The solution structure of the prototype foamy virus RNase H domain indicates an important role of the basic loop in substrate binding

Retrovirology

Birgitta M Wöhrl¹  Maximilian J Hartl¹  Paul Rösch¹  Kristian Schweimer¹  Berit Leo¹ 

[1] Universität Bayreuth, Lehrstuhl Biopolymere, Universitätsstr. 30, D-95447, Bayreuth, Germany

关键词: Substrate binding;    Solution structure;    Basic protrusion;    NMR;    Basic loop;    C-helix;    Retroviral RNase H;    Foamy virus;   

Others  :  1209269
DOI  :  10.1186/1742-4690-9-73

 received in 2012-06-14, accepted in 2012-08-10,  发布年份 2012

【 摘 要 】

Background

The ribonuclease H (RNase H) domains of retroviral reverse transcriptases play an essential role in the replication cycle of retroviruses. During reverse transcription of the viral genomic RNA, an RNA/DNA hybrid is created whose RNA strand needs to be hydrolyzed by the RNase H to enable synthesis of the second DNA strand by the DNA polymerase function of the reverse transcriptase. Here, we report the solution structure of the separately purified RNase H domain from prototype foamy virus (PFV) revealing the so-called C-helix and the adjacent basic loop, which both were suggested to be important in substrate binding and activity.

Results

The solution structure of PFV RNase H shows that it contains a mixed five-stranded β-sheet, which is sandwiched by four α-helices (A-D), including the C-helix, on one side and one α-helix (helix E) on the opposite side. NMR titration experiments demonstrate that upon substrate addition signal changes can be detected predominantly in the basic loop as well as in the C-helix. All these regions are oriented towards the bound substrate. In addition, signal intensities corresponding to residues in the B-helix and the active site decrease, while only minor or no changes of the overall structure of the RNase H are detectable upon substrate binding. Dynamic studies confirm the monomeric state of the RNase H domain. Structure comparisons with HIV-1 RNase H, which lacks the basic protrusion, indicate that the basic loop is relevant for substrate interaction, while the C-helix appears to fulfill mainly structural functions, i.e. positioning the basic loop in the correct orientation for substrate binding.

Conclusions

The structural data of PFV RNase H demonstrate the importance of the basic loop, which contains four positively charged lysines, in substrate binding and the function of the C-helix in positioning of the loop. In the dimeric full length HIV-1 RT, the function of the basic loop is carried out by a different loop, which also harbors basic residues, derived from the connection domain of the p66 subunit. Our results suggest that RNases H which are also active as separate domains might need a functional basic loop for proper substrate binding.

【 授权许可】

   
2012 Leo et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Moelling K, Bolognesi DP, Bauer H, Büsen W, Plassmann HW, Hausen P: Association of retroviral reverse transcriptase with an enzyme degrading the RNA moiety of RNA-DNA hybrids. Nat New Biol 1971, 234:240-243.
• [2]Hartl MJ, Bodem J, Jochheim F, Rethwilm A, Rösch P, Wöhrl BM: Regulation of foamy virus protease activity by viral RNA - a novel and unique mechanism among retroviruses. J Virol 2011, 85:4462-4469.
• [3]Hartl MJ, Mayr F, Rethwilm A, Wöhrl BM: Biophysical and enzymatic properties of the simian and prototype foamy virus reverse transcriptases. Retrovirology 2010, 7:5. BioMed Central Full Text
• [4]Das D, Georgiadis MM: The crystal structure of the monomeric reverse transcriptase from moloney murine leukemia virus. Structure 2004, 12:819-829.
• [5]Leo B, Hartl MJ, Schweimer K, Mayr F, Wöhrl BM: Insights into the structure and activity of the prototype foamy virus RNase H. Retrovirology 2012, 9:14. BioMed Central Full Text
• [6]Tanese N, Goff SP: Domain structure of the moloney murine leukemia virus reverse transcriptase: Mutational analysis and separate expression of the DNA polymerase and RNase H activities. Proc Natl Acad Sci U S A 1988, 85:1777-1781.
• [7]Schultz SJ, Champoux JJ: RNase H domain of moloney murine leukemia virus reverse transcriptase retains activity but requires the polymerase domain for specificity. J Virol 1996, 70:8630-8638.
• [8]Zhan X, Crouch RJ: The isolated RNase H domain of murine leukemia virus reverse transcriptase. Retention of activity with concomitant loss of specificity. J Biol Chem 1997, 272:22023-22029.
• [9]Kanaya S, Katsuda-Nakai C, Ikehara M: Importance of the positive charge cluster in Escherichia coli ribonuclease HI for the effective binding of the substrate. J Biol Chem 1991, 266:11621-11627.
• [10]Telesnitsky A, Blain SW, Goff SP: Defects in moloney murine leukemia virus replication caused by a reverse transcriptase mutation modeled on the structure of Escherichia coli RNase H. J Virol 1992, 66:615-622.
• [11]Lim D, Orlova M, Goff SP: Mutations of the RNase H C helix of the moloney murine leukemia virus reverse transcriptase reveal defects in polypurine tract recognition. J Virol 2002, 76:8360-8373.
• [12]Lim D, Gregorio GG, Bingman C, Martinez-Hackert E, Hendrickson WA, Goff SP: Crystal structure of the moloney murine leukemia virus RNase H domain. J Virol 2006, 80:8379-8389.
• [13]Zhou D, Chung S, Miller M, Grice SF, Wlodawer A: Crystal structures of the reverse transcriptase-associated ribonuclease H domain of xenotropic murine leukemia-virus related virus. J Struct Biol 2012, 177:638-645.
• [14]Paprotka T, Delviks-Frankenberry KA, Cingoz O, Martinez A, Kung HJ, Tepper CG, Hu WS, Fivash MJ, Coffin JM, Pathak VK: Recombinant origin of the retrovirus XMRV. Science 2011, 333:97-101.
• [15]Nowotny M, Gaidamakov SA, Crouch RJ, Yang W: Crystal structures of RNase H bound to an RNA/DNA hybrid: Substrate specificity and metal-dependent catalysis. Cell 2005, 121:1005-1016.
• [16]Evans DB, Brawn K, Deibel MR, Tarpley WG, Sharma SK: A recombinant ribonuclease H domain of HIV-1 reverse transcriptase that is enzymatically active. J Biol Chem 1991, 266:20583-20585.
• [17]Hostomsky Z, Hostomska Z, Hudson GO, Moomaw EW, Nodes BR: Reconstitution in vitro of RNase H activity by using purified N- terminal and C-terminal domains of human immunodeficiency virus type 1 reverse transcriptase. Proc Natl Acad Sci U S A 1991, 88:1148-1152.
• [18]Smith JS, Roth MJ: Purification and characterization of an active human immunodeficiency virus type 1 RNase H domain. J Virol 1993, 67:4037-4049.
• [19]Smith JS, Gritsman K, Roth MJ: Contributions of DNA polymerase subdomains to the RNase H activity of human immunodeficiency virus type 1 reverse transcriptase. J Virol 1994, 68:5721-5729.
• [20]Davies JF, Hostomska Z, Hostomsky Z, Jordan SR, Matthews DA: Crystal structure of the ribonuclease H domain of HIV-1 reverse transcriptase. Science 1991, 252:88-95.
• [21]Mueller GA, Pari K, DeRose EF, Kirby TW, London RE: Backbone dynamics of the RNase H domain of HIV-1 reverse transcriptase. Biochemistry 2004, 43:9332-9342.
• [22]Kirby KA, Marchand B, Ong YT, Ndongwe TP, Hachiya A, Michailidis E, Leslie MD, Sietsema DV, Fetterly TL, Dorst CA, Singh K, Wang Z, Parniak MA, Sarafianos SG: Structural and inhibition studies of the RNase H function of xenotropic murine leukemia virus-related virus reverse transcriptase. Antimicrob Agents Chemother 2012, 56:2048-2061.
• [23]Wöhrl BM, Volkmann S, Moelling K: Mutations of a conserved residue within HIV-1 ribonuclease H affect its exo-and endonuclease activities. J Mol Biol 1991, 220:801-818.
• [24]Tisdale M, Schulze T, Larder BA, Moelling K: Mutations within the RNase H domain of human immunodeficiency virus type 1 reverse transcriptase abolish virus infectivity. J Gen Virol 1991, 72:59-66.
• [25]Sarafianos SG, Das K, Tantillo C, Clark AD, Ding J, Whitcomb JM, Boyer PL, Hughes SH, Arnold E: Crystal structure of HIV-1 reverse transcriptase in complex with a polypurine tract RNA:DNA. EMBO J 2001, 20:1449-1461.
• [26]Nowotny M, Gaidamakov SA, Ghirlando R, Cerritelli SM, Crouch RJ, Yang W: Structure of human RNase H1 complexed with an RNA/DNA hybrid: Insight into HIV reverse transcription. Mol Cell 2007, 28:264-276.
• [27]Julias JG, McWilliams MJ, Sarafianos SG, Alvord WG, Arnold E, Hughes SH: Mutation of amino acids in the connection domain of human immunodeficiency virus type 1 reverse transcriptase that contact the template-primer affects RNase H activity. J Virol 2003, 77:8548-8554.
• [28]Kay LE, Torchia DA, Bax A: Backbone dynamics of proteins as studied by 15 N inverse detected heteronuclear NMR spectroscopy: Application to staphylococcal nuclease. Biochemistry 1989, 28:8972-8979.
• [29]Farrow NA, Muhandiram R, Singer AU, Pascal SM, Kay CM, Gish G, Shoelson SE, Pawson T, Forman-Kay JD, Kay LE: Backbone dynamics of a free and phosphopeptide-complexed src homology 2 domain studied by 15 N NMR relaxation. Biochemistry 1994, 33:5984-6003.
• [30]Cornilescu G, Delaglio F, Bax A: Protein backbone angle restraints from searching a database for chemical shift and sequence homology. J Biomol NMR 1999, 13:289-302.
• [31]Schwieters CD, Kuszewski JJ, Tjandra N, Clore GM: The Xplor-NIH NMR molecular structure determination package. J Magn Reson 2003, 160:66-74.
• [32]Laskowski RA, Rullmann JAC, MacArthur MW, Kaptein R, Thornton JM: AQUA and PROCHECK-NMR: Programs for checking the quality of protein structures solved by NMR. J Biomol NMR 1996, 8:477-486.
• [33]Schrödinger L: The PyMOL molecular graphics system, version 1.3. Mannheim, Germany, Schrödinger, LLC; 2010.
• [34]Hartl MJ, Kretzschmar B, Frohn A, Nowrouzi A, Rethwilm A, Wöhrl BM: AZT resistance of simian foamy virus reverse transcriptase is based on the excision of AZTMP in the presence of ATP. Nucleic Acids Res 2008, 36:1009-1016.
• [35]Emsley P, Cowtan K: Coot: Model-building tools for molecular graphics. Acta Chrystallogr D 2004, 60:2126-2132.