63ª Reunião Anual da SBPC

C. Ciências Biológicas - 1. Biofísica - 3. Biofísica Molecular

Search for functional regions in Platelet-Activating Factor (PAF) Receptor by surface-mapping of evolutionary conservation among metazoan homologs

Daniel Fábio Kawano 1 Ivone Carvalho 2

1. Departamento de Ciências Farmacêuticas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto - FCFRP, Universidade de São Paulo - USP 2. Profa. Dra./ Orientadora - Departamento de Ciências Farmacêuticas, FCFRP - USP

INTRODUÇÃO:

Platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a potent lipid mediator of inflammation, platelet aggregation and immunological response, playing a role in important physiological process such as anaphylaxis and cerebral ischemia. Though PAF and its analogs have been used as tools to study mechanisms and pharmacological targets of several diseases, there is little information concerning on the specific binding site of the PAF receptor since the crystal structure of the ligand/receptor complex is unavailable. In order to gain some knowledge on which residues in the sequence of PAF receptor play a role in ligand binding and in the folding of the protein into its 3D structure, we performed an analysis of the evolutionary relationships among PAF receptors from 25 different animal species and mapped the conserved regions into the tertiary structure of human PAF receptor.

METODOLOGIA:

Sequences of PAF receptors from 25 different animal species were retrieved from Uniprot and ENSEMBL: 19 sequences of receptors from mammals, 4 from fishes, one from amphibian and one from bird. Multiple sequence alignments (MSAs) were produced using MUSCLE and the phylogenetic analyses were carried out with MEGA 4.0 using the neighbor-joining algorithm. Bootstrap support values for the optimal trees were calculated using 1,000 replicates with heuristic search settings identical to those for the original search. The evolutionary distances were computed using the Jones-Taylor-Thornton (JTT) matrix-based method with respect to the units of the number of amino acid mutations per site; indels were treated as missing data. The sequences corresponding to the seven transmembrane domains of PAF receptors were inferred via MSAs based on the data reported for the human PAF receptor. Conservational analysis was performed in Consurf using the tertiary structure of human PAF receptor as the 3D scaffold (PDB code 2B0X). Based on the MSAs of the 25 PAF receptors, the Bayesian method was applied for the calculations of the conservation scores using the JTT matrix as the model of substitution for proteins.

RESULTADOS:

From the evolutionary point of view, PAF receptors split into two main lineages: one cluster containing the mammals plus the amphibian/bird clades, and the other comprising exclusively fishes. Based on the MSAs of PAF receptors, we observed that 77 amino acids were totally conserved among the species. These residues are primarily localized (almost 82%) in the transmembrane spans. When residues partially conserved among species are also considered, it is again evident the prevalence of conserved sites in the transmembrane domains, with the exception perhaps of the fourth domain, the most variable transmembrane span. The highest degree of conservation was observed for Domains I, II, III, VI and VII. This observation is consistent with molecular modeling studies which suggest that the ligand-binding site of human PAF receptor is composed fundamentally by residues in the membrane-spanning regions. Moreover, transmembrane domains tend to be more conserved among GPCRs while extra and intracellular loop regions and C-terminus are typically much more divergent.

CONCLUSÃO:

Knowledge of a protein’s functionally important sites has immediate relevance for predicting function, guiding experimental analysis, analyzing molecular mechanisms and understanding protein interactions. Our results suggest that the transmembrane domains of PAF receptors are highly conserved from the evolutionary point of view, highlighting the importance of these regions in the process of ligand binding.

Palavras-chave: PAF, GPCR, Bioinformatics.