEWSLVAPLSTRRSGVGVAVL
NGLIYAVGGYDGSPDYSTGTHLNSVEAYDPERNEWSLVAPLSTRRSGVGVAVL
NGLIYAVGGYDGSPDYSTGTHLNSVEAYDPERNEWSLVAPLSTRRSGVGVAVL
NGLIYAVGGYDGSPDYSTGTHLNSVEAYDPERNEWSLVAPLSTRRSGVGVAVL
S6BE-L3 [SEQ ID NO: 15]
GATATCCATATGAACGGGCTTATCTATGCTGTTGGCGGTTATGATGGCTCCCCGGATGGGTCTACCCATC
TGAATTCGGTCGAAGCCTATGACCCCGAGCGCAACGAATGGTCCTTAGTCGCCCCGCTGAGTACACGTCG
TAGCGGAGTAGGTGTTGCGGTGCTTAACGGATTGATCTATGCGGTTGGTGGGTTTGCAACTCTGGAAACC
GAATCCGGAGAGTTGGTCCCGACAGAACTTAACAGTGTAGAAGCGTATGACCCTGAACGTAATGAGTGGT
CGTTAGTAGCTCCTTTATCAACGCGCCGTAGTGGGGTTGGAGTTGCAGTTTTAAATGGACTGATCTACGC
AGTAGGAGGATATGATGGGTCGCCTGACGGGAGTACGCACCTTAATTCGGTAGAGGCGTACGATCCAGAG
CGTAACGAGTGGAGTCTTGTAGCGCCACTTTCAACACGTCGTTCGGGTGTCGGAGTGGCCGTTCTGAATG
GGTTAATTTATGCTGTCGGAGGGTTTGCCACACTTGAGACCGAGAGTGGAGAGCTTGTGCCAACGGAGTT
AAACTCAGTGGAAGCTTATGATCCTGAGCGCAATGAGTGGTCACTGGTCGCTCCCCTGAGTACTCGCCGC
AGCGGAGTGGGAGTAGCAGTGTTAAATGGACTGATTTATGCCGTGGGCGGCTATGACGGGAGTCCTGATG
GTAGCACGCATCTGAATTCGGTGGAGGCCTACGATCCCGAGCGTAACGAGTGGTCCCTGGTAGCGCCTCT
TTCGACTCGTCGCTCAGGCGTGGGGGTAGCCGTCTTAAACGGCCTTATTTACGCCGTCGGAGGTTTTGCC
ACCTTAGAGACTGAATCTGGCGAATTAGTCCCAACCGAGCTGAACTCCGTCGAAGCGTATGATCCAGAAC
GCAATGAGTGGTCTTTGGTTGCTCCTCTGTCGACTCGTCGTTCAGGGGTGGGAGTGGCGGTATTATAACT
CGAG              CGG
S6BE-L3 [SEQ ID NO: 16]
MNGLIYAVGGY DGSPDGSTH      LNSVEAYDPERNEWSLVAPLSTRRSGVGVAVL
NGLIYAVGGF ATLETESGELVPTE LNSVEAYDPERNEWSLVAPLSTRRSGVGVAVL
NGLIYAVGGY DGSPDGSTH      LNSVEAYDPERNEWSLVAPLSTRRSGVGVAVL
NGLIYAVGGF ATLETESGELVPTE LNSVEAYDPERNEWSLVAPLSTRRSGVGVAVL
NGLIYAVGGY DGSPDGSTH      LNSVEAYDPERNEWSLVAPLSTRRSGVGVAVL
NGLIYAVGGF ATLETESGELVPTE LNSVEAYDPERNEWSLVAPLSTRRSGVGVAVL
S6BR [SEQ ID NO: 17]
GGGAATTCCATATGGGGGGGCTTTTATATGCTGTTGGTGGATATGATGGAAACACTCATCTTAATTCTCT
TGAATGCTATAACCCCGAGACCAACGAATGGAGTCCAGTAGCTCCTCTTTCCGTGCCACGTTCTGGTATT
GGTGTTGCTGTTTTGGGGGGACTTTTGTATGCAGTCGGGGGATACGACGGCAATACCCATCTGAATTCAT
TAGAGTGTTATAACCCTGAGACCAATGAATGGTCTCCAGTAGCTCCTCTGTCAGTGCCACGTTCCGGTAT
CGGGGTAGCGGTGCTGGGTGGTCTGCTGTACGCGGTGGGAGGATACGATGGCAATACGCATTTAAATTCT
TTGGAGTGCTATAACCCCGAAACAAACGAGTGGTCTCCCGTCGCACCCCTGTCGGTACCGAGATCAGGCA
TAGGCGTCGCTGTGCTTGGGGGACTGTTATACGCTGTCGGTGGTTATGATGGAAATACGCACCTTAACAG
CCTTGAATGTTACAATCCGGAAACAAATGAATGGTCCCCGGTAGCTCCCTTGTCGGTCCCCCGTTCGGGC
ATAGGGGTAGCTGTGTTAGGAGGATTGTTGTATGCAGTGGGTGGGTATGACGGTAATACACACCTGAACT
CATTAGAGTGCTATAACCCCGAGACCAACGAGTGGTCGCCAGTTGCTCCCTTGTCAGTTCCCCGGTCCGG
AATCGGGGTCGCTGTCTTGGGAGGGCTTTTGTATGCGGTCGGGGGCTATGATGGTAATACTCACCTGAAT
TCGCTTGAATGCTATAATCCGGAAACTAACGAATGGTCTCCGGTTGCACCTCTGTCGGTACCGCGTAGTG
GCATTGGCGTGGCAGTATTATAACTCGAGCGG
S6BR [SEQ ID NO: 18]
MGGLLYAVGGYDGNTHLNSLECYNPETNEWSPVAPLSVPRSGIGVAVL
GGLLYAVGGYDGNTHLNSLECYNPETNEWSPVAPLSVPRSGIGVAVL
GGLLYAVGGYDGNTHLNSLECYNPETNEWSPVAPLSVPRSGIGVAVL
GGLLYAVGGYDGNTHLNSLECYNPETNEWSPVAPLSVPRSGIGVAVL
GGLLYAVGGYDGNTHLNSLECYNPETNEWSPVAPLSVPRSGIGVAVL
GGLLYAVGGYDGNTHLNSLECYNPETNEWSPVAPLSVPRSGIGVAVL
S6BC [SEQ ID NO: 19]
GGGAATTCCATATGAATGGACACATATATGCAGTTGGTGGCTATGACGGACATACCCATTTAAACTCAGT
TGAGGCTTATGACCCGGAGACCGATGAATGGCGCTTGGTCGCACCGTTGACGACGCCCAGATCCGGTATG
GGAGTTGCTGTTCTTAACGGCCATATATATGCGGTAGGCGGGTATGATGGTCATACCCACCTGAATTCCG
TAGAGGCCTATGATCCAGAGACTGACGAGTGGAGACTTGTTGCCCCTTTAACGACACCACGGTCGGGCAT
GGGGGTCGCCGTCCTTAACGGTCACATATACGCAGTCGGCGGCTATGATGGTCATACCCACCTGAATAGT
GTGGAAGCTTACGATCCAGAAACGGACGAATGGCGCTTAGTTGCTCCGTTGACGACACCCAGAAGCGGGA
TGGGTGTGGCTGTGCTGAACGGCCATATATATGCAGTAGGTGGGTACGATGGACACACACATCTTAATTC
CGTGGAAGCCTATGATCCCGAAACTGATGAGTGGCGGCTTGTAGCTCCACTGACGACCCCACGCAGCGGG
ATGGGGGTAGCTGTTCTGAACGGCCATATTTACGCGGTGGGGGGCTATGACGGCCATACGCATTTAAATT
CTGTCGAGGCATACGACCCAGAGACAGATGAATGGCGCCTGGTAGCACCTCTTACTACACCTAGATCTGG
CATGGGAGTCGCGGTTTTGAATGGGCACATCTACGCCGTGGGGGGCTACGATGGACATACACATTTGAAT
TCTGTCGAAGCTTATGATCCCGAGACAGATGAATGGAGACTGGTTGCCCCATTGACCACGCCCCGGAGTG
GAATGGGCGTAGCTGTACTTTAACTCGAGCGG
S6BC [SEQ ID NO: 20]
MNGHIYAVGGYDGHTHLNSVEAYDPETDEWRLVAPLTTPRSGMGVAVL
NGHIYAVGGYDGHTHLNSVEAYDPETDEWRLVAPLTTPRSGMGVAVL
NGHIYAVGGYDGHTHLNSVEAYDPETDEWRLVAPLTTPRSGMGVAVL
NGHIYAVGGYDGHTHLNSVEAYDPETDEWRLVAPLTTPRSGMGVAVL
NGHIYAVGGYDGHTHLNSVEAYDPETDEWRLVAPLTTPRSGMGVAVL
NGHIYAVGGYDGHTHLNSVEAYDPETDEWRLVAPLTTPRSGMGVAVL
Specific embodiments are polypeptides with SEQ ID NO: 2, 4, 6, 8, 10,
12, 14, 16, 18, and 20, wherein the Nterminal methionine is absent.
keap1 [SEQ ID NO: 21]
GRLIYTAGG-----YFRQSLSYLEAYNPSNGTWLRLADLQVPRSGLAGCVV (blade 1)
GGLLYAVGGRNNSPDGNTDSSALDCYNPMTNQWSPCAPMSVPRNRIGVGVI (blade 2)
DGHIYAVGG----SHGCIHHNSVERYEPERDEWHLVAPMLTRRIGVGVAVL (blade 3)
NRLLYAVGG----FDGTNRLNSAECYYPERNEWRMITAMNTIRSGAGVCVL (blade 4)
HNCIYAAGG----YDGQDQLNSVERYDVETETWTFVAPMKHRRSALGITVH (blade 5)
QGRIYVLGG----YDGHTFLDSVECYDPDTDTWSEVTRMTSGRSGVGVAVT (blade 6)
NGLIYAVGG----YDGNTHLNSVEAYDPERNEWSLVAPLSTRRSGVGVAVL [consensus] [SEQ ID NO: 33]
S6AE (S6_2E) [SEQ ID NO: 22]
DGRIYAVGGYDGSPDGHTHLNSVEAYDPETDTWSLVAPMKTRRSGVGVAVL
S6AR (S6_2R) [SEQ ID NO: 23]
NRLLYAVGGYDGSPDGHTHLNSVECYDPETNEWSLVAPMNTRRSGVGVAVL
S6AC (S6_2C) [SEQ ID NO: 24]
NGRIYAVGGYDGS PDGHTHLNSVEAYDPETDEWSFVAPMTTPRSGVGVAVL
S6BE (S6_6E) [SEQ ID NO: 25]
NGLIYAVGGYDGNTHLNSVEAYDPERNEWSLVAPLSTRRSGVGVAVL
S6BE-3HH (S6_6E-3HH) [SEQ ID NO: 26]
NGLIYAVGGYDGNTHLNSVEAYDPERNEWSLVAPLSTRRSGVGVAVL (blade 1)
NGLIYAVGGYDGNTHLNSVEAYDPHHNEWSLVAPLSTRRSGVGVAVL (blade 2)
S6BE-L1 (S6_6E-L1) [SEQ ID NO: 27]
NGLIYAVGGYDGTGYNTHLNSVEAYDPERNEWSLVAPLSTRRSGVGVAVL
S6BE-L2 (S6_6E-L2) [SEQ ID NO: 28]
NGLIYAVGGYDGSPDYSTGTHLNSVEAYDPERNEWSLVAPLSTRRSGVGVAVL
S6BE-L3 (S6_6E-L3) [SEQ ID NO: 29]
NGLIYAVGGYDGSPDGSTHLNSVEAYDPERNEWSLVAPLSTRRSGVGVAVL (blade 1)
NGLIYAVGGFATLETESGELVPTELNSVEAYDPERNEWSLVAPLSTRRSGVGVAVL (blade 2)
S6BR (S6_6R) [SEQ ID NO: 30]
GGLLYAVGGYDGNTHLNSLECYNPETNEWSPVAPLSVPRSGIGVAVL
S6BC (does not express) (S6_6C) [SEQ ID NO: 31]
NGHIYAVGGYDGHTHLNSVEAYDPETDEWRLVAPLTTPRSGMGVAVL
NGRIYAVGGXnLNSVEAYDPETDEWSFVAPMTTPRSGVGVAVL [SEQ ID NO: 32] wherein X n is
1 to 10 or 15 amino acids.
SEQ ID NO: 33
NGLIYAVGGY DGNTHLNSVE AYDPERNEWS LVAPLSTRRS GVGVAVL
SEQ ID NO: 34
NGRIX5AVGG Xn LNSVEAX16DX18ETDEWSF VAPMTTPRSG VGVAVL
Xn = is any of 1-15 amino acids that can be incorporated in a polypeptide
X5 = is any of 1-15 amino acids that can be incorporated in a polypeptide
X14 = is any of 1-15 amino acids that can be incorporated in a polypeptide
X16 = is any of 1-15 amino acids that can be incorporated in a polypeptide
X18 = is any of 1-15 amino acids that can be incorporated in a polypeptide
In specific embodiments Xn, X5, X16, X18 are each independently selected
from the group of the 20 naturally occurring amino acids.
in the conserved sequence motif, the amino acids Xn are not numbered. As the
sequence listing does number the Xn aminoacids, the numbering of amino acids
cterminal of Xn will differ.
Thus X14, X16 and X18 of the sequence motif become respectively positions 29, 31 and
33 in SEQ ID NO 34. Throughout the specification the numbering of the conserved
sequence motif is used.
S6BE-3HR [SEQ ID: 35]
CATATGAATGGCCTGATCTACGCCGTAGGAGGGTACGACGGAAACACTCATTTAAACTCGGTGGAAGCAT
ACGATCCAGAACGTAACGAGTGGAGTTTGGTTGCTCCATTATCTACACGTCGCAGTGGCGTAGGTGTAGC
TGTTTTGAACGGTTTAATTTACGCTGTCGGAGGTTACGACGGTAATACACATCTGAACTCCGTTGAGGCC
TATGACCCTCACCGCAACGAATGGTCGTTGGTAGCACCTCTTTCGACCCGCCGTAGTGGCGTAGGCGTGG
CCGTGTTGAATGGGTTGATTTACGCCGTCGGGGGTTACGACGGAAACACGCATCTTAACTCGGTAGAGGC
GTATGACCCGGAACGCAATGAGTGGAGCTTGGTGGCCCCCCTTTCAACACGCCGTTCGGGCGTCGGAGTT
GCGGTTTTGAACGGCCTGATTTACGCAGTCGGTGGATACGACGGCAATACTCATCTGAACTCAGTAGAAG
CATACGACCCCCACCGTAACGAGTGGTCATTGGTGGCACCTTTGAGTACTCGCCGTTCAGGGGTTGGCGT
TGCCGTGTTGAATGGCTTAATCTATGCGGTGGGGGGATATGATGGCAACACCCACCTTAACTCAGTCGAA
GCCTATGACCCAGAACGTAACGAATGGTCATTAGTTGCTCCGCTTAGTACCCGTCGTTCCGGAGTCGGGG
TTGCCGTATTGAACGGGTTGATTTATGCTGTCGGCGGATACGACGGAAACACTCACTTGAACTCTGTAGA
AGCTTATGATCCACATCGTAATGAGTGGAGCCTGGTTGCGCCGCTGAGTACACGCCGCAGTGGTGTGGGC
GTAGCAGTTTTATAACTCGAG
S6BE-3HR [SEQ ID: 36]
NGLIYAVGGYDGNTHLNSVEAYDPERNEWSLVAPLSTRRSGVGVAVLNGLIYAVGGYDGNTHLNSVEAYD
PHRNEWSLVAPLSTRRSGVGVAVLNGLIYAVGGYDGNTHLNSVEAYDPERNEWSLVAPLSTRRSGVGVAV
LNGLIYAVGGYDGNTHLNSVEAYDPHRNEWSLVAPLSTRRSGVGVAVLNGLIYAVGGYDGNTHLNSVEAY
DPERNEWSLVAPLSTRRSGVGVAVLNGLIYAVGGYDGNTHLNSVEAYDPHRNEWSLVAPLSTRRSGVGVA
VL
S2BE-3HR [SEQ ID: 37]
CATATGAACGGTCTTATTTATGCGGTAGGAGGATACGACGGCAACACCCACTTGAACTCTGTGGAGGCGT
ATGATCCAGAGCGTAATGAATGGTCACTGGTCGCTCCCTTGAGCACGCGCCGTTCCGGCGTGGGAGTAGC
GGTATTGAACGGCTTAATTTATGCGGTTGGGGGATATGATGGAAATACGCATCTGAACTCAGTTGAAGCA
TACGATCCGCATCGTAACGAGTGGTCTTTGGTTGCTCCTTTATCGACGCGCCGTTCAGGTGTAGGCGTGG
CGGTCTTGTAACTCGAG
S2BE-3HR [SEQ ID: 38]
NGLIYAVGGYDGNTHLNSVEAYDPERNEWSLVAPLSTRRSGVGVAVLNGLIYAVGGYDGNTHLNSVEAYD
PHRNEWSLVAPLSTRRSGVGVAVL
S6BE-3CHR [SEQ ID: 39]
catatgaacggtttgatttatgcagttggaggttacgatggcaacacccacttaaatagtgtggaaGCTt
atgatcccgaacgcaatgaatggtcgctggtagcgccgctgtcaactcgccgctctggggtaggcgtggc
cgttttgaatggcctgatctatgcagtcggcgggtatgatggtaatacccatcttaacagtgttgaggca
tattgcccacatcgtaatgagtggagtctggttgcgccgctttcgacccgccgcagcggcgtcggcgtcg
cggtgctgaatggtctgatttacgcagtaggcggctacgacggtAACacgcacctcaattccgtagaagc
gtacgacccggaacgtaatgaatggagcctcgtggcgccgctttcaacccgcaggtcaggcgtgggtgtg
gccgttctgaatggcctgatttacgcggtgggcggctacgatggaaatacgcacctcaactctgttgagg
cctactgcccgcatcgtaacgaatggagcttggtggccccactgagcacccgtcgatcgggcgtaggggt
ggcggttttaaacgggctgatctatgcggtgggcggatatgatggcaacacccatctgaacagcgtggaa
gcttatgatcctgagcgtaacgaatggtccttagttgctcctttaagcacacgccgttccggcgttggcg
tcgcggtgcttaacggactgatttatgctgtcggtggttacgacggtaacacgcatctgaactcggtgga
agcctattgtccgcaccgtaatgaatggagcctagtcgcaccgctgagcactcgccggtctggagtcggt
gtagccgtgttgTAACTCGAG
S6BE-3CHR [SEQ ID: 40]
NGLIYAVGGYDGNTHLNSVEAYDPERNEWSLVAPLSTRRSGVGVAVLNGLIYAVGGYDGNTHLNSVEAYC
PHRNEWSLVAPLSTRRSGVGVAVLNGLIYAVGGYDGNTHLNSVEAYDPERNEWSLVAPLSTRRSGVGVAV
LNGLIYAVGGYDGNTHLNSVEAYCPHRNEWSLVAPLSTRRSGVGVAVLNGLIYAVGGYDGNTHLNSVEAY
DPERNEWSLVAPLSTRRSGVGVAVLNGLIYAVGGYDGNTHLNSVEAYCPHRNEWSLVAPLSTRRSGVGVA
VL
mEm-v22S6BE-3HR [SEQ ID: 41]
CATatggtgtctaagggcgaggAACTGTTTACCGGCGTGGTGCCGATTCTGGTGGAACTGGATGGCGATG
TGAACGGCCATAAATTTAGCGTGAGCGGCGAAGGCGAAGGCGATGCGACCTATGGTAAACTGACCCTGAA
ATTTATTTGCACCACCGGCAAACTGCCGGTGCCGTGGCCTACCTTAGTTACTACCTTAACCTATGGCGTT
CAATGCTTTGCGCGCTATCCGGATCACATGAAACAGCATGATTTTTTCAAGAGCGCGATGCCGGAAGGCT
ATGTGCAGGAACGCACTATTTTTTTTAAAGACGATGGCAACTATAAGACCCGCGCGGAAGTGAAATTTGA
AGGCGATACCCTGGTGAACCGCATTGAACTGAAAGGCATTGATTTTAAGGAGGACGGCAACATCCTGGGC
CATAAACTGGAATATAACTATAACAGCCACAAGGTGTACATCACCGCGGATAAACAGAAAAACGGCATTA
AAGTGAACTTCAAGACCCGCCATAACATCGAAGATGGCAGCGTGCAGCTGGCGGATCATTATCAGCAGAA
CACCCCGATTGGCGATGGCCCGGTTTTACTGCCTGATAACCATTATCTGAGCACCCAGAGCAAACTGAGC
AAAGATCCGAACGAAAAACGCGATCACATGGTGCTGCTGGAATTTGTGACCGCGGCGGGCATTaccctgg
gcatggatgaattatacaaaggcagcGGATCCATGAACACCCATCTGAACAGCGTGGAAGCGTATGATCC
GGAACGCAACGAATGGAGCCTGGTGGCGCCTTTAAGCACCCGTCGCAGCGGTGTTGGTGTTGCGGTTTTA
AATGGTTTAATTTACGCGGTGGGCGGCTATGATGGCAACACCCATTTAAACAGCGTGGAAGCGTATGATC
CGCATCGCAACGAATGGAGCCTGGTGGCGCCTTTAAGCACCCGCCGTAGCGGTGTTGGTGTTGCGGTTTT
AAATGGCCTGATTTATGCGGTGGGCGGCTATGATGGCAACACCCATTTAAACAGCGTGGAAGCGTATGAT
CCGGAACGCAACGAATGGAGCCTGGTGGCGCCTTTAAGCACCCGCCGTAGCGGTGTTGGTGTTGCGGTTT
TAAATGGTTTAATTTACGCGGTGGGCGGCTATGATGGCAACACCCATTTAAACAGCGTGGAAGCGTATGA
TCCGCATCGCAACGAATGGAGCCTGGTGGCGCCTTTAAGCACCCGTCGTAGCGGTGTTGGCGTTGCGGTT
TTAAACGGCTTAATTTATGCGGTGGGCGGCTATGATGGCAACACCCATTTAAACAGCGTGGAAGCGTATG
ATCCGGAACGCAACGAATGGAGCCTGGTGGCGCCATTAAGCACCCGTCGTTCAGGTGTTGGCGTTGCGGT
ATTAAACGGCTTAATTTATGCGGTGGGCGGCTATGATGGCAACACCCATTTAAACAGCGTGGAAGCGTAT
GATCCGCATCGCAACGAATGGAGCCTGGTGGCGCCTTTAAGCACCCGCCGTAGCGGTGTTGGTGTTGCGG
TGTTAAATGGCTTAATTTATGCGGTGGGCGGCTATGATGGCTAACTCGAG
mEm-v22S6BE-3HR [SEQ ID: 42]
VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQC
FARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHK
LEYNYNSHKVYITADKQKNGIKVNFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKD
PNEKRDHMVLLEFVTAAGITLGMDELYKGSGSMNTHLNSVEAYDPERNEWSLVAPLSTRRSGVGVAVLNG
LIYAVGGYDGNTHLNSVEAYDPHRNEWSLVAPLSTRRSGVGVAVLNGLIYAVGGYDGNTHLNSVEAYDPE
RNEWSLVAPLSTRRSGVGVAVLNGLIYAVGGYDGNTHLNSVEAYDPHRNEWSLVAPLSTRRSGVGVAVLN
GLIYAVGGYDGNTHLNSVEAYDPERNEWSLVAPLSTRRSGVGVAVLNGLIYAVGGYDGNTHLNSVEAYDP
HRNEWSLVAPLSTRRSGVGVAVLNGLIYAVGGYDG
S6BE-3HR-L3 [SEQ ID: 43]
CATATGAACGGCCTGATTTATGCGGTGGGCGGCTATGATGGCAGCCCGGATGGTTCAACTCATCTGAATT
CCGTGGAAGCGTATGATCCGGAACGCAACGAATGGAGCCTGGTGGCGCCGTTAAGCACCCGTCGTAGCGG
TGTTGGTGTTGCGGTTTTAAATGGTCTGATTTATGCGGTGGGCGGCTTTGCGACCCTGGAAACCGAATCA
GGTGAACTGGTGCCGACCGAATTAAACAGCGTTGAAGCGTATGATCCGCATCGCAACGAATGGAGCCTGG
TGGCGCCTTTAAGCACCCGTCGTTCAGGTGTTGGTGTTGCGGTTTTAAATGGTTTAATTTATGCGGTGGG
CGGCTATGATGGCAGCCCGGATGGTAGCACCCATCTGAATAGCGTGGAAGCGTATGATCCGGAACGCAAC
GAATGGAGCCTGGTGGCGCCTTTAAGCACCCGTCGTTCAGGCGTTGGCGTTGCGGTTTTAAATGGCCTGA
TTTATGCGGTGGGCGGCTTTGCGACCCTGGAAACCGAATCAGGTGAACTGGTGCCGACCGAACTGAACAG
CGTGGAAGCGTATGATCCGCATCGCAACGAATGGAGCCTGGTGGCGCCTTTAAGCACCCGCCGTAGCGGT
GTTGGTGTTGCGGTTTTAAATGGCCTGATTTATGCGGTGGGCGGCTATGATGGCAGCCCGGATGGTAGCA
CCCATCTGAATTCTGTGGAAGCGTATGATCCGGAACGCAACGAATGGAGCCTGGTGGCGCCTTTAAGCAC
CCGTCGTAGCGGTGTTGGCGTTGCGGTTTTAAATGGTTTAATTTACGCGGTGGGCGGCTTTGCGACCCTG
GAAACCGAAAGCGGTGAACTGGTTCCGACCGAACTGAATAGCGTGGAAGCGTATGATCCGCATCGCAACG
AATGGAGCCTGGTGGCGCCTTTAAGCACCCGTCGTAGCGGTGTTGGTGTTGCGGTTTTATAACTCGAG
S6BE-3HR-L3 [SEQ ID: 44]
NGLIYAVGGYDGSPDGSTHLNSVEAYDPERNEWSLVAPLSTRRSGVGVAVLNGLIYAVGGFATLETESGE
LVPTELNSVEAYDPHRNEWSLVAPLSTRRSGVGVAVLNGLIYAVGGYDGSPDGSTHLNSVEAYDPERNEW
SLVAPLSTRRSGVGVAVLNGLIYAVGGFATLETESGELVPTELNSVEAYDPHRNEWSLVAPLSTRRSGVG
VAVLNGLIYAVGGYDGSPDGSTHLNSVEAYDPERNEWSLVAPLSTRRSGVGVAVLNGLIYAVGGFATLET
ESGELVPTELNSVEAYDPHRNEWSLVAPLSTRRSGVGVAVL
dS6AC [SEQ ID: 45]
CATATGAACGGCCGCATTTATGCGGTTGGCGGCTATGATGGCAGCCCGGATGGTCATACCCATCTGAATA
GCGTGGAAGCGTATGATCCGGAAACCGATGAATGGAGCTTTGTGGCGCCGATGACCACCCCGCGTAGCGG
TGTTGGTGTTGCGGTTTTAAACGGCCGCATTTATGCGGTGGGCGGCTATGATGGCAGCCCGGATGGTCAT
ACCCATCTGAATAGCGTGGAAGCGTATGATCCGGAAACCGATGAATGGAGCTTTGTGGCGCCGATGACCA
CCCCGCGTAGCGGTGTTGGTGTTGCGGTTTTAAATGGTCGCATTTATGCGGTTGGCGGCTATGATGGCAG
CCCGGATGGTCATACCCATCTGAATAGCGTGGAAGCGTATGATCCGGAAACCGATGAATGGAGCTTTGTG
GCGCCGATGACCACCCCGCGTTCAGGTGTTGGTGTTGCGGTTTTAAATGGCCGCATTTATGCGGTGGGCG
GTTATGATGGCAGCCCGGATGGTCATACCCATCTGAACAGCGTGGAAGCGTATGATCCGGAAACCGATGA
ATGGAGCTTTGTGGCGCCGATGACCACCCCGCGTAGCGGTGTTGGTGTTGCGGTTTTAAACGGTCGTATT
TATGCGGTGGGCGGTTATGATGGCAGCCCGGATGGTCATACCCATCTGAATAGCGTGGAAGCGTATGATC
CGGAAACCGATGAATGGAGCTTTGTGGCGCCGATGACCACCCCGCGTAGCGGTGTGGGCGTTGCGGTTTT
AAATGGCCGTATTTATGCGGTTGGCGGCTATGATGGCAGCCCGGATGGTCATACTCATCTGAATAGCGTG
GAAGCGTATGATCCGGAAACCGATGAATGGAGCTTTGTGGCGCCGATGACCACCCCGCGTAGCGGTGTTG
GTGTTGCGGTTTTAAGCGGTGGTCGCATTTATGCGGTGGGCGGCTATGATGGCAGCCCGGATGGTCATAC
CCATCTGAACAGCGTGGAAGCGTATGATCCGGAAACCGATGAATGGAGCTTTGTGGCGCCGATGACCACC
CCGCGTAGCGGTGTTGGTGTTGCGGTGTTAAATGGTCGCATTTATGCGGTGGGCGGCTATGATGGCAGCC
CGGATGGCCATACCCATCTGAATAGCGTGGAAGCGTATGATCCGGAAACCGATGAATGGAGCTTTGTGGC
GCCGATGACCACCCCGCGTTCAGGTGTTGGTGTTGCGGTTCTGAATGGCCGTATTTATGCGGTGGGCGGC
TATGATGGCAGCCCGGATGGTCATACTCATCTGAACAGCGTGGAAGCGTATGATCCGGAAACCGATGAAT
GGAGCTTTGTGGCGCCGATGACCACCCCGCGTAGCGGTGTTGGTGTTGCGGTTTTAAACGGCCGTATTTA
TGCGGTGGGCGGCTATGATGGCAGCCCGGATGGTCATACCCATCTGAACAGCGTGGAAGCGTATGATCCG
GAAACCGATGAATGGAGCTTTGTGGCGCCGATGACCACCCCGCGTAGCGGTGTTGGTGTTGCGGTTTTAA
ACGGTCGCATTTATGCGGTGGGCGGCTATGATGGCAGCCCGGATGGTCATACCCATCTGAACAGCGTGGA
AGCGTATGATCCGGAAACCGATGAATGGAGCTTTGTGGCGCCGATGACCACCCCGCGTTCAGGTGTTGGT
GTTGCGGTTTTAAATGGCCGTATTTATGCGGTGGGCGGCTATGATGGCAGCCCGGATGGTCATACCCATC
TGAATAGCGTGGAAGCGTATGATCCGGAAACCGATGAATGGAGCTTTGTGGCGCCGATGACCACCCCGCG
TAGCGGTGTTGGTGTTGCGGTTTTATAACTCGAG
dS6AC [SEQ ID: 46]
NGRIYAVGGYDGSPDGHTHLNSVEAYDPETDEWSFVAPMTTPRSGVGVAVLNGRIYAVGGYDGSPDGHTH
LNSVEAYDPETDEWSFVAPMTTPRSGVGVAVLNGRIYAVGGYDGSPDGHTHLNSVEAYDPETDEWSFVAP
MTTPRSGVGVAVLNGRIYAVGGYDGSPDGHTHLNSVEAYDPETDEWSFVAPMTTPRSGVGVAVLNGRIYA
VGGYDGSPDGHTHLNSVEAYDPETDEWSFVAPMTTPRSGVGVAVLNGRIYAVGGYDGSPDGHTHLNSVEA
YDPETDEWSFVAPMTTPRSGVGVAVLSGGRIYAVGGYDGSPDGHTHLNSVEAYDPETDEWSFVAPMTTPR
SGVGVAVLNGRIYAVGGYDGSPDGHTHLNSVEAYDPETDEWSFVAPMTTPRSGVGVAVLNGRIYAVGGYD
GSPDGHTHLNSVEAYDPETDEWSFVAPMTTPRSGVGVAVLNGRIYAVGGYDGSPDGHTHLNSVEAYDPET
DEWSFVAPMTTPRSGVGVAVLNGRIYAVGGYDGSPDGHTHLNSVEAYDPETDEWSFVAPMTTPRSGVGVA
VLNGRIYAVGGYDGSPDGHTHLNSVEAYDPETDEWSFVAPMTTPRSGVGVAVL

TABLE 1
Crystallographic information tables for the 4 initial SAKe designs.
	S6AE	S6AR	S6AC	S6BE (normal)
PDB code	7ON6	7ON8	7ONA	7ONC
Wavelength	1.00003	1.00003	1.00003	0.92003
Resolution range	38.12-1.35	(1.398-1.35)	39.12-1.5	(1.554-1.5)	39.77-1.3	(1.347-1.3)	23.4-1.1	(1.139-1.1)
Space group	P 31 2 1	P 21 21 21	I 2 2 2	P 6
Unit cell	82.767 82.767	45.986 63.249	46.932 74.8725	46.8027 46.8027
	90.023 90 90 120	99.567 90 90 90	74.8655 90 90 90	34.9814 90 90 120
Total reflections	1541949	(142973)	618399	(60166)	410214	(40479)	337801	(31921)
Unique	78555	(7776)	47304	(4657)	32753	(3206)	17817	(1780)
reflections
Multiplicity	19.6	(18.4)	13.1	(12.9)	12.5	(12.6)	19.0	(17.9)
Completeness(%)	99.99	(100.00)	99.98	(100.00)	99.70	(98.86)	99.78	(99.83)
Mean I/sigma(I)	50.48	(6.70)	26.77	(2.58)	26.52	(4.59)	39.80	(12.34)
Wilson B-factor	14.63	15.39	15.69	10.9
R-merge	0.03554	(0.5203)	0.06382	(1.053)	0.04844	(0.4897)	0.0397	(0.1398)
R-meas	0.03649	(0.535)	0.06645	(1.097)	0.05057	(0.5099)	0.04116	(0.1441)
R-pim	0.008191	(0.1242)	0.01832	(0.3023)	0.01429	(0.14)	0.01045	(0.03452)
CC1/2	1	(0.966)	1	(0.818)	0.999	(0.97)	0.993	(0.987)
CC*	1	(0.991)	1	(0.948)	1	(0.992)	0.998	(0.997)
Reflections used	78552	(7776)	47302	(4657)	32741	(3201)	17809	(1779)
in refinement
Reflections used	3890	(363)	2325	(238)	1535	(137)	829	(86)
for R-free
R-work	0.1370	(0.1445)	0.1763	(0.2070)	0.1584	(0.2036)	0.1311	(0.0978)
R-free	0.1578	(0.1885)	0.1822	(0.2214)	0.1870	(0.2368)	0.1503	(0.1404)
CC(work)	0.968	(0.954)	0.957	(0.862)	0.966	(0.975)	0.955	(0.976)
CC(free)	0.968	(0.908)	0.944	(0.853)	0.918	(0.948)	0.952	(0.944)
Number of non-	2715	2477	1271	427
hydrogen atoms	2322	2259	1120	374
macromolecules
ligands	30	0	2	0
solvent	363	218	149	53
Protein residues	305	302	149	47
RMS(bonds)	0.009	0.011	0.01	0.019
RMS(angles)	1.1	1.22	1.08	1.65
Ramachandran	97.36	95.3	97.24	97.78
favored (%)
Ramachandran	2.64	4.7	2.76	2.2
allowed (%)
Ramachandran	0.00	0.00	0.00	0.00
outliers (%)
Rotamer outliers (%)	0.00	0.00	0.00	0.00
Clashscore	2.67	1.38	0.93	1.36
Average B-factor	19.94	18.82	21.35	14.52
macromolecules	17.7	17.76	20.38	12.92
solvent	33.49	29.84	28.76	25.79
ligands	29.27		14.3
Number of TLS		6
groups

TABLE 2
Crystallographic information tables for the self-assembled S6BE structure and S6BE loop variants.
	S6BE (self-
	assembled)	S6BEL1	S6BEL2	S6BEL3
PDB code	7ONE	7ONG	7ON7	7ONH
Wavelength	1.1808	0.9763	0.9763	0.9763
Resolution range	35.04-1.2	(1.243-1.2)	37.87-1.95	(2.02-1.95)	39.86-1.95	(2.02-1.95)	40.55-1.55	(1.606-1.55)
Space group	P 6	P 6 2 2	P 1 21 1	P 41 3 2
Unit cell	46.5489 46.5489	43.725 43.725	79.96 45.4	90.668 90.668
	35.0445 90 90 120	72.146 90 90 120	81.62 90 114.72 90	90.668 90 90 90
Total reflections	227910	(14062)	120255	(11712)	257814	(24890)	38062	(3616)
Unique reflections	13612	(1351)	3335	(315)	39042	(873)	19032	(1809)
Multiplicity	16.7	(10.4)	36.1	(37.2)	6.6	(6.5)	2.0	(2.0)
Completeness (%)	99.85	(99.27)	99.88	(99.68)	79.84	(22.49)	99.71	(97.31)
Mean I/sigma(I)	62.70	(27.66)	25.91	(8.83)	7.37	(1.46)	95.39	(19.84)
Wilson B-factor	11.59	22.98	11.38	12.76
R-merge	0.03273	(0.07503)	0.1254	(0.6093)	0.1378	(1.456)	0.003807	(0.03001)
R-meas	0.03382	(0.079)	0.1273	(0.6176)	0.1495	(1.582)	0.005383	(0.04244)
R-pim	0.008205	(0.02438)	0.02115	(0.1001)	0.05752	(0.6145)	0.003807	(0.03001)
CC1/2	0.999	(0.998)	0.999	(0.989)	0.998	(0.782)	1	(0.996)
CC*	1	(0.999)	1	(0.997)	0.999	(0.937)	1	(0.999)
Reflections used in	13624	(1351)	3332	(314)	31325	(873)	19031	(1809)
refinement
Reflections used	676	(72)	178	(13)	1564	(41)	954	(88)
for R-free
R-work	0.1542	(0.1665)	0.1914	(0.1627)	0.2317	(0.2502)	0.1493	(0.1498)
R-free	0.1652	(0.2125)	0.2311	(0.2299)	0.2786	(0.3103)	0.1594	(0.1583)
CC(work)	0.923	(0.954)	0.930	(0.968)	0.855	(0.749)	0.967	(0.947)
CC(free)	0.952	(0.910)	0.952	(0.955)	0.866	(0.809)	0.965	(0.920)
non H atoms	418	391	4644	977
macromolecules	373	363	4061	817
ligands	0	2	0	5
solvent	45	26	583	155
Protein residues	47	50	546	107
RMS(bonds)	0.01	0.01	0.003	0.01
RMS(angles)	1.25	1.06	0.49	1.17
Ramachandran	97.78	95.83	97.68	98.1
favored (%)
Ramachandran	2.22	4.17	2.32	1.9
allowed (%)
Ramachandran	0	0	0	0
outliers (%)
Rotamer outliers (%)	0	0	0.48	0
Clashscore	0	0	3.27	1.25
Average B-factor	15.29	22.68	11.65	17.67
macromolecules	13.87	22.46	10.98	15.29
solvent	27.07	25.49	16.32	29.44
ligands		24.76		41
Number of TLS		1	2	9
groups

TABLE 3
Crystallographic information tables for the various crystal structures of S6BE-3HH.
	S6BE-3HH	S6BE-3HH
	(self-	(alternative
	S6BE-3HH	assembled)	packing)
PDB code	7OP4	7OPU	7OPV
Wavelength	1	0.9763	0.9763
Resolution range	38.15-1.52	(1.574-1.52)	68.33-1.7	(1.761-1.7)	40.54-1.95	(2.02-1.95)
Space group	C 1 2 1	C 1 2 1	P 1
Unit cell	44.049 76.29 67.987	76.4486 44.1528	46.9614 47.1524
	90 90.021 90	68.326 90 90.0535 90	70.876 72.1828
			76.4037 60.1756
Total reflections	234771	(23764)	169862	(16090)	120140	(11837)
Unique reflections	33235	(3298)	25255	(2491)	34968	(3389)
Multiplicity	7.1	(7.2)	6.7	(6.5)	3.4	(3.5)
Completeness (%)	96.01	(95.04)	99.79	(99.52)	96.00	(94.43)
Mean I/sigma(I)	30.43	(8.87)	21.82	(3.29)	35.05	(14.57)
Wilson B-factor	16.85	23.2	19.29
R-merge	0.03691	(0.207)	0.0454	(0.5517)	0.03621	(0.06553)
R-meas	0.03988	(0.2231)	0.04926	(0.6008)	0.04302	(0.08098)
R-pim	0.01493	(0.08249)	0.01892	(0.2351)	0.02296	(0.04661)
CC1/2	0.999	(0.99)	1	(0.878)	0.995	(0.29)
CC*	1	(0.998)	1	(0.967)	0.999	(0.671)
Reflections used in	33207	(3293)	25216	(2479)	34944	(3388)
refinement
Reflections used for	1644	(153)	1241	(143)	1699	(147)
R-free
R-work	0.1855	(0.2014)	0.1853	(0.2425)	0.1585	(0.1383)
R-free	0.2023	(0.2409)	0.2256	(0.2483)	0.2038	(0.2306)
CC(work)	0.961	(0.939)	0.958	(0.872)	0.966	(0.953)
CC(free)	0.947	(0.946)	0.955	(0.884)	0.949	(0.866)
Number of non-	2320	2268	4547
hydrogen atoms	2090	2079	4113
macromolecules
ligands	0	0	0
solvent	230	189	434
Protein residues	280	281	562
RMS(bonds)	0.006	0.006	0.007
RMS(angles)	0.93	0.85	0.79
Ramachandran	97.12	97.49	96.24
favored (%)
Ramachandran	2.88	2.51	3.76
allowed (%)
Ramachandran	0	0	0
outliers (%)
Rotameroutliers(%)	0	0	0.24
Clashscore	5.16	2.72	3.38
Average B-factor	20.72	26.63	21.71
macromolecules	19.64	25.86	20.59
solvent	30.6	35.08	32.25
ligands
Number of TLS		6	18
groups

Example 1. Protein Design

The proteins examined in this research were designed using the RE₃Volutionary protein design method [Voet et al. Proceedings of the National Academy of Sciences 2014, 111, 15102-15107]. The KELCH domain of human Keap1 (PDB code: 1ZGK) was chosen as a template for the SAKe designs. Clustal Omega was used to generate multiple sequence alignments (MSAs) starting from the six repeats of the keap1 β-propeller [Madeira et al. Nucleic acids research 2019, 47, W636-W641]. The MSAs and their accompanying unrooted phylogenetic trees were used to construct lists of putative ancestral sequences using the FastML server [The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrodinger, LLC.], with 250 sequences per node for a total of 10000 sequences for each SAKe construct. Idealized symmetric backbone models were designed using both PyMOL [Ashkenazy et al. Nucleic acids research 2012, 40, W580-W584] and PyRosetta [Chaudhury et al. Bioinformatics 2010, 26, 689-691]. With PyMOL, the second and last blades of the Keap1 Bpropeller were extracted. The first blade was used for the design of type A SAKe (47 amino acids per repeat) and the last blade for type B SAKe (51 amino acids per repeat). The N-termini were truncated to minimize clashing with their symmetry equivalents during the subsequent Rosetta Symmetric Docking procedure [Andre et al. Proc Natl Acad Sci 2007, 104, 17656-17661]. A sixfold rotational symmetry was enforced and generated 20000 Monte Carlo Simulated Annealing (MCSA) optimized models per SAKe type. Results were evaluated on their docking score and RMSD from a manually constructed symmetric backbone. The blades of the best models were reconnected, adding in only the exact amount of amino acids that were removed earlier. The putative ancestral sequences were mapped on their corresponding backbone models using a custom PyRosetta script. For each SAKe type a model was selected with the lowest Talaris2013 energy score (e.g. SAKe6E ‘E’), a model with lowest RMSD from the input backbone (e.g. SAKe6R ‘R’) and a c-optimized model (e.g. SAKe6C ‘C’). For all SAKe constructs except S6AR, cysteine residues were mutated to serine or alanine. Following the interpretation of later experiments, S6BE mutants were rationally designed to have altered self-assembly properties. The S6BE-3HH variant was designed from S6BE via following mutations: E24H-R25H, E118H-R119H and E212H-R213H. Amino acid sequences were reverse translated into DNA sequences, using a codon optimization tool provided by the supplier (Integrated DNA Technologies, Iowa, United States).

Example 2. Production of Proteins

DNA sequences were cloned into pET-28a(+) via NdeI and XhoI restriction sites, adding an N-terminal hexahistidine tag to the constructs. For cloning, the recombinant vectors were transformed into E. coli DH5a via heatshock. The vectors were validated via Sanger Sequencing (LGC Ltd, Teddington, United Kingdom), using T7 promotor and terminator primers provided by LGC. Correct plasmids were transformed into E. coli BL21 via heat shock. 1L cultures were grown in a shaking incubator at 37° C. to an OD₆₀₀ of 0.6. Thereafter, cultures were incubated on ice for 20 min. After adding 1 mM isopropyl β-D-1thiogalactopyranoside (IPTG) incubation was continued at 20° C. for 16-18 h while shaking. Cells were harvested via centrifugation at 3000 g. The supernatant was discarded and pellets were immediately stored at −24° C. Pellets were thawed on ice and suspended in 40 ml of 50 mM NaH₂PO₄ (pH 8), 200 mM NaCl, 10 mM Imidazole, 1 mM phenylmethylsulfonyl fluoride (PMSF) and 30 mg hen eggwhite lysozyme. They were incubated for 30 min at 15° C., while rotating. After, they were lysed via sonication. Lysates were centrifuged at 3000 g for 30 min. The Supernatant was filtered (0.45 μM) and loaded on a Nickel nitrilotriacetic (NINTA) column equilibrated with a 50 mM NaH2PO4 (pH 8), 200 mM NaCl and 10 mM imidazole buffer. The column was washed with 10 mM and 20 mM imidazole and the proteins eluted with 300 mM imidazole. The fractions containing proteins were collected and dialyzed overnight in 50 mM NaH2PO4 (pH 8) and 200 mM NaCl. At the same time, histidine tags were removed via thrombin (100 U per protein). The dialyzed samples were subjected to an additional Ni-NTA chromatography step and then loaded on a Superdex200 pg 16/600 column equilibrated with 20 mM HEPES (pH 8) and 200 mM NaCl.

Abs₂₈₀ peaks were collected, dialyzed in 20 mM HEPES (pH8) and concentrated to stocks of 20 mg/ml or more. These stock proteins were stored at 4° C. The Superdex column was standardized using the Bio-Rad “gel filtration standard 151-1901” protein marker.

Example 3. Crystallization and Xray Diffraction

Proteins were crystallized via sitting-drop vapor diffusion; using Qiagen Nextal Crystal Screening kits, MRC 96-well plates and an ARI Gryphon robot. For native crystallography droplets consisted of 0.3 μL mother liquor and 0.3 μL of 10 mg/ml protein in 20 mM HEPES pH 8.0, 200 mM NaCl. Protein crystals were vitrified after single-step soaking. PEG 400 or glycerol were used as cryoprotectant. Xray diffraction experiments were performed at Diamond Light Source (United Kingdom), Elletra (Italy) and SLS (Switzerland). The diffraction patterns were indexed using XDS or DIALS [Kabsc Acta Crystallographica Section D: Biological Crystallography 2010, 66, 125-132; Winter et al. Acta Crystallographica Section D. 2018, 74, 85-97]. Data reduction was done with Aimless in CCP4 [Evans et al. Acta Crystallographica Section D: Biological Crystallography 2013, 69, 1204-1214; Winn et al. Acta Crystallographica Section D: Biological Crystallography 2011, 67, 235-242]. Molecular Replacement phasing was done with PHASER, using computationally designed models as search ensemble [McCoy et al. Journal of applied crystallography 2007, 40, 658-674]. Refinement was done manually with phenix.refine and Coot [Adams et al. Acta Crystallographica Section D: Biological Crystallography 2010, 66, 213-221; Emsley et al. Acta Crystallographica Section D: Biological Crystallography 2010, 66, 486-501]. The final structures were validated using Molprobity and the PDB validation tool [Chen et al. Acta Crystallographica Section D: Biological Crystallography 2010, 66, 12-21], before being deposited at RCSB PDB. The data for S6BE-L2 was severely anisotropic. The Diffraction Anisotropy Server was used to improve the corresponding MTZ file (merged with Aimless at a 1.95° A cutoff).

TABLE 4
Crystallization conditions for all SAKe crystals deposited in RCSB PDB.
		Final protein	Reservoir
Protein	PDB code	concentration	solution
S6AE	7ON6	5 mg/mL	0.2M tri-potassium citrate, 1.6M AmSO4
S6AR	7ON8	5 mg/mL	58% (v/v) MPD, 20% (v/v) Glycerol, 0.085M
			HEPES
S6AC	7ONA	5 mg/mL	0.19M Calcium chloride, 0.095M HEPES
			sodium salt pH 7.5, 26.6% (v/v) PEG 400,
			5% (v/v) Glycerol
S6BE	7ONC	5 mg/mL	0.2M Potassium formate, 20%(w/v)
			PEG 3350
S6BE (self-	7ONE	5 mg/mL	50 mM acetate - acetic acid, pH 4.0
assembled)
S6BE-3HH	7OP4	5 mg/mL	0.1M MES pH 6.5, 0.9M Na phosphate, 0.9M
			K phosphate
S6BE-3HH (self-	70PU	10 mg/mL	50 mM citrate - citric acid, pH 4.0
assembled)
S6BE-3HH	7OPV	5 mg/mL	0.1M MES pH 6.5, 1.2M tri-Na citrate, 0.995
(alternative			mM Cu(NO3)2
packing)
S6BE-L1	7ONG	5 mg/mL	0.2M Calcium acetate, 0.1M Sodium
			cacodylate pH 6.5, 40% (v/v) PEG 300
S6BE-L2	7ON7	5 mg/mL	0.2M Magnesium chloride, 0.1M Tris pH 8.5,
			30% (w/v) PEG 4000
S6BE-L3	7ONH	5 mg/mL	0.1M Tris pH 8.0, 2.4M Ammonium sulfate

Example 4. Determination of pI and Surface Electrostatics

Surface electrostatics were calculated at pH 4.0 and 8.0 via PDB2PQR, using a complete

SAKe monomer as input. The calculated surfaces were visualized via PyMOL. pI values were calculated via PROPKA [Dolinsky et al. Nucleic acids research 2004, 32, W665-W667; Li et al. Proteins: Structure, Function, and Bioinformatics 2005, 61, 704-721; Dolinsky et al. Nucleic acids research 2007, 35, W522-W525; Olsson et al. Journal of chemical theory and computation 2011, 7, 525-537].

Example 5. CD Spectroscopy

CD spectroscopy was performed with a JASCO J-1500 spectrometer. To measure the CD spectra, protein samples were diluted to 400 μL of 0.1 mg/ml in 20 mM NaH2PO4 (pH 7.6). Ellipticity was measured at 20° C. from 260 nm to 200 nm, using 1 mm cuvettes. 5 Accumulations were averaged. For determination of melting temperatures, samples were diluted to 400 uL of 0.25 mg/ml in 20 mM NaH2PO4 (pH 7.6). The signal at 233 nm was followed from 0 to 95° C. with intervals of 0.2° C., using sealable 2 mm cuvettes. The data was analyzed with a custom Python script, which fits a sigmoidal curve and extracts its midpoint. The positive signal at 233 nm was previously attributed to interactions between aromatic residues. Disappearance of this signal during melting experiments seemed indicative of tertiary structure disruption [Saxena et al. Biochemistry 1996, 35, 15215-15221; Nikkhah et al. Biomolecular engineering 2006, 23, 185-194].

TABLE 5
Melting temperatures obtained via CD measurements.
	Protein	PDB code(s)	Tm (° C.)
	keap1	1ZGK	44.1
	S6AE	7ON6	67.1
	S6AR	7ON8	75.3
	S6AC	7ONA	87.2
	S6BE	7ONC, 7ONE	95
	S6BE-3HH	7OPU, 7OP4, 7OPV	>95
	S6BE-L1	7ONG	60.6
	S6BE-L2	7ON7	56
	S6BE-L3	7ONH	51.7

Example 6. Spontaneous Crystal Assembly

The tipping point of pH induced self-assembly was found to be approximately 4.5. To show reversibility of assembly, 500 μL of 5 mg/mL S6BE was dialyzed at 20° C. in 50 mM citrate-citric acid buffer at pH 4.0, 4.5 and 5.0. To confirm the pH 4.5 tipping point, a similar experiment was repeated for both S6BE and S6BE-3HH. 500 μL Samples of various protein concentrations (5.0 mg/mL, 2.5 mg/ml, 1.0 mg/ml and 0.5 mg/mL) were dialyzed at 20° C. in 50 mM citrate-citric acid buffer (pH 4.5 and 4.0). For each protein, a self-assembled crystal was soaked in cryo-protectant, vitrified and shipped of for Xray diffraction. Pictures of the self-assembled crystals were taken with a Nikon SMZ800N microscope, outfitted with a TV Lens C 0.45× (Nikon, Japan).

DLS

Concentrated Sake6 and Sake6-3HH protein stock solutions (20 mg/ml, HEPES pH 8) were diluted with either 20 mM MES pH 5.6 or MilliQ to a concentration of 1 mg/ml. Metal suspensions (Cu(NO₃)₂ and Zn(NO₃)₂, MilliQ) were titrated into the 100 μl of prepared protein solution to achieve the desired ratio of protein:metal. Size measurements were obtained at 25° C. using a Zetasizer Nano ZS instrument (Malvern Instruments) and quartz cuvette (ZEN2112). Data analysis was performed using the Zetasizer software 7.11 (Malvern Instruments).

AFM

The protein stock solutions (40 mg/ml, HEPES pH 8) were diluted with the imaging buffer (20 mM MES pH 5.6 or MilliQ) to a desired concentration. The metal salts were suspended in MilliQ (Cu(NO₃)₂ and Zn(NO₃)₂, Sigma-Aldrich) and mixed with the protein solutions to obtain the correct ratio of protein:metal, before being left to incubate for 20 minutes. Next, 30 μl of diluted protein/metal solution was drop cast onto freshly cleaved substrates, muscovite mica (Agar Scientific) or HOPG (ZYB grade, Advanced Ceramics Inc.).

An additional 30 μl droplet of the protein/metal solution was then carefully pipetted onto the AFM's cantilever holder, previously loaded with Nanoworld ARROW-UHF AuD20 (Resonance Frequency 0.7-2.0 MHZ) and brought into contact with the surface droplet in the AFM. The sample and AFM system was then left to equilibrate for 1 hour at 25° C. before imaging.

The images were captured on a Cypher ES atomic force microscope (Asylum Research) using the amplitude modulation mode whilst in solution. The imaging force and frequency were both carefully adjusted to reduce any disruption in the self-assembled surface arrays. The AFM data processing was performed with a combination of SPIP and Gwyddion software. The imaging buffer was either 20 mM MES (PH 5.6) or MilliQ, with protein concentrations of 0.5-5 μM and Cu(NO₃)₂/Zn(NO₃)₂ concentrations (5 μM-50 μM) depending on the requirements of the experiment.

Example 7. In Vivo Half-Life of SAKe Cages

In vivo half-life is an important factor in developing pharmaceutical molecules, as it directly effects the duration of adequate therapeutic effect. Herein, size is an important determinant for in vivo half-life of proteins. Below 70 kDa, most proteins are quickly removed via renal clearance. SAKe proteins of the present invention are roughly 30 kDa. To avoid clearance, SAKe proteins can either be genetically fused or the formation of larger complexes can be induced to surpass the glomerular filtration cutoff. Various SAKe mutants have been engineered and characterized to increasing biological size (Table 6). Additionally, these SAKe mutants show potential as bi/multi-specifics.

TABLE 6
SAKe scaffold mutations allowing an increase of biological size.
Protein	Mutation	Effect	Supported by
dS6AC	Genetic fusion via a	Increasing particle size	SDS PAGE
	Ser linker	via genetic fusion of two	SEC
		SAKes	XRD
S6BE-3HR	The three His-His	Increasing particle size	SDS PAGE
	sites of S6BE-3HH	via Zinc-induced cage	SEC
	were mutated to	formation	XRD
	three His-Arg sites
S2BE-3HR	Truncated version	Confirmation that two	SDS PAGE
	of S6BE-3HR,	blades of the S6BE-3HR	SEC
	where only two out	reassemble the six-	XRD
	of six repeats are	bladed propeller
	expressed	structure without loss of
		the ability to assemble a
		cage following the
		addition of Zinc.
S6BE-3HR-MeM	Genetic fusion of	Simple functionalization	SDS PAGE
	mEmerald fluorescent protein	via genetic fusion	SEC
	to S6BE-3HR
S6BE-3HR-L3	L3 loop graft on	loop modifications do not	SDS PAGE
	S6BE-3HR scaffold	interfere with cage	SEC
		assembly
S6BE-3CHR	Mutated of Asp	Improved SAKe cage	SDS PAGE
	near each of three	formation using a zinc-	SEC
	His-Arg sites to Cys	finger metal	XRD
		binding motif

SDS PAGE

S6BE-3CHR (30.4 kDa), S6BE-3 HR-L3 (34.2 kDa) and mEm-v22S6BE-3 HR (57.7 kDa) are soluble and SDS PAGE confirms their expected sizes (FIG. 25). Multiple thicker bands indicate presence of partially/non-denatured species. This has been reported for thermostable SAKes such as S6BE, S2BE and several of their mutants.

DS6AC (65.0 kDa), a fusion of two S6AC units, is soluble and SDS PAGE analysis confirms the expected size (FIG. 26).

S6BE-3 HR (30.5 kDa) is soluble and SDS PAGE analysis confirms the expected size (FIG. 27). Multiple thicker bands indicate presence of partially/non-denatured species. This has been reported for thermostable SAKes such as S6BE, S2BE and several of their mutants.

S2BE-3 HR (10.4 kDa) is soluble and SDS PAGE confirms the expected size

(FIG. 28). Multiple thicker bands indicate presence of partially/non-denatured species. This has been reported for thermostable SAKes such as S6BE, S2BE and several of their mutants. Here, the lower band corresponds with the fully denatured species.

Size Exclusion Chromatography (SEC)

Concentrated protein samples were loaded on HiLoad Superdex 75 pg 16/600 or HiLoad Superdex 200 pg 16/600 SEC columns (Cytiva). SEC can also be used to assess biological size. All samples were run with a HEPES buffer (20 mM HEPES, pH 8.0, 200 mM NaCl). Proteins expected to interact with metals were incubated with at least 5 mM EDTA prior to SEC injection.

Size exclusion chromatography confirms the expected sizes of each SAKe mutant in solution (FIG. 29). Without metals, no assemblies can be observed. Peaks at lower elution volumes, or higher molecular weights, are caused by impurities or domain swapped species.

Xray Diffraction (XRD)

Protein crystals can be diffracted to study the atomic three dimensional structure of the constituents. This way, the mechanism of metal-binding could be unraveled for proteins such as S6BE-3 HR and S6BE-3CHR. Crystals were grown via sitting drop vapor diffusion in MRC 2-well plates (Hampton Research, UK). Droplets were set up using a Gryphon crystallization robot (Art Robbins Instruments, USA): 0.5 μL crystal screening kit buffer was mixed with 0.25 μL Zn(NO₃) (in water) and 0.25 μl from a 20 mg/ml protein stock (20 mM HEPES pH 8.0, 200 mM NaCl) (Table 7).

The crystals were vitrified after single-step soaking using 25% PEG400 or glycerol as a cryoprotectant. Xray diffraction experiments were performed at Diamond Light Source (UK). Diffraction patterns were indexed using XDS or DIALS. Data reduction was done with Aimless in CCP4. Molecular Replacement phasing was done with PHASER, using an in-house model of S6BE. Refinement was done manually with phenix.refine and Coot.

TABLE 7
Co-crystalization conditions for
various Zn-containing SAKe cages.
Protein	Crystallization buffer	Crystallization mixture
S6BE-3HR	0.2M Na malonate pH 7.0,	0.5 μL crystallization
	20% PEG 3350	buffer + 0.25 μL
S2BE-3HR	1M Succinic	Zn(NO3) (4 mM,
	acid pH 7, 0.1M	water) + 0.25 μL protein
	HEPES pH 7, 1% PEG	(20 mg/mL, 20 mM HEPES,
	MME 2000	200 mM NaCl, pH 8.0)
S6BE-3CHR	0.1M Na Citrate	0.5 μL crystallization buffer +
		0.25 μL Zn(NO3) (6.57
		mM, water) + 0.25 μL
		protein (20 mg/ml, 20 mM
		HEPES, 200 mM NaCl, pH
		8.0)

Xray diffraction experiments carried out on S6BE-3 HR and S6BE-3CHR highlight their mechanisms for zinc coordination and subsequent cage assembly (FIG. 30). S6BE-3 HR cages coordinate zinc via 2His-2Asp motives, while S6BE-3CHR cages contain 2His-2Cys zinc finger-like motives on its vertexes.

SEC shows that S2BE-3 HR proteins first self-assemble as six-bladed trimers (FIG. 105). When zinc is added, these trimers assemble tetrahedral cages identical to those formed by S6BE-3 HR (FIG. 31).

Claims

1. A polypeptide comprising 2 to 9 repeats of a sequence, each of said sequences having at least 80% identity with [SEQ ID NO: 34] NGRIX5AVG8G9-Xn-LNSVX14AX16DX18ETDEW23SFVAPM29TTPR33SGVG37VAV40L], wherein Xn are between 1 and 15 amino acids, wherein x can be any amino acid, and wherein repeats are separated from each other from between 0 and 15 amino acids,wherein the amino acids, Gly8, Gly9, and Trp23 in each of said sequences are conserved,and with the proviso that optionally for one of said repeats, an aminoterminal part of said one repeat is located at the carboxyterminal end of said polypeptide and the remaining carboxyterminal part of said one repeat is located at the aminoterminal end of said polypeptide.

2. The polypeptide according to claim 1, wherein, in one or more, or in all of said sequences,X5 is Tyrosine, Phenylalanine, Tryptophane, Histidine or Methionine,X16 is Tyrosine, Phenylalanine, or Tryptophane, andX18 is Proline or Valine.

3. The polypeptide according to claim 1 or 2, wherein, in one or more, or in all of said sequences X14 is Glutamic acid, Aspartic acid, Cysteine, or Serine.

4. The polypeptide according to any one of claims 1 to 3, wherein, in one or more, or in all of said sequences, Arg33 is conserved and/orVal40 is conserved.

5. The polypeptide according to any one of claims 1 to 4, wherein, in one or more, or in all of said sequences, X5 is Tyrosine,X14 is Glutamic acid,X16 is TyrosineX18 is Proline,Arg33 is conserved, andVal40 is conserved.

6. The polypeptide according to any one of claims 1 to 5, wherein, in one or more, or in all of said sequences, the amino acids, Trp23, Met29, and Gly37 are conserved.

7. The polypeptide according to any one of claims 1 to 6, comprising 2, 3 or 6 repeats of said sequence.

8. The polypeptide according to any one of claims 1 to 7, wherein, one or more or all of said sequences has at least 80, 90 or 95% identity with SEQ ID NO: 32 or is identical to SEQ ID NO:32.

9. The polypeptide according to any one of claims 1 to 8, wherein Xn are between 5 to 15 amino acids, or between 5 to 10 amino acids.

10. The polypeptide according to any one claims 1 to 9, wherein each of said sequences in a repeat are separated from each other with 1 up to 5 amino acids.

11. The polypeptide according to any one of claims 1 to 10, wherein one or more of said repeats are immediately adjacent to each other.

12. The polypeptide according to any one of claims 1 to 11, wherein all of said sequences are immediately adjacent to each other.

13. The polypeptide according to any one of claims 1 to 12, wherein the repeats of said sequence in the polypeptide are identical, with exception of the amino acids Xn.

14. The polypeptide according to any one of claims 1 to 9, comprising a first repeat and a second repeat of said sequence wherein said first and second repeat occur alternating in said polypeptide.

15. A multimer polypeptide of polypeptides as defined in any one of claims 1 to 14.

16. The multimeric polypeptide according to claim 15, wherein the polypeptides, as defined in any one of claims 1 to 14, are non-covalently bound to each other.

17. The multimeric polypeptide according to claim 15, wherein within the multimer of polypeptides, as defined in any one of claims 1 to 10, polypeptides are bound by cystine bonds.

18. The multimeric polypeptide according to any one of claims 15 to 17, which is a hexamer of 3 non-covalently bound polypeptides as defined in any one of claims 1 to, having two repeats, or which is a hexamer of 2 non non-covalently bound polypeptides as defined in any one of claims 1 to 9 having 3 repeats.

19. A method of producing a functional protein comprising the steps of: a) providing a polypeptide according to any one of claims 1 to 14,wherein Xn is random selected or designed by an in silico method,b) generating a multimeric protein of said polypeptides,c) testing the multimeric protein for the function, andd) selecting the multimer protein with the function.

20. The method according to claim 19, wherein the function is protein binding, an enzymatic activity, or the binding to an organic molecule.

21. The method according to claim 19 or 20, further comprising the step of determining whether the multimeric protein is stable at pH below 4, or wherein the multimeric protein is resistant against proteolytic degradation.