Through extensive mutagenesis trial-and-error efforts, a stable, homodimeric construct of the catalytic core domain of the HIV1 integrase enzyme has been generated. Although under conditions that closely resemble the physiological environment the enzyme rapidly samples different conformational states, leading to extensive line broadening and disappearance of NMR signals, we have found conditions that include high Mg2+ concentrations (40 mM) where the equilibrium is shifted to what appear to be one major and one minor state, that differ from one another in the structure of the C-terminal helix. We have evaluated the stability of the catalytic core domain by novel methods to measure the rate at which backbone amide hydrogens exchange (HX) with solvent. These HX rates contain valuable information on protein structure and function, but NMR methods for measuring HX rates were quite limited in their applicability to large protein systems. An alternate method for measuring rapid hydrogen exchange has been developed that is well-suited for larger proteins, and we have applied the method to the deuterated, homodimeric 36 kDa HIV-1 integrase catalytic core domain (CCD). Using long mixing times for water-amide magnetization exchange at multiple pH values, HX rates spanning more than four orders of magnitude were measured, as well as NOE cross-relaxation rates to nearby exchangeable protons. HX protection factors for the CCD are found to be large (>104) for residues along the dimer interface, but much smaller in many other regions. Notably, the catalytic helix (residues 152-167) exhibits low HX protection at both ends, indicative of fraying at both termini as opposed to just the N-terminal end, as originally thought. Residues in the LEDGF/p75 binding pocket also show marginal stability, with protection factors in the 10-100 range (1.4-2.7 kcal/mol). Additionally, elevated NOE cross-relaxation rates are identified and, as expected, correspond to proximity of the amide proton to a rapidly-exchanging proton, typically from an OH side chain. Indirect NOE transfer between H2O and the amide proton of I141, a residue in the partially disordered active site of the enzyme, suggests its proximity to the side chain of S147, an interaction seen in the DNA-bound form for a homologous integrase.

Project Start
Project End
Budget Start
Budget End
Support Year
4
Fiscal Year
2012
Total Cost
$100,212
Indirect Cost
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State
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Louis, John M; Tozser, Jozsef; Roche, Julien et al. (2013) Enhanced stability of monomer fold correlates with extreme drug resistance of HIV-1 protease. Biochemistry 52:7678-88
Sgourakis, Nikolaos G; Lange, Oliver F; DiMaio, Frank et al. (2011) Determination of the structures of symmetric protein oligomers from NMR chemical shifts and residual dipolar couplings. J Am Chem Soc 133:6288-98
Fitzkee, Nicholas C; Torchia, Dennis A; Bax, Ad (2011) Measuring rapid hydrogen exchange in the homodimeric 36 kDa HIV-1 integrase catalytic core domain. Protein Sci 20:500-12
Fitzkee, Nicholas C; Bax, Ad (2010) Facile measurement of (1)H- (15)N residual dipolar couplings in larger perdeuterated proteins. J Biomol NMR :
Fitzkee, Nicholas C; Masse, James E; Shen, Yang et al. (2010) Solution conformation and dynamics of the HIV-1 integrase core domain. J Biol Chem 285:18072-84