Function
[1] is a serine protease that is found in the bacteria Vibrio cholerae. The function of VesB is to contribute to intestinal growth and pathogenesis.[2] VesB is also able to cleave peptide bonds in proteins after arginines. This means that the substrate of VesB would be a protein containing arginine(s), (XXRXX) and the product after cleavage is all of the protein through the arginine, and the rest of the protein (XXR + XX), or the rest of the protein through the next arginine(s).
Disease
Cholera is a disease that is caused by the release of cholera toxin into the intestine. When this toxin is released, it causes rapid fluid loss and severe dehydration. VesB has been detected in Vibrio cholerae from patients with clinical cholera. The cholera toxin is activated when VesB cleaves the A subunit of the cholera toxin.
Relevance
Studying VesB is relevant because it is a newly identified serine protease that is secreted by the type II secretion system in V. cholerae. Although it is known where VesB is secreted and that it's part of the chymotrypsin subfamily, it's biological function is still unknown. Finding out what the actual function of VesB is could help understand how VesB exactly works in the promotion of cholera from V. cholerae.
Structural Highlights
VesB's is made up mostly of beta sheets with some alpha helices and random coil. VesB has two that make up it's tertiary structure, along with . It has a N-terminal protease domain (in blue) with a trypsin/chymotrypsin-fold and a C-terminal Ig-fold domain (in green). Although VesB's structure does not seem very large, looking at a of VesB shows a more accurate view of how large VesB is and how much space it actually takes up. VesB has and hydrophilic regions on it's structure. The hydrophobic regions are shown in gray and the hydrophilic regions are shown in purple. This view just shows the ratio of hydrophobic to hydrophilic regions in the structure of VesB, and that there are more hydrophilic regions than hydrophobic regions. This crystal structure was solved without a ligand, but the VesB ligand is known to be any protein containing Arg-X. The of VesB is made up of Asp125-His78-Ser221. In this triad, aspartic acid is deprotonated and proton transfer goes from histidine to aspartic acid. Since histidine is then deprotonated, it grabs the proton from serine's hydroxyl group. This active serine then can attack an incoming substrate, which allows VesB to cleave the substrate. The of VesB is made of the catalytic triad shown in red (Asp125,His78,Ser221), a hydrophobic pocket shown in green (Val159, Val180, Ile164), and a cleavage site shown in blue (Arg32, Ile33). Asp220 (shown in purple) is also in the active site and coordinates the N-terminal group of Ile33 in active VesB. One additional structural feature of VesB is that it has a , but it is not visible because both Cysteine residues are part of disordered regions in VesB. Another structural feature of VesB is it's (shown in red). Although this Ig-fold in VesB currently has no known function, it is predicted that it may be involved in stabilizing the protease domain, helping with substrate specificity, binding to the bacterial surface, or being part of an undefined secretion motif of the T2S system. One last structural feature is that in VesB's solved crystal structure, the Ser221 in the original catalytic triad was mutated to an because the Serine could have caused toxicity in overexpression.
Kinetic Data
After a kinetic protease activity assay using the synthetic peptide Boc-Gln-Ala-Arg-7-amino-4-methylcoumarin as a substrate was carried out, it was found that VesB was efficiently able to cleave the trypsin substrate, but the Boc-Glu-Lys-Lys-AMC and Leu-AME were cleaved with greatly reduced efficiency instead. Also, no cleaveage was observed for the chymotrypsin or elastase substrate. Kinetic values for Boc-Gln-Ala-Arg-AMC were then found and gave a Vmax around 0.137 nmol/min and a Km of about 0.0327 mM. A Vmax and Km for Boc-Glu-Lys-Lys-AMC and Boc-Leu-AMC were undetermined. A inhibition of VesB by serine protease inhibitors benzamide and leupeptin was also observed. All of this kinetic data suggests that VesB is an extracellular trypsin-like protease that has a preference for arginine in the substrate.
