The substrate-binding site in Escherichia coli cyclophilin A preferably recognizes a cis-proline isomer or a highly distorted form of the trans isomer.

The three-dimensional structure of Escherichia coli cytosolic cyclophilin A (CyPA) complexed with a tripeptide (succinyl-Ala-Pro-Ala-p-nitroanilide) was refined at 1.8 A resolution by the multiple isomorphous replacement method to a crystallographic R-factor of 17.6%. As in human CyPA, the peptide binding site in E. coli enzyme is in a cleft created on the surface of the upper sheet of two orthogonal beta-sheets. In this cleft, the walls of the hydrophobic pocket are formed by the side-chains of five non-polar residues, Phe48, Met49, Phe107, Leu108, and Try120, with Phe99 at the bottom. When the cis isomer of the tripeptide binds to the enzyme, a cis-proline ring is inserted into the hydrophobic pocket. Since the binding pocket of CyPAs are largely hydrophobic, the cis isomer of a peptide can be bound more firmly than the trans isomer. Distortion of the trans isomer could lead to better binding, but at an energetic cost of the distortion energy. At the periphery of the upper beta-sheet in E. coli CyPA, conformations of loops L1, L3, and L4 and the segment connecting alpha1 and beta3 with deletions or insertions against human CyPA differ significantly from those in human CyPA. The refined model also shows that steric hindrance to attachment of cyclosporin A (CsA) prevents E. coli CyPA forming a complex with CsA. Thus, the extra amino acid residue of E. coli CyPA, polar Gln89, lies along the pathway to the hydrophobic pocket of CyPA and seems to prevent the access hydrophobic part of CsA to the cleft of CyPA.