Patent Grant
11634699
This disclosure relates to methods and materials useful for forming isopeptide bonds.
Reagents that site-specifically conjugate various molecules (e.g. peptides, drugs, detectable agents and the like) to polypeptides such as proteins have a wide range of potential uses, including among others: the construction of antibody-drug conjugates (ADCs) to treat disease, selective domain labeling for biophysical studies, the construction of immuno-PET reagents for non-invasive imaging, cell specific labeling, and the preparation of multifunctional protein nanoparticles and complexes for industrial purposes. Currently, the sortase A enzyme from Staphylococcus aureus (Sa-SrtA) is perhaps the most widely used bioconjugation reagent. Sa-SrtA is used in the art to generate antibody conjugates, nucleic acid-protein fusions, PEGylation/lipidated proteins, live cell-labeling, protein cyclization, silent labeling, domain labelling, to add proteins to solid supports, and the like.
While conventional Sa-SrtA is a useful bioconjugation reagent, it can only attach molecules to the C- or N-terminus of a protein via a backbone peptide bond. In this context, it would be highly desirable to have new reagents and associated methods that allow artisans to conjugate polypeptide side chains via isopeptide bonds because in principle, this will enable any surface on a polypeptide to be modified. In addition, such linkages are less susceptible to proteolysis and, therefore provide this advantage over other peptide bonds.
For the reasons noted above, there is a need in the art for methods and materials useful for forming isopeptide bonds, for example in methods of site-specific protein modification and engineering.
As discussed in detail below, we have developed new enzyme reagents that are capable of coupling polypeptides together via the formation of isopeptide bonds, as well as a variety of methodologies for using such reagents. The new enzyme reagents comprise bacterial Class C sortase variant polypeptides having one or more amino acid mutations in the lid region of the sortases, mutations that unexpectedly confer the enzymes with an ability to catalyze threonine-lysine isopeptide bond formation in vitro. The new sortase enzymatic activity disclosed herein can be adapted for use in a wide variety of conventional methodologies that are used in the art to modify polypeptides such as proteins.
The new methods and materials disclosed herein are useful, for example, in applications of the type which conventionally use Staphylococcus aureus Sortase A (Sa-SrtA), a transpeptidase that has been widely adopted for site-specific protein modification and engineering, but which only allows for the formation of new amide bonds between a C-terminal sorting motif LPXTG (SEQ ID NO: 5 where X equals any amino acid) and an N-terminal oligoglycine. The key difference between such conventional reagents and the invention is that the new enzymatic reagents disclosed herein can create isopeptide bonds, not peptide bonds. In this context, there are a number of advantages to attaching polypeptides together via isopeptide bonds. These advantages include enabling new sites on polypeptides to be labeled (as Sa-SrtA is only efficient at modifying N- and C-termini). In addition, these new enzymatic reagents can be used in combination with conventional Sa-SrtA in order to enable multiple molecules to be attached to a single protein (e.g. antibody-drug conjugates that contain both a drug as well as a label for tracking tissue distribution). The invention technology disclosed herein can be used in a wide variety of contexts, for example to generate antibody conjugates, nucleic acid-protein fusions, PEGylation/lipidated proteins, live cell-labeling, protein cyclization, silent labeling, domain labeling, to add proteins to solid supports etc. etc. In addition to providing new ways to couple polypeptides together, this isopeptide linkage activity is further desirable because the modification is specific, and the isopeptide linkages may be less susceptible to proteolysis than conventional peptide bonds.
As discussed in detail below, it has been discovered that introducing amino acid mutations (e.g. substitutions and deletions) into the lid region of Class C bacterial sortases can provide these enzymes with a new activity, namely an ability to form isopeptide linkages between a threonine residue in a first polypeptide and a lysine residue in a second polypeptide. Certain working embodiments of the invention disclosed herein utilize an active truncated variant Corynebacterium diphtheriae sortase (SEQ ID NO. 1) having substitution and/or deletion mutations in the lid region of a Cd-SrtA polypeptide. In this context, embodiments of the invention include compositions of matter comprising a variant polypeptide having at least 90% identity to a Corynebacterium diphtheriae Class C sortase polypeptide (e.g. SEQ ID NO. 1, SEQ ID NO. 40 or SEQ ID NO. 41), wherein the Corynebacterium diphtheriae Class C sortase variant polypeptide comprises at least one amino acid substitution mutation or deletion in the lid region of the polypeptide; and the Corynebacterium diphtheriae Class C sortase variant polypeptide exhibits an increased ability to catalyze threonine-lysine isopeptide bond formation in vitro as compared to wild type Corynebacterium diphtheriae Class C sortase.
Typically in the compositions of the invention, the variant polypeptide comprises at least two substitution or deletion mutations in the lid region of the Corynebacterium diphtheriae Class C sortase, for example those selected from the group consisting of D45, W47 and N49 of SEQ ID NO: 1. In certain embodiments of the invention, the variant polypeptide comprises substitution mutations D45G and W47G in SEQ ID NO: 1, and optionally a further substitution mutation in SEQ ID NO: 1 such as N49A. In other embodiments of the invention, the variant polypeptide comprises a deletion mutation in at least one amino acid of residues 43-51 of SEQ ID NO: 1. Optionally, the Corynebacterium diphtheriae Class C sortase variant polypeptide in such compositions exhibits an ability to form isopeptide bonds in vitro that is at least 25% or 50% of the ability to form isopeptide bonds exhibited by a Corynebacterium diphtheriae sortase polypeptide comprising lid region substitution mutations D45G and W47G in the polypeptide of SEQ ID NO: 1 or lid region deletion mutations in amino acids 43-51 the polypeptide of SEQ ID NO: 1.
The compositions of the invention include those used in the methods of the invention discussed below and can include additional reagents pertinent to such methods. For example, in certain embodiments of the invention, compositions can further comprise at least one of a polypeptide (e.g. a polypeptide comprises an amino acid sequence YPKN (SEQ ID NO. 6) or LPLT (SEQ ID NO. 7)), a polynucleotide, a lipid, a sugar, a therapeutic agent or a detectable label. In certain embodiments of the invention, the polypeptide, the polynucleotide, the lipid, the sugar, the therapeutic agent or the detectable label is coupled to a polypeptide comprising an amino acid sequence YPKN (SEQ ID NO. 6) or LPLT (SEQ ID NO. 7). In some embodiments of the invention, the compositions further include a wild type Class C sortase bacterial polypeptide such as a Staphylococcus aureus sortase polypeptide (e.g. SEQ ID NO: 2).
Other embodiments of the invention methods of forming an isopeptide bond in vitro between a threonine residue in a first polypeptide and a lysine residue in a second polypeptide, the method comprising forming a mixture comprising the first polypeptide and the second polypeptide disposed within a bacterial Class C sortase variant polypeptide composition such as those disclosed herein (e.g. using a composition discussed immediately above); and then allowing the sortase variant polypeptide to form an isopeptide bond between the threonine residue in the first polypeptide and the lysine residue in the second polypeptide, so that an isopeptide bond between a threonine residue in a first polypeptide and a lysine residue in a second polypeptide is formed. In certain embodiments of the invention, the methods include using a polypeptide having an amino acid motif comprising at least four contiguous amino acid residues that is present in a SpaA (SEQ ID NO: 3) or a SpaB (SEQ ID NO: 4) polypeptide (e.g. the amino acid motif LPXT (SEQ ID NO: 8)). Optionally, an agent used in such methods comprises a detectable marker (e.g. a fluorophore or a PET label), an antibody, a growth factor or a fluorescent protein. Such methodological embodiments of the invention can be used in a wide variety of common biotechniques including therapeutic agent-polypeptide conjugation, nucleic acid-polypeptide conjugation, detectable marker-polypeptide conjugation, PEGylation or lipidation of proteins, detectably labelling live cells, protein cyclization, silent labeling, domain labeling, covalently attaching polypeptides to solid supports and the like.
Other objects, features and advantages of the present invention will become apparent to those skilled in the art from the following detailed description. It is to be understood, however, that the detailed description and specific examples, while indicating some embodiments of the present invention, are given by way of illustration and not limitation. Many changes and modifications within the scope of the present invention may be made without departing from the spirit thereof, and the invention includes all such modifications.
In the description of embodiments, reference may be made to the accompanying figures which form a part hereof, and in which is shown by way of illustration a specific embodiment in which the invention may be practiced. It is to be understood that other embodiments may be utilized and structural changes may be made without departing from the scope of the present invention. Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.
Many of the aspects of the techniques and procedures described or referenced herein are well understood and commonly employed by those skilled in the art. The following provides a number of illustrative embodiments of the invention.
As disclosed herein, we have discovered that mutations in the lid region of Class C sortases provide these enzymes with a new activity, namely an ability to form isopeptide linkages between a threonine residue in a first polypeptide and a lysine residue in a second polypeptide. Illustrative working embodiments of the invention disclosed herein comprise a variant Corynebacterium diphtherias sortase (Cd-SrtA) having mutations in the lid region of the Cd-SrtA polypeptide, for example an active truncated variant of this enzyme (SEQ ID NO. 1) having at least two substitution mutations selected from the group consisting of D45G, W47G and N49A of SEQ ID NO:1 or at least 2 deletion mutations in amino acids 43-51 the polypeptide of SEQ ID NO: 1. In illustrative embodiments of the invention (see, e.g., the schematics shown in
We have discovered that the Corynebacterium diphtherias sortase (Cd-SrtA) enzyme can be precisely modified in a manner that allows it to be used as a new form of bioconjugation reagent. On the bacterial cell surface, Cd-SrtA functions as a transpeptidase that joins two proteins together. One protein contains a C-terminal LPLTG (SEQ ID NO. 5) amino acid sequence, and the other protein contains a pilin motif (WXXXVXVYPKH (SEQ ID NO: 12)) that harbors a reactive side-chain ε-amine lysine nucleophile (underlined) (
Atomic structures of Cd-SrtA have revealed that its active site is masked by a polypeptide appendage called a “lid”. The lid inhibits the transpeptidation activity of the enzyme by preventing substrate binding. In the examples below we show that the strategic introduction of amino acid mutations into the lid activates the enzyme, enabling it to catalyze lysine isopeptide bond formation in vitro, whereas the wild-type Cd-SrtA protein is not active in vitro. At present, we have created two activated enzymes, Cd-SrtA2M (Cd-SrtA with D81G and W83G mutations) and Cd-SrtA3M (Cd-SrtA with D81G, W83G and N85A mutations). Cd-SrtA3M has the highest level of in vitro activity.
As disclosed herein, we show that Cd-SrtA2M can ligate its natural protein substrates via an isopeptide bond. Its substrates are two autonomously folded domains: CSpaA containing the LPLTG sequence at its C-terminus (residues 491-495 of SpaA) and NSpaA containing the pilin motif (residues 181-191 of SpaA). In work described below, we have extended these studies to demonstrate peptide labeling of proteins and protein-protein ligation.
As discussed in detail below, mutations in the lid region of Class C bacterial sortases can provide these enzymes with a new activity, namely an ability to form isopeptide linkages between a threonine residue in a first polypeptide and a lysine residue in a second polypeptide. The working embodiments of the invention disclosed herein utilize variants Corynebacterium diphtheriae sortase (Cd-SrtA) having deletion or substitution mutations in the lid region of a catalytic domain variant of a Cd-SrtA polypeptide (e.g. amino acids 43-51 of SEQ ID NO: 1). In this context, embodiments of the invention include compositions of matter comprising a variant polypeptide having at least 90% identity to Corynebacterium diphtheriae Class C sortase polypeptide of SEQ ID NO: 1 (or at least 90% identity to SEQ ID NO: 2, or at least 90% identity to SEQ ID NO. 41); wherein the Corynebacterium diphtheriae Class C sortase variant polypeptide comprises at least one amino acid substitution or deletion mutation in amino acids 43-51 of SEQ ID NO: 1; and the Corynebacterium diphtheriae Class C sortase variant polypeptide exhibits an increased ability to catalyze threonine-lysine isopeptide bond formation in vitro as compared to wild type Corynebacterium diphtheriae Class C sortase.
Typically in the compositions of the invention, the variant polypeptide comprises at least two substitution or deletion mutations in the lid region of the Corynebacterium diphtheriae Class C sortase, for example one or more substitution mutations selected from the group consisting of D45, W47 and N49 of SEQ ID NO: 1, and/or a plurality of deletion mutations in residues 43-51 of SEQ ID NO: 1. In certain embodiments of the invention, the variant polypeptide comprises substitution mutations D45G and W47G in SEQ ID NO: 1, and optionally the further substitution mutation N49A in SEQ ID NO: 1. Optionally, the Corynebacterium diphtheriae Class C sortase variant polypeptide in such compositions exhibits an ability to form isopeptide bonds in vitro that is at least 25% or 50% of the ability to form isopeptide bonds exhibited by a Corynebacterium diphtheriae sortase polypeptide comprising lid region substitution mutations D45G and W47G in the polypeptide of SEQ ID NO: 1 or one or more deletion mutations in ILDPWLNRA of SEQ ID NO: 1 (including all amino acids ILDPWLNRA), for example using an assay of isopeptide bond formation that is disclosed herein. In this context, the invention disclosed herein further provides methods of identifying class C sortase enzymes having a modified ability to form isopeptide bonds in vitro, namely by making systematic mutations in the amino acids of the lid region of such enzymes and then testing such mutants for an ability to form isopeptide using an assay of isopeptide bond formation that is disclosed herein.
After discovering that iterative substitution mutations of the occlusionary “lid” structure of CdSrtA yielded incremental increases in transpeptidation product, we reasoned that removal of most or all residues comprising the lid structure would yield an even more active variant. Examination of the crystal structure of CdSrtA WT showed that the lid begins and ends at a similar position in the tertiary structure, providing evidence that complete deletion of the intervening residues would be tolerated and not preclude proper protein folding. Thus, we deleted nine residues from the lid (“ILDPWLNRA”), calling this new construct CdSrtAΔ. Expression of this engineered variant was robust and protein stability was comparable to other constructs. When we then assayed transpeptidation activity of CdSrtAΔ by our gel-based assay (
Illustrative Applications
Creating Isopeptide-Linked Protein-Peptide Conjugates.
Creating isopeptide-linked protein-peptide conjugates is a promising use for the new technology disclosed herein as it enables peptide-based molecules to be attached to proteins via a robust isopeptide bond (see, e.g. the schematics shown in
We have also demonstrated Cd-SrtA3M catalyzed protein labeling with a peptide-fluorophore (see, e.g.
Joining Proteins Together Via Lysine Isopeptide Bonds.
Embodiments of the invention can be used to join proteins together in vitro. In such embodiments, two fusion proteins are typically used. The first contains the protein of interest fused to NSpaA (either at its N- or C-termini). The second protein is modified to contain a LPLTG motif (SEQ ID NO. 5) at the C terminus. The new reagent then ligates the proteins together, forming an isopeptide linkage between the threonine of the LPLTG (SEQ ID NO: 5) sequence (appended to the C-terminus of one protein) and the sidechain lysine residue of NSpaA (fused to the other protein of interest).
We have demonstrated protein-protein isopeptide coupling activity. As discussed in detail below, a GFP (green fluorescent protein) construct containing a C-terminal LPLTG (SEQ ID NO. 5) sequence was ligated to NSpaA (
In addition to the SpaA ligation systems reported above, the GFP-SpaB fusion has use in additional applications or fields. For example, the SpaB protein from C. diphtherias has inherent ability to tightly bind human pharyngeal cells. In this context, GFP-SpaB fusions can be used to label pharyngeal cells in vitro or in vivo. (i.e. labeling cells for bioassay, or labeling pharyngeal cells for medical procedures). Further, the system can be adapted to allow for small molecules (e.g. comprising a LPLTG (SEQ ID NO: 5) motif) to be coupled to SpaB, allowing for targeted small molecule payload delivery directly to pharyngeal cells. This can be useful for targeted delivery of therapeutics to pharyngeal infections, cancers, or other ailments which are presently difficult to treat.
The technology disclosed herein can be adapted for use with conventional applications that utilize Sa-SrtA. For example, embodiments of the invention can be used to generate antibody conjugates, nucleic acid-protein fusions, PEGylation/lipidated proteins, live cell-labeling, protein cyclization, silent labeling, domain labelling, to add proteins to solid supports and the like. A key difference between the invention disclosed herein and the conventional Sa-SrtA technology is that the new reagent unexpectedly creates isopeptide bonds, not peptide bonds. There are a number advantages to attaching proteins via isopeptide bonds including: (1) these linkages may be more resistant to proteolysis and therefore more stable, (2) the new enzyme reagent enables new sites on the protein to be labeled as Sa-SrtA is only efficient at modifying N- and C-six termini and (3) the new reagent can be used in combination Sa-SrtA enabling multiple molecules be to be attached to a single protein (e.g. ADCs that contain both an attached drug and a label for tracking tissue distribution).
Illustrative applications for the new reagents and methods disclosed herein include generating antibody-drug conjugates, generating nucleic acid-protein conjugates (e.g. conjugates useful to deliver the nucleic acid into the cell to modify the genome or modulate gene expression), covalent attachment of proteins and to solid supports and surfaces (e.g. to construct materials for biosensing and biocatalysis, lipid modification of proteins through sortase-catalyzed transpeptidation (e.g. to allow for the preparation of proteins that can be localized to regions of the cell via attached lipids (e.g. attachment of lipids to Ras and Rab proteins), the immobilization of proteins to biacore sensor chips and the like.
It has been reported that the S. aureus SrtA enzyme can form LPXTG ((SEQ ID NO: 5) where X is any amino acid) and lysine isopeptide conjugates (e.g. WO2015130846A2 and Bellucci J J, Bhattacharyya J, Chilkoti A. A non-canonical function of sortase enables site-specific conjugation of small molecules to lysine residues in proteins. Angewandte Chemie (International ed in English). 2015; 54(2):441-445. doi:10.1002/anie.201408126). This however is generally considered a side reaction, as the enzyme preferentially carries out a typical transpeptidation reaction with a primary amine N-terminal glycine nucleophile resulting in a normal peptide bond. Additionally, these studies only demonstrate ligation using canonical pilin motifs. To our knowledge this is the first report for utilization of an engineered sortase enzyme to robustly and specifically generate isopeptide protein bioconjugates with proteins containing a pilin motif (e.g. SpaA-NTD and SpaB). As well as the first reported SpaB bioconjugate. Moreover, the Cd-SrtA the only enzyme reagent available in the art that enables peptide and protein linkage via lysine isopeptide bonds at high yield.
The Examples below provide illustrative methods and materials that can be used in the practice the various embodiments of the invention disclosed herein.
Part 1
Gram-positive sortase enzymes represent two broad functional categories—those that crosslink proteins to the cell wall, and those that can catalyze this reaction and polymerize proteins to build adhesive pilus fibers. In this Example, we disclose an in vitro reproduction of a robust pilus polymerization reaction using a variant of a corynebacterial pilus-specific sortase, in which the catalytic center is unmasked. By molecular modeling, we uncovered a conserved structural element of pilus-specific sortases critical for protein ligation in vitro and further demonstrated that the activated sortase ligates the isolated domains of the pilin harboring the donor and acceptor motifs for ligation. Besides enabling future molecular studies and antibiotic development, our system provides a powerful new platform for bioconjugation and protein engineering.
Adhesive protein polymers, called pili or fimbriae, are expressed on the cell envelope by many Gram-negative and Gram-positive bacteria, and they are critical for bacterial virulence (1). Many types of Gram-negative pili have been reported, including the well-studied retractable type IV, conjugative, and chaperone-assisted pili (2). These pili are formed by distinct pathways (2, 3); however, none of these pili are covalently linked polymers, unlike the sortase-catalyzed pili found in many Gram-positive bacteria, including Actinomyces oris, Enterococcus faecalis, Bacillus cereus, and numerous species of streptococci and lactobacilli (4-6).
One of the well-studied sortase-mediated pilus assembly systems involves Corynebacterium diphtheriae (7), the causative agent of pharyngeal diphtheria (8). C. diphtheriae produces three distinct pilus types (7, 9, 10), each comprised of a pilus tip adhesin, a pilus shaft made of the major pilin, and a base pilin that is covalently anchored to the cell wall (11). The archetypal SpaA-type pilus, which mediates adherence to the pharyngeal epithelium (12), consists of the tip pilin SpaC, shaft pilin SpaA, and pilus base SpaB (13). A pilus-specific sortase named SrtA is required for pilus polymerization (13), performing a repetitive, irreversible transpeptidation reaction that covalently links the pilin subunits via an isopeptide bond (14). Although each Spa pilin harbors a cell wall sorting signal (CWSS (SEQ ID NO. 19)), which starts with a conserved LPXTG (SEQ ID NO: 5) motif, followed by a stretch of hydrophobic amino acids and a positively charged tail (15), SpaA contains a pilin motif with the lysine residue K190 acting as a nucleophile for the aforementioned transpeptidation reaction (13). According to the current model (16), SrtA cleaves the LPXTG (SEQ ID NO: 5) motif of Spa pilins between threonine and glycine, forming acyl-enzyme intermediates between the threonine residue and the SrtA catalytic cysteine residue. This intermediate is then nucleophilically attacked by the reactive K190 of an incoming SpaA subunit. In pilus biogenesis, the SpaC-SrtA acyl enzyme intermediate forms first, resulting in the joining of the ε-amine group of K190 to the threonine carbonyl carbon atom in the LPXT (SEQ ID NO: 8) of SpaC. Pilus polymerization ensues when additional SpaA protomers are joined progressively to the pilus base by the SrtA enzyme via the same lysine-mediated transpeptidation reaction. Polymerization is terminated with the entry of SpaB to the pilus base (11), which is then anchored to the cell wall by the housekeeping sortase SrtF (17). This cell wall anchoring of pilus polymers is likely similar to that of surface proteins catalyzed by the prototype sortase A enzyme from Staphylococcus aureus (18, 19). While most of this biphasic model of pilus assembly in Gram-positive bacteria (6)—the pilus polymerization followed by cell wall anchoring—has experimentally been validated, the molecular determinants that make up a pilus-specific sortase and enables the enzyme to join proteins together remain unknown.
The SrtA enzyme is classified as a member of the class C sortase subgroup within the sortase superfamily that have the unique ability to cross-link proteins via lysine-threonine isopeptide bonds (20, 21). Although all sortases share a canonical β-barrel sortase superfamily fold (22, 23), class C enzymes are distinguished by the presence of a conserved N-terminal region that forms a “lid” that covers the active site structurally and functionally (24-26). In Streptococcus pneumoniae, X-ray crystallographic evidence originally suggested that the lid region was flexible, possibly modulating substrate binding; however, subsequent studies in solution utilizing NMR showed this region to be relatively rigid in the SrtC1 enzyme (24, 27-29). Mutations of the lid region in A. oris SrtC2 or Streptococcus agalactiae SrtC1 did not alter the pilus polymerizing activities in vivo (30, 31); nonetheless, the mutations caused enzyme instability and increased hydrolytic activity in S. agalactiae SrtC1 (30), supporting a regulatory role for the N-terminal lid. However, the unique structural properties that enable class C sortase enzymes to cross-link proteins remain unknown.
We report here the crystal structures of the C. diphtherias class C sortase SrtA lacking the signal peptide and transmembrane domain (referred to as SrtAWT) and a mutant of this protein that has substitutions in the lid interface, which normally masks the catalytic pocket (SrtA2M). Using these recombinant enzymes and a SpaA substrate that is missing the signal peptide and transmembrane domain, we succeeded in reconstituting the SpaA pilus shaft polymerization reaction in vitro, demonstrating that the removal of SrtA's lid not only unmasked the catalytic center structurally, but also enables the polymerizing activity in vitro. Subsequently, by structural modeling, phylogenic and mutational analyses, we identified two structural elements that enable SrtA to cross-link proteins. Importantly, we showed that the activated sortase can ligate the isolated pilin domains, thus defining the donor and acceptor motifs for the ligation reaction. The system we report provides a new platform for in vitro mechanistic investigations of Gram-positive pilus assembly, antibiotic development and biotechnological applications of protein modification and conjugation via a unique transpeptidation reaction.
Results and Discussions
Structure of the C. diphtheriae pillus-specific sortase—The archetypal SpaA pilus polymer produced by corynebacteria is built by the dedicated pilus-specific sortase named SrtA (7, 13). To gain insight into the mechanism of pilus polymerization, we determined the structure of SrtA by X-ray crystallography. We performed crystallization screens using a soluble fragment encompassing the catalytic domain of SrtA (residues 37-257, termed SrtAWT), which was cloned, expressed, and purified from E. coli. SrtAWT crystallized as a homodimer in the P61 2 2 space group. Diffraction data was collected to 2.1 Å resolution and phased by molecular replacement. The electron density for residues 37-248 was well defined enabling their structure to be modeled, while density for the remaining C-terminal residues is missing presumably due to a disordered state.
The overall structure of SrtAWT conforms to the typical sortase fold described previously (22), containing a 7-stranded β-barrel core flanked by several 310 and alpha helices (
To investigate the functional importance of the lid in polymerization, we next generated a recombinant SrtA mutant protein, in which the DPW lid motif (residues 81-83 of SEQ ID NO. 1) was mutated to GPG (SEQ ID NO: 14), hereafter described as SrtA2M. We succeeded in determining the crystal structure of SrtA2M at 1.85-Å resolution using crystallization conditions that differed from those used for the WT protein (see Materials & Methods). In the electron density map for SrtA2M, residues 80-86 that represent the lid were invisible. Presumably, the lid residue substitutions prevented contacts with the active site, causing the mutant lid to adopt a range of conformations. Remarkably, a second major difference between the two structures is the absence of interpretable electron density for the H1 helix in the SrtA2M lid-mutant, which might be caused by flexibility around the hinge between helices H1 and H2 and by the absence of stabilizing interactions with neighboring molecules in the crystals of SrtA2M.
To evaluate the involvement of the predicted catalytic residues and the lid in pilus assembly, corynebacterial cells harboring WT and its isogenic mutants were subjected to cell fractionation, and protein samples were immunoblotted with specific antibodies against SpaA, the cognate substrate of SrtA that forms the pilus shaft (7, 13). As shown in
Thus, the overall structure of the C. diphtheriae SrtA′ enzyme resembles class C sortases, or pilus-specific sortases, which possess a distinguishing feature of this class of enzymes, i.e. the lid region (16, 21). In agreement with previous studies (30, 31), the elimination of the lid's interaction at the catalytic pocket does not dramatically affect pilus polymerization in vivo.
In vitro reconstitution of archetypal C. diphtheriae SpaA pilus polymerization—Previous structural and biochemical studies of pilus-specific sortase enzymes in several streptococcal species implicate that the lid may modulate substrate entry into the active site (24, 25, 28, 30). We envisioned that a loss of lid closure might increase the accessibility of the active site. To test this hypothesis, we used the thiol-reactive reagent 4,4′-dithiodipyridine (DTDP) (32, 33), a to probe the solvent accessibility of the catalytic cysteine residue (C222). Disulfide exchange between thiol side chains of Cys resides and DTDP gives rise to 4-thiopyridone, which shows strong absorption at 324 nm (33). Recombinant proteins SrtAWT or SrtA2M (0.6 mg/ml) were rapidly mixed with 0.32 mM DTDP, and the rate of reaction between DTDP and C222 was monitored as an increase in absorbance at 324 nm. Time-dependent changes in absorbance were fit to single or double exponential equations to derive rates as described in the Materials and Methods. As shown in
The increased DTDP reactivity of the active site cysteine residue in SrtA2M described above raises the possibility that the mutant enzyme may be able to assemble pili in vitro—which has been difficult to reconstitute so far for pilus assembling sortases. We, therefore, sought to reconstitute pilus polymerization in vitro using various recombinant sortase enzymes and a soluble form of SpaA (residues 30-500), which is devoid of the N-terminal signal peptide and C-terminal membrane anchor domain (see a diagram in
To visualize BMW SpaA polymers formed by SrtA2M, the reaction mixtures after 72 h of incubation were subjected to electron microscopy, whereby aliquots were applied to nickel grids; bound proteins were washed and stained with 0.75% uranyl formate prior to viewing with an electron microscope. As shown in
To determine if the recombinant SrtA2M enzyme faithfully catalyzes the pilus transpeptidation reaction, we determined whether the SpaA subunits in the BMW SpaA polymers were linked together via covalent lysine isopeptide bonds, in which the threonine residue of the LPLT (SEQ ID NO. 7) sorting signal was joined to the lysine residue within the pilin motif (13). Indeed, mass spectrometry (MS) analysis of excised HMW SpaA polymer SDS-PAGE bands revealed the presence of an isopeptide bond between the carbonyl carbon of T494 and the sidechain amine of Lys190 (
To further probe the mechanism of SpaA pilus assembly, we dissected the SpaA molecule into two components: the N-terminal domain of SpaA-NSpaA, residues 30-194—encompassing the pilin motif with the K190 nucleophile and the C-terminal domain—CSpaA, residues 350-500—containing the CWSS (SEQ ID NO. 19) with the LPLTG (SEQ ID NO. 5) motif (
Structural elements in a sortase required for protein polymerization—To gain insight into how SrtA joins the SpaA proteins together during polymerization, we performed molecular modeling of the NSpaA-SrtA-CSpaA ternary complex, in which the isopeptide bond is modeled using our previously determined crystal structures of SpaA (PDB: 3HR6) (35) and the isolated SrtA (PDB:5K9A) proteins. We first generated a model of the SrtA-SpaA acyl-intermediate, juxtaposing the C-terminus of the C-terminal SpaA domain with the active site Cys222 residue in SrtA. Because the crystal structure of SpaA lacks the CWSS (SEQ ID NO. 19) that forms the acyl-intermediate with SrtA, we modeled the acyl-intermediate by placing the C-terminal domain of SpaA ˜25 Å away from the active site cysteine to accommodate the 9 missing C-terminal residues that contain the CWSS (SEQ ID NO. 19). To construct the ternary complex we then positioned the coordinates of the SpaA N-terminal domain near the acyl intermediate to juxtapose the reactive lysine Lys190 of the pilin motif with the active site Cys222 residue (
In our model of the ternary reaction intermediate, the side chains of Y225, N228 and 5229 within the TP(Y/L)XIN(S/T)H motif (SEQ ID NO. 10) extend from the enzyme's surface in a position to contact NSpaA. To explore their possible roles in catalysis, we constructed a series of mutants of the lid-opened SrtA2M mutant enzyme, in which each of these residues were individually replaced by alanine. The purified S229A and N228A mutant SrtA2M proteins were each defective in transpeptidation in vitro, as no isopeptide linked SpaA-SpaA product was produced even after 48 hours; the Y225A mutant protein had impaired transpeptidation activity as well, but to a lesser extent than the S229A and N228A mutants (
The model of the ternary complex raises the possibility that critical residues in the β7/β8 loop and the helix H1 may only be required for nucleophile recognition during the transpeptidation reaction, but not for the other step of catalysis in which the LPXTG (SEQ ID NO. 5) sorting motif is cleaved to form the thioacyl enzyme-substrate intermediate (4, 37). To test this hypothesis, we determined the importance of these structural elements in thioacyl intermediate formation, using an established HPLC-based assay (38, 39) and a SpaA derived peptide KNAGFELPLTGGSGRI (SEQ ID NO. 9) (SpaApep) as the substrate. The enzymes and SpaApep substrate were mixed at 1:10 molar ratio, and the loss of the intact peptide was monitored by HPLC with the hydrolysis activity of SrtA2M set as 100%. Consistent with a selective role in nucleophile recognition, none of the mutants exhibited any significant defect in cleaving the LPXTG (SEQ ID NO. 5) motif (
In conclusion, we reported here the high-resolution crystal structures of the C. diphtherias pilus-specific sortase SrtA enzyme (SrtAWT) and a mutant form of the enzyme with mutations in the lid region (SrtA2M) and through these, illuminated some of the basic features of the sortase that functions to polymerize pilus proteins in Gram-positive bacteria. The structure of the wild-type enzyme displayed a characteristic “closed” configuration of a class C sortase with its catalytic site occluded by a molecular lid (
Materials and Methods
Bacterial Strains, Plasmids, and Media
C. diphtherias strains were grown in Heart Infusion (HI) broth (Becton Dickinson) or on HI agar plates at 37° C. When needed, kanamycin was added to the concentration of 25 μg ml-1. E. coli DH5a and BL21 (DE3), used for cloning and protein expression and purification, respectively, were grown either in Luria-Bertani (LB) or 2TY media at 37° C. in the presence of ampicillin (Amp) at 100 μg ml−1.
In Vitro Reconstitution of Pilus Polymerization
In vitro reactions were carried out at room temperature and proteins were dissolved in assay buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 1 mM DTT). All reactions used a fixed 100 μM concertation of SrtA enzyme and 300 μM SpaA substrate (either full length, or each individual domain). Reactions were stirred gently by continuous rotation. Time points were taken at 0 h, 24 h, 48 h, and 72 h, and reactions were quenched by addition of two volumes of SDS-loading dye.
Probing Accessibility of the SrtA Active Site
Reaction rates of 4,4′-dithiodipyridine (DTDP) and SrtA proteins via the cysteine C222 residue were determined by stopped-flow experiments, which were performed at 23° C. using an Applied Photophysics Ltd. (Leatherhead, UK) Model SX.18 MV sequential stopped-flow spectrofluorimeter with a 150 watt Xe/Hg lamp, and a dead time of 1.7 ms. All triplicate reactions were carried out in Reaction Buffer (50 mM MOPS, 200 mM KCl, 1 mM EDTA, pH 7.5). Absorbance was monitored at 325 nm after rapidly mixing solutions in syringe A, which contained 0.6 mg/ml protein, and syringe B containing 0.32 mM DTDP. Reaction rates (k) were derived by fitting data to the following equations with 1 (equation 1) or two (equation 2) rates:
A=A max*(1−e−kt) equation 1
A=A max*(1−e−klt)+A max 2*(1−e−k2t) equation 2
where A is absorbance at 324 nm at time, t, and Amax is the maximum absorbance.
Cell Fractionation and Western Blotting
Cell fractionation and Western blotting were followed according to published procedures with some modifications (41, 42). Briefly, mid-log phase cultures of C. diphtherias strains grown at 37° C. were normalized an OD600 of 1.0, and subject to cell wall protein extraction using mutanolysin (300 U/ml). Protein samples obtained from culture medium (S) and cell wall (W) were TCA precipitated and acetone washed. The protoplasts after the cell wall extraction were used for analysis of cell membrane-bound proteins. Protein samples were resuspended in SDS sample buffer containing 3% urea and heated at 100° C. for 10 min prior to SDS-PAGE analysis using 3-12% or 3-20% Tris-glycine gradient gels. Detection of proteins was performed by immunoblotting with specific antibodies (1:20,000 for α-SpaA; 1:4,000, α-SpaH; 1:5,000, α-MdbA, and 1:4,000, α-SrtA).
Mass Spectrometry of Pilus Polymers
Protein digestion and isopeptide bond identification were performed according to previous protocols (35, 43). Specifically, proteins entrapped in gel bands were reduced with 10 mM dithiothreitol (Sigma) at 60° C. for an hour and then alkylated with 50 mM iodoacetamide (Sigma) at 45° C. for a few minutes in the dark. These reduction and alkylation steps were skipped for the acyl intermediate samples. Samples were digested with 200 ng trypsin (Thermo Scientific) at 37° C. overnight. At the end of trypsin digestion, 200 ng of Asp-N endoproteinase (Thermo Scientific) were added for another overnight incubation.
Digested peptides were extracted from the gel bands in 50% acetonitrile/49.9% water/0.1% trifluoroacetic acid (TFA) and cleaned with C18 StageTip (44) before mass spectrometry analysis. Digested peptides were separated on EASY-Spray column (25 cm×75 μm ID, PepMap RSLC C18, 2 μm, Thermo Scientific) connected to an EASY-nLC 1000 nUPLC (Thermo Scientific) using a gradient of 5-35% acetonitrile in 0.1% formic acid and a flow rate of 300 nl/min for 30 minutes. Tandem mass spectra were acquired in a data-dependent manner with an Orbitrap Q Exactive mass spectrometer (Thermo Fisher Scientific) interfaced to a nanoelectrospray ionization source.
The raw MS/MS data were converted into MGF format by Thermo Proteome Discoverer 1.4 (Thermo Scientific). We wrote in-house programs to search for the isopeptides in two different approaches. The first approach was performed as previously described (35) and used calculated masses of predicted peptides containing the isopeptide linkage to guide the search. The second approach was based on the observation of published spectra as well as our own on the presence of ions specific for the fragments of ELPLT (m/z 215.138, 225.122, 243.132, 294.180, 312.190, 322.174 and 340.186). The in-house programs sifted through tens of thousands of mass spectra looking for this information specifically and pulled out mass spectra of interest for further analysis and manual validation.
Determination of SrtA Hydrolysis by an HPLC-Based Assay
In vitro hydrolysis reactions were performed based on the method developed by Kruger et al. (45). 50 μM SrtA (wild-type or mutant) was incubated with 500 μM KNAGFELPLTGGSGRI (SEQ ID NO. 9) (SpaApep) in 100 μl reactions at 37° C. for 48 h. The reactions were quenched by adding 50 μl of 1 M HCl and injected onto a Waters XSelect HSS C18 reversed phase HPLC column. Peptides were eluted by applying a gradient from 3 to 23% acetonitrile (in 0.1% trifluoroacetic acid) over 25 minutes at a flow rate of 1 ml/min. Elution of the peptides was monitored by absorbance at 215 nm. Peak fractions were collected and their identities were confirmed by MALDI-TOF mass spectrometry.
Electron Microscopy
For visualization of in vitro pilus polymers, 7 μl aliquots of pilus polymerization reactions diluted in half with water were applied onto nickel grids, washed 5 times with distilled water, and stained with 0.75% uranyl formate for 1 min prior to viewing by a JEOL JEM-1400 electron microscope.
pSUMO-SrtAWT and its derivatives—To generate a recombinant plasmid expressing His tagged SrtAWT (residues 37 to 257), the srtA gene sequence without N-terminal signal peptide and C-terminal membrane spanning domains was PCR-amplified from the genomic DNA of C. diphtherias NCTC 13129 with appropriate primers and inserted into the pE-SUMO (LifeSensors) expression vector using the Gibson assembly method (New England BioLabs). pSUMO-SrtAWT was used as a template to generate D81A and W83A mutations (pSUMOSrtA2M) as well as Y225A, S229A or N228A mutation, using site-directed mutagenesis carried out by QuickChange method (Agilent). Resulting plasmids were then transformed into XL10 for amplification prior to DNA sequence confirmation. Similarly, pSUMO ΔSrtA2M were generated using pSUMO-SrtA2M as a template, in which H1 helix (residues 37-54) was removed. The resulting plasmid was introduced into E. coli BL21 (DE3) after verification by DNA sequencing.
pMCSG-SrtAWT and its derivatives—For protein crystallization, the same srtA fragment as the above was cloned into the pMCSG7 expression vector by ligation-independent cloning (LIC) as previously reported (1). The resulting plasmid was introduced into E. coli DH5a for selection and DNA sequencing, and then E. coli BL21 (DE3). To generate pMCSG-SrtA2M, pMCSG-SrtAWT was used as a template for inverse PCR amplification with a pair of phosphorylated primers carrying the intended mutation as previously described (1). The resulting linear PCR product was ligated before introduced into E. coli DH5a. The generated plasmid was verified by DNA sequencing prior to introduce into E. coli BL21 (DE3).
Recombinant SpaA plasmids—A plasmid expressing recombinant His-tagged SpaA protein of C. diphtheriae lacking the N-terminal signal peptide and C-terminal transmembrane domain (residues 30 to 500) was generated using the LIC method described above. The resulting plasmid (pMCSG-SpaA) was introduced into E. coli BL21 (DE3) after verification by DNA sequencing. Using the above site-directed mutagenesis method, pMCSG-SpaA was then used as a template to generate pMCSG-SpaAK190A that expresses the same SpaA molecule with lysine 190 replaced by alanine. To generate plasmids pSUMO-NSpaA and pMBP-CSpaA, which express the N-terminal (residues 30 to 194) and C-terminal (residues 350 to 500) domains of SpaA, pE-SUMO and pE-MAPLE were used, respectively, in the same cloning protocol as described for pSUMO-SrtAWT above. pSrtA and its derivatives—For srtA expression in C. diphtheriae, the E. coli/Corynebacterium shuttle vector pCGL0243 was used (2). pSrtA (3), a pCGL0243 derivative that expresses C. diphtheriae srtA under control of the spaA promoter, served as a template for site-directed mutagenesis, as described above, to generate various SrtA variants used in this study. The resulting plasmids were introduced into E. coli DH5a for DNA sequencing prior to electroporation into C. diphtheriae strains.
Protein Expression and Purification
For in vitro pilus polymerization, His-tagged proteins were purified according to a published procedure (4). Briefly, E. coli BL21 (DE3) cells harboring pSUMO-SrtAWT, pSUMO-SrtA2M, pMCSG-SpaA, pMCSG-SpaAK190A, pSUMO-NSpaA, or pMBP-CSpaA were grown in LB supplemented with ampicillin at 100 μg ml-1 at 37° C. until OD600 of ˜0.6. Cells were equilibrated to 17° C. and treated with 1 mM IPTG before they were allowed to grow overnight at 17° C. to induce protein expression. Cells were then harvested by centrifugation (8,000 RPM for 20 min) and stored at −80° C. for further processing. SrtA and SpaA-derived proteins were purified His6x-SUMO-fusion using HisPure Co2+ IMAC resin (Thermo) per the manufacturer's instructions. Briefly, cell pellets were resuspended in 50 mM Tris-HCl pH 8.0, 300 mM NaCl, and 5 mM CaCl2 (lysis buffer) and lysed by sonication. Subsequent cell lysate was then fractionated by centrifugation (15,000 RPM for 1 hr) and the supernatant was loaded onto HisPure Co2+ IMAC resin. Proteins were then eluted from the resin using lysis buffer supplemented with 200 mM Imidazole. The His6x-SUMO tag was removed by the addition of His6x-Ulp1 protease, and subsequent HisPure Co2+ purification. Protein purity was determined by SDS-PAGE analysis.
For crystallization, recombinant proteins were purified according to a published procedure (5). Briefly, E. coli BL21 (DE3) cells harboring pMCSG-SrtA′ or pMCSG-SrtA2M was cultured in 2TY medium containing ampicillin at 100 μg ml-1 at 37° C. with shaking until OD600 of ˜1.0. The culture was later induced with 0.4 mM IPTG and allowed to grow overnight at 18° C. with shaking. Cells were harvested and disrupted by sonication. The lysate containing SrtA was purified by using Ni-NTA (Qiagen) affinity chromatography with the addition of 5 mM β-mercaptoethanol in all buffers. The N-terminal His-tag and TEV restriction sequence of the protein was removed by the TEV protease (0.15 mg for 20 mg purified protein) incubated for 16 h at 4° C., and then passed through a Ni-NTA column to remove both the TEV protease and cleaved N-terminal tags. The final step of purification was gel-filtration on HiLoad 16/60 Superdex 200pg column (GE Healthcare) in crystallization buffer 10 mM HEPES buffer pH 7.5, 200 mM NaCl and 1 mM DTT. The protein was concentrated on Amicon Ultracel 10K centrifugal filters (Millipore) up to 24 mg/ml concentration.
Protein Crystallization, Data Collection, Structure Determination and Refinement
The initial crystallization condition was determined with a sparse crystallization matrix at 4° C. and 16° C. temperatures using the sitting-drop vapor-diffusion technique as reported (5). The best crystallization condition for the SrtAWT protein was found in 0.1 M MES:NaOH buffer pH 6.5, 1.6 M ammonium sulfate and 10% dioxane at 4° C. The SrtA2M protein was crystallized in 0.2 M sodium chloride and 20% PEG 3350 at 4° C. The SrtAWT and SrtA2M protein crystals selected for data collection were soaked in the crystallization buffer supplemented with either 28% sucrose or 25% glycerol, respectively, and flash-cooled in liquid nitrogen. Single-wavelength X-ray diffraction data were collected at 100 K temperature at the 19-ID beamline using the program SBCcollect. The intensities were integrated and scaled with the HKL3000 suite (6). The SrtA structures were determined by molecular replacement using the HKL3000 suite incorporating following programs: MOLREP (7), SOLVE/RESOLVE (8), and ARP/wARP (9). The coordinates for the A. oris sortase SrtC-1 (10) (PDB:2XWG) were used as the starting model for the SrtAWT structure. Several rounds of manual adjustments of structure models using COOT (11) and refinements with Refmac program (12) from CCP4 suite (13) were performed. The stereochemistry of the structure was validated with PHENIX suite (6) incorporating MOLPROBITY (14) tools. Atomic coordinates and structure factors of SrtAWT and SrtA2M were deposited into the Protein Data Bank as 5K9A and 6BWE, respectively.
Proteins that are site-specifically modified with peptides and chemicals have applications as therapeutics, imaging tools, diagnostic reagents and novel materials. Here we show that the sortase enzyme from Corynebacterium diphtheriae can be used to attach peptides to a protein via a specific lysine-isopeptide bond. Using rational mutagenesis we created CdSrtA3M, an activated cysteine transpeptidase that catalyzes in vitro isopeptide bond formation. CdSrtA3M mediates bioconjugation to a specific lysine residue within a fused domain derived from the SpaA protein. Modifications yields greater than >95% can be achieved. We show that CdSrtA3M can be used with the S. aureus SrtA enzyme, enabling dual, orthogonal protein labeling via lysine isopeptide- and backbone peptide-bonds.
Methods that site-specifically functionalize proteins are of significant interest for applications including therapeutics, imaging tools, diagnostic reagents and novel materials. The first sortase to be developed into a protein engineering tool was from Staphylococcus aureus (SaSrtA)1-4. It catalyzes a transpeptidation reaction that covalently modifies the protein via a backbone peptide bond, by joining peptide segments that contain a LPXTG ‘sort-tag’ and an N-terminal oligoglycine amine group5,6. Several groups have now optimized the reaction to modify proteins with a range of molecules, including drugs, lipids, sugars, fluorophores, and peptides7-14. While SaSrtA is powerful tool for protein engineering, it is almost exclusively used to modify target proteins at their N- or C-termini. Although SaSrtA labeling of internal lysine side chains can occur, this process is a side reaction and has low sequence specificity9,15,16 In this example we show that a mutationally activated sortase enzyme from Corynebacterium diphtheriae (CdSrtA) can be used to site-specifically attach peptides to a protein via a lysine-isopeptide bond. CdSrtA and SaSrtA have orthogonal activities, enabling dual peptide-fluorophore labeling of a protein via lysine isopeptide- and backbone peptide-bonds, respectively. Pathogenic Gram-positive bacteria use specialized sortase enzymes to construct pili. Pili are thin crosslinked fibers (0.2-3.0 μm×2-6 nm) that project from the cell surface to mediate bacterial adherence to host tissues, biofilm formation and host immunity modulation17-19. These structures are distinct from pili produced by Gram negative bacteria because their protein subunits (called pilins) are covalently linked to one another via lysine-isopeptide bonds, which confers enormous tensile strength.
Recently, we reconstituted in vitro the assembly which builds the archetypal SpaA-pilus in C. diphtheriae, the causative agent of pharyngeal diphtheria20. CdSrtA functions as a pilin polymerase, performing a repetitive transpeptidation reaction that covalently links adjacent SpaA pilin subunits together via lysine-isopeptide bonds. As shown in scheme 1 in
Toward the goal of creating a lysine modifying bioconjugation reagent we improved the ligation activity of CdSrtA2M by introducing additional mutations into its inhibitory lid structure and defined substrate determinants required for catalysis. In addition to the aforementioned D81 and W83 mutations in CdSrtA2M, inspection of the crystal structure reveals three residues within the lid that may stabilize its positioning over the active site (179, N85, K89). The ligation activities of three triple mutants of CdSrtA containing the D81G and W83G alterations, as well I79R, N85A or K89A substitutions were determined. A D81G/W83G/N85A triple mutant, hereafter called CdSrtA3M, has the highest level of ligation activity (
To elucidate determinants required for recognition of the K190 nucleophile, CdSrtA3M was incubated with a peptide containing the pilin motif (DGWLQDVHVYPKHQALS (SEQ ID NO. 11)) and either CSpaA or a peptide containing its C-terminal sorting signal (KNAGFELPLTGGSGRI (SEQ ID NO. 9)) (
We next demonstrated that CdSrtA3M can be used to label a target protein via an isopeptide bond with either a peptide fluorophore or another protein. In the labeling reaction the target protein is first expressed as a fusion with the NSpaA domain containing the pilin motif (PM), and then reacted with a LPLTG(SEQ ID NO. 5)-containing biomolecule and CdSrtA3M (
The CdSrtA and SaSrtA sortase transpeptidases recognize chemically distinct nucleophiles and sorting signals, suggesting that they can be used orthogonally to selectively label a single target protein at different sites. In the orthogonal modification strategy a target protein is produced that contains the N-terminal oligoglycine and pilin K190 nucleophiles that are recognized by SaSrtA and CdSrtA3M, respectively. To demonstrate orthogonal labeling we created a fusion protein that contained the Small Ubiquitin-like Modifier (SUMO) protein harboring pentaglycine peptide and the pilin motif at its N- and C-termini, respectively (G5-SUMOPM). The fusion protein was then sequentially reacted with each enzyme and peptide fluorophores containing the cognate sorting signal (
Separation of the reaction products by SDS-PAGE confirms dual labeling, as the appropriate fluorescence for each probe is detected during the procedure at ˜33 kD (
The bioconjugation chemistry catalyzed by CdSrtA3M enables site specific lysine labeling of a protein, creating isopeptide linkages that may be less susceptible to proteolysis than conventional peptide bonds. Transglutaminases also can modify protein lysine residues, but unlike CdSrtA3M, these enzymes exhibit minimal substrate specifity26,27. SaSrtA can also modify lysines, but this is a side reaction that occurs with minimal specificity and at low efficiency because the lysine ε-amine is not SaSrtA's natural substrate9,15,16. Bioconjugation catalyzed by CdSrtA3M is functionally similar to the non-enzymatic SpyTag/SpyCatcher system, but it is unique because it is enzyme catalyzed and can therefore be better controlled. CdSrtA3M can be used in combination SaSrtA chemoenzymatic labeling, providing a new and powerful avenue for protein bioconjugation.
Protein Expression and Purification
His6-tagged proteins were purified according to a published procedure. Briefly, E. coli BL21 (DE3) cells harboring pSUMO-CdSrtAWT, pSUMO-CdSrtA2M, pSUMO-CdSrtA3M, pSUMO-SaSrtA WT, pSUMO-SaSrtA 4M, pSUMO-NSpaA, or pSUMO-SpaA were grown in LB supplemented with kanamycin at 500 μg/ml at 37° C. until OD600 of ˜0.6. Cells were induced with 1 mM IPTG and protein expression was allowed to proceed overnight at 17° C. Cells were then harvested by centrifugation (7,000 RPM for 10 min). All proteins were purified as a His6x-SUMO-fusion using HisPure Co2+ IMAC resin (Thermo) per the manufacturer's instructions. Briefly, cell pellets were resuspended in 50 mM Tris-HCl pH 8.0, 300 mM NaCl (lysis buffer) and lysed by high pressure homogenization. Subsequent cell lysate was then fractionated by centrifugation (15,000 RPM for 40 min) and the supernatant was loaded onto HisPure Co2+ IMAC resin. Proteins were then eluted from the resin using lysis buffer supplemented with 200 mM Imidazole. The His6x-SUMO tag was removed by the addition of His6x-Ulp1 protease, and subsequent HisPure Co2+ purification. Protein purity was determined by SDS-PAGE analysis.
In Vitro of Pilin Domain Coupling Assays
In vitro reactions were carried out at room temperature and proteins were dissolved in assay buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 5 mM DTT). All reactions used a fixed 100 μM concentration of SrtA enzyme and 300 μM SpaA substrate (SpaA and NSpaA). Reactions were stirred gently by continuous rotation. Time points were taken at 0 h, 24 h, 48 h and reactions were quenched by addition of two volumes of SDS-loading dye. Quantitative analysis of transpeptidation was carried out by analyzing band intensities of the gel images using ImageJ (NIH)2.
Sortase-Catalyzed Peptide Conjugation Reactions
In general, 100 μM CdSrtA3M was incubated with 1 mM peptide and 100 μM of the pilin domain (NSpaA or SpaA) in 50 μl reactions, in the presence of 5 mM DTT for 24 hours. For SaSrtA mediated peptide conjugation, the improved variant “4M” (bearing the following point mutations: P94S/D160N/D165A/K196T) was used to improve transpeptidation speed3. 15 minutes incubation with 25 μM SaSrtA 4M resulted in partial labeling, and extended incubation of 2 hours yields almost complete labeling. After the incubations, the reactions were diluted 10× with ddH2O and mixed with 2,5 dihydroxybenzoic acid matrix (DHB) for analysis by matrix-assisted laser desorption ionization mass spectrometry (MALDI) or diluted with 25 μl SDS and separated by SDS-PAGE for further analysis.
Determination of SrtA Hydrolysis by an HPLC-Based Assay
In vitro hydrolysis reactions were performed based on the method developed by Kruger et al4. The sorting signal peptide was synthesized by Peptide2.0 and used without further purification. Lyophilized peptide was dissolved in assay buffer to 2 mM. 50 μM CdSrtA or SaSrtA (wild-type or mutant) was incubated with 500 μM KNAGFELPLTGGSGRI (SEQ ID NO. 9) (LPLTGpep) or KNAGFELPATGGSGRI (SEQ ID NO. 17) (LPATGpep) and 5 mM DTT in 100 μl reactions at 37° C. for 24 h and 48h. The reactions were quenched by adding 50 μl of 1 M HCl and injected onto a Waters)(Bridge Peptide BEH C18 reversed phase HPLC column. Peptides were eluted by applying a gradient from 5 to 51% acetonitrile (in 0.1% trifluoroacetic acid) over 30 minutes at a flow rate of 1 ml/min. Elution of the peptides was monitored by absorbance at 215 nm. Quantitative analysis of the HPLC traces was facilitated by integration of individual peak areas using Graphical Analysis (Vernier).
Fluorescent Peptides and Fluorescent Gel Imaging
FITC-KNAGFELPLTGGSGRI (SEQ ID NO. 9) was synthesized by Peptide 2.0 and used without further purification. AlexaFluor546-CNAGFELPATGGSRI (SEQ ID NO. 18) was created by expressing Psumo-CNAGFELPATGGSRI (SEQ ID NO. 18) in BL21 (DE3) cells and purifying the His-tagged fusion protein by Co2+ IMAC, as described previously. The desired peptide was cleaved from SUMO by treating with His6x-ULP1 protease and the peptide was purified by an additional Co2+ IMAC purification. The resultant peptide was then reduced with 1 mM TCEP and selectively modified by AlexaFluor546-maliemide (Invitrogen) via the N-terminal cysteine residue on the peptide. The fluorescent peptide was purified again by separation with a Waters XSelect HSS C18 reversed phase HPLC column. The fluorescent peptide conjugate was confirmed by absorbance at 554 nm and MALDI and lyophilized. Both peptides were dissolved into assay buffer at 2 mM. Sortase-catalyzed peptide modifications were performed as described, and samples were loaded onto pre-cast 12% linear Bis-Tris NuPAGE gels and run at 170V for 70 min to achieve separation between apo NSpaA and NSpaA modified by LPLTGpep. The gels were then washed in ddH2O and fluorescence data was acquired with a Pharos FX gel imager (BioRad). Fluorescein isothiocyanate (FITC) was detected by excitation with a 488 nm laser line and detection with a 515-545 nm emission filter. AlexaFluor546 was detected by excitation with a 532 nm laser line and detection by an emission filter at 580-630 nm.
Tandem Mass Spectrometry of NSpaA-LPLTpep Complex
Protein digestion and isopeptide bond identification were performed according to previous protocols. Specifically, proteins entrapped in gel bands were reduced with 10 mM dithiothreitol (Sigma) at 60° C. for an hour and then alkylated with 50 mM iodoacetamide (Sigma) at 45° C. for a few minutes in the dark. These reduction and alkylation steps were skipped for the acyl intermediate samples. Samples were digested with 200 ng trypsin (Thermo Scientific) at 37° C. overnight. At the end of trypsin digestion, 200 ng of Asp-N endoproteinase (Thermo Scientific) were added for another overnight incubation. Digested peptides were extracted from the gel bands in 50% acetonitrile/49.9% water/0.1% trifluoroacetic acid (TFA) and cleaned with C18 StageTip before mass spectrometry analysis.
Digested peptides were separated on EASY-Spray column (25 cm×75 μm ID, PepMap RSLC C18, 2 μm, Thermo Scientific) connected to an EASY-nLC 1000 nUPLC (Thermo Scientific) using a gradient of 5-35% acetonitrile in 0.1% formic acid and a flow rate of 300 nl/min for 30 minutes. Tandem mass spectra were acquired in a data-dependent manner with an Orbitrap Q Exactive mass spectrometer (Thermo Fisher Scientific) interfaced to a nanoelectrospray ionization source.
The raw MS/MS data were converted into MGF format by Thermo Proteome Discoverer 1.4 (Thermo Scientific). We wrote in-house programs to search for the isopeptides in two different approaches. The first approach was performed as previously described and used calculated masses of predicted peptides containing the isopeptide linkage to guide the search. The second approach was based on the observation of published spectra as well as our own on the presence of ions specific for the fragments of ELPLT (m/z 215.138, 225.122, 243.132, 294.180, 312.190, 322.174 and 340.186). The in-house programs sifted through tens of thousands of mass spectra looking for this information specifically and pulled out mass spectra of interest for further analysis and manual validation.
Corynebacterium diphtheriae CdSrtA (active truncated variant sequence with lid residues
Corynebacterium diphtheriae CdSrtA variant of SEQ ID NO. 1 (active truncated variant sequence
Staphylococcus aureus Sortase
Corynebacterium Diphtheriae Major Pilin SpaA
Corynebacterium diphtheriae surface-anchored fimbrial subunit SpaB
faecium, SrtC); WP_014569086 (Lactobacillus rhamnosus, SrtC1); WP_060958109 (Actinomyces
oris, SrtC1); WP_060956887 (Actinomyces oris, SrtC2); WP_000047114 (Streptococcus
pneumoniae, SrtC 1); WP_050148456 (Streptococcus agalactiae, SrtC 1); WP_000746885
Corynebacterium diphtheriae SrtA
Corynebacterium diphtheriae SrtC
Corynebacterium diphtheriae SrtB
Corynebacterium diphtheriae SrtD
Corynebacterium diphtheriae SrtE
Lactobacillus rhamnosus SrtC1
faecium class C sortase 1
faecium class C sortase 2
Actinomyces oris SrtC2
Streptococcus pneumoniae SrtC1
Streptococcus agalactiae SrtC1
Streptococcus agalactiae SrtC2
Bacillus cereus SrtD
Ruminococcus albus class C sortase
Staphylococcus aureus SrtA
Streptococcus pyogenes SrtB
Clostridium perfringens SrtD
Streptomyces coelicolor
Corynebacterium diphtheriae SrtF
Embodiments and aspects of the present invention disclosed herein were found in manuscript disclosure that was included with the priority application disclosure (U.S. Provisional Patent Application Ser. No. 62/665,076, filed on May 1, 2018) before the manuscripts were published. The manuscripts were later published as Chang et al., PNAS Jun. 12, 2018 115 (24) E5477-E5486 and McConnell et al., J. Am. Chem. Soc., 2018, 140 (27), pp 8420-8423.
All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. Publications cited herein are cited for their disclosure prior to the filing date of the present application. Nothing here is to be construed as an admission that the inventors are not entitled to antedate the publications by virtue of an earlier priority date or prior date of invention. Further, the actual publication dates may be different from those shown and require independent verification.
This concludes the description of the illustrative embodiments of the present invention. The foregoing description of one or more embodiments of the invention has been presented for the purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise form disclosed. Many modifications and variations are possible in light of the above teaching.
This application is the National Stage of International Application No. PCT/US19/30206 (International Publication No. WO 2019/213262), filed May 1, 2019, which claims the benefit under 35 U.S.C. Section 119(e) of commonly-assigned U.S. Provisional Patent Application Ser. No. 62/665,076, filed on May 1, 2018, and entitled “REAGENT TO LABEL PROTEINS VIA LYSINE ISOPEPTIDE BONDS” which application is incorporated by reference herein.
This invention was made with government support under Grant Number DE017382/DE025015, awarded by the National Institutes of Health and DE-FC02-02ER63421 awarded by the U.S. Department of Energy. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2019/030206 | 5/1/2019 | WO |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2019/213262 | 11/7/2019 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 20030022178 | Schneewind et al. | Jan 2003 | A1 |
| 20040029129 | Wang et al. | Feb 2004 | A1 |
| 20170226495 | Guimaraes et al. | Aug 2017 | A1 |
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| Number | Date | Country | |
|---|---|---|---|
| 20210115424 A1 | Apr 2021 | US |
| Number | Date | Country | |
|---|---|---|---|
| 62665076 | May 2018 | US |
