Open discovery: An integrated live Linux platform of Bioinformatics tools

Historically, live linux distributions for Bioinformatics have paved way for portability of Bioinformatics workbench in a platform independent manner. Moreover, most of the existing live Linux distributions limit their usage to sequence analysis and basic molecular visualization programs and are devoid of data persistence. Hence, open discovery ‐ a live linux distribution has been developed with the capability to perform complex tasks like molecular modeling, docking and molecular dynamics in a swift manner. Furthermore, it is also equipped with complete sequence analysis environment and is capable of running windows executable programs in Linux environment. Open discovery portrays the advanced customizable configuration of fedora, with data persistency accessible via USB drive or DVD.     

Deciphering the role of the electrostatic interactions in the alpha-tropomyosin head-to-tail complex

Skeletal alpha-tropomyosin (Tm) is a dimeric coiled-coil protein that forms linear assemblies under low ionic strength conditions in vitro through head-to-tail interactions. A previously published NMR structure of the Tm head-to-tail complex revealed that it is formed by the insertion of the N-terminal coiled-coil of one molecule into a cleft formed by the separation of the helices at the C-terminus of a second molecule. To evaluate the contribution of charged residues to complex stability, we employed single and double-mutant Tm fragments in which specific charged residues were changed to alanine in head-to-tail binding assays, and the effects of the mutations were analyzed by thermodynamic double-mutant cycles and protein-protein docking. The results show that residues K5, K7, and D280 are essential to the stability of the complex. Though D2, K6, D275, and H276 are exposed to the solvent and do not participate in intermolecular contacts in the NMR structure, they may contribute to head-to-tail complex stability by modulating the stability of the helices at the Tm termini.     

Mutagenesis study of rice nonspecific lipid transfer protein 2 reveals residues that contribute to structure and ligand binding

Plant nonspecific lipid transfer protein 2 (nsLTP2) is a small (7 kDa) protein that binds lipid-like ligands. An inner hydrophobic cavity surrounded by alpha-helices is the defining structural feature of nsLTP2. Although nsLTP2 structures have been reported earlier, the detailed mechanisms of ligand binding and lipid transfer remain unclear. In this study, we used site-directed mutagenesis to determine the role of various hydrophobic residues (L8, I15, F36, F39, Y45, Y48, and V49) in the structure, stability, ligand binding, and lipid transfer activity of rice nsLTP2. Three single mutations (L8A, F36A, and V49A) drastically alter the native tertiary structure and perturb ligand binding and lipid transfer activity. Therefore, these three residues are structurally important. The Y45A mutant, however, retains a native-like structure but has decreased lipid binding affinity and lipid transfer activity, implying that this aromatic residue is critical for these biological functions. The mutants, I15A and Y48A, exhibit quite different ligand binding affinities. Y48 is involved in planar sterol binding but not linear lysophospholipid association. As for I15A, it had the highest dehydroergosterol binding affinity in spite of the lower lipid binding and transfer abilities. Our results suggest that the long alkyl side chain of I15 would restrict the flexibility of loop I (G13-A19) for sterol entry. Finally, F39A can markedly increase the exposed hydrophobic surface to maintain its transfer efficiency despite reduced ligand binding affinity. These findings suggest that the residues forming the hydrophobic cavity play various important roles in the structure and function of rice nsLTP2.     

Structure-based mutagenesis of Penicillium griseofulvum xylanase using computational design

Penicillium griseofulvum xylanase (PgXynA) belongs to family 11 glycoside hydrolase. It exhibits unique amino acid features but its three-dimensional structure is not known. Based upon the X-ray structure of Penicillium funiculosum xylanase (PfXynC), we generated a three-dimensional model of PgXynA by homology modeling. The native structure of PgXynA displayed the overall beta-jelly roll folding common to family 11 xylanases with two large beta-pleated sheets and a single alpha-helix that form a structure resembling a partially closed right hand. Although many features of PgXynA were very similar to previously described enzymes from this family, crucial differences were observed in the loop forming the "thumb" and at the edge of the binding cleft. The robustness of the xylanase was challenged by extensive in silico-based mutagenesis analysis targeting mutations retaining stereochemical and energetical control of the protein folding. On the basis of structural alignments, modeled three-dimensional structure, in silico mutations and docking analysis, we targeted several positions for the replacement of amino acids by site-directed mutagenesis to change substrate and inhibitor specificity, alter pH profile and improve overall catalytic activity. We demonstrated the crucial role played by Ser44(PgXynA) and Ser129(PgXynA), two residues unique to PgXynA, in conferring distinct specificity to P. griseofulvum xylanase. We showed that the pH optimum of PgXynA could be shifted by -1 to +0.5 units by mutating Ser44(PgXynA) to Asp and Asn, respectively. The S44D and S44N mutants showed only slight alteration in K(m) and V(max) whereas a S44A mutant lost both pH-dependence profile and activity. We were able to produce PgXynA S129G mutants with acquired sensitivity to the Xylanase Inhibitor Protein, XIP-I. The replacement of Gln121(PgXynA), located at the start of the thumb, into an Arg residue resulted in an enzyme that possessed a higher catalytic activity.     

Flexible protein-flexible ligand docking with disrupted velocity simulated annealing

By docking flexible balanol to a rigid model of protein kinase A (PKA), we found that a new simulated annealing protocol termed disrupted velocity simulated annealing (DIVE-SA) outperformed the replica-exchange method and the traditional simulated annealing method in identifying the correct docking pose. In this protocol, the atomic velocities were reassigned periodically to encourage the system to sample a large conformational space. We also found that scaling potential energy surface to reduce structural transition barriers could further facilitate docking. The DIVE-SA method was then evaluated on its ability to perform flexible ligand-flexible protein docking of three ligands (balanol, a balanol analog, and ATP) to PKA. To reduce computational time and to avoid possible unphysical structural changes resulting from the use of nonoptimal force fields, a soft restrain was applied to keep the root-mean-square-deviation (RMSD) between instantaneous protein structures and a chosen reference structure small. Because the restrain was applied to the overall RMSD rather than to individual atoms, a protein could still experience relatively large conformational changes during docking. To examine the impact of applying such a restrain on docking, we constructed two semi-flexible protein models by choosing two different crystal structures as reference. Both the balanol analog and ATP were able to dock to either one of these semi-flexible protein models. On the other hand, balanol could only dock well to one of them. Further analysis indicated that the restrain on the glycine-rich loop was too strong, preventing it to adjust its structure to accommodate balanol in the binding pocket of PKA. Removing the restrain on the glycine-rich loop resulted in much better docking poses. This finding demonstrates the important role that the flexibility of the glycine-rich loop play in accepting different ligands and should profitably not be restrained in molecular docking so that more diverse ligands can be studied.     

High quality binding modes in docking ligands to proteins

Multiple near-optimal conformations of protein-ligand complexes provide a better chance for accurate representation of biomolecular interactions, compared with a single structure. We present ISE-dock--a docking program which is based on the iterative stochastic elimination (ISE) algorithm. ISE eliminates values that consistently lead to the worst results, thus optimizing the search for docking poses. It constructs large sets of such poses with no additional computational cost compared with single poses. ISE-dock is validated using 81 protein-ligand complexes from the PDB and its performance was compared with those of Glide, GOLD, and AutoDock. ISE-dock has a better chance than the other three to find more than 60% top single poses under RMSD = 2.0 A and more than 80% under RMSD = 3.0 A from experimental. ISE alone produced at least one 3.0 A or better solutions among the top 20 poses in the entire test set. In 98% of the examined molecules, ISE produced solutions that are closer than 2.0 A from experimental. Paired t-tests (PTT) were used throughout to assess the significance of comparisons between the performances of the different programs. ISE-dock provides more than 100-fold docking solutions in a similar time frame as LGA in AutoDock. We demonstrate the usefulness of the large near optimal populations of ligand poses by showing a correlation between the docking results and experiments that support multiple binding modes in p38 MAP kinase (Pargellis et al., Nat Struct Biol 2002;9:268-272] and in Human Transthyretin (Hamilton, Benson, Cell Mol Life Sci 2001;58:1491-1521).     

A knowledge-based forcefield for protein-protein interface design

A distance-dependent knowledge-based potential for protein-protein interactions is derived and tested for application in protein design. Information on residue type specific C(alpha) and C(beta) pair distances is extracted from complex crystal structures in the Protein Data Bank and used in the form of radial distribution functions. The use of only backbone and C(beta) position information allows generation of relative protein-protein orientation poses with minimal sidechain information. Further coarse-graining can be done simply in the same theoretical framework to give potentials for residues of known type interacting with unknown type, as in a one-sided interface design problem. Both interface design via pose generation followed by sidechain repacking and localized protein-protein docking tests are performed on 39 nonredundant antibody-antigen complexes for which crystal structures are available. As reference, Lennard-Jones potentials, unspecific for residue type and biasing toward varying degrees of residue pair separation are used as controls. For interface design, the knowledge-based potentials give the best combination of consistently designable poses, low RMSD to the known structure, and more tightly bound interfaces with no added computational cost. 77% of the poses could be designed to give complexes with negative free energies of binding. Generally, larger interface separation promotes designability, but weakens the binding of the resulting designs. A localized docking test shows that the knowledge-based nature of the potentials improves performance and compares respectably with more sophisticated all-atoms potentials.     

Conformational flexibility and kinetic complexity in antibody-antigen interactions

The kinetics of dissociation of three structurally characterized anti-hen egg white lysozyme antibodies (H8, H10, and H26), with hen egg white lysozyme (HEL) and the avian variant Japanese quail lysozyme (JQL) were examined. These antibodies share over 90% sequence identity and recognize the same epitope, but differ in their degree of cross-reactivity and predicted combining site rigidity. Competitive dissociation induced by the addition of excess unlabeled HEL after varied periods of antibody-antigen association was followed in real time using fluorescence anisotropy. Dissociation was in many cases non-single-exponential, and the observed off-rates became slower as the complex age increased, suggesting multi-step association kinetics consistent with an encounter-docking view of protein-protein interactions. The fully docked fraction of the complexes just prior to inducing dissociation was high for the HEL complexes but was dramatically reduced for JQL complexes, that is final docking was antigen-sensitive. Variations among the systems can be understood in terms of the complexes' differing conformational flexibilities, based on the encounter-docking model of protein-protein associations.     

In silico study of the human rhodopsin and meta rhodopsin II/S-arrestin complexes: impact of single point mutations related to retina degenerative diseases

We propose two models of the human S-arrestin/rhodopsin complex in the inactive dark adapted rhodopsin and meta rhodopsin II form, obtained by homology modeling and knowledge based docking. First, a homology model for the human S-arrestin was built and validated by molecular dynamics, showing an average root mean square deviation difference from the pattern behavior of 0.76 A. Then, combining the human S-arrestin model and the modeled structure of the two human rhodopsin forms, we propose two models of interaction for the human S-arrestin/rhodopsin complex. The models involve two S-arrestin regions related to the N domain (residues 68-78; 170-182) and a third constituent of the C domain (248-253), with the rhodopsin C terminus (330-348). Of the 22 single point mutations related to retinitis pigmentosa and congenital night blindness located in the cytoplasmatic portion of rhodopsin or in S-arrestin, our models locate 16 in the interaction region and relate two others to possible dimer formation. Our calculations also predict that the light activated complex is more stable than the dark adapted rhodopsin and, therefore, of higher affinity to S-arrestin.     

Improved model building and assessment of the Calcium-sensing receptor transmembrane domain

A three-dimensional model of the human Calcium-sensing receptor (CaSR) seven transmembrane domain was built via a novel sequence alignment method based on the conserved contacts in proteins using the crystal structure of bovine rhodopsin as the template. This model was tested by docking NPS 2143, the first identified allosteric antagonist of CaSR. In our model, Glu837 plays a critical role in anchoring the protonated nitrogen atom and hydroxy oxygen atom of NPS 2143. The phenyl moiety of the ligand contacts residues Phe668, Pro672, and Ile841. The naphthalene moiety is surrounded by several hydrophobic residues, including Phe684, Phe688, and Phe821. Our model appears to be consistent with all six residues that have been demonstrated to be critical for NPS 2143 binding, in contrast with existing homology models based on traditional sequence alignment of CaSR to rhodopsin. This provides validation of our sequence alignment method and the use of the rhodopsin backbone as the initial structure in homology modeling of other G protein-coupled receptors that are not members of the rhodopsin family.