Malate dehydrogenase (MDH) exists in multimeric form in normal and extreme solvent conditions where residues of the interface are involved in specific interactions. The interface salt-bridge (ISB) and its microenvironment (ME) residues may have a crucial role in the stability and specificity of the interface. To gain insight into this, we have analyzed 218 ISBs from 42 interfaces of 15 crystal structures along with their sequences. Comparative analyses demonstrate that the ISB strength is similar to 30 times greater in extremophilic cases than that of the normal one. To this end, the interface residue propensity, ISB design and pair selection, and ME-residue's types, i.e., type-I and type-II, are seen to be intrinsically involved. Although Type-I is a common type, Type-II appears to be extremophile-specific, where the net ME-residue count is much lower with an excessive net ME-energy contribution, which seems to be a novel interface compaction strategy. Furthermore, the interface strength can be enhanced by selecting the desired mutant from the net-energy profile of all possible mutations of an unfavorable ME-residue. The study that applies to other similar systems finds applications in protein-protein interaction and protein engineering.Communicated by Ramaswamy H. Sarma

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.

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.

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.

Residues in allelic positions, in the local segment of aligned sequences of proteins show wide variations. Here, we describe PROPAB that computes the propensity tables for helix, strand and coil types from multiple 3D structure files following ab initio statistical procedure. It also classifies them in range specific and chain specific manners. It further computes percentage composition and physicochemical properties along with residues propensities. It also prepares FASTA files for different segments (helix, strand and coil) in the exact order that they follow in the sequence. Representative analyses on orthologous (homologous across species) proteins demonstrate wide segmental variations of physicochemical properties. Such variations provide insights to relate the adaptation of these proteins in a given functional constraint under diverse environmental conditions. Thus, the program finds applications in the structural and evolutionary analysis of proteins.

Biological systems are made of complex networks non-covalent interactions observed among protein-protein, protein-DNA, protein-lipid 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.

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.

Chikungunya virus (CHIKV) is a female Aedes mosquito-borne alphavirus that recently caused large-scale epidemics of arthritic disease in tropics of Asian region. Molecular substitutions in E1 envelope glycoprotein as well as in other structural and non-structural proteins cause variation in genotypes, infectivity and severity in clinical symptoms. GenBank Isolates AY424803 and KF818473 were associated with epidemics/outbreaks occurred in 1965 and 2012, in Maharashtra and West Bengal states of India respectively. Although E1 glycoprotein based phylogenetic analyses established the former as Asian and the latter as ECSA (East- Central-South-African) genotypes, detailed insight into atomic structures and the effect of acquired mutations of these proteins remain to be worked out. In this study we applied in silico approaches on E1 protein to investigate detailed differential analyses of sequence, atomic model structure and acquired substitutions for their effects on stability and lethality. Physicochemical and sequence studies of these proteins show fine tuning of these properties in reference to the template (3N43). Atomic models remarkably show similar structural features as template including domains topologies, secondary structure arrangements and disulfide bonds. Side chain conformations of models and template, are seen to be similar as both form salt-bridges with almost identical specificity. In silico site directed mutagenesis shows, out of 14 and 2 acquired substitutions in AY424803 and KF818473, only 3 and none are lethal respectively. Our studies provide detailed structural analyses of E1 glycoprotein, new approaches to assess side chain specificity and judgment of lethality of acquired substitutions that have potential applications in interdisciplinary virology.

Citrate Synthase is an enzyme and most often responsible for catalyzing the first reaction of the citric acid cycle: the condensation of acetyle-CoA and oxaloacetate to form citrate. The protein sequence of mitochondrial citrate synthase having Uniprot Id O75390, has no 3D structure in protein data bank (PDB) till now. We know protein structure play an important role in their function. Our present work is based on primary and secondary structure analysis and production of high quality 3D structure of mitochondrial citrate synthase and its salt-bridge analysis. The protein was retrieved from UniProt date base. Physicochemical property has been calculated with the help of PHYSICO software. Homology modeling was performed by using MODELLER 9v11 and final model was then undergoing NAMD minimization and successfully passes through PROCHEAK, ERRAT, VERIFY-3D, Pro-Q and RAMPAGE. This model is successfully submitted in Protein Model Database having PMDB id PM0080060.

Specific electrostatics (i.e. salt-bridge) includes both local and non-local interactions that contribute to the overall stability of proteins. It has been shown that a salt-bridge could either be buried or exposed, networked or isolated, hydrogen-bonded or non-hydrogen bonded, in secondary-structure or in coil, formed by single or multiple bonds. Further it could also participates either in intra- or inter-dipole interactions with preference in orientation either for basic residue at N-terminal (orientation-I) or acidic residue at N-terminal (orientation-II). In this context SBION2 is unique in that it reports above mentioned binary items in excel format along with details on intra and inter-dipole interactions and orientations. These results are suitable for post run statistical analyses involving large datasets. Reports are also made on protein-protein interactions, intervening residue distances and general residue specific salt-bridge details. A ready to use compact supplementary table is also produced. The program runs in three alternative modes. Each mode works on any number of structure files with any number of chains at any given atomic distance of ion-pair. Thus SBION2 provides intricate details on salt-bridges and finds application in structural bioinformatics.