Background: HIV-1 reverse transcriptase (RT) is a major target for the treatment of acquired immunodeficiency syndrome (AIDS). Resistance mutations in RT compromise treatment, however. Efforts to understand the enzymatic mechanism of RT and the basis for mutational resistance to anti-RT drugs have been hampered by the failure to crystallize a catalytically informative RT-substrate complex.
Results: We present here experiments that allow us to understand the reason for the failure to crystallize such a complex. Based on this understanding, we have devised a new approach for using a combinatorial disulfide cross-linking strategy to trap a catalytic RT*template:primer*dNTP ternary complex, thereby enabling the growth of co-crystals suitable for high-resolution structural analysis. The crystals contain a fully assembled active site poised for catalysis. The cross-link itself appears to be conformationally mobile, and the surrounding region is undistorted, suggesting that the cross-link is a structurally passive device that merely acts to prevent dissociation of the catalytic complex.
Conclusions: The new strategy discussed here has resulted in the crystallization and structure determination of a catalytically relevant RT*template:primer*dNTP complex. The structure has allowed us to analyze possible causes of drug resistance at the molecular level. This information will assist efforts to develop new classes of nucleoside analog inhibitors, which might help circumvent current resistance profiles. The covalent trapping strategy described here may be useful with other protein-DNA complexes that have been refractory to structural analysis.