Explain the process of manipulation that Harrison use to create the closed compl

Question

Explain the process of manipulation that Harrison use to create the closed complex in HIV-1 RT p66.

see reference: https://www.ncbi.nlm.nih.gov/pubmed/9831551

RESEARCH ARTICLES Rogers, P. J. DeMott, Geophys. Res. Lett. 25,139 ed with RT inhibitors is a major limitation of 199 Heintzenberg K. OkdaJ Strom, Atmos. a Helmer. J.M. C. Plane, J. Olan, C. S.Gardner.J. Geophys Res. 103, 10.913 (19984 Cgyis obtained from their flux after corecting the weight fraction of Fe in their calculation. An additional uncertainty in meteorisic 36 U. M. Biermann et al, Geophys, Res. Lett 23, 1693 near the po fuxes is introduced because the actual mass available for incorporation into stratospheric aerosols may be 37. G. S. Kent, M. P. McCormick, S. K. Schaffner, J. Geo- polypeptide segment connecting palm and different from the incident mass because the oxygen bound into minerals and oxides is released during abla- 3. R. Dixon, in preparation tion and then accumulated during later oxidation of 39 I.J. Zieman et al., J. Geophys. Res. 100, 25.975 antiviral therapy (4, 5. Al NNRTIs bind phobic pocket created by displacement of the thumb. Viral mutations conferring resistance phys. Res. 96, 5249 (1991) to can in (1995: J G. Watson et al., Aer. Sci. Technol, 21,terms of alterations in their common binding 1994 Range shown is from primary geological site. In contrast, the positions of altered res- ron, silicon, and other elements 34. D. J. Hofmann, J. M. Rosen, J. M. Kiernan, J. Laby, Atmos. Sol. 33, 1783 (1976): M. Chin and D. D. Davis, idues in viruses resistant to nucleoside ana- J. Geophys Res. 100, 8993 (1995): D. K. Weisenstein 4 The assistance of the pillots, ground crews, and staff et al., ibid. 102, 13,019 (1997) M. J. Mills, thess of the WB-S7F is gratefully acknowledged. This work ogs do not follow so clear a pattern (5). Lack University of Colorado, Boulder (1996). Condensible was funded by NOAA base funding the NASA Atmo of a structure for a catalytic complex of RT spheric Effects of Aviation Project, and the NASA Upper Atmospheric Research Progyam. B. Gary assist- ed with the MTP data analysis. with has hindered template:primer and dNTP substrates B. Gary assist sulfur, from oxidation of OCS and so). with the assumption that 70% of the aerosol by weight is SI Previously determined RT structures in- and the unliganded enzyme (3, 6-10). The only pub lished structure of RT with a bound template: primer is an RT template:primer Fab terna ry complex (6), which shows that the primer 35. Y. Chen, S. M. Kreidenweis, L M Mcinnes, D. C 16 September 1998; accepted 2 November 1998 clude a number of NNRTI complexes Structure of a Covalently Trapped Catalytic Complex of HIV-1 Reverse Transcriptase: aspartic acid residues in the palm and that the the ward the RNase H. The position of fingers and thumb define a deep cleft, with the poly- merase active site at its base. This feature, also present in crystals of RT with bound NNRTIs, has led to a model in which the 5 Implications for Drug Resista te tde nt det last, ih te pules Huifang Huang. Rajiv Chopra, Gregory L. Verdine,t son ers are mutated in A combinatorial disulfide cross-linking strategy was used to prepare a stalled complex of human immunodeficiency virus-type 1 (HIV-1) reverse transcrip- tase with a DNA template:primer and a deoxynucdeoside triphosphate (dNTP). and the crystal structure of the complex was determined at a resolution of 3.2 and palm that strains (3, 6). Other DNA tain a similar cleft, but recent structures of catalytic complexes show that it closes down when substrates bind and that it does not serve as a channel for polynucleotide chains con at allows capture of a state in which the substrates are poised for attack on the dNTP. Conformational changes that accompany formation of the catalytic complex produce distinct clusters of the residues that are altered in viruses resistant to nucleoside analog drugs. The positioning of these residues in the neighborhood of the dNTP helps to resolve some long-standing puzzles about the molecular basis of resistance. The resistance mutations are likely to influ- ence binding or reactivity of the inhibitors, relative to normal dNTPs, and the clustering of the mutations correlates with the chemical structure of the drug. stalled, covalently tethered complexes of RT with template:primer, and we have crystal- one scribed here at 3.2 A resolution, the primer terminus is a dideoxynucleotide and thus un- The reverse transcriptase (RT) of HIV-1 is an cleoside analogs [for example, 3'-azido-23' of this catalytic complex contains bound important target of antiviral therapy in the dideoxythymidine (AZT), 2',3'-dideoxyinosine dTTP in precisely the expected position for treatment of acquired immunodeficiency syn (ddl, and 2-deoxy-3'-thiacytidine (3TC)). The attack by the (missing) 3' OH. The fingers drome (AIDS) (). RT has two distinct other two inhibitors are members of a chemi domain bends, relative to other RT structures, enzymatic activities, an RNA- or DNA- cally diverse group of nonnucleoside RT inhib- so that various residues near the fingertips form part of the dNTP-binding site. This clease (RNase H, but current agents areHIV-1 RT is a dimer of two related conformational adjustment defines the com- directed only against the polymerase. Five chains, a 66-kD subunit (p66) and a 51-kD plete catalytic site and leads to a revised of the seven inhibitors currently licensed in subunit (p51) derived from p66 by proteolytic interpretation of the mechanism by which the United States are chain-terminating nu cleavage (2). The two chains have in common various mutations confer resistance to nucle- dependent DNA polymerase and a ribonu- itors (NNRTIs). four domains [referred to as "fingers,” oside analog drugs. H. Huang and G. L Verdine are in the Department of Chemistry and Chemical Biology, Harvard University, p66 also has a COOH-terminal RNase H. The Trapping of a Catalytic HIV-1 RT Cambridge, MA 02138, USA. R. Chopra and S. C p66 subunit has both the polymerase and Substrate Complex Harrison are at the Howard Hughes Medical Institute RNase H active sites. The palm contains res- The difficulty in obtaining good crystals of an idues critical for polymerase catalytic activ RT.template:primer dNTP complex stems ty, and its folded structure resembles that of a from RT's relatively modest specificity in corresponding catalytic domain in other DNA binding to polymerizable versus nonpolymer- izable sites in DNA, even in the presence of Emergence of resistance in patients treat- a dNTP. To overcome this problem, we chose and Department of Molecular and Cellular Biology. These authors contributed equally to this work tTo whom correspondence should be addressed. E and schadmine and RNA polymerases. www.sciencemag.org SCIENCE VOL 282 27 NOVEMBER 1998 1669

Explanation / Answer

HIV-1 RT is a heterodimer composed of two subunits: The larger, p66, is 560 amino acids long, and the smaller, p51, contains the first 440 residues of p66.Three-dimensional structures that do and do not contain bound substrates and inhibitors have been quite helpful in understanding the functions of HIV-1 RT. The p66 subunit is composed of two domains: polymerase and RNase H. The polymerase domain contains the fingers, palm, and thumb subdomains; the connection subdomain links the polymerase domain to the RNase H domain. p51 is folded into similar subdomains (fingers, palm, thumb, and connection), but the relationships of those subdomains to each other are different in p66 and p51 .A comparison of the available structures has made it clear that HIV-1 RT is quite flexible and that this flexibility plays an important role in the behavior of the enzyme.

The structure of unliganded RT has the thumb subdomain in the ‘closed’  with the thumb moving to accommodate the nucleic acid. Subsequent binding of an incoming dNTP forms the E+/DNA/dNTP complex that is believed to have the fingers in the open configuration . There is no crystal structure that corresponds to this complex. The conversion of E+/DNA/dNTP to the activated closed ternary complex (E*/DNA/dNTP) involves movement of the p66 fingers subdomain , a conformational change thought to be the rate-limiting step in polymerization.n all of the complexes, the primer terminus is positioned at what we call the ‘priming site’ (P site). This allows the incoming dNTP to bind at the nucleotide-binding site (N site).

RT containing the C280S mutation in both p66 and p51 subunits and the Q258C mutation only in the p66 subunit was prepared using an expression system . with the p66 and p51 subunits expressed from two separate coding regions.

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