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1 Molecular Structural Biology Unit, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland 20892, USA
2 Department of Biological Chemistry, and the Wolfson Centre for Applied Structural Biology, The Institute of Life Sciences, Hebrew University of Jerusalem, Jerusalem 91904, Israel
3 Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA
Reprint requests to: Dennis A. Torchia, Bldg. 30, Room 113, MSC4307, Bethesda, Maryland 20892; e-mail: dtorchia{at}dir.nidcr.nih.gov; fax: (301) 480-0240.
Crystal structures have shown that the HIV-1 protease flaps, domains that control access to the active site, are closed when the active site is occupied by a ligand. Although flap structures ranging from closed to semi-open are observed in the free protease, crystal structures reveal that even the semi-open flaps block access to the active site, indicating that the flaps are mobile in solution. The goals of this paper are to characterize the secondary structure and fast (sub-ns) dynamics of the flaps of the free protease in solution, to relate these results to X-ray structures and to compare them with predictions of dynamics calculations. To this end we have obtained nearly complete backbone and many sidechain signal assignments of a fully active free-protease construct that is stabilized against autoproteolysis by three point mutations. The secondary structure of this protein was characterized using the chemical shift index, measurements of 3hJNC` couplings across hydrogen bonds, and NOESY connectivities. Analysis of these measurements indicates that the protease secondary structure becomes irregular near the flap tips, residues 49–53. Model-free analysis of 15N relaxation parameters, T1, T2 (T1
) and 15N-{1H} NOE, shows that residues in the flap tips are flexible on the sub-ns time scale, in contrast with previous observations on the inhibitor-bound protease. These results are compared with theoretical predictions of flap dynamics and the possible biological significance of the sub-ns time scale dynamics of the flap tips is discussed.
Keywords: AIDS; NMR; secondary structure; relaxation; hydrogen bonds
Abbreviations: HIV, human immunodeficiency virus • ms, millesecond • µs, microsecond • ns, nanosecond • ps, picosecond • NOE, nuclear Overhauser effect • HSQC, heteronuclear single quantum coherence • NOESY, NOE spectroscopy • CSI, Chemical Shift Index • CPMG, Carr-Purcell-Meiboom-Gill • TMPR and PMPR, triple- and penta-mutant HIV-1 protease constructs, respectively
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