Sortase enzymes are cell wall-associated transpeptidases that facilitate the attachment of proteins to the peptidoglycan. Exclusive to Gram positive bacteria, sortase enzymes contribute to many processes, including virulence and pilus attachment, but their role in Streptomyces coelicolor biology remained elusive. Previous work suggested that the sortases anchored a subset of a group of hydrophobic proteins known as the long chaplins. The chaplins are important in aerial hyphae development, where they are secreted from the cells and coat the emerging aerial hyphae to reduce the surface tension at the air-aqueous interface. Two sortases (SrtE1 and SrtE2) were predicted to anchor these long chaplins to the cell wall of S. coelicolor. Deletion of both sortases or long chaplins revealed that although the long chaplins were dispensable for wild type-like aerial hyphae formation, the sortase mutant had a severe defect in growth. These two sortases were found to be nearly redundant, as deletion of individual enzymes led to only a modest change in phenotype. In vitro analysis of sortase cleavage activity showed that both sortases recognized the unique LAXTG pentapeptide sequence found in the long chaplins, and 11 other putative substrate proteins. Transcriptional analysis revealed that a number of genes typically expressed during aerial hyphae development were not expressed in a sortase deletion mutant. This suggests that the sortases have a role in transcriptional regulation, a phenomenon that has not been described previously. Current work is focused on addressing the mechanism(s) by which sortases affect transcriptional regulation, with a specific focus on the role of the proteins that they anchor to the cell wall (sortase substrates) in aerial growth. / Thesis / Master of Science (MSc)

M13 filamentous bacteriophage has been used as a biotemplate for the nucle- ation of materials. Phage is an ideal and diverse scaffold with its large aspect ratio and ability to display biomolecules to bind a range of targets. To form more complex patterned materials, interactions between the phage must be specific and reliable. We develop a phage labeling method using sortase enzymes to create multi-phage nanostructures. We exploit two sortases and functionalize the N-termini of the pIII, pIX, and pVIII proteins with small and large moieties. For the pVIII, we show a 100 fold improvement in display of GFP molecules on the phage surface. Taking advantage of orthogonal sortases, we simultaneously label two capsid proteins on a single phage particle. Using these N-terminal labeling techniques, we demonstrate fluorescent staining of cells and construct a lampbrush phage structure linking the pIII of one phage to the pVIII of another using a biotin-streptavidin linkage. To further expand our labeling repertoire, C-terminal sortase labeling of phage was pursued. To achieve this goal, we transfer a loop structure from cholera toxin to pIII and label it with a fluorophore and a multi-domain protein. With this archi- tecture, we form end-to-end dimers using sortase to conjugate the loop structure to phage containing the nucleophile motif. Lastly, we investigate DNA hybridization as a method for crosslinking phage. Using sortase, we label the pVIII on two sets of phage: one with ssDNA and the other with a complementary DNA oligonucleotide. We anneal these phages together and observe phage networks that are dispersed by heat and reform upon cooling.

Understanding protein function is fundamental to the fields of protein engineering and drug design. While most of the previous efforts in this direction have focused on the sequence-structure-function paradigm, recent studies have pointed to protein dynamics as being integral to its activity. The work in the current thesis follows this overall theme of obtaining insights into protein function from its structure and dynamics. It can be broadly divided into two sections. In the first section, the thesis candidate has tried to elucidate the residues modulating the substrate specificity of a particular family of enzymes, known as sortases, through structural and computational studies (including dynamics simulations) on a novel member in the family. This work has been carried out in collaboration with Dr. R.P. Roy, National Institute of Immunology, New Delhi (biochemical characterization was performed by Mr. Vijay Pawale at Dr. Roy‟s laboratory). In the second half of this thesis, the candidate has described a structure-based method involving the use of cis-peptide containing segments for the function annotation of proteins. The incorporation of dynamics information leads to an improvement of our annotation approach, which is also demonstrated. This part of the work has been carried out in collaboration with Dr. Debnath Pal, Department of Computational and Data Sciences, Indian Institute of Science. Following is a chapter-wise description of the overall layout of the thesis. Section I: Insight into substrate specificity in sortase enzymes from structural studies on a novel housekeeping sortase of class E (SrtE) Chapter 1| A brief account of sortases: This chapter provides a brief survey of the literature on sortases and the scope of the work presented in the thesis. Many surface proteins in Gram-positive bacteria are incorporated into the cell wall through covalent ligation by a class of cysteine transpeptidases known as Sortase. These surface proteins contain a cell wall sorting signal (CWSS) which is recognized by sortase, enzymatically cleaved and subsequently joined covalently to the pentaglycine branch of lipid II (a peptidoglycan precursor) in general, which is finally incorporated into the peptidoglycan cell wall. Six classes of sortases have been identified on the basis of their sequence. These sortases differ in the substrate motif that they recognize and the function performed. The class A sortase (SrtA) is expressed ubiquitously in Gram-positive bacteria. It is involved in the cell surface anchoring of a large number of functionally distinct proteins which contain an LPXTG recognition motif in their CWSS, and is referred to as the „house-keeping‟ sortase. Sortases of other types are not ubiquitous and are meant to perform specialized functions. Sortase B is involved in iron acquisition, sortase C in pilus formation and sortase D in sporulation. The substrate motifs recognized by these sortases are, in general, different from the recognition motif in SrtA substrates. Several Gram-positive bacteria with a high GC content in their genome have been suggested to use a sortase E (SrtE) instead of SrtA to perform the housekeeping activity. These sortase sequences share low identity with sortases of classes A-D. The substrates of SrtE have been proposed to contain an LAXTG recognition motif instead of LPXTG based on genomic analyses. Class F consists of sortases from several Actinobacteria. However, the biological function of these sortases is not well understood. To date, structures of sortases from classes A-D have been determined, all of which display an eight-stranded beta barrel fold (termed the sortase fold), a conserved catalytic triad of His-Cys-Arg and a TLXTC motif at the active site (C: catalytic Cysteine; X varies across the different classes of sortases). Sortase B and C are augmented by additional secondary structure features which are absent in sortase A. SrtA from Staphylococcus aureus is the most well studied among sortases of known structure. Several of the surface proteins attached by sortases are responsible for bacterial virulence. SrtA deletion mutants have been found to exhibit reduced virulence without affecting cell viability. Moreover, the localization of sortase in the cell membrane and the absence of eukaryotic homologs have made sortase an attractive target for the development of novel therapeutics. In addition, the transpeptidase activity of sortase has found extensive applications in biotechnology. The prototype SrtA from Staphylococcus aureus is commonly used for these applications; however, its use is limited by its obligate Ca2+ ion-dependent activity and the stringent preference for an LPXTG motif. Hence, characterization of new sortases with altered substrate recognition profiles and rational modification of known sortases has tremendous potential for biotechnological applications and advancements. While sortases of classes A-D have been studied extensively to date and their structures determined, no structural data is available for a class E sortase. The thesis candidate has solved the first high resolution crystal structure of a putative housekeeping Sortase E in Streptomyces avermitilis (SavSrtE), a bacterium with a GC rich genome. Biochemical experiments performed by our collaborator on this protein have demonstrated Ca2+ independent transpeptidase activity and a preference for LAXTG-containing peptides as its cognate substrate over the LPXTG motif that is recognized by sortase A. Moreover, the protein exhibits a preference for small uncharged residues in the position succeeding the penta-peptide motif. This thesis documents the results of crystal structure analyses, molecular docking studies and dynamics simulations to understand the structural basis for these experimental findings. Finally, sequence analyses were performed to detect possible residues which modulate substrate specificity. Based on these analyses, mutations were performed. The thesis also documents the crystal structure solution and analysis of an active site mutant (residue T196 at the position X in the TLXTC motif). Chapter 2| Methods for the analyses of Sortase E from S. avermitilis (SavSrtE): This chapter provides a description of the procedures used to carry out the thesis work. An N-terminus truncated construct (∆N50) of wild type SavSrtE and its mutant T196V were cloned, expressed and purified in the laboratory of our collaborator, Dr. R.P. Roy (NII, New Delhi), and provided to us for structure and sequence analyses. Initially, crystallization trials were carried out on the wild type protein using commercially available screening kits and the sitting drop vapor diffusion method. The condition which gave crystals was optimized further. Finally, diffraction quality crystals were obtained in a drop containing 1μL of protein (4 mg/mL in 10 mM Tris-HCl buffer pH 7.2, 100 mM NaCl and 2 mM beta-mercaptoethanol) mixed with 1μL solution of the crystallization condition containing 1.6 M ammonium sulfate, 0.1 M citric acid at pH 3.75 using the hanging drop vapor diffusion method. The crystals were cryo-protected in a 10% sucrose solution and diffraction data collected at the European Synchrotron Radiation Facility (BM-14, ESRF). The crystals diffracted to 1.65Å. The protein crystallized in the P3221 space group with unit cell parameters a = b = 85.84Å, c = 48.20Å, α = β = 90°, γ = 120°. Calculation of Matthews coefficient indicated the presence of one molecule in the asymmetric unit. T196V mutant protein yielded diffraction quality crystals in the same condition as the wild type protein. The crystals were cryo-protected using sucrose and diffraction data were collected at the BM-14 beamline. The mutant crystals diffracted to 1.70Å. The protein crystallized in the P3221 space group with unit cell parameters a = b = 84.98Å, c = 48.00Å, α = β = 90°, γ = 120° and one molecule in the asymmetric unit. The quality of the datasets was assessed by SFCHECK and data were found to be of appropriate quality for structure solution. SavSrtE has low sequence identity (25 – 34%) to other class A sortases of known structure. Hence the scaled data, sequence information and model coordinates (sortase A from Streptococcus agalactiae, PDB ID: 3rcc) were submitted to the MR (molecular replacement) phasing option in the EMBL-Hamburg AutoRickshaw pipeline. The model generated from the server was used as input to PHASER for MR. The MR solution was subjected to one cycle of rigid body refinement followed by several cycles of restrained refinement using REFMAC from the CCP4 suite, with alternate rounds of inspection and manual model building in COOT for model improvement. The convergence of the refinement procedure was checked from the reduction in R-factors. The most essential refinement statistics for the final models of the wild type protein and T196V mutant are tabulated below. Table 1 Wild type (5GO5) T196V (5GO6) Resolution 1.65 Å 1.70 Å Rwork / Rfree (%) 16.11 / 19.05 17.31 / 20.82 R.M.S. bond lengths (Å) 0.012 0.019 R.M.S. bond angles (°) 1.53 1.89 Average B-factors (Å2) Protein 19.1 32.5 Water 32.6 42.4 SO42- 58.7 60.8 Gly 36.0 - Ramachandran map statistics Most favoured region (%) 86.8 89.8 Additional allowed region (%) 13.2 10.2 Generously allowed region (%) 0.0 0.0 Outliers (%) 0.0 0.0 The genome of S. avermitilis was searched using the ScanProsite tool to identify putative substrates, details of which are also documented in this chapter. Additionally, the thesis candidate performed Mutual Information analysis on an alignment of 1569 sortase sequences from different classes to identify the residues possibly regulating substrate specificity. Based on this analysis, mutations were performed of which the T196V mutant has been studied in this thesis. Finally, this chapter describes the protocol used to perform protein peptide docking and subsequent molecular dynamics simulations to understand how dynamics may influence substrate specificity. Chapter 3| Analyses of SavSrtE sequence and structure: This chapter provides a description of the analyses on the wild type SavSrtE and the T196V mutant. The overall fold of SavSrtE is very similar to that observed in the structures of other sortases, although the sequence similarity to other classes is low. Variations are observed in the loop regions (longer β1/β2 and β6/β7 loops). The active site is comprised by residues from the β2/H1 loop, β3/β4 loop, β4 strand, β6/β7 loop, β7 strand, β7/β8 loop and β8 strand. It also does not carry any cluster of electronegative residues close to the active site and therefore, is expected to have Ca2+ ion independent activity, which is observed in biochemical experiments (Dr. R.P. Roy‟s lab). Comparison with other housekeeping sortases showed that the β6/β7 loop in SavSrtE is in a closed conformation, indicating the presence of a preformed binding pocket for the LAXTG substrate binding, contrary to the prototype SrtA from Staphylococcus aureus which requires a Ca2+ ion to stabilize the closed conformation. Moreover, a small pocket is observed adjacent to the catalytic triad which contained electron density fitting a Gly molecule. This pocket is proposed to be the binding site for the second substrate that resolves the protein-peptide intermediate through a nucleophilic attack. Our docking simulations showed that a Gly of a triglycine moiety can be positioned in this pocket. Biochemical experiments established that SavSrtE recognizes the substrate motif LAXTG instead of LPXTG which is preferred by class A sortases. It also prefers Gly based nucleophiles as the second substrate. Additionally, the protein is found to prefer neutral residues over charged residues in the position succeeding the Gly of the LAXTG motif. Structure analyses showed the presence of a bulky Tyr residue (Y112) at the active site pocket which, according to molecular docking studies, hinders the productive binding of Pro-containing peptides (LPXTG) over Ala-containing ones (LAXTG). The OH group of Y112 is involved in a hydrogen bond with the backbone nitrogen of the second Ala in the ALANT peptide but not in the Pro-containing peptide. Y112 is held rigidly in place via interactions with neighbouring residues and a network of hydrogen-bonded water molecules in the crystal structure. A Tyr residue is found to be present in an equivalent position in several sortase sequences of Class E, and may be a general feature responsible for the specificity of sortase Es to putative LAXTG-containing substrates in their genomes. It may be mentioned that class D sortases, which contain a Phe residue at the equivalent position, recognize the LPXTA substrate motif. The side chain of this Phe displays different rotamers in the NMR structure of Bacillus anthracis SrtD, pointing to its flexibility, whereas Y112 in S. avermitilis SrtE is rigid. In addition, molecular dynamics simulations on the models of protein-peptide complex (obtained from docking) showed that the two peptides have similar backbone dynamics, unlike the case of S. aureus SrtA where the Ala-containing peptide does not maintain a kinked conformation similar to the Pro-containing cognate peptide. Hence the Tyr at the active site appears to be the main factor behind the discrimination of the two peptides. Substrate sequences in the S. avermitilis genome contain small neutral residues in the position succeeding the Thr-Gly peptide bond in the substrate. This preference is also observed in biochemical assays. Docking calculations showed that the protein cannot accommodate large side chains in the site where this residue is positioned. To detect the residues involved in altering the substrate specificity of SavSrtE, we performed a multiple sequence alignment using 1569 sortase sequences and carried out mutual information (MI) analysis on this data. Our analysis implicated several residue pairs lining the active site pocket in modulating substrate specificity. These included the aforementioned Tyr residue as well as the position X (T196 in SavSrtE) in the TLXTC motif at the active site. Mutations were performed at these positions and crystallization trials performed. We could successfully crystallize and solve the structure of the T196V mutant, which has been documented in this thesis. The mutant protein has the same overall structure as the wild type. Moreover, the catalytic Cys residue was observed to be unmodified in this structure, compared to the wild type which was presumably altered by β-mercaptoethanol added during protein purification. The mutated residue (Val) was found to have a different side chain rotamer than T196. Moreover, the absence of any polar atom in the side chain of V196 disrupted the hydrogen-bonded network of water molecules observed at the active site in the wild type structure. Experiments on the mutant showed a reduction in activity, implying that T196 is important for substrate recognition. The altered side chain orientation of V196 is expected to be responsible for the reduction in activity, though a peptide-bound crystal structure would be necessary to clearly understand the mechanism. In this respect, future crystallization trials may be performed with modified peptides that bind covalently to the active site Cys residue, similar to the strategy employed for S. aureus SrtA and Bacillus anthracis SrtA. Our structure and sequence analyses have pointed to some residue positions responsible for the modified substrate specificity. While only one mutant has been characterized, the other mutants also need to be studied (through biochemical asssays and structure analysis) to understand how they contribute to substrate recognition. In this context, double mutants may also be generated to understand the combined effect. For example, single mutations of E105 and E108 were found to reduce the activity of Staphylococcus aureus SrtA, while the double mutant resulted in Ca2+ ion independent activity. Additional structure and sequence analysis coupled with experiments are necessary to detect residues which may be mutated to enhance the activity of SavSrtE, similar to what has been performed for S. aureus SrtA. To summarize, our studies show that the substrate specificity of SavSrtE is different from that of class A sortases, and provide an explanation for it using structure analyses and computation. This altered specificity profile, orthogonal to that of S. aureus SrtA, and Ca2+ ion independent activity make it a potential candidate for use in simultaneous conjugation of multiple peptide substrates to their target. Moreover, this structure may be used firstly as a model to design inhibitors for housekeeping srtEs from pathogenic organisms like Corynebacterium diphtheriae and Tropheryma whipplei. Secondly, most of the previous studies on inhibitor design for sortases documented small molecules or peptidomimetics binding to the pocket of the first substrate. Since distinct binding pockets have been observed for the two substrates in SavSrtE, this information may be used to build inhibitors targeting the second pocket or spanning both the pockets. Section II: Identifying functionally important cis-peptide containing segments in proteins and their utility in molecular function annotation Chapter 4| Functionally important cis-peptide fragments in proteins: detection and relevance: This chapter describes the relevance of cis-peptides to protein function and a method to detect such functionally important cis-peptides in proteins. Cis-peptide bonds are comparatively rare in proteins due to the steric strain associated with the 1,4-atomic clash in the peptide chain. Consequently, only about 0.03% of Xaa-Xnp (Xaa: any amino acid; Xnp: any amino acid other than Pro) peptide bonds occur in the cis conformation; the occurrence is somewhat higher (5%) for imino peptide-containing Xaa-Pro cases. Despite their low occurrence, cis-peptides have been found to be evolutionarily conserved, pointing to their important role in structure and function. Cis-Xnp peptide bonds exhibit a significant disposition towards ligand-binding sites and dimerization interfaces, whereas cis-Pro bonds have been found to occur in a rare „touch-turn‟ motif at functional sites. Cis-trans isomerization is expected to play a regulatory role in many cellular processes. Non-conservation of these peptides is implicated in the evolution of different function among similar protein folds. Hence, there has been a renewed interest in detecting cis-peptides from residue patterns and linking them to molecular function. The importance of proteins as molecular 'workhorses' makes it imperative to understand how they function. However, a vast majority of the proteins catalogued in public sequence and structure databases do not have experimentally verified functional annotation. Experimental approaches are inadequate to manually curate these large numbers of un-annotated proteins. This necessitates the use of computational function prediction tools. The simplest prediction methods involve the assessment of similarity in sequence and three-dimensional structure with homologous proteins of known function. The presence of high overall similarity, however, does not predict function unambiguously since certain protein folds are associated with multiple functions while proteins with different folds may share functional traits. Often proteins with different global structure are found to have structural similarity at the local level of segments of residues that are responsible for the similarity in function. This has given rise to fragment-based (FB) function annotation methods. FB methods may involve locating functionally relevant surface patches or cavities formed by sequentially distant residues, or the presence of structurally conserved, contiguous residue fragments with proven relevance to function. The direct relevance of the cis-peptide bond to protein function suggests its use for the purpose of function annotation in a FB approach, yet no method exists to exploit it. This chapter describes a method using geometric clustering and level-specific Gene Ontology (GO) molecular-function (MF) terms to identify, in a statistically significant manner, cis-peptide embedded fragments (henceforth referred to as cis-fragments) in a protein linked to its molecular function. Such fragments were associated with GO MF based propensity value ≥ 20 at p-value ≤ 0.05, indicating the statistical significance of our results. The relevance of the identified cis-fragments to protein function was further verified through a literature survey. The features of these fragments are discussed in this chapter. Some of these fragments do not overlap with known PROSITE patterns, depicting the utility of these fragments as sequence patterns. Moreover, the thesis candidate identified contiguous stretches of functionally important trans-peptide fragments and cis-fragments forming extended structure-based functional signatures. Chapter 5| Use of functionally important cis-fragments in annotation: In this chapter, the candidate describes how a library of cis-peptide embedded fragments with proven association to molecular function can be useful for annotating proteins with known structure (and having cis-peptide) but unknown function. The functionally important fragments detected in the previous chapter were searched for exact matches in sequence and cis-peptide in a test set of PDB entries of known function at different thresholds of sequence redundancy and p-value. Additionally, the match or mis-match in GO MF term between the functionally important fragment and the test protein was also evaluated. To assess the efficiency of our method in annotation, true positive rate (TPR) and false positive rate (FPR) were calculated at each threshold as follows: TPR  TP and FPR  FP TP  FN FP TN The following table explains how the numbers of cases with TP, FP, etc. were assigned. Cases with match in Match in cis-peptide No match in cis- sequence peptide Match in GO MF TP FN No match in GO MF FP TN The cis-fragments alone were sufficient to identify other proteins with similar function. Over different thresholds, TPR >0.91 and FPR <0.23 were observed. Annotation recall benchmarks interpreted using receiver-operator-characteristic-plot returned >0.9 area-under-curve, corroborating the utility of the annotation method. Further, the applicability of our method in fragment-based function annotation is illustrated for cases where homology-based annotation transfer is not possible. The work presented here adds to the repertoire of function annotation approaches and also facilitates engineering, design and allied studies around the cis-peptide neighbourhood of proteins. The results presented in chapters 4 and 5 have already been published (reprint enclosed) with the thesis candidate as the first author. Chapter 6| Molecular dynamics information improves cis-peptide based function annotation of proteins: The preceding chapters have demonstrated the use of functionally relevant cis-peptide segments in a homology-independent, fragment match-based protein function annotation method. However, proteins are not static molecules; their dynamics is integral to their activity. Hence we have incorporated the dynamics (obtained using an in-house coarse-grained forcefield) of functionally important cis-peptide segments in our annotation method. This is the first study to include both static and dynamics information to improve the prediction of protein molecular function. To ascertain the improvement upon incorporating dynamics, the ACV-based dynamics profiles (details in chapter) were compared in a dataset consisting of 102 pairs each of positive data (PDB entries with match in fragment sequence and cis-peptide) and negative data (PDB entries with match in fragment sequence but no match in cis-peptide). Our analyses depicted that using only cis-peptide information gave less false positives and a low FPR (0.11), which is desirable, but also a relatively low TPR (0.72). This is due to large FN (trans-peptide with matching GO MF), which can arise when the cis-fragment undergoes cis-trans isomerization to accomplish its function and coordinates have been obtained for the segment in the test data in the trans-state, or if there is an error in assignment of the omega angle during structure solution. On the other hand, using only dynamics information increases the numbers of both true and false positives and hence the TPR (0.95) and FPR (0.51). This is due to false-positive matches for cases where fragments with similar secondary structure show similar dynamics, but the proteins do not share a common function. Combining the predictions from the two methods reduces errors while detecting the true matches, thereby enhancing the utility of our method in function annotation (TPR: 0.95 and FPR: 0.07). Subsequently, we have combined static and dynamics information to annotate proteins of unknown function. A combined approach, therefore, opens up new avenues of improving existing automated function annotation methodologies. The work described in this chapter has been submitted to a peer reviewed journal. Future prospects include the development of a web server to facilitate the application of our method by a wide research community. A possible improvement includes identification and comparison of the dynamics of additional sites close to the identified cis-fragment, in an automated manner, to improve the accuracy of our annotation. Appendix 1 gives a description of the results of biochemical experiments performed in the laboratory of our collaborator Dr. R.P. Roy, NII, New Delhi. Appendix 2 contains additional data supplementary to chapter 4. Appendix 3 provides additional data supplementary to chapter 5. Appendix 4 provides additional data supplementary to chapter 6. Appendix 5 contains reprints of publications.

In the past two decades, the field of protein engineering has evolved rapidly to include new genetic and chemical techniques to alter protein function. Protein engineering seeks to improve enzyme properties through powerful methods that specifically incorporate novel or improved function in proteins. One such method is protein ligation, which is used to selectively link synthetic and recombinant polypeptides. Due to the limitations of current protein labeling techniques, simple site-specific modification methods remain in high demand. Use of enzyme-based labeling has been the focus of various studies because of its substrate specificity. Sortase-mediated transpeptidation is one approach that has been well documented. Staphylococcus aureus sortase A (SrtAstaph), a membrane-anchored cysteine transpeptidase present in gram-positive bacteria, covalently anchors virulence-associated surface proteins to the peptidoglycan cross bridge of the cell wall. SrtAstaph, one of the most characterized sortases, has found numerous applications in the semi-synthesis of protein and peptide conjugates. While current studies have demonstrated the growing range of applications for sortase A, the enzyme itself has seen very few improvements. In steady-state kinetic analysis, the calculated K cat value of SrtAstaph was 2.27 × 10−5 s−1 indicative of its slow in-vitro turnover rate. Due to sortase’s relative inefficiency, several studies documented the use of excessive amounts of the enzyme in vitro (>30μM) or reactions were incubated for long periods. Through the use of directed evolution, we aimed to improve the catalytic activity of sortase A. Using random mutagenesis and an in vivo bacterial-based screen we isolated a variant that showed a 13-fold increase in its catalytic efficiency when compared to wild-type. This sortase mutant will enable more efficient labeling of LPETG-tagged substrates and will provide further insight into the enzyme’s molecular mechanism of catalysis, which is currently limited.

La variabilité du système protéolytique de surface a été étudiée chez 30 souches de St. thermophilus. Cette variabilité consiste en la présence ou l’absence du gène prtS, en la présence de deux allèles différents de ce gène, en la présence d'une protéase PrtS ancrée et/ou soluble et enfin en l'expression variable, due à une variabilité de la régulation du système protéolytique, du gène prtS et d'autres gènes qui interviennent, pour la plupart, dans le métabolisme azoté. L’expression des gènes prtS, pepX, pepC, pepN, amiA1CDEF, dtpT, livJHMGF, ilvC, ilvDBN, bcaT, ackA, ldh, codY et relA a été quantifiée chez les souches PB302 et PB18O en lait et en milieu M17. La souche PB302 est représentative des souches qui se développent rapidement en lait alors que la souche PB18O l’est de celles qui ont une croissance intermédiaire dans ce milieu. Alors que l’expression des gènes étudiés est peu différente en milieu M17 où les deux souches ont une croissance similaire, cette expression diverge lorsque les deux souches sont cultivées en lait.Globalement, la différence de croissance observée en lait entre les deux souches pourrait résulter d'une variabilité de la capacité protéolytique et de l’expression, entre autres, des gènes codant PrtS, le régulateur CodY, les transporteurs des oligopeptides (Ami), des di-tripeptides (DtpT) et des acides aminés ramifiés (LivJ) et de ceux codant des enzymes impliquées dans la voie de biosynthèse des acides aminés ramifiés (IlvC, IlvB et BcaT), ces derniers étant nécessaires pour la croissance en lait. Tous ces gènes possèdent en amont de leur promoteur une boîte CodY potentielle et pourraient donc appartenir au régulon CodY / The variability of the cell envelope-associated proteolytic system was studied in 30 strains of St. thermophilus. Variations in strains consist in the presence or absence of the gene prtS, the presence of two allelic forms of prtS, the presence of an anchored and/or soluble form of the protease PrtS and in the variable expression of the gene prtS and other genes involved mainly in nitrogen metabolism, thus in the variability of the regulation genetic of this system. Expression of the genes prtS, pepX, pepC, pepN, amiA1CDEF, dtpT, livJHMGF, ilvC, ilvDBN, bcaT, ackA, ldh, codY and relA was quantified in the PB302 and PB18O strains. The strain PB302 is representative of strains which exhibit a rapid growth in milk. The strain PB18O is representative those with intermediate growth in milk. In M17 medium, where both strains have similar growth, little difference in the expression of genes tested was observed. Conversely, the two strains did not express the selected genes in the same way when grown in milk. Overall, the difference in growth observed between strains in milk could result from variable proteolytic activities and variable expression of genes encoding, for example, the proteinase PrtS, the regulator CodY, transporters of oligo- or di-tri- peptides (Ami or DtpT) or branched chain amino acids, or BCAA (LivJ) and enzymes in the biosynthetic pathway of BCAA (IlvC, IlvB et BcaT) which are necessary for growth in milk. All these genes have a potential CodY box at the upstream of their promoter and could therefore belong to the regulon CodY

Les bactéries lactiques (BL), communément employées en industrie agroalimentaire, font à présent l’objet d’études visant à les utiliser pour de nouvelles applications telles que le développement de vaccins vivants ou la délivrance de molécules d’intérêt biothérapeutique chez l’hôte. Dans cette optique, différents systèmes de présentation de protéines à la surface des bactéries à Gram positif ont été développés. L’un d’entre eux est basé sur l’activité d’enzymes, les sortases, liant de façon covalente les protéines à la paroi bactérienne. Nous avons utilisé la BL modèle, Lactococcus lactis, afin d’étudier les sortases, jusqu’alors étudiées essentiellement chez les bactéries pathogènes. La sortase A (SrtA) est responsable de l’ancrage d’au moins cinq protéines à motif LPxTG à la surface. Une seconde sortase, de classe C (SrtC), a été identifiée et caractérisée. Nous avons mis en évidence la capacité de L. lactis à produire des pili à sa surface qui sont polymérisés par SrtC et ancrés à la paroi par SrtA. Ces pili résultent de la polymérisation de la piline majeure YhgE qui peut être surplombée par la piline mineure de coiffe YhgD. La production de pili chez L. lactis entraîne un changement de comportement des cellules résultant à des phénotypes particuliers. Nous avons pu l’associer à l’auto-agrégation des cellules en culture liquide, à la formation de biofilms hétérogènes et aériens, et à l’adhésion à la mucine gastrique de porc. Plus précisément, YhgE a été impliquée dans l’auto-agrégation et les biofilms atypiques, et une troisième piline, dont l’appartenance au pilus n’a pas été démontrée, semble aussi impliquée dans la production de biofilms atypiques. / Lactic acid bacteria (LAB), which are commonly used in food industry, are now being studied for their use in new applications such as biotherapeutic molecule delivery vehicules in human host or as live vaccines. Recently, surface protein delivery systems have been developed in Gram positive bacteria and one of them is based on enzymes, the sortases which covalently bind proteins to the cell wall. We used the LAB model, Lactococcus lactis, in order to study the sortases of these non-pathogenic bacteria. This work has functionally characterized the sortase A (SrtA) responsible for cell wall anchoring of at least five LPxTG proteins. A second sortase, from class C (SrtC), has been identified and characterized. We demonstrated the ability of L. lactis to produce pili on its surface that are polymerized by SrtC and cell wall anchored by SrtA. These pili result from polymerization of the YhgE major pilin and can be topped by the YhgD tip minor pilin. Pili production in L. lactis leads a change in cell behavior resulting in individual phenotypes. We were able to associate it with the self-aggregation of cells in liquid cultures, heterogeneous and aerial biofilm formation and bacterial adhesion onto pig gastric mucin. Specifically, YhgE was involved in both self-aggregation and atypical biofilm formation, while a third pilin, whose pilus membership has not been established, was also involved in the production of atypical biofilms.

Nature employs complex networks of protein-tailoring enzymes to effect the post-translational modification of proteins in vivo. By comparison, modern chemical methods rely upon either nonspecific labeling techniques or upon the genetic incorporation of bioorthogonal handles. To develop truly robust bioconjugates it is necessary to develop methods which possess the exquisite activity and specificity observed in biological catalysts. One attractive strategy to achieve this is the engineering of protein-tailoring enzymes possessing user-defined specificity and high catalytic efficiency. / Chemistry and Chemical Biology

<p>Gram-positive pathogens, such as <italic>Streptococcus pyogenes</italic> and <italic>Staphylococcus aureus</italic>, are etiological agents of a large array of human diseases. Unfortunately, our ability to treat these infections is increasingly limited due to the development of bacterial resistance to many existing therapies. Thus, novel targets for antimicrobial development are urgently needed. An attractive candidate for a new class of anti-virulence chemotherapeutics is the sortase class of enzymes. Sortases are extracellular transpeptidases unique to Gram-positive bacteria. Their function is to covalently attach secreted virulence factors to the bacterial cell wall. Deletion or inhibition of sortases results in severe attenuation of bacteria for infection. In order to develop novel effective antimicrobial agents, a robust understanding of the biological and chemical mechanisms of the target are required. To this end, this dissertation endeavors to further illuminate the biochemical mechanism of sortase enzymes and to extend the current knowledge of the roles of sortases and their substrates during infection.</p><p>Through steady-state kinetics, active site reactivity measurements, three-dimensional structure determination via X-ray crystallography, and computational modeling of substrate binding, the basic enzyme mechanism of <italic>S. pyogenes</italic> sortase A (SrtA) has been revealed. In general, <italic>S. pyogenes</italic> SrtA displays many of the same mechanistic characteristics as previously studied sortases, including a reverse protonation mechanism, a conserved tertiary structure arrangement, and utilization of similar substrate binding interfaces and conserved active site residue functions. These findings suggest a general sortase mechanism, conserved among classes and species.</p><p>Initial steps have also been taken to characterize <italic>S. pyogenes</italic> sortase C (SrtC). SrtC enzymes are unique in that they covalently polymerize secreted proteins, rather than attach them to peptidoglycan. Full length and truncation mutant constructs of SrtC and its substrate, T3, and peptide substrate mimics have been produced in soluble form for use in kinetic assays. Additionally, initial crystallization conditions have been identified for <italic>S. pyogenes</italic> SrtC towards the goal of three-dimensional structure determination. A homology model of the structure has also been produced, displaying many of the general features observed for other sortase enzymes.</p><p>Additionally, a computational analysis of the mechanism of isopeptide bond formation in <italic>S. pyogenes</italic> SPy0128, a substrate of <italic>S. pyogenes</italic> SrtC, has been performed. Isopeptide bonds have previously been found in structural studies of Gram-positive bacterial adhesins in each domain of these multi-domain proteins. The bonds are typically formed between conserved lysine and asparagine residues, and formation is likely catalyzed by adjacent conserved glutamates. A direct nucleophilic attack mechanism, starting from an inverse protonation state, is supported in this study. Of note, there appears to be temporal regulation of isopeptide bond formation in the different domains of <italic>S. pyogenes</italic> SPy0128, with the C-terminal domain isopeptide bond forming prior to or simultaneously with the N-terminal domain isopeptide bond.</p><p>Previous studies suggest that SrtA activity is required for <italic>S. aureus</italic> to survive phagocytosis by a macrophage. The production of reactive oxygen species by professional phagocytes could lead to inhibition of SrtA via oxidation of a conserved nucleophilic cysteine residue in the active site. Through determination of inhibition kinetics, identification of oxidative modifications, reduction potential measurements, and analyses of SrtA in vivo activity in the presence of reactive oxygen species, it has been demonstrated that <italic>S. aureus</italic> SrtA is resistant to oxidative inhibition. These findings support SrtA activity inside the phagolysosome of a professional phagocyte and likely contribute to the ability of <italic>S. aureus</italic> to evade the innate immune system.</p><p>The roles of sortases and their substrates during <italic>S. aureus</italic> survival inside professional phagocytes have not been thoroughly investigated. Through analysis of the regulation of these surface proteins under phagolysosomal conditions and macrophage phagocytosis survival assays, initial characterization of the functions of sortases and their substrates in this environment has been completed. Previous studies have suggested a role for SrtA and its substrate, Protein A, and these genes and two other sortase-substrates were upregulated in response to phagolysosomal conditions. However, neither sortases nor their substrates demonstrated a direct function in phagocytosis survival. These findings imply a complex interplay between <italic>S. aureus</italic> and professional phagocytes. Further studies are necessary to delineate the direct activities of surface anchored proteins during phagocytosis of <italic>S. aureus</italic> by professional phagocytes.</p> / Dissertation

Indiana University-Purdue University Indianapolis (IUPUI) / Stiffening and softening of extracellular matrix (ECM) are critical processes governing many aspects of biological processes. The most common practice used to investigate these processes is seeding cells on two-dimensional (2D) surfaces of varying stiffness. In recent years, cell-laden three-dimensional (3D) scaffolds with controllable properties are also increasingly used. However, current 2D and 3D culture platforms do not permit spatiotemporal controls over material properties that could influence tissue processes. To address this issue, four-dimensional (4D) hydrogels (i.e., 3D materials permitting time-dependent control of matrix properties) are proposed to recapitulate dynamic changes of ECM properties. The goal of this thesis was to exploit orthogonal enzymatic reactions for on-demand stiffening and/or softening of cell-laden hydrogels. The first objective was to establish cytocompatible hydrogels permitting enzymatic crosslinking and stiffening using enzymes with orthogonal reactivity. Sortase A (SrtA) and mushroom tyrosinase (MT) were used sequentially to achieve initial gelation and on-demand stiffening. In addition, hydrogels permitting reversible stiffening through SrtA-mediated peptide ligation were established. Specifically, poly(ethylene glycol) (PEG)-peptide hydrogels were fabricated with peptide linkers containing pendent SrtA substrates. The hydrogels were stiffened through incubation with SrtA, whereas gel softening was achieved subsequently via addition of SrtA and soluble glycine substrate. The second objective was to investigate the role of dynamic matrix stiffening on pancreatic cancer cell survival, spheroid formation, and drug responsiveness. The crosslinking of PEG-peptide hydrogels was dynamically tuned to evaluate the effect of matrix stiffness on cell viability and function. Specifically, dynamic matrix stiffening inhibited cell proliferation and spheroid formation, while softening the cell-laden hydrogels led to significant increase in spheroid sizes. Matrix stiffness also altered the expression of chemoresistance markers and responsiveness of cancer cells to gemcitabine treatment. markers and responsiveness of cancer cells to gemcitabine treatment.

Peptide und Proteine sind wichtige Untersuchungsobjekte der biochemischen Forschung. Es wurden in den letzten Jahren eine Reihe von Ligationsmethoden entwickelt, um weitgehend entschützte, gereinigte Peptidsequenzen im wässrigen Milieu zu koppeln. Vor diesem Hintergrund von besonderem Interesse für einen möglichen Einsatz bei Ligationen ist die bakterielle Transpeptidase Sortase A. Dieses Enzym ist in vivo an der Anknüpfung von Proteinen an das bakterielle Peptidoglycan beteiligt, wobei es Substrate an einem LPXTG-Motiv zwischen Threonin und Glycin spaltet und auf ein Oligoglycin-Nucleophil überträgt. Zur Untersuchung der Enzymaktivität wurde in dieser Arbeit ein einfacher HPLC-basierter Assay etabliert. Die an Peptidmodellen gewonnenen Resultate wurden schließlich für den Aufbau eines löslichen Rezeptors genutzt. Ein Schlüsselschritt war die Sortase-vermittelte Ligation des in E. coli exprimierten, gefalteten Rezeptor-N-Terminus an ein 3-Loop-Konstrukt. Das erhaltene 23 kDa große Rezeptormimetikum war nach chromatographischer Reinigung homogen gemäß HPLC und MS. Es zeigte eine spezifische, hoch affine Bindung zu natürlichen Peptidliganden des CRF1-Rezeptors. Weiterhin konnte demonstriert werden, dass sich Sortase für die selektive Markierung von Proteinen eignet. So wurde ein Fluoreszenzlabel C-terminal an das 50 kDa Protein NEMO geknüpft. Als weiteres Anwendungsbeispiel der Sortase-vermittelten Ligation diente die Darstellung von PNA–CPP-Konjugaten. Die Verwendung von Überschüssen des Peptides und die Entfernung der niedermolekularen Abgangsgruppe durch Dialyse erwies sich als sehr effektiv und gestattete gute bis hervorragende Kupplungsausbeuten von bis zu 94%. Die biologische Wirkung der erhaltenen CPP–PNA-Konjugate konnte in Aufnahmeuntersuchungen an Zellen gezeigt werden. / Peptides and proteins are important research objects in biochemical research. Therefore, several ligation methods to couple unprotected, purified peptide sequences in aqueous media have been developed during the last years. At a special interest in this case is the bacterial transpeptidase sortase A. This enzyme couples proteins in vivo to the bacterial peptidoglycan by cleavage at a LPXTG-recognition motif between threonine and glycine and subsequent transfer to an oligoglycin nucleophile. In order to investigate the enzymatic activity, a simple HPLC-based assay was established in this work. Results obtained with model peptides were used for the assembly of a soluble receptor. A key step was the sortase-mediated ligation of the folded receptor N-terminus (expressed in E. coli) to the 3-loop-construct. The resulting receptor mimic of 23 kDa was homogeneous according to HPLC and MS. It showed specific binding to natural peptide ligands of the CRF1-receptor with high affinity. Furthermore, it could be shown that sortase is usable for selective protein labeling. For this purpose, a fluorescence label was attached C-terminally to the 50 kDa protein NEMO. As a further example of sortase-mediated ligation served the synthesis of PNA-CPP-conjugates. The use of an excess of the peptide and dialyzing away the small leaving group proved to be very effective and coupling yields up to 94% could be achieved. The biological activity of the CPP-PNA-conjugates could be shown by uptake studies in cells.