Three-dimensional models of Mycobacterium tuberculosis proteins Rv1555, Rv1554 and their docking analyses with sildenafil, tadalafil, vardenafil drugs, suggest interference with quinol binding likely to affect protein's function.

BACKGROUND: Earlier based on bioinformatics analyses, we had predicted the Mycobacterium tuberculosis (M.tb) proteins; Rv1555 and Rv1554, among the potential new tuberculosis drug targets. According to the 'TB-drugome' the Rv1555 protein is 'druggable' with sildenafil (Viagra), tadalafil (Cialis) and vardenafil (Levitra) drugs. In the present work, we intended to understand via computer modeling studies, how the above drugs are likely to inhibit the M.tb protein's function. RESULTS: The three-dimensional computer models for M.tb proteins; Rv1555 and Rv1554 constructed on the template of equivalent membrane anchor subunits of the homologous E.coli quinol fumarate reductase respiratory protein complex, followed by drug docking analyses, suggested that the binding of above drugs interferes with quinol binding sites. Also, we experimentally observed the in-vitro growth inhibition of E.coli bacteria containing the homologous M.tb protein sequences with sildenafil and tadalafil drugs. CONCLUSIONS: The predicted binding sites of the drugs is likely to affect the above M.tb proteins function as quinol binding is known to be essential for electron transfer function during anaerobic respiration in the homologous E.coli protein complex. Therefore, sildenafil and related drugs currently used in the treatment of male erectile dysfunction targeting the human phosphodiesterase 5 enzyme may be evaluated for their plausible role as repurposed drugs to treat human tuberculosis.

Structural complexity and functional diversity of plant NADPH oxidases.

Plant NADPH oxidases also known as respiratory burst oxidase homologs (Rbohs) are a family of membrane-bound enzymes that play diverse roles in the defense response and morphogenetic processes via regulated generation of reactive oxygen species. Rbohs are associated with a variety of functions, although the reason for this is not clear. To evaluate using bioinformatics, the possible mechanisms for the observed functional diversity within the plant kingdom, 127 Rboh protein sequences representing 26 plant species were analyzed. Multiple clusters were identified with gene duplications that were both dicot as well as monocot-specific. The N-terminal sequences were observed to be highly variable. The conserved cysteine (equivalent of Cys890) in C-terminal of AtRbohD suggested that the redox-based modification like S-nitrosylation may regulate the activity of other Rbohs. Three-dimensional models corresponding to the N-terminal domain for Rbohs from Arabidopsis thaliana and Oryza sativa were constructed and molecular dynamics studies were carried out to study the role of Ca2+ in the folding of Rboh proteins. Certain mutations indicated possibly affect the structure and function of the plant NADPH oxidases, thereby providing the rationale for further experimental validation.

Mutations in human SARS-CoV-2 spike proteins, potential drug binding and epitope sites for COVID-19 therapeutics development.

The comparison of 303,250 human SARS-CoV-2 spike protein sequences with the reference protein sequence Wuhan-Hu-1, showed ∼96.5% of the spike protein sequence has undergone the mutations till date, since outbreak of the COVID-19 pandemic disease that was first reported in December 2019. A total of 1,269,629 mutations were detected corresponding to 1,229 distinct mutation sites in the spike proteins comprising 1,273 amino acid residues. Thereby, ∼3.5% of the human SARS-CoV-2 spike protein sequence has remained invariant in the past two years. Considering different mutations occur at the same mutation site, a total of 4,729 distinct mutations were observed and are catalogued in the present work. The WHO/CDC, U.S.A., classification and definitions for the current variants being monitored (VBM) and variant of concern (VOC) are assigned to the SARS-CoV-2 spike protein mutations identified in the present work along with a list of other amino acid substitutions observed for the variants. All 195 amino acid residues in receptor binding domain (Thr333-Pro527) were associated with mutations in SARS-CoV-2 spike protein sequence including Lys417, Tyr449, Tyr453, Ala475, Asn487, Thr500, Asn501 and Gly502 that make interactions with the ACE-2 receptor ≤3.2 â€‹Å distance as observed in the crystal structure complex available in the Protein Data Bank (PDB code:6LZG). However, not all these residues were mutated in the same spike protein. Especially, Gly502 mutated only in two spike protein sequences and Tyr449 mutated only in seven spike protein sequences among the spike protein sequences analysed constitute potential sites for the design of suitable inhibitors/drugs. Further, forty-four invariant residues were observed that correspond to ten domains/regions in the SARS-CoV-2 spike protein and some of the residues exposed to the protein surface amongst these may serve as epitope targets to develop monoclonal antibodies.

Conformational analysis corresponding to intra-chain disulfide bridged peptides in proteins of known three-dimensional structure.

We have carried out a systematic analysis in order to evaluate whether Intra-Chain Disulfide Bridged Peptides (ICDBPs) observed in proteins of known three-dimensional structure adopt structurally similar conformations as they may correspond to structural/functional motifs. 406 representative ICDBPs comprising between 3 to 17 amino acid residues could be classified according to peptide sequence length and main-chain secondary structure conformation into 146 classes. ICDBPs comprising 6 amino acid residues are maximally represented in the Protein Data Bank. They also represent the maximum number of main-chain secondary structure conformational classes. Individual ICDBPs in each class represent different protein superfamilies and correspond to different amino acid sequences. We identified 145 ICDBP pairs that had not less-than 0.5 A root mean square deviation value corresponding to their equivalent peptide backbone atoms. We believe these ICDBPs represent structural motifs and possible candidates in order to further explore their structure/function role in the corresponding proteins. The common conformational classes observed for ICDBPs defined according to the main-chain secondary structure conformations; H (helix), B (residue in a isolated beta bridge), C (coil), E (extended beta strand), G (3(10) helix), I (pi helix), S (bend), T (hydrogen-bonded turn) were; "CHHH", "CTTC", "CSSS" and "CSSC" (for ICDBP length 4), "CSSCC" (length 5), "EETTEE", "CCSSCC", "CCSSSC" (length 6), "EETTTEE" (length 7), "EETTTTEE" (length 8), "EEEETTEEEE" (length 10), "EEEETTTEEEE" (length 11) and "EEEETTTTEEEE" (length 12).

PSSARD (2.0): a database server for making flexible queries relating amino acid sequences to main-chain secondary structure conformations for proteins of known three-dimensional structure and certain useful applications.

We have updated the Protein Sequence-Structure Analysis Relational Database (PSSARD) first published in the Int. J. Biol. Macromol. 36 (2005) 259-262 corresponding to 1573 representative protein chains selected from the Protein Data Bank (PDB). In this, the updated and revised PSSARD (Version 2.0), we have included all proteins in the Protein Data Bank available at the time of developing this database including the NMR PDB entries. The current database corresponds to 22,752 XRAY PDB entries and 3977 NMR PDB entries and is separated accordingly in order to facilitate the appropriate database search. The representative protein chains can also be separately accessed within the current database. We have made a provision to combine more than one field to query the database and the results of any search can be used to carry out further nested searches using a combination of queries. We have provided hyperlinks to the individual PDB entries obtained as the result of any search in PSSARD in order to obtain additional details relevant to the protein structure. Certain applications useful to identify domains and structural motifs are discussed.

Juxtaposed half-cystines as disulphide bridged partners in protein tertiary structure.

Disulphide bridges involving juxtaposed half-cystines are observed in a number of protein three-dimensional structures analyzed from the Protein Data Bank. These disulphide bridges comprise a 'ring of 8-atoms' corresponding to Calpha1-C'-N-Calpha2-Cbeta2-Sgamma2-Sgamma1-Cbeta1-Calpha1 in the two half-cystines. The presence of such disulphide bridges introduces a 'bend' or 'kink' in the protein polypeptide chain.

Analysis of disulphide bond connectivity patterns in protein tertiary structure.

The analysis of disulphide bond containing proteins in the Protein Data Bank (PDB) revealed that out of 27,209 protein structures analyzed, 12,832 proteins contain at least one intra-chain disulphide bond and 811 proteins contain at least one inter-chain disulphide bond. The intra-chain disulphide bond containing proteins can be grouped into 256 categories based on the number of disulphide bonds and the disulphide bond connectivity patterns (DBCPs) that were generated according to the position of half-cystine residues along the protein chain. The PDB entries corresponding to these 256 categories represent 509 unique SCOP superfamilies. A simple web-based computational tool is made freely available at the website that allows flexible queries to be made on the database in order to retrieve useful information on the disulphide bond containing proteins in the PDB. The database is useful to identify the different SCOP superfamilies associated with a particular disulphide bond connectivity pattern or vice versa. It is possible to define a query based either on a single field or a combination of the following fields, i.e., PDB code, protein name, SCOP superfamily name, number of disulphide bonds, disulphide bond connectivity pattern and the number of amino acid residues in a protein chain and retrieve information that match the criterion. Thereby, the database may be useful to select suitable protein structural templates in order to model the more distantly related protein homologs/analogs using the comparative modeling methods.

Comparative analyses of the proteins from Mycobacterium tuberculosis and human genomes: Identification of potential tuberculosis drug targets.

Tuberculosis, one of the major infectious diseases affecting human beings is caused by the bacillus Mycobacterium tuberculosis. Increased resistance to known drugs commonly used for the treatment of tuberculosis has created an urgent need to identify new targets for validation and to develop drugs. In this study, we have used various bioinformatics tools, to compare the protein sequences from twenty-three M. tuberculosis genome strains along with the known human protein sequences, in order to identify the 'conserved' M. tuberculosis proteins absent in human. Further, based on the analysis of protein interaction networks, we selected one-hundred and forty proteins that were predicted as potential M. tuberculosis drug targets and prioritized according to the ranking of 'clusters' of interacting proteins. Comparison of the predicted 140 TB targets with literature indicated that 46 of them were previously reported, thereby increasing the confidence in our predictions of the remaining 94 targets too. The analyses of the structures and functions corresponding to the predicted potential TB drug targets indicated a diverse range of proteins that included ten 'druggable' targets with some of the known drugs.

The automatic detection of known beta-propeller structural motifs from protein tertiary structure.

Following our previous work on the analysis of 'structural plasticity' associated with the beta-propeller structural motifs, we have now developed a simple method that can automatically detect all the known beta-propellers in protein tertiary structure, given a list of Protein Data Bank (PDB) codes as input to the computer program. Our beta-propeller detection (BPD) method identifies the location of beta-propellers in the protein structure, specifies the beta-propeller type, the beta-sheet associated beta-strand pattern and the structurally similar beta-propellers observed in other proteins. When tested on 21,566 proteins in the PDB, the BPD method was capable of correctly identifying all the known 245 beta-propellers described in the structural classification of proteins (SCOP) with the number of false positives detected being less than 0.2%. Forty-one false positives were detected that correspond to eight known protein families. When compared with some of the popular web-based programs that can automatically detect 'structural similarities' between the query and target proteins, our method has the advantage of also being capable of detecting beta-propellers associated with 'structural plasticity' and in situations where the target and query proteins differ in amino acid sequence length.

PSSARD: protein sequence-structure analysis relational database.

We have implemented a relational database comprising a representative dataset of amino acid sequences and their associated secondary structure. The representative amino acid sequences were selected according to the PDB_SELECT program by choosing proteins corresponding to protein crystal structure data deposited in the protein data bank that share less than 25% overall pair-wise sequence identity. The secondary structure was extracted from the protein data bank website. The information content in the database includes the protein description, PDB code, crystal structure resolution, total number of amino acid residues in the protein chain, amino acid sequence, secondary structure conformation and its summary. The database is freely accessible from the website mentioned below and is useful to query on any of the above fields. The database is particularly useful to quickly retrieve amino acid sequences that are compatible to any super-secondary structure conformation from several proteins simultaneously.

Computational tools for the analysis of heteroatom groups and their neighbours in protein tertiary structure.

A number of Protein Data Bank (PDB) entries contain heteroatoms defined as HETATM. These include the atomic co-ordinates mainly for heteroatom groups, such as cofactors, coenzymes, prosthetic groups, metal ions, sugars, drugs, peptides, heavy-atom derivatives, non-standard amino acid residues/nucleotides, water molecules and so on. In order to evaluate the different heteroatom (Het) groups and their distribution in protein tertiary structure, we have extracted these from all proteins in the PDB and provided the data in an easily accessible format at the following website. The data can be queried on the PDB code, protein name/description, Het Group code or Het Group name. Further, we have also developed a web-based software application that reports neighbouring atoms evaluated by a "user-defined" distance cut-off value (in Angstrom units), either between a specific Het Group or all Het Groups in a given PDB with amino acid residues and water molecules in the corresponding protein, or neighbours for only all the amino acid residues in the given PDB with respect to Het Groups and water molecules. Together, the database and software applications are useful to gather information that can be further analyzed in order to obtain insights into the preferred interactions of heteroatom groups in proteins, study their binding mode, design novel molecules or to annotate protein function.

Analyses of the Sequence and Structural Properties Corresponding to Pentapeptide and Large Palindromes in Proteins.

The analyses of 3967 representative proteins selected from the Protein Data Bank revealed the presence of 2803 pentapeptide and large palindrome sequences with known secondary structure conformation. These represent 2014 unique palindrome sequences. 60% palindromes are not associated with any regular secondary structure and 28% are in helix conformation, 11% in strand conformation and 1% in the coil conformation. The average solvent accessibility values are in the range between 0-155.28 Å2 suggesting that the palindromes in proteins can be either buried, exposed to the solvent or share an intermittent property. The number of residue neighborhood contacts defined by interactions ≤ 3.2 Ǻ is in the range between 0-29 residues. Palindromes of the same length in helix, strand and coil conformation are associated with different amino acid residue preferences at the individual positions. Nearly, 20% palindromes interact with catalytic/active site residues, ligand or metal ions in proteins and may therefore be important for function in the corresponding protein. The average hydrophobicity values for the pentapeptide and large palindromes range between -4.3 to +4.32 and the number of palindromes is almost equally distributed between the negative and positive hydrophobicity values. The palindromes represent 107 different protein families and the hydrolases, transferases, oxidoreductases and lyases contain relatively large number of palindromes.

Serotype 1 and 8 Pneumococci Evade Sensing by Inflammasomes in Human Lung Tissue.

Streptococcus pneumoniae is a major cause of pneumonia, sepsis and meningitis. The pore-forming toxin pneumolysin is a key virulence factor of S. pneumoniae, which can be sensed by the NLRP3 inflammasome. Among the over 90 serotypes, serotype 1 pneumococci (particularly MLST306) have emerged across the globe as a major cause of invasive disease. The cause for its particularity is, however, incompletely understood. We therefore examined pneumococcal infection in human cells and a human lung organ culture system mimicking infection of the lower respiratory tract. We demonstrate that different pneumococcal serotypes differentially activate inflammasome-dependent IL-1ß production in human lung tissue and cells. Whereas serotype 2, 3, 6B, 9N pneumococci expressing fully haemolytic pneumolysins activate NLRP3 inflammasome-dependent responses, serotype 1 and 8 strains expressing non-haemolytic toxins are poor activators of IL-1ß production. Accordingly, purified haemolytic pneumolysin but not serotype 1-associated non-haemolytic toxin activates strong IL-1ß production in human lungs. Our data suggest that the evasion of inflammasome-dependent innate immune responses by serotype 1 pneumococci might contribute to their ability to cause invasive diseases in humans.

Three-dimensional models corresponding to the C-terminal domain of human alphaA- and alphaB-crystallins based on the crystal structure of the small heat-shock protein HSP16.9 from wheat.

We propose three-dimensional models corresponding to the C-terminal domain of human alphaA- and alphaB-crystallins by using the comparative modeling program Modeler and the more closely related crystal structure of the small heat-shock protein (sHSP) belonging to the eukaryotic species from wheat HSP16.9 as template structure. The sequence alignments differ slightly from alignments that were used previously to construct alpha-crystallin models based on homology and the crystal structure of the more distantly related small heat-shock protein from archaeal species; Methanococcus jannaschii Mj HSP16.5, the only related structure then available as a template. The alpha-crystallin models based on HSP16.9 show better 3-D profile scores and reflect the relative shifts in the beta-strands corresponding to the beta-sandwich associated with the core C-terminal domain that is common to small heat-shock proteins and the alpha-crystallins. The loop between the equivalent beta5-beta7 strands corresponds to a region of seven amino acid residues deletion in alpha-crystallins and defines the new set of amino acid residues likely to be associated with a dimer interface. The models may be useful to examine sites of mutations that are known to affect chaperone-like activity and provide the structural basis for dimerization in alpha-crystallins.

Structural plasticity associated with the beta-propeller architecture.

The beta-propeller architecture observed in protein tertiary structure and classified into the five different types according to number of 'blades' (or beta-sheets) and a sixth type classified according to the secondary structure composition of the blades (the beta beta alpha beta-molecular unit) is characterized by variations (or plasticity) in the structure. These correspond to the number of beta-strands associated with the blade, the number of amino acid residues associated with equivalent beta-strands in the different blades and the presence of alpha-helices and twisted beta-strands. We have generated a beta-sheet associated beta-strand pattern that may be important for protein structure prediction and modeling. Analysis of the beta-propellers extracted primarily from the SCOP database revealed there are 179 beta-propellers. The examination of the secondary structure corresponding to the beta-propeller using PDBsum that was useful to define the beta-sheet associated beta-strand pattern, combined with visualization on graphics display revealed structural plasticity associated with the beta-propeller architecture. Particularly, the type 6- and 7-bladed beta-propellers known to be associated with sequence and functional diversity are more common and associated with relatively more structural variations compared to the other beta-propeller types.

Three-dimensional models and structure analysis of corynemycolyltransferases in Corynebacterium glutamicum and Corynebacterium efficiens.

The corynemycolyltransferase proteins were identified from Corynebacterium glutamicum and Corynebacterium efficiens genomes using computational tools available in the public domain. Three-dimensional models were constructed for corynemycolyltransferases based on the crystal structures of related mycolyltransferases in Mycobacterium tuberculosis using the comparative modeling methods. The corynemycolyltransferases share overall an alpha/beta-fold characteristic of the mycolyltransferases despite low sequence identity (<20%) shared by some of the corynemycolyltransferases. However, a significant difference is observed in the region between amino acid residues Trp82-Trp97 and Ala222-Asn223 corresponding to mycolyltransferases. The specificity pockets defined by interactions with the trehalose substrate observed in the crystal structure complex of Ag85B mycolyltransferase (PDB code: 1F0P) suggests that trehalose may not bind some corynemycolyltransferases. This is due to critical mutations in corynemycolyltransferase binding subsites that lead to loss of equivalent side-chain interactions with trehalose and unfavorable steric interactions, particularly, in the case of cmytC gene and the protein corresponding to the gene identifier CE0356 with the equivalent Ala222-Asn223 "long insertion loop". Further, the fibronectin binding region (Phe58-Val69), in mycolyltransferases associated with mediating host-pathogen interactions in M. tuberculosis comprises amino acid residue mutations in the corresponding region in the soil bacterium--Corynebacterium corynemycolyltransferases, that suggest a different epitope and therefore possible lack of binding to fibronectin. The corynemycolyltransferase cmytA responsible for the cell shape formation and for maintaining the cell surface integrity is associated with a C-terminal domain that we have recently shown to comprise tandem amino acid sequence repeats that is likely to be associated with a regular secondary structural motif.

Can natural proteins designed with 'inverted' peptide sequences adopt native-like protein folds?

We have carried out a systematic computational analysis on a representative dataset of proteins of known three-dimensional structure, in order to evaluate whether it would possible to 'swap' certain short peptide sequences in naturally occurring proteins with their corresponding 'inverted' peptides and generate 'artificial' proteins that are predicted to retain native-like protein fold. The analysis of 3,967 representative proteins from the Protein Data Bank revealed 102,677 unique identical inverted peptide sequence pairs that vary in sequence length between 5-12 and 18 amino acid residues. Our analysis illustrates with examples that such 'artificial' proteins may be generated by identifying peptides with 'similar structural environment' and by using comparative protein modeling and validation studies. Our analysis suggests that natural proteins may be tolerant to accommodating such peptides.

Versatile roles of plant NADPH oxidases and emerging concepts.

NADPH oxidase (NOX) is a key player in the network of reactive oxygen species (ROS) producing enzymes. It catalyzes the production of superoxide (O2(-)), that in turn regulates a wide range of biological functions in a broad range of organisms. Plant Noxes are known as respiratory burst oxidase homologs (Rbohs) and are homologs of catalytic subunit of mammalian phagocyte gp91(phox). They are unique among other ROS producing mechanisms in plants as they integrate different signal transduction pathways in plants. In recent years, there has been addition of knowledge on various aspects related to its structure, regulatory components and associated mechanisms, and its plethora of biological functions. This update highlights some of the recent developments in the field with particular reference to important members of the plant kingdom.

Analysis of bortezomib inhibitor docked within the catalytic subunits of the Plasmodium falciparum 20S proteasome.

The three-dimensional fold of Plasmodium falciparum (Pf) 20S proteasome is similar to yeast Saccharomyces cerevisiae 20S proteasome. The twenty eight subunits complex corresponding to two copies of seven distinct α and seven distinct ß subunits shares >35% sequence identity with equivalent subunits of the yeast 20S proteasome. Bortezomib (Velcade®) - a known inhibitor of the three catalytic subunits; ß1, ß2, ß5 of the yeast 20S proteasome can bind in the equivalent subunits of the Pf 20S proteasome and is in agreement with experimental results. The model defines the binding mode of the bortezomib inhibitor within the catalytic subunits of the Pf 20S proteasome and provides the structural basis for the design of Pf 20S proteasome-specific inhibitors. The substitutions associated within the catalytic subunits of Pf 20S proteasome relative to yeast 20S proteasome; Thr21-Ser, Thr22-Ser, Thr31-Ser, Thr35-Asn, Ala49-Ser (in ß1 subunit), Ser20-Ala, Gln22-Glu (ß2) and Thr21-Ser, Ala22-Met, Gln53-Leu (ß5) may influence the relative caspase-like, tryptic-like and chymotryptic-like activities of the Pf 20S proteasome. The plasmodia-specific 'large' insert comprising fifty four amino acid residues (in ß1 subunit) of the Pf 20S proteasome is distant from the catalytic sites.

Certain heptapeptide and large sequences representing an entire helix, strand or coil conformation in proteins are associated as chameleon sequences.

Helices, strands and coils in proteins of known three-dimensional structure, corresponding to heptapeptide and large sequences ('probe' peptides), were scanned against peptide sequences of variable length, comprising seven or more residues that correspond to a different conformation ('target' peptides) in protein crystal structures available from the Protein Data Bank (PDB). Where the 'probe' and 'target' peptide sequences exactly match, they correspond to 'chameleon' sequences in protein structures. We observed ∼548 heptapeptide and large chameleon sequences that included peptides in the coil conformation from 53,794 PDB files that were analyzed. However, after excluding several chameleon peptides based on the quality of protein structure data, redundancy and peptides associated with cloning artifacts, such as, histidine-tags, we observed only ten chameleon peptides in structurally different proteins and the maximum length comprised seven amino acid residues. Our analysis suggests that the quality of protein structure data is important for identifying possibly, the 'true chameleons' in PDB. Majority of the chameleon sequences correspond to an entire strand in one protein that is observed as part of helix sequence in another protein. The heptapeptide chameleons are characterized with a high propensity of alanine, leucine and valine amino acid residues. The total hydropathy values range between -11.2 and 22.9, the difference in solvent accessibility between 2.0 Å(2) and 373 Å(2) units and the difference in total number of residue neighbor contacts between 0 and 7 residues. Our work identifies for the first time heptapeptide and large sequences that correspond to a single complete helix, strand or coil, which adopt entirely different secondary structures in another protein.