期刊
PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS
卷 78, 期 2, 页码 295-308出版社
WILEY
DOI: 10.1002/prot.22542
关键词
HIV fusion inhibitor; chemokine; GAG binding; quaternary state; competition fusion assay
资金
- NIH [R21 A1079777]
- Robert Welch Foundation [A1472]
The N-terminal region of the chemokine RANTES is critical for its function. A synthesized N-terminally modified analog of RANTES, P2-RANRES, was discovered using a phage display selection against living CCR5-expressing cells, and has been reported to inhibit HIV-1 env-mediated cell-cell fusion at subnanomolar levels (Hartley et al. J Virol 2003;77:6637-6644). In the present study we produced this protein using E. coli overexpression and extensively studied its structure and function. The x-ray crystal structure of P2-RANTES was solved and refined at 1.7 angstrom resolution. This protein was found to be predominantly a monomer in solution by analytical ultracentrifugation, but a tetramer in the crystal. in studies of glycosaminoglycan binding, P2-RANTES was found to be significantly, less able to bind heparin than wild type RANTES. We also tested this protein for receptor internalization where it was shown to be functional, in cell-cell fusion assays where recombinant P2-RANTES was a potent fusion inhibitor (IC50 = 2.4 +/- 0.8 nM), and in single round infection assays where P2-RANTES inhibited at subuanomolar levels. Further, in a modified fusion assay designed to test specificity of inhibition, P2-RANTES was also highly effective, with a 65-fold improvement over the fusion inhibitor C37, which is closely related to the clinically approved inhibitor T-20. These studies provide detailed structural and functional information for this novel N-terminally modified chemokine mutant. This information will be very useful in the development of more potent anti-HIV agents. PDB Accession Number: 2vxw. Proteins 2010: 79:295-308. (C) 2009 Wiley-Liss, Inc.
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