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Mutations in the Flap Domain of the HIV Protease Can Compensate for M46F R. MARTIN1, J. COLLINS2, L. EVERITT3, and A. KAPLAN3. 1Univ. of California at Los Angeles Sch. of Med.; 2NCI, Frederick, MD; and 3Univ. of North Carolina Sch. of Med., Chapel Hill One of the common structural features of retroviral proteases is the flap domain. Structural studies of bound and native enzyme indicate that the flaps move approximately 7 Å during the course of binding, cleavage and release of the substrate. Position 46 of the flap is of particular interest as an Ile substitution at this position appears frequently during selection for protease inhibitor resistance both in vitro and in vivo even though it makes no direct contact with substrate or inhibitor. In addition, this substitution appears to stabilize the flaps in a closed position. Overall, these observations suggest that the role of position 46 as a "hinge" of the mobile flap has important consequences for the development of protease inhibitor resistant enzymes as well as the mechanics of flap motion and substrate recognition. We constructed an infectious clone containing an M46F substitution. Upon culture, the mutant reverted to wild type growth kinetics. Sequencing and subsequent construction of recombinants confirmed that either a same site mutation (M46N) or a double mutation (M46F/K55N) were responsible for the wild type growth kinetics. Although it has been suggested that position 46 forms a hydrophobic pocket that interacts with the substrate, the ability of the enzyme to function with an asparagine at this position makes this unlikely. Structural modeling suggests that the K55N substitution allows the Phe at position 46 to rotate, thereby avoiding a steric interaction between F46 and F53. In addition, we have constructed a protease dimer composed of two monomers joined by a 5 amino acid linker. Expression of this construct in E. coli was toxic to the bacterial cells and almost completely prevented cell growth. Using this system, we introduced mutations at positions 46 and 55. Our data demonstrate that a functional protease dimer may contain genetically heterogeneous flaps, as long as both are functional. Key Words: flap, HIV protease, resistance |
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© 7th
Conference on Retroviruses and Opportunistic Infections, |