title: An in silico approach to understand the structure-function properties of a serine protease (Bacifrinase) from Bacillus cereus and experimental evidence to support the interaction of Bacifrinase with fibrinogen and thrombin.
abstract: Microbial fibrinogenolytic serine proteases find therapeutic applications in the treatment of thrombosis- and hyperfibrinogenemia-associated disorders. However, analysis of structure-function properties of an enzyme is utmost important before its commercial application. In this study, an attempt has been made to understand the structure of a fibrinogenolytic protease enzyme, "Bacifrinase" from Bacillus cereus strain AB01. From the molecular dynamics trajectory analysis, the modelled three-dimensional structure of the protease was found to be stable and the presence of a catalytic triad made up of Asp102, His83 and Ser195 suggests that it is a serine protease. To understand the mechanism of enzyme-substrate and enzyme-inhibitor interactions, the equilibrated protein was docked with human fibrinogen (the physiological substrate of this enzyme), human thrombin and with ten selective protease inhibitors. The Bacifrinase-chymostatin interaction was the strongest among the selected protease inhibitors. The serine protease inhibitor phenyl methane sulphonyl fluoride was found to interact with the Ser134 residue of Bacifrinase. Furthermore, protein-protein docking study revealed the fibrinogenolytic property of Bacifrinase and its interaction with Aα-, Bβ- and Cγ-chains human fibrinogen to a different extent. However, biochemical analysis showed that Bacifrinase did not hydrolyse the γ-chain of fibrinogen. The in silico and spectrofluorometric studies also showed interaction of Bacifrinase with thrombin as well as fibrinogen with a Kd value of 16.5 and .81 nM, respectively. Our findings have shed light on the salient structural features of Bacifrinase and confirm that it is a fibrinogenolytic serine protease.
authors.affiliation: a Microbial Biotechnology and Protein Research Laboratory, Department of Molecular Biology and Biotechnology, School of Sciences , Tezpur University , Tezpur 784028 , Assam , India.
authors.forename: Bandana
authors.initials: B
authors.lastname: Bora
authors.affiliation: b Molecular Modelling and Simulation Lab, Department of Molecular Biology and Biotechnology, School of Sciences , Tezpur University , Tezpur 784028 , Assam , India.
authors.forename: Akash Deep
authors.initials: AD
authors.lastname: Biswas
authors.affiliation: c Department of Biotechnology and Bioinformatics , North Eastern Hill University , Shillong 793022 , Meghalaya , India.
authors.forename: Arun Bahadur
authors.initials: AB
authors.lastname: Gurung
authors.affiliation: c Department of Biotechnology and Bioinformatics , North Eastern Hill University , Shillong 793022 , Meghalaya , India.
authors.forename: Atanu
authors.initials: A
authors.lastname: Bhattacharjee
authors.affiliation: b Molecular Modelling and Simulation Lab, Department of Molecular Biology and Biotechnology, School of Sciences , Tezpur University , Tezpur 784028 , Assam , India.
authors.forename: Venkata Satish Kumar
authors.initials: VS
authors.lastname: Mattaparthi
authors.affiliation: a Microbial Biotechnology and Protein Research Laboratory, Department of Molecular Biology and Biotechnology, School of Sciences , Tezpur University , Tezpur 784028 , Assam , India.
authors.forename: Ashis K
authors.initials: AK
authors.lastname: Mukherjee
mesh_terms: D000595:Amino Acid Sequence; D001409:Bacillus cereus / Q000201:enzymology*; D005340:Fibrinogen / Q000737:chemistry*; D008024:Ligands; D008958:Models, Molecular; D062105:Molecular Docking Simulation; D056004:Molecular Dynamics Simulation; D011480:Protease Inhibitors / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D057057:Serine Proteases / Q000737:chemistry*; D013057:Spectrum Analysis; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013917:Thrombin / Q000737:chemistry*
chemical_list: D008024:Ligands; D011480:Protease Inhibitors; D005340:Fibrinogen; D057057:Serine Proteases; D013917:Thrombin
keywords: Bacillus cereus; I-TASSER; PATCH DOCK; YASARA; molecular dynamics; protease–fibrinogen interaction; protein–chymostatin; serine protease