Biomedical subjects

Andras Fiser

Publications and source records attributed to Andras Fiser.

10 recordsLinked to original sources

PubMed2006

Structural basis of inducible costimulator ligand costimulatory function: determination of the cell surface oligomeric state and functional mapping of the receptor binding site of the protein.

Inducible costimulator (ICOS) ligand (ICOSL), a B7-related transmembrane glycoprotein with extracellular IgV and IgC domains, binds to ICOS on activated T cells and delivers a positive costimulatory signal for optimal T cell function. Toward determining the structural features of ICOSL crucial for its costimulatory function, the present study shows that ICOSL displays a marked oligomerization potential, resembling more like B7-1 than B7-2. Use of ICOSL constructs lacking either the IgC or IgV domain demonstrates that receptor binding is mediated solely by the IgV domain but requires the IgC domain for maintaining the structural integrity of the protein. To map further the receptor recognition surface on ICOSL, a homology-based protein structure model of the ICOS:ICOSL complex was constructed. Based on predictions from the model, a series of mutations were generated targeting the potential receptor binding surface on ICOSL, and the mutants were tested for their biological function in terms of ICOS binding and T cell costimulation ability. The results provide experimental validation of the model and show that the receptor binding site on ICOSL is constituted chiefly by aromatic/hydrophobic residues. Critical ICOSL residues essential for ICOS binding map to the GFCC'C'' beta-sheet face of the IgV domain and approximately overlap with the B7-1/B7-2 motif(s) that recognize CD28/CTLA-4. Altogether, similar structural features of ICOSL and B7 isoforms suggest a close evolutionary relationship between these costimulatory ligands, yet differences at the same time explain their unique specificity for the cognate binding partners, ICOS and CD28/CTLA-4, respectively.

Amino Acid Sequence↗

PubMed2006

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 alpha/beta-tubulin heterodimer, GTP bound to beta-tubulin is hydrolyzed to GDP reaching a steady-state equilibrium between free tubulin dimers and microtubules. The binding of Taxol to beta-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 beta- and alpha-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 beta-and alpha-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.

Animals↗

PubMed2006

Pentapeptide repeat proteins.

The pentapeptide repeat protein (PRP) family has more than 500 members in the prokaryotic and eukaryotic kingdoms. These proteins are composed of, or contain domains composed of, tandemly repeated amino acid sequences with a consensus sequence of [S,T,A,V][D,N][L,F][S,T,R][G]. The biochemical function of the vast majority of PRP family members is unknown. The three-dimensional structure of the first member of the PRP family was determined for the fluoroquinolone resistance protein (MfpA) from Mycobacterium tuberculosis. The structure revealed that the pentapeptide repeats encode the folding of a novel right-handed quadrilateral beta-helix. MfpA binds to DNA gyrase and inhibits its activity. The rod-shaped, dimeric protein exhibits remarkable similarity in size, shape, and electrostatics to DNA.

Bacterial Proteins↗

PubMed2005

Transactivation of Abl by the Crk II adapter protein requires a PNAY sequence in the Crk C-terminal SH3 domain.

To gain a better understanding of how Crk II regulates the function of the Abl tyrosine kinase, we explored the function of the C-terminal linker and SH3 domain, a region of Crk II that is still poorly understood. Molecular modeling, tryptophan fluorescence, and covariation sequence alignment indicate that the Crk-SH3-C has a unique binding groove and RT loop not observed in typical SH3 domains. Based on these models, we made a series of mutations in the linker and in residues predicted to destabilize the putative binding pocket and RT loop. In Abl transactivation assays, Y222F and P225A mutations in the linker resulted in strong transactivation of Abl by Crk II. However, mutations predicted to be at the surface of the Crk SH3-C were not activators of Abl. Interestingly, combinations of activating mutations of Crk II with mutations in the highly conserved PNAY sequence in the SH3-C inactivated the activating mutations, suggesting that the SH3-C is necessary for activation. Our data provide insight into the role of highly conserved residues in the Crk-SH3-C, suggesting a mechanism for how the linker and the Crk-SH3-C function in the transactivation of the Abl tyrosine kinase.

Amino Acid Sequence↗

PubMed2005

The ybeY protein from Escherichia coli is a metalloprotein.

The three-dimensional crystallographic structure of the ybeY protein from Escherichia coli (SwissProt entry P77385) is reported at 2.7 A resolution. YbeY is a hypothetical protein that belongs to the UPF0054 family. The structure reveals that the protein binds a metal ion in a tetrahedral geometry. Three coordination sites are provided by histidine residues, while the fourth might be a water molecule that is not seen in the diffraction map because of its relatively low resolution. X-ray fluorescence analysis of the purified protein suggests that the metal is a nickel ion. The structure of ybeY and its sequence similarity to a number of predicted metal-dependent hydrolases provides a functional assignment for this protein family. The figures and tables of this paper were prepared using semi-automated tools, termed the Autopublish server, developed by the New York Structural GenomiX Research Consortium, with the goal of facilitating the rapid publication of crystallographic structures that emanate from worldwide Structural Genomics efforts, including the NIH-funded Protein Structure Initiative.

Amino Acid Sequence↗

PubMed2005

New York-Structural GenomiX Research Consortium (NYSGXRC): a large scale center for the protein structure initiative.

Structural GenomiX, Inc. (SGX), four New York area institutions, and two University of California schools have formed the New York Structural GenomiX Research Consortium (NYSGXRC), an industrial/academic Research Consortium that exploits individual core competencies to support all aspects of the NIH-NIGMS funded Protein Structure Initiative (PSI), including protein family classification and target selection, generation of protein for biophysical analyses, sample preparation for structural studies, structure determination and analyses, and dissemination of results. At the end of the PSI Pilot Study Phase (PSI-1), the NYSGXRC will be capable of producing 100-200 experimentally determined protein structures annually. All Consortium activities can be scaled to increase production capacity significantly during the Production Phase of the PSI (PSI-2). The Consortium utilizes both centralized and de-centralized production teams with clearly defined deliverables and hand-off procedures that are supported by a web-based target/sample tracking system (SGX Laboratory Information Data Management System, LIMS, and NYSGXRC Internal Consortium Experimental Database, ICE-DB). Consortium management is provided by an Executive Committee, which is composed of the PI and all Co-PIs. Progress to date is tracked on a publicly available Consortium web site (http://www.nysgxrc.org) and all DNA/protein reagents and experimental protocols are distributed freely from the New York City Area institutions. In addition to meeting the requirements of the Pilot Study Phase and preparing for the Production Phase of the PSI, the NYSGXRC aims to develop modular technologies that are transferable to structural biology laboratories in both academe and industry. The NYSGXRC PI and Co-PIs intend the PSI to have a transforming effect on the disciplines of X-ray crystallography and NMR spectroscopy of biological macromolecules. Working with other PSI-funded Centers, the NYSGXRC seeks to create the structural biology laboratory of the future. Herein, we present an overview of the organization of the NYSGXRC and describe progress toward development of a high-throughput Gene-->Structure platform. An analysis of current and projected consortium metrics reflects progress to date and delineates opportunities for further technology development.

Cloning, Molecular↗

PubMed2004

MODBASE, a database of annotated comparative protein structure models, and associated resources.

MODBASE (http://salilab.org/modbase) is a relational database of annotated comparative protein structure models for all available protein sequences matched to at least one known protein structure. The models are calculated by MODPIPE, an automated modeling pipeline that relies on the MODELLER package for fold assignment, sequence-structure alignment, model building and model assessment (http:/salilab.org/modeller). MODBASE uses the MySQL relational database management system for flexible querying and CHIMERA for viewing the sequences and structures (http://www.cgl.ucsf.edu/chimera/). MODBASE is updated regularly to reflect the growth in protein sequence and structure databases, as well as improvements in the software for calculating the models. For ease of access, MODBASE is organized into different data sets. The largest data set contains 1,26,629 models for domains in 659,495 out of 1,182,126 unique protein sequences in the complete Swiss-Prot/TrEMBL database (August 25, 2003); only models based on alignments with significant similarity scores and models assessed to have the correct fold despite insignificant alignments are included. Another model data set supports target selection and structure-based annotation by the New York Structural Genomics Research Consortium; e.g. the 53 new structures produced by the consortium allowed us to characterize structurally 24,113 sequences. MODBASE also contains binding site predictions for small ligands and a set of predicted interactions between pairs of modeled sequences from the same genome. Our other resources associated with MODBASE include a comprehensive database of multiple protein structure alignments (DBALI, http://salilab.org/dbali) as well as web servers for automated comparative modeling with MODPIPE (MODWEB, http://salilab. org/modweb), modeling of loops in protein structures (MODLOOP, http://salilab.org/modloop) and predicting functional consequences of single nucleotide polymorphisms (SNPWEB, http://salilab. org/snpweb).

Amino Acid Sequence↗

PubMed2004

High-throughput computational and experimental techniques in structural genomics.

Structural genomics has as its goal the provision of structural information for all possible ORF sequences through a combination of experimental and computational approaches. The access to genome sequences and cloning resources from an ever-widening array of organisms is driving high-throughput structural studies by the New York Structural Genomics Research Consortium. In this report, we outline the progress of the Consortium in establishing its pipeline for structural genomics, and some of the experimental and bioinformatics efforts leading to structural annotation of proteins. The Consortium has established a pipeline for structural biology studies, automated modeling of ORF sequences using solved (template) structures, and a novel high-throughput approach (metallomics) to examining the metal binding to purified protein targets. The Consortium has so far produced 493 purified proteins from >1077 expression vectors. A total of 95 have resulted in crystal structures, and 81 are deposited in the Protein Data Bank (PDB). Comparative modeling of these structures has generated >40,000 structural models. We also initiated a high-throughput metal analysis of the purified proteins; this has determined that 10%-15% of the targets contain a stoichiometric structural or catalytic transition metal atom. The progress of the structural genomics centers in the U.S. and around the world suggests that the goal of providing useful structural information on most all ORF domains will be realized. This projected resource will provide structural biology information important to understanding the function of most proteins of the cell.

Algorithms↗

PubMed2003

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.

Internet↗

PubMed2003

The zebrafish forkhead transcription factor Foxi1 specifies epibranchial placode-derived sensory neurons.

Vertebrate epibranchial placodes give rise to visceral sensory neurons that transmit vital information such as heart rate, blood pressure and visceral distension. Despite the pivotal roles they play, the molecular program underlying their development is not well understood. Here we report that the zebrafish mutation no soul, in which epibranchial placodes are defective, disrupts the fork headrelated, winged helix domain-containing protein Foxi1. Foxi1 is expressed in lateral placodal progenitor cells. In the absence of foxi1 activity, progenitor cells fail to express the basic helix-loop-helix gene neurogenin that is essential for the formation of neuronal precursors, and the paired homeodomain containing gene phox2a that is essential for neuronal differentiation and maintenance. Consequently, increased cell death is detected indicating that the placodal progenitor cells take on an apoptotic pathway. Furthermore, ectopic expression of foxi1 is sufficient to induce phox2a-positive and neurogenin-positive cells. Taken together, these findings suggest that Foxi1 is an important determination factor for epibranchial placodal progenitor cells to acquire both neuronal fate and subtype visceral sensory identity.

Amino Acid Sequence↗