Molecular dynamics analysis of antibody recognition and escape by human H1N1 influenza hemagglutinin.

The antibody immunoglobulin (Ig) 2D1 is effective against the 1918 hemagglutinin (HA) and also known to cross-neutralize the 2009 pandemic H1N1 influenza HA through a similar epitope. However, the detailed mechanism of neutralization remains unclear. We conducted molecular dynamics (MD) simulations to study the interactions between Ig-2D1 and the HAs from the 1918 pandemic flu (A/South Carolina/1/1918, 18HA), the 2009 pandemic flu (A/California/04/2009, 09HA), a 2009 pandemic flu mutant (A/California/04/2009, 09HA_mut), and the 2006 seasonal flu (A/Solomon Islands/3/2006, 06HA). MM-PBSA analyses suggest the approximate free energy of binding (ΔG) between Ig-2D1 and 18HA is -74.4 kcal/mol. In comparison with 18HA, 09HA and 06HA bind Ig-2D1 ∼6 kcal/mol (ΔΔG) weaker, and the 09HA_mut bind Ig-2D1 only half as strong. We also analyzed the contributions of individual epitope residues using the free-energy decomposition method. Two important salt bridges are found between the HAs and Ig-2D1. In 09HA, a serine-to-asparagine mutation coincided with a salt bridge destabilization, hydrogen bond losses, and a water pocket formation between 09HA and Ig-2D1. In 09HA_mut, a lysine-to-glutamic-acid mutation leads to the loss of both salt bridges and destabilizes interactions with Ig-2D1. Even though 06HA has a similar ΔG to 09HA, it is not recognized by Ig-2D1 in vivo. Because 06HA contains two potential glycosylation sites that could mask the epitope, our results suggest that Ig-2D1 may be active against 06HA only in the absence of glycosylation. Overall, our simulation results are in good agreement with observations from biological experiments and offer novel mechanistic insights, to our knowledge, into the immune escape of the influenza virus.

Molecular-level simulation of pandemic influenza glycoproteins.

Computational simulation of pandemic diseases provides important insight into many disease features that may benefit public health. This is especially true for the influenza virus, a continuing global pandemic threat. Molecular or atomic-level investigation of influenza has predominantly focused on the two major virus glycoproteins, neuraminidase (NA) and hemagglutinin (HA). In this chapter, we walk the readers through major considerations for studying pandemic influenza glycoproteins, from choosing the most useful choice of system(s) to avoiding common pitfalls in experimental design and execution. While a brief discussion of several potential simulation and docking techniques is presented, we emphasize molecular dynamics (MD) and Brownian dynamics (BD) simulation techniques and molecular docking, within the context of biologically outstanding questions in influenza research.

Mechanism of 150-cavity formation in influenza neuraminidase.

The recently discovered 150-cavity in the active site of group-1 influenza A neuraminidase (NA) proteins provides a target for rational structure-based drug development to counter the increasing frequency of antiviral resistance in influenza. Surprisingly, the 2009 H1N1 pandemic virus (09N1) neuraminidase was crystalized without the 150-cavity characteristic of group-1 NAs. Here we demonstrate, through a total sum of 1.6 µs of biophysical simulations, that 09N1 NA exists in solution preferentially with an open 150-cavity. Comparison with simulations using avian N1, human N2 and 09N1 with a I149V mutation and an extensive bioinformatics analysis suggests that the conservation of a key salt bridge is crucial in the stabilization of the 150-cavity across both subtypes. This result provides an atomic-level structural understanding of the recent finding that antiviral compounds designed to take advantage of contacts in the 150-cavity can inactivate both 2009 H1N1 pandemic and avian H5N1 viruses.

Web servers and services for electrostatics calculations with APBS and PDB2PQR.

APBS and PDB2PQR are widely utilized free software packages for biomolecular electrostatics calculations. Using the Opal toolkit, we have developed a Web services framework for these software packages that enables the use of APBS and PDB2PQR by users who do not have local access to the necessary amount of computational capabilities. This not only increases accessibility of the software to a wider range of scientists, educators, and students but also increases the availability of electrostatics calculations on portable computing platforms. Users can access this new functionality in two ways. First, an Opal-enabled version of APBS is provided in current distributions, available freely on the web. Second, we have extended the PDB2PQR web server to provide an interface for the setup, execution, and visualization of electrostatic potentials as calculated by APBS. This web interface also uses the Opal framework which ensures the scalability needed to support the large APBS user community. Both of these resources are available from the APBS/PDB2PQR website: http://www.poissonboltzmann.org/.

Opal web services for biomedical applications.

Biomedical applications have become increasingly complex, and they often require large-scale high-performance computing resources with a large number of processors and memory. The complexity of application deployment and the advances in cluster, grid and cloud computing require new modes of support for biomedical research. Scientific Software as a Service (sSaaS) enables scalable and transparent access to biomedical applications through simple standards-based Web interfaces. Towards this end, we built a production web server (http://ws.nbcr.net) in August 2007 to support the bioinformatics application called MEME. The server has grown since to include docking analysis with AutoDock and AutoDock Vina, electrostatic calculations using PDB2PQR and APBS, and off-target analysis using SMAP. All the applications on the servers are powered by Opal, a toolkit that allows users to wrap scientific applications easily as web services without any modification to the scientific codes, by writing simple XML configuration files. Opal allows both web forms-based access and programmatic access of all our applications. The Opal toolkit currently supports SOAP-based Web service access to a number of popular applications from the National Biomedical Computation Resource (NBCR) and affiliated collaborative and service projects. In addition, Opal's programmatic access capability allows our applications to be accessed through many workflow tools, including Vision, Kepler, Nimrod/K and VisTrails. From mid-August 2007 to the end of 2009, we have successfully executed 239,814 jobs. The number of successfully executed jobs more than doubled from 205 to 411 per day between 2008 and 2009. The Opal-enabled service model is useful for a wide range of applications. It provides for interoperation with other applications with Web Service interfaces, and allows application developers to focus on the scientific tool and workflow development. Web server availability: http://ws.nbcr.net.

SMAP-WS: a parallel web service for structural proteome-wide ligand-binding site comparison.

The proteome-wide characterization and analysis of protein ligand-binding sites and their interactions with ligands can provide pivotal information in understanding the structure, function and evolution of proteins and for designing safe and efficient therapeutics. The SMAP web service (SMAP-WS) meets this need through parallel computations designed for 3D ligand-binding site comparison and similarity searching on a structural proteome scale. SMAP-WS implements a shape descriptor (the Geometric Potential) that characterizes both local and global topological properties of the protein structure and which can be used to predict the likely ligand-binding pocket [Xie,L. and Bourne,P.E. (2007) A robust and efficient algorithm for the shape description of protein structures and its application in predicting ligand-binding sites. BMC bioinformatics, 8 (Suppl. 4.), S9.]. Subsequently a sequence order independent profile-profile alignment (SOIPPA) algorithm is used to detect and align similar pockets thereby finding protein functional and evolutionary relationships across fold space [Xie, L. and Bourne, P.E. (2008) Detecting evolutionary relationships across existing fold space, using sequence order-independent profile-profile alignments. Proc. Natl Acad. Sci. USA, 105, 5441-5446]. An extreme value distribution model estimates the statistical significance of the match [Xie, L., Xie, L. and Bourne, P.E. (2009) A unified statistical model to support local sequence order independent similarity searching for ligand-binding sites and its application to genome-based drug discovery. Bioinformatics, 25, i305-i312.]. These algorithms have been extensively benchmarked and shown to outperform most existing algorithms. Moreover, several predictions resulting from SMAP-WS have been validated experimentally. Thus far SMAP-WS has been applied to predict drug side effects, and to repurpose existing drugs for new indications. SMAP-WS provides both a user-friendly web interface and programming API for scientists to address a wide range of compute intense questions in biology and drug discovery. SMAP-WS is available from the URL http://smap.nbcr.net.

Role of secondary sialic acid binding sites in influenza N1 neuraminidase.

Within influenza viral particles, the intricate balance between host cell binding and sialic acid receptor destruction is carefully maintained by the hemagglutinin (HA) and neuraminidase (NA) glycoproteins, respectively. A major outstanding question in influenza biology is the function of a secondary sialic acid binding site on the NA enzyme. Through a series of Brownian dynamics (BD) simulations of the avian N1, human pandemic N2, and currently circulating pandemic (H1)N1 enzymes, we have probed the role of this secondary sialic acid binding site in the avian N1 subtype. Our results suggest that electrostatic interactions at the secondary and primary sites in avian NA may play a key role in the recognition process of the sialic acid receptors and catalytic efficiency of NA. This secondary site appears to facilitate the formation of complexes with the NA protein and the sialic acid receptors, as well as provide HA activity to a lesser extent. Moreover, this site is able to steer inhibitor binding as well, albeit with reduced capacity in N1, and may have potential implications for drug resistance or optimal inhibitor design. Although the secondary sialic acid binding site has previously been shown to be nonconserved in swine NA strains, our investigations of the currently circulating pandemic H1N1 strain of swine origin appears to have retained some of the key features of the secondary sialic acid binding site. Our results indicate possible lowered HA activity for this secondary sialic acid site, which may be an important event in the emergence of the current pandemic strain.

Emerging methods for ensemble-based virtual screening.

Ensemble based virtual screening refers to the use of conformational ensembles from crystal structures, NMR studies or molecular dynamics simulations. It has gained greater acceptance as advances in the theoretical framework, computational algorithms, and software packages enable simulations at longer time scales. Here we focus on the use of computationally generated conformational ensembles and emerging methods that use these ensembles for discovery, such as the Relaxed Complex Scheme or Dynamic Pharmacophore Model. We also discuss the more rigorous physics-based computational techniques such as accelerated molecular dynamics and thermodynamic integration and their applications in improving conformational sampling or the ranking of virtual screening hits. Finally, technological advances that will help make virtual screening tools more accessible to a wider audience in computer aided drug design are discussed.

Mechanism of glycan receptor recognition and specificity switch for avian, swine, and human adapted influenza virus hemagglutinins: a molecular dynamics perspective.

Hemagglutinins (HA's) from duck, swine, and human influenza viruses have previously been shown to prefer avian and human glycan receptor analogues with distinct topological profiles, pentasaccharides LSTa (alpha-2,3 linkage) and LSTc (alpha-2,6 linkage), in comparative molecular dynamics studies. On the basis of detailed analyses of the dynamic motions of the receptor binding domains (RBDs) and interaction energy profiles with individual glycan residues, we have identified approximately 30 residue positions in the RBD that present distinct profiles with the receptor analogues. Glycan binding constrained the conformational space sampling by the HA. Electrostatic steering appeared to play a key role in glycan binding specificity. The complex dynamic behaviors of the major SSE and trimeric interfaces with or without bound glycans suggested that networks of interactions might account for species specificity in these low affinity and high avidity (multivalent) interactions between different HA and glycans. Contact frequency, energetic decomposition, and H-bond analyses revealed species-specific differences in HA-glycan interaction profiles, not readily discernible from crystal structures alone. Interaction energy profiles indicated that mutation events at the set of residues such as 145, 156, 158, and 222 would favor human or avian receptor analogues, often through interactions with distal asialo-residues. These results correlate well with existing experimental evidence, and suggest new opportunities for simulation-based vaccine and drug development.

MEME SUITE: tools for motif discovery and searching.

The MEME Suite web server provides a unified portal for online discovery and analysis of sequence motifs representing features such as DNA binding sites and protein interaction domains. The popular MEME motif discovery algorithm is now complemented by the GLAM2 algorithm which allows discovery of motifs containing gaps. Three sequence scanning algorithms--MAST, FIMO and GLAM2SCAN--allow scanning numerous DNA and protein sequence databases for motifs discovered by MEME and GLAM2. Transcription factor motifs (including those discovered using MEME) can be compared with motifs in many popular motif databases using the motif database scanning algorithm TOMTOM. Transcription factor motifs can be further analyzed for putative function by association with Gene Ontology (GO) terms using the motif-GO term association tool GOMO. MEME output now contains sequence LOGOS for each discovered motif, as well as buttons to allow motifs to be conveniently submitted to the sequence and motif database scanning algorithms (MAST, FIMO and TOMTOM), or to GOMO, for further analysis. GLAM2 output similarly contains buttons for further analysis using GLAM2SCAN and for rerunning GLAM2 with different parameters. All of the motif-based tools are now implemented as web services via Opal. Source code, binaries and a web server are freely available for noncommercial use at http://meme.nbcr.net.

Multiscale modeling in rodent ventricular myocytes.

There is a growing body of experimental evidence suggesting that the Ca(2+) signaling in ventricular myocytes is characterized by a high gradient near the cell membrane and a more uniform Ca(2+) distribution in the cell interior [1]--[7]. An important reason for this phenomenon might be that in these cells the t-tubular system forms a network of extracellular space, extending deep into the cell interior. This allows the electrical signal, that propagates rapidly along the cell membrane, to reach the vicinity of the sarcoplasmic reticulum (SR), where intracellular Ca(2+) required for myofilament activation is stored [1], [8]--[11]. Early studies of cardiac muscle showed that the t-tubules are found at intervals of about 2 lm along the longitudinal cell axis in close proximity to the Z-disks of the sarcomeres [12]. Subsequent studies have demonstrated that the t-tubular system has also longitudinal extensions [9]--[11], [13].

Distinct glycan topology for avian and human sialopentasaccharide receptor analogues upon binding different hemagglutinins: a molecular dynamics perspective.

Hemagglutinin (HA) binds to sialylated glycans exposed on the host cell surface in the initial stage of avian influenza virus infection. It has been previously hypothesized that glycan topology plays a critical role in the human adaptation of avian flu viruses, such as the potentially pandemic H5N1. Comparative molecular dynamics studies are complementary to experimental techniques, including glycan microarray, to understand the mechanism of species-specificity switch better. The examined systems comprise explicitly solvated trimeric forms of avian H3, H5, and swine H9 in complex with avian and human glycan receptor analogues--LSTa (alpha-2,3-linked lactoseries tetrasaccharide a) and LSTc (alpha-2,6-linked lactoseries tetrasaccharide c), respectively. The glycans adopted distinct topological profiles with inducible torsional angles when bound to different HAs. The corresponding receptor binding domain amino acid contact profiles were also distinct. Avian H5 was able to accommodate LSTc in a tightly "folded umbrella"-like topology through interactions with all five sugar residues. After considering conformational entropy, the relative binding free-energy changes, calculated using the molecular mechanics-generalized Born surface area technique, were in agreement with previous experimental findings and provided insights on electrostatic, van der Waals, desolvation, and entropic contributions to HA-glycan interactions. The topology profile and the relative abundance of free glycan receptors may influence receptor binding kinetics. Glycan composition and topological changes upon binding different HAs may be important determinants in species-specificity switch.

Ensemble-based virtual screening reveals potential novel antiviral compounds for avian influenza neuraminidase.

Avian influenza virus subtype H5N1 is a potential pandemic threat with human-adapted strains resistant to antiviral drugs. Although virtual screening (VS) against a crystal or relaxed receptor structure is an established method to identify potential inhibitors, the more dynamic changes within binding sites are neglected. To accommodate full receptor flexibility, we use AutoDock4 to screen the NCI diversity set against representative receptor ensembles extracted from explicitly solvated molecular dynamics simulations of the neuraminidase system. The top hits are redocked to the entire nonredundant receptor ensemble and rescored using the relaxed complex scheme (RCS). Of the 27 top hits reported, half ranked very poorly if only crystal structures are used. These compounds target the catalytic cavity as well as the newly identified 150- and 430-cavities, which exhibit dynamic properties in electrostatic surface and geometric shape. This ensemble-based VS and RCS approach may offer improvement over existing strategies for structure-based drug discovery.

MEME: discovering and analyzing DNA and protein sequence motifs.

MEME (Multiple EM for Motif Elicitation) is one of the most widely used tools for searching for novel 'signals' in sets of biological sequences. Applications include the discovery of new transcription factor binding sites and protein domains. MEME works by searching for repeated, ungapped sequence patterns that occur in the DNA or protein sequences provided by the user. Users can perform MEME searches via the web server hosted by the National Biomedical Computation Resource (http://meme.nbcr.net) and several mirror sites. Through the same web server, users can also access the Motif Alignment and Search Tool to search sequence databases for matches to motifs encoded in several popular formats. By clicking on buttons in the MEME output, users can compare the motifs discovered in their input sequences with databases of known motifs, search sequence databases for matches to the motifs and display the motifs in various formats. This article describes the freely accessible web server and its architecture, and discusses ways to use MEME effectively to find new sequence patterns in biological sequences and analyze their significance.

A comparative proteomics resource: proteins of Arabidopsis thaliana.

Using an integrative genome annotation pipeline (iGAP) for proteome-wide protein structure and functional domain assignment, we analyzed all the proteins of Arabidopsis thaliana. Three-dimensional structures at the level of the domain are assigned by fold recognition and threading based on a novel fold library that extends common domain classifications. iGAP is being applied to proteins from all available proteomes as part of a comparative proteomics resource. The database is accessible from the web.

Use of conserved key amino acid positions to morph protein folds.

By using three-dimensional (3D) structure alignments and a previously published method to determine Conserved Key Amino Acid Positions (CKAAPs) we propose a theoretical method to design mutations that can be used to morph the protein folds. The original Paracelsus challenge, met by several groups, called for the engineering of a stable but different structure by modifying less than 50% of the amino acid residues. We have used the sequences from the Protein Data Bank (PDB) identifiers 1ROP, and 2CRO, which were previously used in the Paracelsus challenge by those groups, and suggest mutation to CKAAPs to morph the protein fold. The total number of mutations suggested is less than 40% of the starting sequence theoretically improving the challenge results. From secondary structure prediction experiments of the proposed mutant sequence structures, we observe that each of the suggested mutant protein sequences likely folds to a different, non-native potentially stable target structure. These results are an early indicator that analyses using structure alignments leading to CKAAPs of a given structure are of value in protein engineering experiments.

CKAAPs DB: a Conserved Key Amino Acid Positions DataBase.

The Conserved Key Amino Acid Positions DataBase (CKAAPs DB) provides access to an analysis of structurally similar proteins with dissimilar sequences where key residues within a common fold are identified. CKAAPs may be important in protein folding and structural stability and function, and hence useful for protein engineering studies. This paper provides an update to the initial report of CKAAPs DB [Li et al. (2001) Nucleic Acids Res., 29, 329-331]. CKAAPs DB contains CKAAPs for the representative set of polypeptide chains derived from the CE and FSSP databases, as well as subdomains (conserved regions of the order of 100 residues within a domain) identified by CE. The new version now offers different perspectives on the CKAAPs. First, CKAAPs are mapped onto their respective Protein Data Bank (PDB) structures rendered by Molscript, providing a spatial context for the CKAAPs. Secondly, CKAAPs may be highlighted within a structure-based sequence alignment, as well as secondary structure alignment. Thirdly, the resulting sequence homologs from the structure alignment may be viewed in alignments colorized based on identities and property groups using Mview. New search capabilities have also been provided for searching by keyword combinations, PDB IDs, EC numbers, GI numbers, LocusLink ID, taxonomy, gene ontology and pathways. A new custom CKAAPs analysis interface has been implemented where a user may change the criteria for inclusion of chains, initiate CKAAPs analysis and retrieve results. CKAAPs DB is accessible through the web at http://ckaaps.sdsc.edu/. Plain text analysis results are available by FTP at ftp://ftp.sdsc.edu/pub/sdsc/biology/ckaap.