Structural and functional insights of itaconyl-CoA hydratase from Pseudomonas aeruginosa highlight a novel N-terminal hotdog fold.

Itaconyl-CoA hydratase in Pseudomonas aeruginosa (PaIch) converts itaconyl-CoA to (S)-citramalyl-CoA upon addition of a water molecule, a part of an itaconate catabolic pathway in virulent organisms required for their survival in humans host cells. Crystal structure analysis of PaIch showed that a unique N-terminal hotdog fold containing a 4-residue short helical segment α3-, named as an "eaten sausage", followed by a flexible loop region slipped away from the conserved ß-sheet scaffold, whereas the C-terminal hotdog fold is similar to all MaoC. A conserved hydratase motif with catalytic residues provides mechanistic insights into catalysis, and existence of a longer substrate binding tunnel may suggest the binding of longer CoA derivatives.

Exploring structural features and potential lipid interactions of Pseudomonas aeruginosa type three secretion effector PemB by spectroscopic and calorimetric experiments.

Type Three Secretion System (T3SS) is a sophisticated nano-scale weapon utilized by several gram negative bacteria under stringent spatio-temporal regulation to manipulate and evade host immune systems in order to cause infection. To the best of our knowledge, this present study is the first report where we embark upon characterizing inherent features of native type three secretion effector protein PemB through biophysical techniques. Herein, first, we demonstrate binding affinity of PemB for phosphoinositides through isothermal calorimetric titrations. Second, we shed light on its strong homo-oligomerization propensity in aqueous solution through multiple biophysical methods. Third, we also employ several spectroscopic techniques to delineate its disordered and helical conformation. Lastly, we perform a phylogenetic analysis of this new effector to elucidate evolutionary relationship with other organisms. Taken together, our results shall surely contribute to our existing knowledge of Pseudomonas aeruginosa secretome.

Diversity in the ligand binding pocket of HapR attributes to its uniqueness towards several inhibitors with respect to other homologues - A structural and molecular perspective.

Vibrio cholerae is a prolific bacterium. Cumulative studies clearly demonstrate the key role of quorum sensing on the lifecycle of this bacterium. Of the sensory network components, HapR is known as high cell density master regulator. Until now, no information is available on native HapR ligand despite the protein having a ligand binding pocket. Interestingly, function of SmcR, a HapR homologue of Vibrio vulnificus is inhibited by a small molecule Qstatin. Structural analysis of SmcR with Qstatin identifies key interacting residues in SmcR ligand binding domain. Despite bearing significant homology with SmcR, HapR function remained unabated by Qstatin. Sequence alignment indicates divergence in the key residues of ligand binding pocket between these two regulators. A series of ligand binding domain mutants of HapR was constructed where only HapR quadruple mutant responded to Qstatin and newly synthesized IMT-VC-212. Crystal structure analysis revealed four key residues are responsible for changes in the volume of ligand binding pocket of HapR quadruple mutant compared to the wild type counterpart, thereby increasing the accessibility of Qstatin and its derivative in case of the former. The mechanistic insights exuberating from this study will remain instrumental in designing inhibitors against wild type HapR.

Delineating specific regions of N- terminal domain of T3SS ATPase YsaN of Yersinia enterocolitica governing its different oligomerization states.

Oligomerization of YsaN, a putative T3SS-ATPase is a necessary and crucial event for T3SS functioning in Y. enterocolitica. Different oligomeric states have been proposed for similar ATPases, yet, the true nature of its activation and formation of different oligomers is still poorly understood. In-vitro studies of YsaN reveal that its activation and oligomerization depend on its N-terminal region and occur as a result of active catalysis of ATP in an ATP concentration-dependent manner following two-step cooperative kinetics. Also, the N-terminal 83 amino acid residues of YsaN are crucial for higher-order oligomer formation while YsaN∆83 is capable of hexamer formation upon oligomerization. Enzyme kinetics study shows reduced ATPase activity of YsaN∆83 (3.19 ± 0.09 µmol/min/mg) in comparison to YsaN (9.076 ± 0.72 µmol/min/mg). Negative-TEM study of YsaN and YsaN∆83 oligomer suggests that the formation of higher-order oligomer (probably dodecamer) occurs by stacking of two hexamers through their N-terminal faces involving N-terminal 83 amino acid residues which have been further supported by the docking of two hexamers during the in-silico study. These results suggest that YsaN is an oligomerization-activated T3SS ATPase, where distinct regions of its N-terminal domain regulate its different oligomeric nature and is essential for its activation.

Biophysical and Computational Approaches to Unravel pH-Dependent Conformational Change of PspA Assist PspA-PspF Complex Formation in Yersinia enterocolitica.

In enteropathogen, Yersinia enterocolitica, the genes encoding phage shock proteins are organized in an operon (pspA-E), which is activated at the various types of cellular stress (i.e., extracytoplasmic or envelop stress) whereas, PspA negatively regulates PspF, a transcriptional activator of pspA-E and pspG, and is also involved in other cellular machinery maintenance processes. The exact mechanism of association and dissociation of PspA and PspF during the stress response is not entirely clear. In this concern, we address conformational change of PspA in different pH conditions using various in-silico and biophysical methods. At the near-neutral pH, CD and FTIR measurements reveal a ß-like conformational change of PspA; however, AFM measurement indicates the lower oligomeric form at the above-mentioned pH. Additionally, the results of the MD simulation also support the conformational changes which indicate salt-bridge strength takes an intermediate position compared to other pHs. Furthermore, the bio-layer interferometry study confirms the stable complex formation that takes place between PspA and PspF at the near-neutral pH. It, thus, appears that PspA conformational change in adverse pH conditions abandons PspF from having a stable complex with it, and thus, the latter can act as a trans-activator. Taken together, it seems that PspA alone can transduce adverse signals by changing its conformation.

Crystallographic and biophysical analyses of Pseudomonas aeruginosa ketopantoate reductase: Implications of ligand induced conformational changes in cofactor recognition.

Ketopantoate reductases (KPRs) catalyse NADPH-dependent reduction of ketopantoate to pantoate, the rate-limiting step of pantothenate biosynthetic pathway. In our recent study, we showed KPRs are under dynamic evolutionary selection and highlighted the possible role of ordered substrate binding kinetics for cofactor selection. To further delineate this at molecular level, here, we perform X-ray crystallographic and biophysical analyses of KPR in presence of non-canonical cofactor NAD+. In our structure, NAD+ was found to be highly dynamic in catalytic pocket of KPR, which could attain stable conformation only in presence of ketopantoate. Further, isothermal calorimetric (ITC) titrations showed that affinity of KPR for ketopantoate is higher in presence of NADP+ than in presence of NAD+ and lowest in absence of redox cofactors. In sum, our results clearly depict two modes of redox cofactor selections in KPRs, firstly by specific salt bridge interactions with unique phosphate moiety of NADP+ and secondly via ordered sequential heterotrophic cooperative binding of substrate ketopantoate.

Arginine 37 of Glycine Linker Dictates Regulatory Function of HapR.

HapR is designated as a high cell density quorum sensing master regulatory protein of Vibrio cholerae. It is a member of the TetR family protein and functions both as an activator and a repressor by directly communicating with cognate promoters, thus controlling the expression of a plethora of genes in a density-dependent manner. Molecular insights reveal the domain architecture and further unveil the significance of a cross talk between the DNA binding domain and the dimerization domain for the functionality of the wild-type protein. The DNA binding domain is made up of three α-helices, where a helix-turn-helix motif spans between the helices α2 and α3. The essentiality of the glycine-rich linker linking helices α1 and α2 came into prominence while unraveling the molecular basis of a natural non-functional variant of HapR. Subsequently, the importance of linker length was demonstrated. The present study, involving a series of biochemical analyses coupled with molecular dynamics simulation, has illustrated the indispensability of a critical arginine within the linker at position 37 contributing to HapR-DNA binding activity.

Deciphering the structural intricacy in virulence effectors for proton-motive force mediated unfolding in type-III protein secretion.

Given that the protein unfolding requisite for type-III secretion system (T3SS)-mediated secretion is an energetically unfavorable process, the question of how do pathogenic bacteria unfold and secrete hundreds of toxic proteins in seconds remain largely unknown. In this study, a systematic effort combining experimental and computational approaches has been employed to get some mechanistic insights on the unfolding of effectors in T3SS secretion. The in-depth analysis of pH-dependent folding and stability of a T3SS effector ExoY revealed that proton-concentration gradient (~pH 5.8-6.0) generated by proton-motive force (PMF) had significantly affected folding and structural stability of this protein without significant loss of the free energy of unfolding. Importantly, the lower energetic cost associated with the global unfolding of ExoY was mainly due to its inherent stereo-chemical frustrations embedded within its native-like structure as observed from its core structural analysis. These observations suggest that the cooperation between the evolved structural features of ExoY and pH-mediated unfolding is crucial for PMF-mediated T3SS secretion. From a comprehensive computational analysis of 371 T3SS effectors it was concluded that many of these effectors belong to the category of intrinsically disordered proteins (IDPs) and have similar conserved structural archetypes to facilitate early-stage unfolding process as observed in ExoY. We had also provided details of folding, stability, and molecular evolution in T3SS effectors and established the role of evolved structural archetypes in early-stage unfolding events of this effector for maintaining balance in secretion and function trade-off.

Comparative studies on ion-pair energetic, distribution among three domains of life: Archaea, eubacteria, and eukarya.

Salt-bridges play a unique role in the structural and functional stability of proteins, especially under harsh environments. How these salt-bridges contribute to the overall thermodynamic stability of protein structure and function across different domains of life is elusive still date. To address the issue, statistical analyses on the energies of salt-bridges, involved in proteins' structure and function, are performed across three domains of life, that is, archaea, eubacteria, and eukarya. Results show that although the majority of salt-bridges are stable and conserved, yet the stability of archaeal proteins (∆∆Gnet = -5.06 ± 3.8) is much more than that of eubacteria (∆∆Gnet = -3.7 ± 2.9) and eukarya (∆∆Gnet = -3.54 ± 3.1). Unlike earlier study with archaea, in eukarya and eubacteria, not all buried salt-bridge in our dataset are stable. Buried salt-bridges play surprising role in protein stability, whose variations are clearly observed among these domains. Greater desolvation penalty of buried salt-bridges is compensated by stable network of salt-bridges apart from equal contribution of bridge and background energy terms. On the basis proteins' secondary structure, topology, and evolution, our observation shows that salt-bridges when present closer to each other in sequence tend to form a greater number. Overall, our comparative study provides insight into the role of specific electrostatic interactions in proteins from different domains of life, which we hope, would be useful for protein engineering and bioinformatics study.

Genome-wide survey and crystallographic analysis suggests a role for both horizontal gene transfer and duplication in pantothenate biosynthesis pathways.

Pantothenate is the metabolic precursor of Coenzyme A, an indispensable cofactor for many fundamental cellular processes. In this study, we show that many bacterial species have acquired multiple copies of pantothenate biosynthesis pathway genes via horizontal and vertical gene transfer events. Some bacterial species were also found to lack panE and panD genes, and depended on alternative enzymes/metabolic sources for pantothenate production. To shed light on the factors responsible for such dynamic evolutionary selections, the structural and functional characteristics of P. aeruginosa ketopantoate reductase (KPR), an enzyme that catalyzes the rate-limiting step and also the most duplicated, was investigated. A comparative analysis of apo and NADP+ bound crystal structures of P. aeruginosa KPR with orthologs, revealed that the residues involved in the interaction with specific phosphate moiety of NADP+ are relatively less conserved, suggesting dynamic evolutionary trajectories in KPRs for redox cofactor selection. Our structural and biochemical data also show that the specific conformational changes mediated by NADPH binding facilitate the cooperative binding of ketopantoate. From drastically reduced catalytic activity for NADH catalyzed the reaction with significantly higher KM of ketopantoate, it appears that the binding of ketopantoate is allosterically regulated to confer redox cofactor specificity. Altogether, our results, in compliance with earlier studies, not only depict the role of lateral gene transfer events in many bacterial species for enhancing pantothenate production but also highlight the possible role of redox cofactor balance in the regulation of pantothenate biosynthesis pathways.

Structural and functional characterization of type three secretion system ATPase PscN and its regulator PscL from Pseudomonas aeruginosa.

Type Three Secretion Systems (T3SS) from many gram-negative bacteria utilize ATPases for the translocation of effector proteins into the eukaryotic host cells through injectisome. Cytosolic regulators effectively control the action of these ATPases. PscN from Pseudomonas aeruginosa was an ATPase which was regulated by an uncharacterized PscL. Here we have bioinformatically, biochemically, and biophysically characterized PscN as a T3SS ATPase and PscL as its regulator. In solution, PscN exists predominantly as oligomer and hydrolyzes ATP with Vmax of 3.9 ± 0.2 µmol/min/mg and K m 0.93 ± 0.06 mM. Hexameric structure of PscN was observed under AFM and TEM in the presence of ATP. PscL was dimeric in solution and interacted with PscN strongly in Ni-NTA pull-down assay and SPR analysis. PscL was shown to downregulate PscN ATPase activity up to 80% when mixed with PscN in 1:2 ratio (PscN:PscL). SEC data reconfirm the PscN-PscL interaction stoichiometry (ie, 1:2 ratio) which can also be visualized under AFM. In the present study, we have also found out the existence of an oligomeric form of the PscN-PscL heterotrimeric complex. PscL being the regulator of PscN and interacts to form this conformation, which may play an important role too in the regulation of T3SS utilized by Pseudomonas aeruginosa. For structural aspect, three dimensional in silico models of PscN, PscL, and PscN-PscL were generated. So, in short, present study tried to enlighten both the structural, functional and mechanistic insights into the action of PscN-PscL complex in T3SS mediated pathogenic pathway.

ASBAAC: Automated Salt-Bridge and Aromatic-Aromatic Calculator.

Biological systems are made of complex networks non-covalent interactions observed among protein-protein, protein-DNA, proteinlipid complexes using hydrogen-bonds, salt-bridges, aromatic-aromatic, van der Waals (vdW), hydrophobic-interactions and several others using distance criteria. Hence, large-scale data analysis is required to understand the principles of biological complex formation. Therefore, it is of interest to analyze non-covalent interaction namely, salt-bridge and aromatic-aromatic contacts in known and modeled protein complex structures. Here, we describe ASBAAC for automatic calculation of salt-bridges and aromatic-aromatic contacts in protein complexes. This software tool is fast, robust and user-friendly for large-scale analysis of inter-chain salt bridges and aromatic-aromatic contact in protein complexes. AVAILABILITY: ASBAAC is available for free at http://sourceforge.net/projects/asbaac.

Crystal structure and substrate specificity of ExoY, a unique T3SS mediated secreted nucleotidyl cyclase toxin from Pseudomonas aeruginosa.

BACKGROUND: The nucleotidyl cyclase toxin ExoY is an important virulence determinant of Pseudomonas aeruginosa that causes severe acute and chronic infections in immune-compromised individuals. Additionally, this unique T3SS effector shows a striking preference for cUMP, a newly identified non-canonical secondary messenger. Thereby, ExoY is also considered as a potential tool to study unexplored cUMP signaling pathways. METHODS: The crystal structure of ExoY was determined at 2.2 Šresolutions by in-situ proteolysis assisted crystallization and Rosetta-molecular replacement method. Additionally, isothermal calorimetric (ITC) and molecular dynamic (MD) simulation studies were also carried out to gain molecular insights into its substrate specificity and catalysis. RESULTS AND CONCLUSION: ExoY is a partially unfolded protein with higher propensity to form soluble higher-order oligomers. However, with meticulous attempts of removing of disordered regions by proteases, the recalcitrant ExoY could be successfully crystallized. The crystal structure of ExoY revealed similar overall structural fold present in other anthrax toxA family of nucleotidyl cyclases, with two-to-three distinctly conserved regions conferring specificity to eukaryotic binding partner. The in-vitro catalytic preference of ExoY is in the following order: cGMP > cUMP > cAMP > cCMP. The substrate specificity of ExoY mainly depends on its ability to bind NTP in proper geometrical orientations. ExoY also seems to prefer one-metal-ion dependent catalysis than two-metal-ion dependent catalysis. GENERAL SIGNIFICANCE: Our results provide much needed structural insight on ExoY, an important virulence determinant of Pseudomonas aeruginosa and an exciting tool to study non-canonical cNMP signaling pathways. ACCESSION NUMBERS: The structure factors and coordinate files have been deposited in the Protein Data Bank with accession number 5XNW.

Quantum mechanical electronic structure calculation reveals orientation dependence of hydrogen bond energy in proteins.

Hydrogen bond plays a unique role in governing macromolecular interactions with exquisite specificity. These interactions govern the fundamental biological processes like protein folding, enzymatic catalysis, molecular recognition. Despite extensive research work, till date there is no proper report available about the hydrogen bond's energy surface with respect to its geometric parameters, directly derived from proteins. Herein, we have deciphered the potential energy landscape of hydrogen bond directly from the macromolecular coordinates obtained from Protein Data Bank using quantum mechanical electronic structure calculations. The findings unravel the hydrogen bonding energies of proteins in parametric space. These data can be used to understand the energies of such directional interactions involved in biological molecules. Quantitative characterization has also been performed using Shannon entropic calculations for atoms participating in hydrogen bond. Collectively, our results constitute an improved way of understanding hydrogen bond energies in case of proteins and complement the knowledge-based potential. Proteins 2017; 85:1046-1055. © 2017 Wiley Periodicals, Inc.

Transition of phosphopantetheine adenylyltransferase from catalytic to allosteric state is characterized by ternary complex formation in Pseudomonas aeruginosa.

BACKGROUND: Phosphopantetheine adenylyltransferase (PPAT) is a rate limiting enzyme which catalyzes the conversion of ATP and pantetheine to dephosphocoenzyme and pyrophosphate. The enzyme is allosteric in nature and regulated by Coenzyme A (CoA) through feedback inhibition. So far, several structures have been solved to decipher the catalytic mechanism of this enzyme. METHODS: To address catalytic and inhibitory mechanisms of PPAT, structural insights from single crystal X-ray diffraction method were primarily used, followed by biophysical and biochemical analysis. RESULTS: We have solved the structures of PPAT from Pseudomonas aeruginosa with its substrate analogue AMP-PNP and inhibitor CoA. For the first time, a co-crystal structure of PPAT with Acetyl-CoA (AcCoA) was determined. Enzymatic analysis was performed to decipher the catalytic, allosteric and inhibitory mechanisms involved in regulation of PPAT. Binding affinities of PPAT with its substrates and inhibitors were determined by SPR. CONCLUSION: Previous studies from Escherichia coli and Arabidopsis indicated the inhibitory activity of AcCoA. PPAT-AcCoA structure along with some biochemical methods established AcCoA as an inhibitor to PPAT and illustrated its inhibitory mechanism. Transition from catalytic to allosteric state involves formation of ternary complex. We have studied the structural features of the ternary complex of PPAT along with its product pyrophosphate and inhibitor CoA and validated it with other biophysical and biochemical methods. Extensive analysis of all these 3D structures indicates that changes in side chains R90 and D94 are responsible for transition between catalytic and allosteric inhibitory states. GENERAL SIGNIFICANCE: These enzymatic studies provide new insights into the allosteric mechanism of PPAT.

Structural analysis of inter-genus complexes of V-antigen and its regulator and their stabilization by divalent metal ions.

Gram-negative bacteria like Yersinia, Pseudomonas, and Aeromonas need type III secretion system (T3SS) for their pathogenicity. V-antigen and its regulator are essential for functioning of T3SS. There is significant functional conservation amongst V-antigen and its regulator belonging to the Ysc family. In this study, we have structurally characterized the inter-genus complexes of V-antigen and its regulator. ConSurf analysis demonstrates that V-antigens belonging to the Ysc family show high structural identity predominantly confined to the two long helical regions. The regulator of V-antigen shows high conservation in its first intramolecular coiled-coil domain, responsible for interaction with V-antigen. ∆LcrG(1-70) localizes within the groove formed by long helices of LcrV, as observed in PcrV-∆PcrG(13-72) interaction. Inter-genus complexes of LcrV-PcrG and PcrV-LcrG exhibited elongated conformation and 1:1 heterodimeric state like the native complex of PcrV-PcrG and LcrV-LcrG. Both native and inter-genus complexes showed rigid tertiary structure, solvent-exposed hydrophobic patches, and cooperative melting behavior with high melting temperature. LcrV-PcrG and PcrV-LcrG showed nanomolar affinity of interaction, identical to PcrV-PcrG interaction, but stronger than LcrV-LcrG interaction. Calcium (a secretion blocker of T3SS) propels all the complexes towards a highly monodisperse form. Calcium and magnesium increase the helicity of the native and inter-genus complexes, and causes helix-helix stabilization. Stabilization of helices leads to a slight increase in the melting temperature by 1.5-2.0 °C. However, calcium does not alter the affinity of interaction of V-antigen and its regulator, emphasizing the effect of divalent of cations at the structural level without any regulatory implications. Therefore, the structural conservation of these inter-genus complexes could be the basis for their functional complementation.

Binding mode analysis of a major T3SS translocator protein PopB with its chaperone PcrH from Pseudomonas aeruginosa.

Pseudomonas aeruginosa, a Gram-negative pathogen uses a specialized set of Type III secretion system (T3SS) translocator proteins to establish virulence in the host cell. An understanding of the factors that govern translocation by the translocator protein-chaperone complex is thus of immense importance. In this work, experimental and computational techniques were used to probe into the structure of the major translocator protein PopB from P. aeruginosa and to identify the important regions involved in functioning of the translocator protein. This study reveals that the binding sites of the common chaperone PcrH, needed for maintenance of the translocator PopB within the bacterial cytoplasm, which are primarily localized within the N-terminal domain. However, disordered and flexible residues located both at the N- and C-terminal domains are also observed to be involved in association with the chaperone. This intrinsic disorderliness of the terminal domains is conserved for all the major T3SS translocator proteins and is functionally important to maintain the intrinsically disordered state of the translocators. Our experimental and computational analyses suggest that a "disorder-to-order" transition of PopB protein might take place upon PcrH binding. The long helical coiled-coil part of PopB protein perhaps helps in pore formation while the flexible apical region is involved in chaperone interaction. Thus, our computational model of translocator protein PopB and its binding analyses provide crucial functional insights into the T3SS translocation mechanism.

PcrG protects the two long helical oligomerization domains of PcrV, by an interaction mediated by the intramolecular coiled-coil region of PcrG.

BACKGROUND: PcrV is a hydrophilic translocator of type three secretion system (TTSS) and a structural component of the functional translocon. C-terminal helix of PcrV is essential for its oligomerization at the needle tip. Conformational changes within PcrV regulate the effector translocation. PcrG is a cytoplasmic regulator of TTSS and forms a high affinity complex with PcrV. C-terminal residues of PcrG control the effector secretion. RESULT: Both PcrV and PcrG-PcrV complex exhibit elongated conformation like their close homologs LcrV and LcrG-LcrV complex. The homology model of PcrV depicts a dumbbell shaped structure with N and C-terminal globular domains. The grip of the dumbbell is formed by two long helices (helix-7 and 12), which show high level of conservation both structurally and evolutionary. PcrG specifically protects a region of PcrV extending from helix-12 to helix-7, and encompassing the C-terminal globular domain. This fragment ∆PcrV(128-294) interacts with PcrG with high affinity, comparable to the wild type interaction. Deletion of N-terminal globular domain leads to the oligomerization of PcrV, but PcrG restores the monomeric state of PcrV by forming a heterodimeric complex. The N-terminal globular domain (∆PcrV(1-127)) does not interact with PcrG but maintains its monomeric state. Interaction affinities of various domains of PcrV with PcrG illustrates that helix-12 is the key mediator of PcrG-PcrV interaction, supported by helix-7. Bioinformatic analysis and study with our deletion mutant ∆PcrG(13-72) revealed that the first predicted intramolecular coiled-coil domain of PcrG contains the PcrV interaction site. However, 12 N-terminal amino acids of PcrG play an indirect role in PcrG-PcrV interaction, as their deletion causes 40-fold reduction in binding affinity and changes the kinetic parameters of interaction. ∆PcrG(13-72) fits within the groove formed between the two globular domains of PcrV, through hydrophobic interaction. CONCLUSION: PcrG interacts with PcrV through its intramolecular coiled-coil region and masks the domains responsible for oligomerization of PcrV at the needle tip. Also, PcrG could restore the monomeric state of oligomeric PcrV. Therefore, PcrG prevents the premature oligomerization of PcrV and maintains its functional state within the bacterial cytoplasm, which is a pre-requisite for formation of the functional translocon.

Interfacial residues of SpcS chaperone affects binding of effector toxin ExoT in Pseudomonas aeruginosa: novel insights from structural and computational studies.

ExoT belongs to the family of type 3 secretion system (T3SS) effector toxins in Pseudomonas aeruginosa, known to be one of the major virulence determinant toxins that cause chronic and acute infections in immuno-compromised individuals, burn victims and cystic fibrosis patients. Here, we report the X-ray crystal structure of the amino terminal fragment of effector toxin ExoT, in complex with full-length homodimeric chaperone SpcS at 2.1 Å resolution. The full-length dimeric chaperone SpcS has the conserved α-ß-ß-ß-α-ß-ß-α fold of class I chaperones, the characteristic hydrophobic patches for binding effector proteins and a conserved polar cavity at the dimeric interface. The stable crystallized amino terminal fragment of ExoT consists of a chaperone binding domain and a membrane localization domain that wraps around the dimeric chaperone. Site-directed mutagenesis experiments and a molecular dynamics study complement each other in revealing Asn65, Phe67 and Trp88 as critical dimeric interfacial residues that can strongly influence the effector-chaperone interactions.

Identification and molecular characterization of YsaL (Ye3555): a novel negative regulator of YsaN ATPase in type three secretion system of enteropathogenic bacteria Yersinia enterocolitica.

Type Three Secretion (T3S) ATPases are involved in delivery of virulent factors from bacteria to their hosts (through injectisome) in an energy (ATP) dependent manner during pathogenesis. The activities of these ATPases are tightly controlled by their specific regulators. In Yersinia enterocolitica, YsaN was predicted as a putative ATPase of the Ysa-Ysp Type Three Secretion System (T3SS) based on sequence similarity with other T3S ATPases. However detailed study and characterization of YsaN and its regulation remains largely obscure. Here, in this study, we have successfully cloned, over-expressed, purified and characterized the molecular properties of YsaN from Yersinia enterocolitica. YsaN acts as a Mg(2+) dependent ATPase and exists in solution as higher order oligomer (dodecamer). The ATPase activity of oligomeric YsaN is several fold higher than the monomeric form. Furthermore, by employing in silico studies we have identified the existence of a negative regulator of YsaN--a hypothetical protein YE3555 (termed 'YsaL'). To verify the functionality of YsaL, we have evaluated the biochemical and biophysical properties of YsaL. Purified YsaL is dimeric in solution and strongly associates with YsaN to form a stable heterotrimeric YsaL-YsaN complex (stoichiometry--2∶1). The N terminal 6-20 residues of YsaN are invariably required for stable YsaL-YsaN complex formation. YsaL inhibited the ATPase activity of YsaN with a maximum inhibition at the molar ratio 2∶1 (YsaL: YsaN). In short, our studies provide an insight into the presence of YsaN ATPase in Yersinia enterocolitica and its regulator YsaL. Our studies also correlate the functionality of one of the existing protein interaction networks that possibly is indispensable for the energy dependent process of Ysa-Ysp T3SS in pathogenic Yersinia enterocolitica.