A salt-bridge stabilized C-terminal hook is critical for the dimerization of a Bowman Birk inhibitor

Abstract

Legume Bowman-Birk inhibitors (BBIs) that inhibit mammalian proteases exist as dimers in solution. The structural basis governing dimerization of HGI-III (horsegram seed BBI) was investigated. An intra-monomer salt bridge (D76–K71) stabilizes an atypical hook-like conformation at the C-terminus. We postulate that this hook, positions D75 to enable an inter-monomer salt-bridge D75a–K24b, which results in dimerization. We verify this by K71A and D76A mutations of HGI-III. The mutants were both monomers, likely due to destabilization of the C-terminal hook. Dimerization was sustained in a double mutant K71D/D76K that was anticipated to form a similar hook critical for dimerization. Conversely, K24b that interacts with D75a of the loop is the specificity determining residue that interacts with trypsin to inhibit its activity. The inter-monomer salt bridge D75a–K24b must be disrupted for the inhibition of trypsin, requiring HGI-III to transition into a monomer. Size exclusion studies and a model of HGI-III-trypsin complex support this notion. Interestingly, isoforms of the inhibitor present in germinated seeds (HGGIs) are monomers; and most strikingly, the C-termini of these inhibitors are truncated with the loss the C-terminal hook critical for dimerization. The tendency of HGI-III to self-associate seems to relate to its physiological function of a storage protein.