PDBsum entry 1rtd

Transferase/DNA

PDB id

1rtd

Contents

			Protein chains

					554 a.a. *


					414 a.a. *

			DNA/RNA

			Ligands

					TTP ×2

			Metals

					_MG ×6

* Residue conservation analysis

References listed in PDB file

Key reference

Title

Structure of a covalently trapped catalytic complex of HIV-1 reverse transcriptase: implications for drug resistance.

Authors

H.Huang, R.Chopra, G.L.Verdine, S.C.Harrison.

Ref.

Science, 1998, 282, 1669-1675. [DOI no: 10.1126/science.282.5394.1669]

PubMed id

9831551

Abstract

A combinatorial disulfide cross-linking strategy was used to prepare a stalled complex of human immunodeficiency virus-type 1 (HIV-1) reverse transcriptase with a DNA template:primer and a deoxynucleoside triphosphate (dNTP), and the crystal structure of the complex was determined at a resolution of 3.2 angstroms. The presence of a dideoxynucleotide at the 3'-primer terminus allows capture of a state in which the substrates are poised for attack on the dNTP. Conformational changes that accompany formation of the catalytic complex produce distinct clusters of the residues that are altered in viruses resistant to nucleoside analog drugs. The positioning of these residues in the neighborhood of the dNTP helps to resolve some long-standing puzzles about the molecular basis of resistance. The resistance mutations are likely to influence binding or reactivity of the inhibitors, relative to normal dNTPs, and the clustering of the mutations correlates with the chemical structure of the drug.



	Figure 1. Fig. 1. RT-DNA tethering reaction. Chemistry of disulfide bond formation between the side chain of an engineered cysteine residue (blue) in helix H (gold) of RT to a thiol group in the minor groove of DNA (activated as the mixed disulfide), which is tethered to N2 of a dG (green) in the template:primer.		Figure 6. Fig. 6. Sites of mutations conferring resistance to various nucleoside analog drugs. (A) "Front" view, corresponding to the orientation in Fig. 4. The polypeptide backbone of the fingers and palm domains (residues 1 to 235) is shown as a red worm, and locations of resistance mutations are indicated by colored squares. The substrates are shown in Corey-Pauling-Koltun representation, with colors as in Fig. 3. The color code for mutations is as follows: light blue for resistance to ddI, ddC, and 3TC; blue for resistance to AZT; and violet for cross resistance to AZT and ddI or ddC. The location of the NNRTI binding site is shown by an arrow. Side chains of the residues at which mutations affect dideoxynucleotide sensitivity project forward: L74 bears on the templating base, and V at this position will also shift Q151 and R72 and hence the dNTP itself; M184 contacts the backbone and base at the primer terminus, and mutation to I or V will also generate a contact to the sugar ring of the dNTP; K65 contacts the -phosphate; and T69D (resistance to ddC) can probably best be explained by assuming a conformational effect on the fingers loop, transmitted to the dNTP by contacts from other fingers residues. (B) "Back" view, from the direction opposite to the one in (A). Side chains of AZT resistance mutations project toward this surface. One of the earliest mutations that appears in patients on AZT monotherapy is K70R. The Lys70 residue projects directly outward in the current model, but mutation to arginine (with five hydrogen-bond donors in fixed orientations on the guanidinium group) could readily induce side-chain reorientation, with contacts to Asp113 or the -phosphate. Subsequent appearance of T215Y/F confers higher levels of resistance. This mutation, likely to affect the rear of the 3' pocket, is frequently "tuned" by appearance of others: K210W (which probably stabilizes the alteration at 215), M41L, and D67N and K219Q (which likely affect the interaction of fingers and palm and hence the formation of the 3' pocket during the polymerization cycle) (16). Figure 4A was prepared with GRASP (50), and Figs. 3, 4B, 5, and 6, with RIBBONS (51).

PROCHECK

	Headers