title: 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 D75(a)-K24(b), 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, K24(b) that interacts with D75(a) of the loop is the specificity determining residue that interacts with trypsin to inhibit its activity. The inter-monomer salt bridge D75(a)-K24(b) 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.
authors.affiliation: Department of Protein Chemistry and Technology, CSIR-Central Food Technological Research Institute, Mysore 570020, India.
authors.forename: Vinod
authors.initials: V
authors.lastname: Kumar
authors.affiliation: Department of Biological Sciences & BioEngineering, Indian Institute of Technology, Kanpur 208016, India.
authors.forename: Saravanan
authors.initials: S
authors.lastname: Murugeson
authors.affiliation: Department of Biological Sciences & BioEngineering, Indian Institute of Technology, Kanpur 208016, India.
authors.forename: Neha
authors.initials: N
authors.lastname: Vithani
authors.affiliation: Department of Biological Sciences & BioEngineering, Indian Institute of Technology, Kanpur 208016, India; Center of Excellence for Chemical Biology, Indian Institute of Technology, Kanpur 208016, India.
authors.forename: Balaji
authors.initials: B
authors.lastname: Prakash
authors.affiliation: Department of Protein Chemistry and Technology, CSIR-Central Food Technological Research Institute, Mysore 570020, India. Electronic address: lrgowda2k11@gmail.com.
authors.forename: Lalitha R
authors.initials: LR
authors.lastname: Gowda
mesh_terms: D000818:Animals; D001665:Binding Sites; D002417:Cattle; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D007887:Fabaceae / Q000737:chemistry; D015870:Gene Expression; D056004:Molecular Dynamics Simulation; D009154:Mutation; D011480:Protease Inhibitors / Q000737:chemistry* / Q000302:isolation & purification; D011485:Protein Binding; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D014357:Trypsin / Q000737:chemistry*
chemical_list: D011480:Protease Inhibitors; D011994:Recombinant Proteins; D014357:Trypsin
keywords: Knob-in-the-hole; Molecular dynamics simulations; Salt bridge; Site-directed mutagenesis; Substrate-induced monomerisation; Trypsin/chymotrypsin inhibitor