Underlying features for the enhanced electrostatic strength of the extremophilic malate dehydrogenase interface salt-bridge compared to the mesophilic one.

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 ∼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.

Intrinsic basis of thermostability of prolyl oligopeptidase from Pyrococcus furiosus.

Salt-bridges play a key role in the thermostability of proteins adapted in stress environments whose intrinsic basis remains to be understood. We find that the higher hydrophilicity of PfP than that of HuP is due to the charged but not the polar residues. The primary role of these residues is to enhance the salt-bridges and their ME. Unlike HuP, PfP has made many changes in its intrinsic property to strengthen the salt-bridge. First, the desolvation energy is reduced by directing the salt-bridge towards the surface. Second, it has made bridge-energy more favorable by recruiting energetically advantageous partners with high helix-propensity among the six possible salt-bridge pairs. Third, ME-residues that perform intricate interactions have increased their energy contribution by making major changes in their binary properties. The use of salt-bridge partners as ME-residues, and ME-residues' overlapping usage, predominant in helices, and energetically favorable substitution are some of the favorable features of PfP compared to HuP. These changes in PfP reduce the unfavorable, increase the favorable ME-energy. Thus, the per salt-bridge stability of PfP is greater than that of HuP. Further, unfavorable target ME-residues can be identified whose mutation can increase the stability of salt-bridge. The study applies to other similar systems.

Screening and molecular characterization of lethal mutations of human homogentisate 1, 2 dioxigenase.

Alkaptonuria (AKU) is an autosomal recessive disorder, which is caused by a site-specific mutation(s) and thus, impaired the function of Homogentisate-1, 2-dioxygenase (HGD), an essential enzyme for the catabolism of phenylalanine and tyrosine. Among frameshift, intronic, splice-site and missense mutations, the latter has been the most common form of genetic variations for the disease. How do the acquired mutations in HGD correlate with the disease? Systematic staged-screening of some sixty-five mutations, which are known to have a relation with the disease, by GVGD, SIFT, SNAP, PANTHER, SDM, PHD-SNP, Meta-SNP, Pmut and Mutpred methods, showed that mutations, W60G, A122D and V300G are potentially related with the severity of AKU. Detailed analyses on molecular docking and molecular dynamics simulation (MDS) of these mutants against the wild-type HGD reveal the loss of structural and molecular dynamic properties of the enzyme. Further, the observed conformational flexibility in mutants at targeted peptide segments seems to have a relation with the impairment of the function of HGD. Taken together, the study involves a designed computational methodology to analyse the disease-associated nsSNPs for AKU, the knowledge of which seems to have potential applications in drug therapies for the disease in particular and other similar systems in general.Communicated by Ramaswamy H. Sarma.

Structural insights from water-ferredoxin interaction in mesophilic algae and halophilic archaea.

We analyzed the water-ferredoxin interaction in mesophilic (moderate temperature) algae (PDB ID: 1AWD) and halophilic (salt-tolerant) archaea (PDB ID: 1DOI) using POWAIND version 2.0 (a protein-water interactions calculation program). It is found that the shell water (SW) is 2.5 fold greater in halophilic ferredoxin than mesophilic ferredoxin. Water-ferredoxin interactions in the core and cavity are the signature of stability. The normalized frequency of such interactions is less in halophilic relative to mesophilic ferredoxin and the halophilic signature for stability by such interactions is negligible. However, the surface dominated with such interactions seems to be important for ferredoxin and oxido-reductase recognition.

Insights from the salt bridge analysis of malate dehydrogenase from H. salinarum and E.coli.

Halophilic proteins have greater abundance of acidic over basic residues in sequence. In structure, the surface is decorated by negative charges, with lower content of Lysine. Using sequence BLOCKs and 3D model of malate dehydrogenase from halophilic archaea (Halobacterium salinarum; hsMDH) and X-ray structure from mesophilic bacteria (E. coli; ecMDH), we show that not only acidic and basic residues have higher mean relative abundance (MRA) and thus, impart higher polarity to the sequences, but also show their presence in the surface of the structure of hsMDH relative to its mesophilic counterpart. These observations may indicate that both the acidic and the basic residues have a concerted role in the stability of hsMDH. Analysis on salt bridges from hsMDH and ecMDH show that in the former, salt bridges are highly intricate, newly engineered and global in nature. Although, these salt bridges are abundant in hsMDH, in the active site the design remains unperturbed. In high salt where hydrophobic force is weak, these salt bridges seem to play a major role in the haloadaptation of the tertiary structure of hsMDH. This is the first report of such an observation.

Stability of buried and networked salt-bridges (BNSB)in thermophilic proteins.

Thermophilic proteins function at high temperature, unlike mesophilic proteins. Thermo-stability of these proteins is due to the unique buried and networked salt-bridge (BNSB). However, it is known that the desolvation cost of BNSB is too high compared to other favorable energy terms. Nonetheless, it is known that stability is provided generally by hydrophobic isosteres without the need for BNSB. We show in this analysis that the BNSB is the optimal evolutionary design of salt-bridge to offset desolvation cost efficiently. Hence, thermophilic proteins with a high level of BNSB provide thermo-stability.

Analysis of salt-bridges in prolyl oligopeptidase from Pyrococcus furiosus and Homo sapiens.

Hyper thermophilic archaea not only tolerate high temperature but also operate its biochemical machineries, normally under these conditions. However, the structural signatures in proteins that answer for the hyper thermo-stability relative to its mesophilic homologue remains poorly understood. We present comparative analyses of sequences, structures and salt-bridges of prolyl-oligopeptidase from Pyrococcus furiosus (pfPOP - PDB ID: 5T88) and human (huPOP - PDB ID: 3DDU). A similar level of hydrophobic and hydrophilic residues in pfPOP and huPOP is observed. A low level of interactions between shell-waters and atom-types in pfPOP indicated hyper thermophilic features are negligible. Salt-bridge-forming-residues (sbfrs) are high in pfPOP's core and surface (pfPOP). Increased sbfrs largely indicate specific-electrostatic is important for thermo stability in pfPOP. Four classes of sbfrs are found namely positionally non-conservative (PNCS), conservative (PCS), unchanged (PU) and interchanged (PIC) type of substitutions. PNCS-sbfrs constitutes 28% and it is associated with the topology of pfPOP at high temperature. PCS helps to increase the salt-bridge population. It is also found that PU maintains similar salt-bridges at the active site and other binding sites while PIC abolishes mesophilic patterns.

PROPAB: Computation of Propensities and Other Properties from Segments of 3D structure of Proteins.

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. AVAILABILITY: PROPAB is freely available at http://sourceforge.net/projects/propab/for worldwide user.

AUTOMINv1.0: an automation for minimization of Protein Data Bank files and its usage.

Global minimal structure of protein/enzyme is energetically compromised that maintains an intricate balance between the rigidity and the flexibility. Such a state makes it interactive to its ligand molecules. Although protein data bank files (PDB) may have achieved the state, in many situations minimization has been crucial to overcome unwanted steric clashes, and other conformational strains. It is more so, when orthologous PDB structures that are intended in a given study, show variations in resolution, R-factor, shell-water contents, loop characteristics etc. Here, a fully automated procedure of minimization would be highly useful. AUTOMINv1.0 is such an automation of minimization that runs on any number of structure files with any number of chains in them along with the inclusion of selective/non-selective shell-waters interacting with polar and or non-polar atom-types of protein. Comparison of the mean binaryitems of salt-bridges of minimized and un-minimized structures (chains > 100) of nucleoside diphosphate kinase from mimi virus shows dramatic improvements in the earlier. Again, the mean steric clashes of 2AZ3.pdb are reduced upon minimization. Remarkably, the observed steric clashes between shell-waters and atom-types of protein are seen to be removed upon minimization. Taken together, AUTOMINv1.0 is an automation of minimization that finds applications in structural bioinformatics.

POWAINDv1.0: A Program for Protein-Water Interactions Determination.

Protein is the most exposed biomolecule in the aqueous environment of the cell. Its structure maintains a delicate balance between the rigidity and the flexibility that imparts binding specificity to its substrate/ligand, etc. Intramolecular interactions of polar and non-polar groups of amino acid residues and intermolecular weak interactions between these groups and shell-waters may contribute to the overall stability of the tertiary structure. However, the question as to what are the dynamics of interactions of shell-water with respect to weak forces and atom-groups of protein (AGP), requires systematic investigations. In this end, we have developed a procedure POWAINDv1.0 that analyzes interactions of crystallographic shell-waters (CSH) in residues and AGP specific manner. The shell-water and AGP specific bridge-interactions are also extracted. Further, the program analyzes favorable and unfavorable nature of each interaction based on the actual and 75% of the sum of van der Waals (vdW) radii of interacting atoms. The EXCEL-outputs are useful in understanding the profile for AGP-CSH interactions and contribution of each component in AGP. Taken together, the program provides intricate details on CSHprotein interactions and finds application in the structural Bioinformatics.