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

Vinod, Kumar and Saravanan, Murugeson and Neha, Vithani and Balaji, Prakash and Lalitha Gowda, R. (2015) A salt-bridge stabilized C-terminal hook is critical for the dimerization of a Bowman Birk inhibitor. Archives of Biochemistry and Biophysics, 566. pp. 15-25.

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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 mono- mers, 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.

Item Type:	Article
Uncontrolled Keywords:	Knob-in-the-hole, Site-directed mutagenesis, Molecular dynamics simulations, Salt bridge, Substrate-induced monomerisation, Trypsin/chymotrypsin inhibitor
Subjects:	500 Natural Sciences and Mathematics > 04 Chemistry and Allied Sciences > 29 Protein Chemistry
Divisions:	Protein Chemistry and Technology Molecular Nutrition
Depositing User:	Somashekar K S
Date Deposited:	10 Mar 2025 08:45
Last Modified:	10 Mar 2025 08:45
URI:	http://ir.cftri.res.in/id/eprint/19240

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