Measuring rapid hydrogen exchange in the homodimeric 36 kDa HIV‐1 integrase catalytic core domain
Open Access
- 6 January 2011
- journal article
- research article
- Published by Wiley in Protein Science
- Vol. 20 (3), 500-512
- https://doi.org/10.1002/pro.582
Abstract
Measurements of rapid hydrogen exchange (HX) of water with protein amide sites contain valuable information on protein structure and function, but current NMR methods for measuring HX rates are limited in their applicability to large protein systems. An alternate method for measuring rapid HX is presented that is well‐suited for larger proteins, and we apply 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.Keywords
This publication has 65 references indexed in Scilit:
- Structure-based modeling of the functional HIV-1 intasome and its inhibitionProceedings of the National Academy of Sciences of the United States of America, 2010
- Facile measurement of 1H–15N residual dipolar couplings in larger perdeuterated proteinsJournal of Biomolecular NMR, 2010
- Solution Conformation and Dynamics of the HIV-1 Integrase Core DomainOnline Journal of Public Health Informatics, 2010
- Retroviral intasome assembly and inhibition of DNA strand transferNature, 2010
- Indirect Detection of Labile Solute Proton Spectra via the Water Signal Using Frequency-Labeled Exchange (FLEX) TransferJournal of the American Chemical Society, 2010
- Comparison of metal-dependent catalysis by HIV-1 and ASV integrase proteins using a new and rapid, moderate throughput assay for joining activity in solutionAIDS Research and Therapy, 2009
- LEDGF/p75 functions downstream from preintegration complex formation to effect gene-specific HIV-1 integrationGenes & Development, 2007
- Intrinsically unstructured proteins and their functionsNature Reviews Molecular Cell Biology, 2005
- NMRPipe: A multidimensional spectral processing system based on UNIX pipesJournal of Biomolecular NMR, 1995
- Dictionary of protein secondary structure: Pattern recognition of hydrogen‐bonded and geometrical featuresPeptide Science, 1983