16 research outputs found
Alignment of protein structures in the presence of domain motions
Abstract Background Structural alignment is an important step in protein comparison. Well-established methods exist for solving this problem under the assumption that the structures under comparison are considered as rigid bodies. However, proteins are flexible entities often undergoing movements that alter the positions of domains or subdomains with respect to each other. Such movements can impede the identification of structural equivalences when rigid aligners are used. Results We introduce a new method called RAPIDO (Rapid Alignment of Proteins in terms of Domains) for the three-dimensional alignment of protein structures in the presence of conformational changes. The flexible aligner is coupled to a genetic algorithm for the identification of structurally conserved regions. RAPIDO is capable of aligning protein structures in the presence of large conformational changes. Structurally conserved regions are reliably detected even if they are discontinuous in sequence but continuous in space and can be used for superpositions revealing subtle differences. Conclusion RAPIDO is more sensitive than other flexible aligners when applied to cases of closely homologues proteins undergoing large conformational changes. When applied to a set of kinase structures it is able to detect similarities that are missed by other alignment algorithms. The algorithm is sufficiently fast to be applied to the comparison of large sets of protein structures.</p
iSPOT: A Web Tool for the Analysis and Recognition of Protein Domain Specificity
Methods that aim at predicting interaction partners are very likely to play an important
role in the interpretation of genomic information. iSPOT (iSpecificity Prediction Of
Target) is a web tool (accessible at http://cbm.bio.uniroma2.it/iSPOT) developed for the
prediction of protein-protein interaction mediated by families of peptide recognition
modules. iSPOT accesses a database of position specific residue-residue interaction
frequencies for members of the SH3 and PDZ protein domain families. The software
utilises this database to provide a score for any potential domain peptide interaction
Development of Computational Tools for the Inference of Protein Interaction Specificity Rules and Functional Annotation Using Structural Information
Relatively few protein structures are known, compared to the enormous amount of sequence data produced in the sequencing of different genomes, and relatively few
protein complexes are deposited in the PDB with respect to the great amount of
interaction data coming from high-throughput experiments (two-hybrid or affinity
purification of protein complexes and mass spectrometry). Nevertheless, we can rely
on computational techniques for the extraction of high-quality and information-rich
data from the known structures and for their spreading in the protein sequence space.
We describe here the ongoing research projects in our group: we analyse the protein
complexes stored in the PDB and, for each complex involving one domain belonging
to a family of interaction domains for which some interaction data are available, we
can calculate its probability of interaction with any protein sequence. We analyse the
structures of proteins encoding a function specified in a PROSITE pattern, which
exhibits relatively low selectivity and specificity, and build extended patterns. To
this aim, we consider residues that are well-conserved in the structure, even if their
conservation cannot easily be recognized in the sequence alignment of the proteins
holding the function. We also analyse protein surface regions and, through the
annotation of the solvent-exposed residues, we annotate protein surface patches via a
structural comparison performed with stringent parameters and independently of the
residue order in the sequence. Local surface comparison may also help in identifying
new sequence patterns, which could not be highlighted with other sequence-based
methods
Distinct binding specificity of the multiple PDZ domains of INADL, a human protein with homology to INAD from Drosophila melanogaster
PDZ domains are protein-protein interaction modules that typically bind to short peptide sequences at the carboxyl terminus of target proteins. Proteins containing multiple PDZ domains often bind to different trans-membrane and intracellular proteins, playing a central role as organizers of multimeric complexes. To characterize the rules underlying the binding specificity of different PDZ domains, we have assembled a novel repertoire of random peptides that are displayed at high density at the carboxyl terminus of the capsid D protein of bacteriophage lambda. We have exploited this combinatorial library to determine the peptide binding preference of the seven PDZ domains of human INADL, a multi-PDZ protein that is homologous to the INAD protein of Drosophila melanogaster. This approach has permitted the determination of the consensus ligand for each PDZ domain and the assignment to class I, class II, and to a new specificity class, class IV, characterized by the presence of an acidic residue at the carboxyl-terminal position. Homology modeling and site-directed mutagenesis experiments confirmed the involvement of specific residues at contact positions in determining the domain binding preference. However, these experiments failed to reveal simple rules that would permit the association of the chemical characteristics of any given residue in the peptide binding pocket to the preference for specific amino acid sequences in the ligand peptide. Rather, they suggested that to infer the binding preference of any PDZ domain, it is necessary to simultaneously take into account all contact positions by using computational procedures. For this purpose we extended the SPOT algorithm, originally developed for SH3 domains, to evaluate the probability that any peptide would bind to any given PDZ domain
Improved cancer immunotherapy by a CD25-mimobody conferring selectivity to human interleukin-2
Interleukin-2 (IL-2) immunotherapy is an attractive approach in treating advanced cancer. However, by binding to its IL-2 receptor Ī± (CD25) subunit, IL-2 exerts unwanted effects, including stimulation of immunosuppressive regulatory T cells (Tregs) and contribution to vascular leak syndrome. We used a rational approach to develop a monoclonal antibody to human IL-2, termed NARA1, which acts as a high-affinity CD25 mimic, thereby minimizing association of IL-2 with CD25. The structure of the IL-2-NARA1 complex revealed that NARA1 occupies the CD25 epitope of IL-2 and precisely overlaps with CD25. Association of NARA1 with IL-2 occurs with 10-fold higher affinity compared to CD25 and forms IL-2/NARA1 complexes, which, in vivo, preferentially stimulate CD8(+) T cells while disfavoring CD25(+) Tregs and improving the benefit-to-adverse effect ratio of IL-2. In two transplantable and one spontaneous metastatic melanoma model, IL-2/NARA1 complex immunotherapy resulted in efficient expansion of tumor-specific and polyclonal CD8(+) T cells. These CD8(+) T cells showed robust interferon-Ī³ production and expressed low levels of exhaustion markers programmed cell death protein-1, lymphocyte activation gene-3, and T cell immunoglobulin and mucin domain-3. These effects resulted in potent anticancer immune responses and prolonged survival in the tumor models. Collectively, our data demonstrate that NARA1 acts as a CD25-mimobody that confers selectivity and increased potency to IL-2 and warrant further assessment of NARA1 as a therapeutic
ATP-gated ionotropic P2X7 receptor controls follicular T helper cell numbers in Peyer's patches to promote host-microbiota mutualism.
Microbial colonization of the gut induces the development of gut-associated lymphoid tissue (GALT). The molecular mechanisms that regulate GALT function and result in gut-commensal homeostasis are poorly defined. T follicular helper (Tfh) cells in Peyer's patches (PPs) promote high-affinity IgA responses. Here we found that the ATP-gated ionotropic P2X7 receptor controls Tfh cell numbers in PPs. Lack of P2X7 in Tfh cells enhanced germinal center reactions and high-affinity IgA secretion and binding to commensals. The ensuing depletion of mucosal bacteria resulted in reduced systemic translocation of microbial components, lowering B1 cell stimulation and serum IgM concentrations. Mice lacking P2X7 had increased susceptibility to polymicrobial sepsis, which was rescued by Tfh cell depletion or administration of purified IgM. Thus, regulation of Tfh cells by P2X7 activity is important for mucosal colonization, which in turn results in IgM serum concentrations necessary to protect the host from bacteremia