Structural and functional characterization of an RNase HI domain from the bifunctional protein Rv2228c from Mycobacterium tuberculosis.
The open reading frame Rv2228c from Mycobacterium tuberculosis is predicted to encode a protein composed of two domains, each with individual functions, annotated through sequence similarity searches. The N-terminal domain is homologous with prokaryotic and eukaryotic RNase H domains and the C-terminal domain with alpha-ribazole phosphatase (CobC). The N-terminal domain of Rv2228c (Rv2228c/N) and the full-length protein were expressed as fusions with maltose binding protein (MBP). Rv2228c/N was shown to have RNase H activity with a hybrid RNA/DNA substrate as well as double-stranded RNase activity. The full-length protein was shown to have additional CobC activity. The crystal structure of the MBP-Rv2228c/N fusion protein was solved by molecular replacement and refined at 2.25-A resolution (R = 0.182; R(free) = 0.238). The protein is monomeric in solution but associates in the crystal to form a dimer. The Rv2228c/N domain has the classic RNase H fold and catalytic machinery but lacks several surface features that play important roles in the cleavage of RNA/DNA hybrids by other RNases H. The absence of either the basic protrusion of some RNases H or the hybrid binding domain of others appears to be compensated by the C-terminal CobC domain in full-length Rv2228c. The double-stranded-RNase activity of Rv2228c/N contrasts with classical RNases H and is attributed to the absence in Rv2228c/N of a key phosphate binding pocket.
Watkins
Harriet A
HA
Maurice Wilkins Centre for Molecular Biodiscovery and School of Biological Sciences, 3A Symonds Street, Private Bag 92019, Auckland, New Zealand.
Baker
Edward N
EN
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain / Q000235:genetics / Q000502:physiology; D018360:Crystallography, X-Ray; D007700:Kinetics; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000378:metabolism*; D033902:Periplasmic Binding Proteins / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary / Q000235:genetics / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016914:Ribonuclease H / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D000637:Transaminases / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D058619:Maltose-Binding Proteins; D033902:Periplasmic Binding Proteins; D011993:Recombinant Fusion Proteins; C079403:CobC protein, Bacteria; D000637:Transaminases; D016914:Ribonuclease H; C078856:ribonuclease HI