Conserved Conformational Dynamics Reveal a Key Dynamic Residue in the Gatekeeper Loop of Human Cyclophilins

Cyclophilins are ubiquitous human enzymes that catalyze peptidyl-prolyl cis-trans isomerization in protein substrates. Of the 17 unique isoforms, five closely related isoforms (CypA-E) are found in various environments and participate in diverse cellular processes, yet all have similar structures and the same core catalytic function. The question is what key residues are behind the conserved function of these enzymes. Here, conformational dynamics are compared across these isoforms to detect conserved dynamics essential for the catalytic activity of cyclophilins. A set of key dynamic residues, defined by the most dynamically conserved positions, are identified in the gatekeeper 2 region. The highly conserved glycine (Gly80) in this region is predicted to underlie the local flexibility, which is further tested by molecular dynamics simulations performed on mutants (G80A) of CypE and CypA. The mutation leads to decreased flexibility of CypE and CypA during substrate binding but increased flexibility during catalysis. Dynamical changes occur in the mutated region and a distal loop downstream of the mutation site in sequence. Examinations of the mutational effect on catalysis show that both mutated CypE and CypA exhibit shifted binding free energies of the substrate under distinct isomer conformations. The results suggest a loss of function in the mutated CypE and CypA. These catalytic changes by the mutation are likely independent of the substrate sequence, at least in CypA. Our work presents a method to identify function-related key residues in proteins.