A conserved cis peptide bond is necessary for the activity of Bowman-Birk inhibitor protein.
The Bowman-Birk inhibitor (BBI) family of protease inhibitors has an inhibitory region comprising a disulfide-linked nine-residue loop that adopts the characteristic canonical motif found in many serine protease inhibitors. A unique feature of the BBI loop is the presence of a cis peptide bond at the edge of the inhibitory loop. BBI-related protein fragments that encapsulate this loop retain the structure and inhibitory activity of the parent protein. The most common BBI loop sequence has a proline-proline element with a cis-trans geometry at P3'-P4'. We have examined this element by analysis of the inhibitory activity and structure for a series of synthetic fragments where each of these proline residues has been systematically replaced with alanine. The results show that only when a proline is present at P3' are potent inhibition and a cis peptide bond at that position in the solution structure observed, suggesting that this conformation is required for biological activity. Though a P4' proline is not essential for activity, it effectively stabilizes the cis conformation at P3' by suppressing alternative conformations. This is most evident from the Pro-Ala variant, which comprises a 1:1 mixture of slowly exchanging and structurally different cis and trans isomers. Monitoring the action of trypsin on this mixture by NMR shows that this protease interacts selectively with the cis P3' structure, providing direct evidence for the link between activity and the nativelike structure of the cis isomer. This is, to the best of our knowledge, the first example where cis isomer selectivity can be demonstrated for a proteinase.
Departments of Chemistry and Biological Sciences, Imperial College of Science, Technology and Medicine, South Kensington, London SW7 2AY, U.K.
Arnd B E
AB
Brauer
Gonzalo J
GJ
Domingo
Robert M
RM
Cooke
Stephen J
SJ
Matthews
Robin J
RJ
Leatherbarrow
D000409:Alanine / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D007536:Isomerism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D011392:Proline / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013816:Thermodynamics; D014357:Trypsin / Q000378:metabolism; D014358:Trypsin Inhibitor, Bowman-Birk Soybean / Q000138:chemical synthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D014358:Trypsin Inhibitor, Bowman-Birk Soybean; D011392:Proline; D014357:Trypsin; D000409:Alanine