Ab initio molecular dynamics-based assignment of the protonation state of pepstatin A/HIV-1 protease cleavage site

A recent C-13 NMR experiment (Smith et al. Nature Struct. Biol. 1996. 3, 94 6-950) on the Asp 25-Asp25 ' dyad in pepstatin A/HIV-1 protease measured tw o separate resonance lines. which were interpreted as being a singly proton ated dyad. We address this issue by performing ab initio molecular dynamics calculations on models for this site accompanied by calculations of C-13 N MR chemical shifts and isotopic shifts. We find that already on the picosec ond time-scale the model proposed by Smith et al. is not stable and evolves toward a different monoprotonated form whose NMR pattern differs from the experimental one. We suggest, instead a different protonation state in whic h both aspartic groups are protonated. Despite the symmetric protonation st ate. the calculated C-13 NMR properties are in good agreement with the expe riment. We rationalize this result using a simple valence bond model, which explains the chemical inequality of the two C sites. The model calculation s, together with our calculations on the complex, allow also the rationaliz ation of C-13 NMR properties on other HIV-1 PR/inhibitor complexes. Both pu tative binding of the substrate to the free enzyme, which has the dyad sing ly protonated (Piana. S.; Carloni, P. Proteins: Struct., Funct., Genet. 200 0. 39, 26-36), and pepstatin A binding to the diprotonated form are consist ent with the inverse solvent isotope effect on the onset of inhibition of p epsin by pepstatin and the kinetic iso-mechanism proposed for aspartic prot eases.