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Crystal structure of xenotropic murine leukaemia virus-related virus (XMRV) ribonuclease H.

Kim JH, Kang S, Jung SK, Yu KR, Chung SJ, Chung BH, Erikson RL, Kim BY, Kim SJ - Biosci. Rep. (2012)

Bottom Line: RNase H (retroviral ribonuclease H) cleaves the phosphate backbone of the RNA template within an RNA/DNA hybrid to complete the synthesis of double-stranded viral DNA.In the present study we have determined the complete structure of the RNase H domain from XMRV (xenotropic murine leukaemia virus-related virus) RT (reverse transcriptase).The basic protrusion motif of the XMRV RNase H domain is folded as a short helix and an adjacent highly bent loop.

View Article: PubMed Central - PubMed

Affiliation: Medical Proteomics Research Center, Korea Research Institute of Bioscience and Biotechnology, Yuseong-Gu, Daejeon, Republic of Korea.

ABSTRACT
RNase H (retroviral ribonuclease H) cleaves the phosphate backbone of the RNA template within an RNA/DNA hybrid to complete the synthesis of double-stranded viral DNA. In the present study we have determined the complete structure of the RNase H domain from XMRV (xenotropic murine leukaemia virus-related virus) RT (reverse transcriptase). The basic protrusion motif of the XMRV RNase H domain is folded as a short helix and an adjacent highly bent loop. Structural superposition and subsequent mutagenesis experiments suggest that the basic protrusion motif plays a role in direct binding to the major groove in RNA/DNA hybrid, as well as in establishing the co-ordination among modules in RT necessary for proper function.

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Superposition of XMRV RNase H with complexed forms of HIV-1 RT (A, B) and human RNase H (C)(A, B) The p66/p51 heterodimer of HIV-1 is represented as a surface diagram, and DNA (blue)/RNA (red) template bound to HIV-1 is represented in backbone form. The p51 subunit is shown in light grey, whereas different colours are used to denote distinct domains in the p66 subunit. The primer grip (residues 349–367) in thumb and connection domains corresponding to the basic protrusion in XMRV RNase H is shown as a ribbon. The basic protrusion of RNase H of XMRV (residues 593–612) is shown in green and the remainder is coloured magenta. The DEDD motif and magnesium ion of XMRV RNase H, shown as sticks and a sphere, respectively, indicate the location of the catalytic core. (C) Ribbons of human RNase H are coloured grey whereas those of XMRV RNase H are coloured magenta. DNA and RNA strands bound to human RNase H are shown in red and blue, respectively. The residues that participate in binding to the DNA/RNA hybrid are coloured black (XMRV) or red (human).
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Figure 3: Superposition of XMRV RNase H with complexed forms of HIV-1 RT (A, B) and human RNase H (C)(A, B) The p66/p51 heterodimer of HIV-1 is represented as a surface diagram, and DNA (blue)/RNA (red) template bound to HIV-1 is represented in backbone form. The p51 subunit is shown in light grey, whereas different colours are used to denote distinct domains in the p66 subunit. The primer grip (residues 349–367) in thumb and connection domains corresponding to the basic protrusion in XMRV RNase H is shown as a ribbon. The basic protrusion of RNase H of XMRV (residues 593–612) is shown in green and the remainder is coloured magenta. The DEDD motif and magnesium ion of XMRV RNase H, shown as sticks and a sphere, respectively, indicate the location of the catalytic core. (C) Ribbons of human RNase H are coloured grey whereas those of XMRV RNase H are coloured magenta. DNA and RNA strands bound to human RNase H are shown in red and blue, respectively. The residues that participate in binding to the DNA/RNA hybrid are coloured black (XMRV) or red (human).

Mentions: Overall, the conformation of this protrusion in XMRV RNase H is highly similar to that of E. coli (Figure 2B). It includes four arginine residues (Arg599, Arg600, Arg601 and Arg609) (Figures 1C and 2A), hence the name, basic protrusion. All these residues are exposed to solvent with no contact with other residues. A comparison of the structure of XMRV RNase H with that of the HIV-1 RT RNase H domain and human RNase H, shown with RNA/DNA hybrid substrates, showed a good alignment in both cases (Figure 3). We took advantage of this to identify the crucial residues responsible for binding to the RNA/DNA hybrid. The superposition showed that residues 606–612 of XMRV RNase H have the potential to make contact with the RNA/DNA hybrid molecule present in the HIV-1 RT model. It is approx.−6 to −9 base pairs away from the scissile phosphate position (between −1 and +1 nucleotides in the RNA strand). Notably, Arg609 and Lys612, in particular, make good contact with the substrate (Figure 3B). The side chains of three consecutive arginine residues point towards the connection domain. Therefore it is reasonable to infer that the basic protrusion motif may stabilize the RT structure and binding with an RNA/DNA hybrid, supporting proper enzymatic function. In HIV-1 RT lacking the basic protrusion motif, the residues Gly359, Ala360 and His361 in the connection domain make alternative interactions with the corresponding region and compensate for the absence of a basic protrusion motif.


Crystal structure of xenotropic murine leukaemia virus-related virus (XMRV) ribonuclease H.

Kim JH, Kang S, Jung SK, Yu KR, Chung SJ, Chung BH, Erikson RL, Kim BY, Kim SJ - Biosci. Rep. (2012)

Superposition of XMRV RNase H with complexed forms of HIV-1 RT (A, B) and human RNase H (C)(A, B) The p66/p51 heterodimer of HIV-1 is represented as a surface diagram, and DNA (blue)/RNA (red) template bound to HIV-1 is represented in backbone form. The p51 subunit is shown in light grey, whereas different colours are used to denote distinct domains in the p66 subunit. The primer grip (residues 349–367) in thumb and connection domains corresponding to the basic protrusion in XMRV RNase H is shown as a ribbon. The basic protrusion of RNase H of XMRV (residues 593–612) is shown in green and the remainder is coloured magenta. The DEDD motif and magnesium ion of XMRV RNase H, shown as sticks and a sphere, respectively, indicate the location of the catalytic core. (C) Ribbons of human RNase H are coloured grey whereas those of XMRV RNase H are coloured magenta. DNA and RNA strands bound to human RNase H are shown in red and blue, respectively. The residues that participate in binding to the DNA/RNA hybrid are coloured black (XMRV) or red (human).
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Related In: Results  -  Collection

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Figure 3: Superposition of XMRV RNase H with complexed forms of HIV-1 RT (A, B) and human RNase H (C)(A, B) The p66/p51 heterodimer of HIV-1 is represented as a surface diagram, and DNA (blue)/RNA (red) template bound to HIV-1 is represented in backbone form. The p51 subunit is shown in light grey, whereas different colours are used to denote distinct domains in the p66 subunit. The primer grip (residues 349–367) in thumb and connection domains corresponding to the basic protrusion in XMRV RNase H is shown as a ribbon. The basic protrusion of RNase H of XMRV (residues 593–612) is shown in green and the remainder is coloured magenta. The DEDD motif and magnesium ion of XMRV RNase H, shown as sticks and a sphere, respectively, indicate the location of the catalytic core. (C) Ribbons of human RNase H are coloured grey whereas those of XMRV RNase H are coloured magenta. DNA and RNA strands bound to human RNase H are shown in red and blue, respectively. The residues that participate in binding to the DNA/RNA hybrid are coloured black (XMRV) or red (human).
Mentions: Overall, the conformation of this protrusion in XMRV RNase H is highly similar to that of E. coli (Figure 2B). It includes four arginine residues (Arg599, Arg600, Arg601 and Arg609) (Figures 1C and 2A), hence the name, basic protrusion. All these residues are exposed to solvent with no contact with other residues. A comparison of the structure of XMRV RNase H with that of the HIV-1 RT RNase H domain and human RNase H, shown with RNA/DNA hybrid substrates, showed a good alignment in both cases (Figure 3). We took advantage of this to identify the crucial residues responsible for binding to the RNA/DNA hybrid. The superposition showed that residues 606–612 of XMRV RNase H have the potential to make contact with the RNA/DNA hybrid molecule present in the HIV-1 RT model. It is approx.−6 to −9 base pairs away from the scissile phosphate position (between −1 and +1 nucleotides in the RNA strand). Notably, Arg609 and Lys612, in particular, make good contact with the substrate (Figure 3B). The side chains of three consecutive arginine residues point towards the connection domain. Therefore it is reasonable to infer that the basic protrusion motif may stabilize the RT structure and binding with an RNA/DNA hybrid, supporting proper enzymatic function. In HIV-1 RT lacking the basic protrusion motif, the residues Gly359, Ala360 and His361 in the connection domain make alternative interactions with the corresponding region and compensate for the absence of a basic protrusion motif.

Bottom Line: RNase H (retroviral ribonuclease H) cleaves the phosphate backbone of the RNA template within an RNA/DNA hybrid to complete the synthesis of double-stranded viral DNA.In the present study we have determined the complete structure of the RNase H domain from XMRV (xenotropic murine leukaemia virus-related virus) RT (reverse transcriptase).The basic protrusion motif of the XMRV RNase H domain is folded as a short helix and an adjacent highly bent loop.

View Article: PubMed Central - PubMed

Affiliation: Medical Proteomics Research Center, Korea Research Institute of Bioscience and Biotechnology, Yuseong-Gu, Daejeon, Republic of Korea.

ABSTRACT
RNase H (retroviral ribonuclease H) cleaves the phosphate backbone of the RNA template within an RNA/DNA hybrid to complete the synthesis of double-stranded viral DNA. In the present study we have determined the complete structure of the RNase H domain from XMRV (xenotropic murine leukaemia virus-related virus) RT (reverse transcriptase). The basic protrusion motif of the XMRV RNase H domain is folded as a short helix and an adjacent highly bent loop. Structural superposition and subsequent mutagenesis experiments suggest that the basic protrusion motif plays a role in direct binding to the major groove in RNA/DNA hybrid, as well as in establishing the co-ordination among modules in RT necessary for proper function.

Show MeSH
Related in: MedlinePlus