Cyclophilin A catalyzes proline isomerization by an electrostatic handle mechanism

Abstract

Proline isomerization is a ubiquitous process that plays a key role in the folding of proteins and in the regulation of their functions. Different families of enzymes, known as "peptidyl-prolyl isomerases" (PPIases), catalyze this reaction, which involves the interconversion between the cis and trans isomers of the N-terminal amide bond of the amino acid proline. However, complete descriptions of the mechanisms by which these enzymes function have remained elusive. We show here that cyclophilin A, one of the most common PPIases, provides a catalytic environment that acts on the substrate through an electrostatic handle mechanism. In this mechanism, the electrostatic field in the catalytic site turns the electric dipole associated with the carbonyl group of the amino acid preceding the proline in the substrate, thus causing the rotation of the peptide bond between the two residues. We identified this mechanism using a combination of NMR measurements, molecular dynamics simulations, and density functional theory calculations to simultaneously determine the cis-bound and trans-bound conformations of cyclophilin A and its substrate as the enzymatic reaction takes place. We anticipate that this approach will be helpful in elucidating whether the electrostatic handle mechanism that we describe here is common to other PPIases and, more generally, in characterizing other enzymatic processes.enzyme catalysis | NMR spectroscopy D ifferent families of enzymes, often referred to as "peptidylprolyl isomerases" (PPIases), catalyze proline isomerization, a process that involves the interconversion between the cis and trans isomers of the N-terminal amide bond of the amino acid proline (1-3). This isomerization process is an intrinsically slow reaction, typically occurring on the time scale of several minutes under physiological conditions. Hence it often represents a ratelimiting step in biochemical reactions and indeed is used ubiquitously as a molecular switch in regulation (1-7).The possible mechanisms by which PPIases speed up this reaction have been the subject of intense scrutiny (8-16), although consensus descriptions of such mechanisms have not yet emerged. A question of particular relevance is the specific manner in which the electrostatic field in the catalytic site may facilitate the isomerization reaction. To investigate this problem, we considered the case of cyclophilin A, a member of the cyclophilin family of . Previous studies have suggested that conformations resembling those typical of the cis-bound and the trans-bound states are populated through conformational fluctuations in the free state of the enzyme and therefore functional insights into its mechanism of action might be obtained from the study of the free state (21-23).The approach that we followed in studying the mechanism of action of cyclophilin A is based on the simultaneous determination of the structures of the cis-bound and trans-bound states of the complex between the enzyme and its substrate as the catalytic process takes place. Our results re...

Structure of a low-population binding intermediate in protein-RNA recognition

“…As predicted, we observe a slight decrease in dissociate rate (K off , −17%) for the 2-thio-U25 mutant compared with the wild-type complex (Fig. 4 B and E), consistent with the weakening of the hydrogen bond induced by the oxygen-to-sulfur substitution (29) in the intermediate, resulting in an overall decrease in K d (−250%). Also in the R5K mutant, we observed a decrease in K off (−800%) (Fig.…”

Unfolding of CPR3 Gets Initiated at the Active Site and Proceeds via Two Intermediates

“…The results of the interaction study of CPR3 with a peptide using NMR show weak binding, as in other cyclophilins (13). The interacting residues of CPR3 were found in six main clusters in b3, b4, b5, the loops between b5 and b6 and b6 and b7, and the helical turn (Fig.…”

“…Several efforts to understand the cis-trans isomerization of the peptidyl-prolyl bond have been made using different approaches such as NMR line-shape analysis, monitoring changes in R 2 upon substrate binding, crystal structure studies with different peptides, molecular dynamics simulations, and density functional theory calculations (9)(10)(11)(12)(13). Eisenmesser and co-workers proposed that the cis conformer of the peptide interacts with residues A101, N102, A103, K82, and R55 of the enzyme; afterward, the enzyme catalyzes rotation of the peptide-prolyl bond by 180 to produce the trans conformation and makes contacts around residues L98 and S99.…”

“…They also proposed that the isomerization is driven by weakening of the double bond character of the peptide bond (11,12). Camilloni and co-workers proposed the socalled ''electrostatic handle mechanism,'' wherein the enzyme creates an electrostatic environment at the catalytic site that rotates a peptide bond in the substrate by pulling the electric dipole associated with the carbonyl group of the residue preceding proline in the peptide bond (13).…”

Conformational Changes and Free Energies in a Proline Isomerase

“…17 This motion is also observed in the free enzyme, where CypA may adopt at least two distinct conformations including a 4 minor state that has a ~6% population. 18 While previous computational studies on CypA have mostly focused on and provided insight into a potential link between protein motions and overall enzyme function, [19][20][21][22][23][24] we here instead aimed to investigate to what extent simulations can describe quantitatively the free energy surface of the free protein in solution.…”