Backbone Dynamics of the RNase H Domain of HIV-1 Reverse Transcriptase

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• 作者：Geoffrey A. Mueller ; Koteppa Pari ; Eugene F. DeRose ; Thomas W. Kirby ; and Robert E. London
• 刊名：Biochemistry
• 出版年：2004
• 出版时间：July 27, 2004
• 年：2004
• 卷：43
• 期：29
• 页码：9332 - 9342
• 全文大小：255K
• 年卷期：v.43,no.29(July 27, 2004)
• ISSN：1520-4995

文摘

Previous NMR relaxation studies of the isolated RNase H domain of HIV-1 reverse transcriptaseat low pH have revealed that it is substantially more dynamic and less ordered than the relatively stableand catalytically active E. coli RNase HI. Using more recently developed techniques, we have investigatedthe dynamic behavior of the RNase H domain of HIV-1 reverse transcriptase at a more physiological pH(6.8), under a variety of solution conditions: no Mg²⁺, 80 mM Mg²⁺, and 80 mM Mg²⁺ plus AMPligand. In addition, we have repeated the previous measurements on a sample containing 100 mM sodiumacetate, pH 5.4. Under all conditions studied, the order parameters from NMR relaxation analysis areuniformly high (>0.8) for most of the domain with the exception of the C-terminal region. Subtle differencescan be found among the conditions studied, although the statistical significance of the differences ismarginal. Residues 71-114 show a slight increase in order parameter with the addition of 5'-AMP.Conformational exchange, measured with CPMG relaxation dispersion experiments in the presence ofMg and AMP, were detected for some NH sites, predominantly located in the N-terminal region of theprotein near strands ages/gifchars/beta2.gif" BORDER=0 ALIGN="middle">2 and ages/gifchars/beta2.gif" BORDER=0 ALIGN="middle">3 and helix ages/gifchars/alpha.gif" BORDER=0>_A (residues 28-69). In contrast with earlier studies indicatingpathologically extreme dynamic behavior that apparently correlated with inactivity of the isolated domain,the relaxation analysis under the conditions of the present study yielded parameters that are more similarto those of the active E. coli RNase HI. A comparison of the order parameters obtained from a model-free analysis of the relaxation data with the B-factors in the crystal structures of the RNase H domain,both for the isolated domain and for the full HIV-1 reverse transcriptase structure, suggests that the dynamicbehavior is similar in all cases.