Human granzyme B (hGzmB), which is present in various immune cells, has
attracted much attention due to its role in various pathophysiological
conditions. The hGzmB activity is triggered at a catalytic triad (His59,
Asp103, Ser198), cleaving its specific substrates. To date, the drug
design strategy against hGzmB mainly targets the catalytic triad, which
causes the non-specificity problem of inhibitors due to the highly
conserved active site in serine proteases. In the present work,
microsecond classical molecular dynamics simulations are devoted to
exploring the structural dynamics of the hGzmB catalytic cycle in the
presence of Ac-IEPD-AMC, a known substrate (active hGzmB), and
Ac-IEPD-CHO, a known inhibitor (inactive hGzmB). By comparing active and
inactive forms of hGzmB in the six different stages of the hGzmB
catalytic cycle, we revealed, for the very first time, an additional
network of interactions involving Arg216, a residue located outside the
conventional binding site. Upon activation, the His59∙∙∙Asp103 hydrogen
bond is broken due to the formation of the Asp103∙∙∙Arg216 salt bridge,
expanding the active site to facilitate the substrate-binding. On the
contrary, the binding of inhibitor Ac-IEPD-CHO to hGzmB prevents the
Arg216-mediated interactions within the catalytic triad, thus preventing
hGzmB activity. In silico Arg216Ala mutation confirms the role of Arg216
in enzyme activity, as the substrate Ac-IEPD-AMC failed to bind to the
mutated hGzmB. Importantly, as Arg216 is not conserved amongst the
various granzymes, the current findings can be a major step to guide the
design of hGzmB specific therapeutics.
Local frustration determines loop opening during the catalytic cycle of an oxidoreductase
