SEQUENCE REQUIREMENTS OF THE HIV-1 PROTEASE FLAP REGION DETERMINED BYSATURATION MUTAGENESIS AND KINETIC-ANALYSIS OF FLAP MUTANTS

The retroviral proteases (PRs) have a structural feature called the fl ap, which consists of a short antiparallel beta-sheet with a turn, The flap extends over the substrate binding cleft and must be flexible to allow entry and exit of the polypeptide substrates and products, We a nalyzed the sequence requirements of the amino acids within the flap r egion (positions 46-56) of the HIV-1 PR, The phenotypes of 131 substit ution mutants were determined using a bacterial expression system, Fou r of the mutant PRs with mutations in different regions of the flap we re selected for kinetic analysis, Our phenotypic analysis, considered in the context of published structures of the HIV-1 PR with a bound su bstrate analogs, shows that: (i) Met-46 and Phe-53 participate in hydr ophobic interactions on the solvent-exposed face of the flap; (ii) Ile -47, Ile-54, and Val-56 participate in hydrophobic interactions on the inner face of the flap; (iii) Ile-50 has hydrophobic interactions at the distance of both the delta and gamma carbons; (iv) the three glyci ne residues in the beta-turn of the flap are virtually intolerant of s ubstitutions, Among these mutant PRs, we have identified changes in bo th k(cat) and K-m. These results establish the nature of the side chai n requirements at each position in the flap and document a role for th e flap in both substrate binding and catalysis.