title: Structure/function studies of human immunodeficiency virus type 1 reverse transcriptase. Alanine scanning mutagenesis of an alpha-helix in the thumb subdomain.
abstract: Human immunodeficiency virus type 1 reverse transcriptase has subunits of 66 and 51 kDa (p66 and p51, respectively). Structural studies indicate that each subunit consists of common subdomains. The polymerase domain of p66 forms a nucleic acid binding cleft, and, by analogy with a right hand, the subdomains are referred to as fingers, palm, and thumb (Kohlstaedt, L. A., Wang, J., Friedman, J. M., Rice, P. A., and Steitz, T. A. (1992) Science 256, 1783-1790). Residues 257-266 correspond to a highly conserved region of primary structure among retroviral pol genes. Crystallographic evidence indicates that these residues are in the thumb subdomain and form part of an alpha-helix (alpha H), which interacts with DNA (Jacobo-Molina, A., Ding, J., Nanni, R. G., Clark, A. D., Jr., Lu, X., Tantillo, C., Williams, R. L., Kamer, G., Ferris, A. L., Clark, P., Hizi, A., Hughes, S. H., and Arnold, E. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 6320-6324). To define the role of this region during catalytic cycling, we performed systematic site-directed mutagenesis from position 253 through position 271 by changing each residue, one by one, to alanine. Each mutant protein was expressed and purified, and their substrate-specific activities were surveyed. The results are consistent with alpha H (residues 255-268) of p66 interacting with the template and/or primer strand. The core of alpha H appears to play an important role in template-primer binding (residues Gln-258, Gly-262, and Trp-266), and in protein-protein interactions (residues Val-261 and Leu-264). The periodicity of the effects observed suggest that a segment of one face of alpha H interacts with the template-primer. The lower fidelity observed with alanine mutants of Gly-262 and Trp-266 correlated with an over 200-fold increase in the dissociation rate constant for template-primer relative to wild type enzyme and suggests that enzyme-DNA interactions in the template-primer stem are important fidelity determinants.
authors.affiliation: Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston 77555-1068.
authors.forename: W A
authors.initials: WA
authors.lastname: Beard
authors.forename: S J
authors.initials: SJ
authors.lastname: Stahl
authors.forename: H R
authors.initials: HR
authors.lastname: Kim
authors.forename: K
authors.initials: K
authors.lastname: Bebenek
authors.forename: A
authors.initials: A
authors.lastname: Kumar
authors.forename: M P
authors.initials: MP
authors.lastname: Strub
authors.forename: S P
authors.initials: SP
authors.lastname: Becerra
authors.forename: T A
authors.initials: TA
authors.lastname: Kunkel
authors.forename: S H
authors.initials: SH
authors.lastname: Wilson
mesh_terms: D000409:Alanine; D000595:Amino Acid Sequence; D001483:Base Sequence; D003062:Codon / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000201:enzymology*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D017433:Protein Structure, Secondary; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D013329:Structure-Activity Relationship
chemical_list: D003062:Codon; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; D000409:Alanine