Environment-Dependent Dimerization as a Functional Switch in Leech Cystatin CysHv

Cystatins from hematophagous organisms modulate host proteases and are promising biotechnological tools. CysHv, a secreted cystatin from the Brazilian leech Haementeria vizottoi, was previously characterized as a potent inhibitor of papain and cathepsin L. In this work, it is shown that its inhibitory activity is tightly linked to conformational state. CysHv is purified as an active monomer but forms inactive dimers in a time-, temperature-, and concentration-dependent manner. A domain-swapping-like mechanism was hypothesized, and a disulfide-engineered mutant restricting local flexibility remained predominantly monomeric, supporting the role of conformational plasticity. Dimer formation correlates with loss of inhibitory activity and is accelerated at acidic pH, suggesting that pH modulates the kinetics rather than triggering dimerization. Comparative structural analysis of cystatins from hematophagous organisms revealed recurrent pairs of oppositely charged residues in β-strands flanking the inhibitory loop in secreted cystatins, but these pairs are generally absent in intracellular homologs. Although causality cannot be established and additional determinants likely contribute to dimerization, this pattern is consistent with pH-sensitive modulation of conformational behavior. Together, these findings identify CysHv as a dimer-prone cystatin for which oligomerization, potentially via a domain-swapping-like mechanism, could act as an environment-dependent regulator of inhibitory activity.