Evolutionarily conserved functional mechanics across pepsin-like and retroviral aspartic proteases

The biological function of the aspartic protease from HIV-1 has recently been related to the conformational flexibility of its structural scaffold. Here, we use a multistep strategy to investigate whether the same mechanism affects the functionality in the pepsin-like fold. (i) We identify the set of conserved residues by using sequence-alignment techniques. These residues cluster in three distinct regions: near the cleavage-site cavity, in the four -sheets cross-linking the two lobes, and in a solvent-exposed region below the long -hairpin in the N-terminal lobe. (ii) We elucidate the role played by the conserved residues for the enzymatic functionality of one representative member of the fold family, the human -secretase, by means of classical molecular dynamics (MD). The conserved regions exhibit little overall mobility and yet are involved into the most important modes of structural fluctuations. These modes influence the substratecatalytic aspartates distance through a relative rotation of the N- and C-terminal lobes. (iii) We investigate the effects of this modulation by estimating the reaction free energy at different representative substrate/ enzyme conformations. The activation free energy is strongly affected by large-scale protein motions, similarly to what has been observed in the HIV-1 enzyme. (iv) We extend our findings to all other members of the two eukaryotic and retroviral fold families by recurring to a simple, topology-based, energy functional. This analysis reveals a sophisticated mechanism of enzymatic activity modulation common to all aspartic proteases. We suggest that aspartic proteases have been evolutionarily selected to possess similar functional motions despite the observed fold variations.