Local and spatial factors determining HIV-1 protease substrate recognition

Insertional mutagenesis of the Escherichia coli thymidylate synthase (TS) w as used to address substrate recognition of HIV-1 protease in a well charac terized structural context. By modifying the TS conformation while maintain ing its enzymic activity, we investigated the influence of protein folding on protease-substrate recognition. A slight destabilization of the TS struc ture permitted the cleavage of a target site, which was resistant in the na tive TS. This result supports a dynamic interpretation of HIV-1 protease sp ecificity. Exposure time of the potential cleavage site, which depends on t he stability of the global conformation, must be compatible with the cleava ge kinetics, which are determined by the local sequence. Cleavage specifici ty has been described as the consequence of cumulative interactions, global ly favourable, between at least six amino acids around the cleavage site. T o investigate influence of local sequence, we introduced insertions of vari able lengths in two exposed loops of the TS. In both environments, insertio n of only two amino acids could determine specific cleavage. We then insert ed libraries of dipeptides naturally cleaved by the HIV-1 protease in order to assess the limitations of established classifications of substrates in different conformational contexts.