Human Immunodeficiency Virus Ribonuclease H (HIV RNase H)
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| X-ray HIV RNaseH Structure PDBID:1HRH, Davies et al 1991 Science 252:88 |
| X-ray HIV Reverse Transcriptase Structure
PDBID:1RTD, H. Huang et al 1998 Science 282:1669 The p66 domain is colored yellow and the P51 domain is colored red. The RNase H domain within p66 is colored blue. The The DNA is shown as a wire drawing and the protein is shown as a ribbon. |
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The humam immunodeficiency virus (HIV-1) reverse transcriptase is a multifunctional enzyme, which plays a crucial role in the life cycle of the virus. The enzyme exhibits both DNA- and RNA-dependent DNA polymerase and ribonuclease H (RNase H) activity, which is essential for reverse transcription of the viral genomic RNA. Unlike in E. coli, RNase H activity is required for viral replication, as evident by the failure of mutant provirus defective for RNase H function to produce infective virus particles. The RNase H domain catalyzes the cleavage of the RNA portion of a DNA/RNA hybrid, a process which is required at several stages during reverse transcription, and displays both endonuclease and 3'-> 5' exonuclease activity. The native form of the enzyme is a heterodimer consisting of p66 and p51 subunits. This arises from asymmetric processing of the p66 homodimer by the HIV protease. The RNase H activity resides in the 15-kDa C-terminal region of the p66 subunit.
There exists a significant degree of structural and sequence homology between E. coli RNase H and the C-terminal region of reverse transcriptase from HIV. But, the 15-kDa C-terminal segment of HIV reverse transcriptase does not exhibit RNase H activity when expressed separately. The full HIV RNase H activity can be reconstituted by recombining the RNase H and DNA polymerase domains, by the addition of a C-terminal His tag or by the formation of a chimera containing the C-terminal a-helix from E. coli RNase H.
We determined the 1H, 15N, 13C and 13CO backbone resonance assignments and secondary structure of the HIV RNase H domain (Trp-113 -> Ala mutant) using 3D heteronuclear NMR spectroscopy. The NMR secondary structure was found to be in agreement with the high-resolution X-ray crystal structure. The principal difference between the two structures is located at the C-terminus of the protein. The C-terminal a-helix observed in the crystal structure is absent, and residues Val-114 to GLy-133 are ill-defined in the NMR structure. Some of this disorder is also observed in the crystal structure, where a putative loop from residues 116 to 120 is not visible in the electron density map.
Dynamic analysis of the HIV RNase H domain by NMR indicates that the RNase H domain experiences extensive mobility throughout its structure as evidence by the 93 residues that exhibit multiple modes of motion. Distinctly mobile region of the protein are identified by large decreases in the overall order parameter (S2) and corresponding to the C-terminal residues and the loop regions between b-strands b1 and b2 and between a-helix aB and b-strand b4. The high mobility of the C-terminus is of particular interest since one stretch of the sequence in this region of the protein consitiutes part of the proposed substrate binding site. Thus, a highly flexible or partially folded binding pocket could explain the lack of enzymatic activity observed for this particular HIV-1 RNase H domain.
(from: Biochemistry (1992) 31(38), 9150-9157. & Journal of Molecular Biology (1991) 221, 1081-1090.)