Mutagenesis of key residues identifies the connection subdomain of HIV-1 reverse transcriptase as the site of inhibition by heme

We have recently demonstrated that metalloporphyrins are potent inhibitors of both human immunodeficiency virus type 1 (HIV-1) and human immunodeficie ncy virus type 2 (HIV-2) reverse transcriptases (RTs) [Argyris, E.G., Vande rkooi, J.M., Venkateswaran, P.S., Kay, B.K., and Paterson, Y. (1999) J. Bio l. Chem. 274, 1549-1556]. In addition, by screening a phage peptide library we discovered that a peptide with sequence similarity to residues 398-407 from the connection subdomain of HIV RTs binds heme. These findings suggest ed that this highly conserved region may be the binding site for metallopor phyrins and a novel site for inhibition of enzymatic activity. Our most rec ent data presented here confirm this suggestion. Screening of HIV-1 RT 398- 407 peptide analogs by fluorescence assays demonstrates that Trp residues a t positions 401 and 402 are important for heme binding. Furthermore, site-d irected mutagenesis of these residues verified these findings and indicated that heme inhibits HIV-1 RT by binding on the connection subdomain of the p66 subunit of the enzyme but not on the p51 subunit. This was also confirm ed by analyzing the binding affinities of heme for mutant HIV-1 RT heterodi mers, using intrinsic fluorescence assays. The clear identification of the connection domain as a novel inhibition site is crucial in understanding th e mechanism of heme binding and enzymatic inhibition and will facilitate th e generation of novel porphyrin-based inhibitors of RT.