The structural basis of a conserved P2 threonine in canonical serine proteinase inhibitors.
Bowman-Birk inhibitors (BBIs) are a well-studied family of canonical inhibitor proteins of serine proteinases. In nature, the active region of BBIs possesses a highly conserved Thr at the P2 position. The importance of this residue has been reemphasized by synthetic BBI reactive site loop proteinomimetics. In particular, this residue was exclusively identified for active chymotrypsin inhibitors selected from a BBI template-assisted combinatorial peptide library. A further kinetic analysis of 26 P2 variant peptides revealed that Thr provides both optimal binding affinity and optimal resistance against enzymatic turnover by chymotrypsin. Herein, we report the (1)H-NMR spectroscopic study of a 5-membered sub-set of these reactive site loop peptides representing a stepwise elimination of the Thr side-chain functionalities and inversion of its side-chain chirality. The P2 Thr variant adopts a three-dimensional structure that closely mimics the one of the corresponding region of the complete protein. This validates the use of this template for the investigation of structure-function relationships. While the overall backbone geometry is similar in all studied variants, conformational changes induced by the modification of the P2 side chain have now been identified and provide a rational explanation of the kinetically observed functional differences. Eliminating the gamma-methyl group has little structural effect, whereas the elimination of the gamma-oxygen atom or the inversion of the side-chain chirality results in characteristic changes to the intramolecular hydrogen bond network. We conclude that the transannular hydrogen bond between the P2 Thr side-chain hydroxyl and the P5' backbone amide is an important conformational constraint and directs the hydrophobic contact of the P2 Thr side chain with the enzyme surface in a functionally optimal geometry, both in the proteinomimetic and the native protein. In at least four canonical inhibitor protein families similar structural arrangements for a conserved P2 Thr have been observed, which suggests an analogous functional role. Substitutions at P2 of the proteinomimetic also affect the conformational balance between cis and trans isomers at a distant Pro-Pro motif (P3'-P4'). Presented with a mixture of cis/trans isomers chymotrypsin appears to interact preferably with the conformer that retains the cis-P3' Pro-trans-P4' Pro geometry found in the parent BBI protein.
Brauer
Arnd B E
AB
Department of Chemistry, Imperial College London, South Kensington Campus, London SW7 2AZ, UK.
Nievo
Marco
M
McBride
Jeffrey D
JD
Leatherbarrow
Robin J
RJ
D000595:Amino Acid Sequence; D000818:Animals; D008956:Models, Chemical; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010456:Peptides, Cyclic / Q000737:chemistry; D017434:Protein Structure, Tertiary; D015842:Serine Proteinase Inhibitors / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship; D013912:Threonine / Q000737:chemistry* / Q000502:physiology
D010456:Peptides, Cyclic; D015842:Serine Proteinase Inhibitors; D013912:Threonine