Ternary complex formation

The formation of the ternary RT-DNA-dNTP complex resembles ternary complexes of other DNA polymerases.35-37 A binding pocket is formed by closure of the fingers domain towards the palm and thumb domains to accommodate the 3'-hydroxyl group of an incoming nucleotide. The distal portion of the fingers domain bends inward to the palm domain and the

Figure 8.3 Conformational changes inside HIV-1 RT p66 subunit during DNA polymerization. (a) unliganded form of RT; (b) binary complex RT'/DNAn; (c) ternary complex RT*/DNAn/dNTP; (a ^ b) rotation of the p66 thumb domain away from the fingers domain induced by DNA binding (see the arrow); (b ^ c) hinge movement of the fingers domain to close down to the polymerase active site at the floor of the palm domain induced by nucleotide binding (see the arrow). The thumb domain in a ^ b is in light gray in the absence of DNA, and dark gray in the presence of DNA. The fingers domain in b ^ c is in light gray in the absence of dNTP, and dark gray in the presence of dNTP. RT' denotes the enzyme form after the conformational change induced by DNA binding. RT* represents the enzyme form after the conformational change induced by nucleotide binding. The protein and DNA backbones are shown in ribbon diagrams. The DNA template and primer are in dark gray and light gray, respectively. (Courtesy of Drs. K. Das, S. Sarafianos, and E. Arnold, Center for Advanced Biotechnology and Medicine, Rutgers University, Piscataway, New Jersey.)

Figure 8.3 Conformational changes inside HIV-1 RT p66 subunit during DNA polymerization. (a) unliganded form of RT; (b) binary complex RT'/DNAn; (c) ternary complex RT*/DNAn/dNTP; (a ^ b) rotation of the p66 thumb domain away from the fingers domain induced by DNA binding (see the arrow); (b ^ c) hinge movement of the fingers domain to close down to the polymerase active site at the floor of the palm domain induced by nucleotide binding (see the arrow). The thumb domain in a ^ b is in light gray in the absence of DNA, and dark gray in the presence of DNA. The fingers domain in b ^ c is in light gray in the absence of dNTP, and dark gray in the presence of dNTP. RT' denotes the enzyme form after the conformational change induced by DNA binding. RT* represents the enzyme form after the conformational change induced by nucleotide binding. The protein and DNA backbones are shown in ribbon diagrams. The DNA template and primer are in dark gray and light gray, respectively. (Courtesy of Drs. K. Das, S. Sarafianos, and E. Arnold, Center for Advanced Biotechnology and Medicine, Rutgers University, Piscataway, New Jersey.)

polymerase active site (see Figure 8.3), inducing a repositioning of the primer 3'-terminus with respect to the rest of the polymerase active site. The palm domain shifts slightly to rearrange the side chains of the active site residues.

The incoming nucleotide also brings in two divalent metal ions, one catalytic and the other for nucleotide binding, to coordinate the oxygen ions of the triphosphate of the nucleotide; the side chains of strictly conserved Asp185 and Asp110; and the backbone carbonyl oxygen of Val111. The metal ions are positioned closely to the 3'-hydroxyl group of the primer terminus. The conformational change induced upon nucleotide binding specifically positions Asp110 for metal chelation. The nucleotide base stacks against the primer terminus and residues Arg72 and Gln151. The triphosphate portion is contacted by Lys65 (g phosphate), Arg72 (a phosphate), two main-chain NH groups, and two metal ions.

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