Structural, biochemical, and in vivo characterization of the first virally encoded cyclophilin from the Mimivirus.
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ABSTRACT:
SUBMITTER: Thai V
PROVIDER: S-EPMC2884007 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The genome sequence of the giant virus Acanthamoeba polyphaga mimivirus revealed the presence of two putative cytochrome P450 (CYP) genes. The product of one of the two predicted CYP genes (YP_143162) showed low-level homology to sterol 14-demethylase (CYP51) and contained a C-terminal polypeptide domain of unknown function. YP_143162 expression (without an N-terminal membrane binding domain) in Escherichia coli yields a CYP protein which gives a reduced CO difference maximum at 448 nm and was formally demonstrated as the first viral cytochrome P450. Analysis of binding of lipid and sterol substrates indicated no perturbation in CYP heme environment, and an absence of activity was seen when 14-methyl sterols were used as a substrate. The function of the CYP protein and its C-terminal domain remain unknown.
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Project description:Peptidyl-prolyl isomerases catalyze the conversion between cis and trans isomers of proline. The cyclophilin family of peptidyl-prolyl isomerases is well known for being the target of the immunosuppressive drug cyclosporin, used to combat organ transplant rejection. There is great interest in both the substrate specificity of these enzymes and the design of isoform-selective ligands for them. However, the dearth of available data for individual family members inhibits attempts to design drug specificity; additionally, in order to define physiological functions for the cyclophilins, definitive isoform characterization is required. In the current study, enzymatic activity was assayed for 15 of the 17 human cyclophilin isomerase domains, and binding to the cyclosporin scaffold was tested. In order to rationalize the observed isoform diversity, the high-resolution crystallographic structures of seven cyclophilin domains were determined. These models, combined with seven previously solved cyclophilin isoforms, provide the basis for a family-wide structure:function analysis. Detailed structural analysis of the human cyclophilin isomerase explains why cyclophilin activity against short peptides is correlated with an ability to ligate cyclosporin and why certain isoforms are not competent for either activity. In addition, we find that regions of the isomerase domain outside the proline-binding surface impart isoform specificity for both in vivo substrates and drug design. We hypothesize that there is a well-defined molecular surface corresponding to the substrate-binding S2 position that is a site of diversity in the cyclophilin family. Computational simulations of substrate binding in this region support our observations. Our data indicate that unique isoform determinants exist that may be exploited for development of selective ligands and suggest that the currently available small-molecule and peptide-based ligands for this class of enzyme are insufficient for isoform specificity.
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Project description:The amoeba-infecting Mimivirus is the largest known double-stranded DNA virus, with a 400 nm particle size, comparable to that of mycoplasma. The complete sequence of its 1.2 Mbp genome has recently been determined [Raoult et al. (2004), Science, 306, 1344-1350] and revealed numerous genes that were not expected to be found in a virus, such as genes encoding translation components, including 4-amino-acyl tRNA synthetases and homologues to various translation initiation, elongation and termination factors. A comprehensive structural and functional study of these Mimivirus gene products was initiated, as they may hold important clues about the origin of DNA viruses. Here, the first preliminary crystallographic and functional results obtained on one of these targets, Mimivirus TyrRS, are reported. Preliminary phasing was obtained using an original combination of homology modelling and normal mode analysis. Experimental evidence that Mimivirus tyrosyl tRNA synthetase recombinant gene product does indeed activate tyrosine is also presented.
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2024-04-09 | GSE246735 | GEO