Andras Fiser

Modeller: Generation and Refinement of Homology-Based Protein Structure Models

Publisher Summary Functional characterization of a protein sequence is one of the most frequent problems in biology. This task is usually facilitated by accurate three-dimensional (3D) structure of the studied protein. In the absence of an experimentally determined structure, comparative or homology modeling can sometimes provide a useful 3D model for a protein (target) that is related to at least one known protein structure (template). A 3D structure of proteins from the same family is more conserved than their primary sequences. Therefore, if similarity between two proteins is detectable at the sequence level, structural similarity can usually be assumed. Comparative modeling usually starts by searching the Protein Data Bank (PDB) of known protein structures using the target sequence as the query. This search is generally done by comparing the target sequence with the sequence of each of the structures in the database. Comparative modeling consists of five steps: (1) search for related protein structures, (2) selection of one or more templates, (3) target–template alignment, (4) model building, and (5) model evaluation. If the model is not satisfactory, some or all of the steps can be repeated. There are several computer programs and Web servers that automate the comparative modeling process. The first Web server for automated comparative modeling was the Swiss-Model server, followed by CPHModels and ModWeb. These servers accept a sequence from a user and return an all-atom comparative model when possible.

ModLoop: automated modeling of loops in protein structures

SUMMARY ModLoop is a web server for automated modeling of loops in protein structures. The input is the atomic coordinates of the protein structure in the Protein Data Bank format, and the specification of the starting and ending residues of one or more segments to be modeled, containing no more than 20 residues in total. The output is the coordinates of the non-hydrogen atoms in the modeled segments. A user provides the input to the server via a simple web interface, and receives the output by e-mail. The server relies on the loop modeling routine in MODELLER that predicts the loop conformations by satisfaction of spatial restraints, without relying on a database of known protein structures. For a rapid response, ModLoop runs on a cluster of Linux PC computers. AVAILABILITY The server is freely accessible to academic users at http://salilab.org/modloop

Tools for comparative protein structure modeling and analysis.

The following resources for comparative protein structure modeling and analysis are described (http://salilab.org): MODELLER, a program for comparative modeling by satisfaction of spatial restraints; MODWEB, a web server for automated comparative modeling that relies on PSI-BLAST, IMPALA and MODELLER; MODLOOP, a web server for automated loop modeling that relies on MODELLER; MOULDER, a CPU intensive protocol of MODWEB for building comparative models based on distant known structures; MODBASE, a comprehensive database of annotated comparative models for all sequences detectably related to a known structure; MODVIEW, a Netscape plugin for Linux that integrates viewing of multiple sequences and structures; and SNPWEB, a web server for structure-based prediction of the functional impact of a single amino acid substitution.

Insights into the mechanism of microtubule stabilization by Taxol

The antitumor drug Taxol stabilizes microtubules and reduces their dynamicity, promoting mitotic arrest and cell death. Upon assembly of the α/β-tubulin heterodimer, GTP bound to β-tubulin is hydrolyzed to GDP reaching a steady-state equilibrium between free tubulin dimers and microtubules. The binding of Taxol to β-tubulin in the polymer results in cold-stable microtubules at the expense of tubulin dimers, even in the absence of exogenous GTP. However, there is little biochemical insight into the mechanism(s) by which Taxol stabilizes microtubules. Here, we analyze the structural changes occurring in both β- and α-tubulin upon microtubule stabilization by Taxol. Hydrogen/deuterium exchange (HDX) coupled to liquid chromatography–electrospray ionization MS demonstrated a marked reduction in deuterium incorporation in both β-and α-tubulin when Taxol was present. Decreased local HDX in peptic peptides was mapped on the tubulin structure and revealed both expected and new dimer–dimer interactions. The increased rigidity in Taxol microtubules was distinct from and complementary to that due to GTP-induced polymerization. The Taxol-induced changes in tubulin conformation act against microtubule depolymerization in a precise directional way. These results demonstrate that HDX coupled to liquid chromatography–electrospray ionization MS can be effectively used to study conformational effects induced by small ligands on microtubules. The present study also opens avenues for locating drug and protein binding sites and for deciphering the mechanisms by which their interactions alter the conformation of microtubules and tubulin dimers.

VISTA, a novel mouse Ig superfamily ligand that negatively regulates T cell responses

VISTA suppresses T cell proliferation and cytokine production and can influence autoimmunity and antitumor responses in mice.

Comparative Protein Structure Modelling

A prerequisite to understand cell functioning on the system level is the knowledge of three-dimensional protein structures that mediate biochemical interactions. The explosion in the number of available gene sequences set the stage for the next step in genome scale projects, to obtain three dimensional structures for each protein. To achieve this ambitious goal, the costly and slow structure determination experiments are boosted with theoretical approaches. The current state and recent advances in structure modelling approaches are reviewed here, with special emphasis on comparative structure modelling techniques.

PSI-2: Structural Genomics to Cover Protein Domain Family Space

One major objective of structural genomics efforts, including the NIH-funded Protein Structure Initiative (PSI), has been to increase the structural coverage of protein sequence space. Here, we present the target selection strategy used during the second phase of PSI (PSI-2). This strategy, jointly devised by the bioinformatics groups associated with the PSI-2 large-scale production centers, targets representatives from large, structurally uncharacterized protein domain families, and from structurally uncharacterized subfamilies in very large and diverse families with incomplete structural coverage. These very large families are extremely diverse both structurally and functionally, and are highly overrepresented in known proteomes. On the basis of several metrics, we then discuss to what extent PSI-2, during its first 3 years, has increased the structural coverage of genomes, and contributed structural and functional novelty. Together, the results presented here suggest that PSI-2 is successfully meeting its objectives and provides useful insights into structural and functional space.

The Immunoglobulin Heavy Chain Constant Region Affects Kinetic and Thermodynamic Parameters of Antibody Variable Region Interactions with Antigen

A central dogma in immunology is that antibody specificity is a function of the variable (V) region. However serological analysis of IgG1, IgG2a, and IgG2b switch variants of murine monoclonal antibody (mAb) 3E5 IgG3 with identical V domains revealed apparent specificity differences for Cryptococcus neoformans glucuronoxylomannan (GXM). Kinetic and thermodynamic binding properties of mAbs 3E5 to a 12-mer peptide mimetic of GXM revealed differences in the affinity of these mAbs for a monovalent ligand, a result that implied that the constant (C) region affects the secondary structure of the antigen binding site, thus accounting for variations in specificity. Structural models of mAbs 3E5 suggested that isotype-related differences in binding resulted from amino acid sequence polymorphisms in the C region. This study implies that isotype switching is another mechanism for generating diversity in antigen binding and that isotype restriction of certain antibody responses may reflect structural constraints imposed by C region on V region binding. Furthermore, isotype affected the polyreactivity of V region identical antibodies, implying a role for C region in determining self-reactivity.

ArchPRED: a template based loop structure prediction server

ArchPRED server () implements a novel fragment-search based method for predicting loop conformations. The inputs to the server are the atomic coordinates of the query protein and the position of the loop. The algorithm selects candidate loop fragments from a regularly updated loop library (Search Space) by matching the length, the types of bracing secondary structures of the query and by satisfying the geometrical restraints imposed by the stem residues. Subsequently, candidate loops are inserted in the query protein framework where their side chains are rebuilt and their fit is assessed by the root mean square deviation (r.m.s.d.) of stem regions and by the number of rigid body clashes with the environment. In the final step remaining candidate loops are ranked by a Z-score that combines information on sequence similarity and fit of predicted and observed [ϕ/ψ] main chain dihedral angle propensities. The final loop conformation is built in the protein structure and annealed in the environment using conjugate gradient minimization. The prediction method was benchmarked on artificially prepared search datasets where all trivial sequence similarities on the SCOP superfamily level were removed. Under these conditions it was possible to predict loops of length 4, 8 and 12 with coverage of 98, 78 and 28% with at least of 0.22, 1.38 and 2.47 Å of r.m.s.d. accuracy, respectively. In a head to head comparison on loops extracted from freshly deposited new protein folds the current method outperformed in a ∼5:1 ratio an earlier developed database search method.

M4T: a comparative protein structure modeling server

Multiple Mapping Method with Multiple Templates (M4T) (http://www.fiserlab.org/servers/m4t) is a fully automated comparative protein structure modeling server. The novelty of M4T resides in two of its major modules, Multiple Templates (MT) and Multiple Mapping Method (MMM). The MT module of M4T selects and optimally combines the sequences of multiple template structures through an iterative clustering approach that takes into account the ‘unique’ contribution of each template, its sequence similarity to other template sequences and to the target sequences, and the quality of its experimental resolution. MMM module is a sequence-to-structure alignment method that is aimed at improving the alignment accuracy, especially at lower sequence identity levels. The current implementation of MMM takes inputs from three profile-to-profile-based alignment methods and iteratively compares and ranks alternatively aligned regions according to their fit in the structural environment of the template structure. The performance of M4T was benchmarked on CASP6 comparative modeling target sequences and on a larger independent test set and showed a favorable performance to current state-of-the-art methods.