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Protein Science (2004), 13:1512-1523. Published by Cold Spring Harbor Laboratory Press. Copyright © 2004 The Protein Society
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The dimerization domain of the HIV-1 capsid protein binds a capsid protein-derived peptide: A biophysical characterization

María T. Garzón1,5, María C. Lidón-Moya1, Francisco N. Barrera1, Alicia Prieto2, Javier Gómez1, Mauricio G. Mateu3 and José L. Neira1,4,5

1 Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, 03202 Elche (Alicante), Spain
2 Centro de Investigaciones Biológicas, CSIC, 28006 Madrid, Spain
3 Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain
4 Instituto de Biofiísica y Física de los sistemas complejos, 50009 Zaragoza, Spain

(RECEIVED December 9, 2003; FINAL REVISION February 17, 2004; ACCEPTED February 26, 2004)



Abstract

The type 1 HIV presents a conical capsid formed by ~1500 units of the capsid protein, CA. Homodimer-ization of CA via its C-terminal domain, CA-C, constitutes a key step in virion assembly. CA-C dimerization is largely mediated by reciprocal interactions between residues of its second {alpha}-helix. Here, we show that an N-terminal-acetylated and C-terminal–amidated peptide, CAC1, comprising the sequence of the CA-C dimerization helix plus three flanking residues at each side, is able to form a complex with the entire CA-C domain. Thermal denaturation measurements followed by circular dichroism (CD), NMR, and size-exclusion chromatography provided evidence of the interaction between CAC1 and CA-C. The apparent dissociation constant of the heterocomplex formed by CA-C and CAC1 was determined by several biophysical techniques, namely, fluorescence (using an anthraniloyl-labeled peptide), affinity chromatography, and isothermal titration calorimetry. The three techniques yielded similar values for the apparent dissociation constant, in the order of 50 µM. This apparent dissociation constant was only five times higher than was the dissociation constant of both CA-C and the intact capsid protein homodimers (10 µM).

Keywords: peptide recognition; binding; calorimetry; fluorescence

Abbreviations: CA, capsid protein of HIV (p24) • CA-C, C-terminal domain of CA • CAC1, peptide comprising the second helix of CA-C • CAC1Aib, the CAC1 peptide with the anthraniloyl moiety attached to the {varepsilon}-group of Lys182 • CD, circular dichroism • IN, integrase • ITC, isothermal titration calorimetry • RT, reverse transcriptase • PR, protease • SEC, size-exclusion chromatography • TSP, 3-(trimethylsilyl) propionic acid-2,2,3,3-2H4-sodium salt • UV, ultraviolet

Reprint requests to: José L. Neira, Instituto de Biología Molecular y Celular, Edificio Torregaitán, Universidad Miguel Hernández, Avda. del Ferrocarril s/n, 03202, Elche (Alicante), Spain; e-mail: jlneira{at}umh.es; fax: 34-966658758.

5 These two authors contributed equally to this work.

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.03555304.


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