Cyclophilin a Catalyzes Proline Isomerization by an Electrostatic Handle Mechanism
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Cyclophilin A catalyzes proline isomerization by an electrostatic handle mechanism Carlo Camillonia, Aleksandr B. Sahakyana, Michael J. Hollidayb, Nancy G. Isernc, Fengli Zhangd, Elan Z. Eisenmesserb, and Michele Vendruscoloa,1 aDepartment of Chemistry, University of Cambridge, Cambridge CB2 1EW, United Kingdom; bDepartment of Biochemistry and Molecular Genetics, University of Colorado, Aurora, CO 80045; cWilliam R. Wiley Environmental Molecular Sciences Laboratory, High Field NMR Facility, Richland, WA 99532; and dNational High Magnetic Field Laboratory, Tallahassee, FL 32310 Edited by Arieh Warshel, University of Southern California, Los Angeles, CA, and approved June 3, 2014 (received for review March 5, 2014) Proline isomerization is a ubiquitous process that plays a key role residue in the substrate and induces the rotation of the electric in the folding of proteins and in the regulation of their functions. dipole corresponding to the carbonyl group of the residue pre- Different families of enzymes, known as “peptidyl-prolyl isomer- ceding the proline. In this sense, the carbonyl group represents ases” (PPIases), catalyze this reaction, which involves the intercon- a handle operated by an electrostatic field and helps overcome version between the cis and trans isomers of the N-terminal amide the isomerization barrier. bond of the amino acid proline. However, complete descriptions of We investigated the conformational properties of cyclophilin the mechanisms by which these enzymes function have remained A during the proline isomerization process by using NMR elusive. We show here that cyclophilin A, one of the most common spectroscopy, which can provide atomic-resolution descriptions PPIases, provides a catalytic environment that acts on the sub- of the motions of macromolecules in solution (24–32). In our strate through an electrostatic handle mechanism. In this mecha- strategy, NMR data are used as replica-averaged structural nism, the electrostatic field in the catalytic site turns the electric restraints in molecular dynamics simulations. Such calculations, 2 dipole associated with the carbonyl group of the amino acid pre- which in general can include NOE-derived distances (29), S - ceding the proline in the substrate, thus causing the rotation of order parameters (29), residual dipolar couplings (33–35), and the peptide bond between the two residues. We identified this chemical shifts (36–38), are particularly suitable when multiple mechanism using a combination of NMR measurements, molecular conformations of a protein are present simultaneously in solu- dynamics simulations, and density functional theory calculations tion, because these conformations can be determined at the to simultaneously determine the cis-bound and trans-bound con- same time (29, 37). formations of cyclophilin A and its substrate as the enzymatic re- action takes place. We anticipate that this approach will be helpful Results and Discussion in elucidating whether the electrostatic handle mechanism that we Simultaneous Determination of the cis-Bound and trans-Bound States. describe here is common to other PPIases and, more generally, in To study the proline isomerization process catalyzed by characterizing other enzymatic processes. cyclophilin A, we considered the model peptide substrate GSFGPDLRAGD (39, 40). We carried out chemical shift enzyme catalysis | NMR spectroscopy measurements in the bound state during the catalytic reaction (SI Text). In addition, we used NOESY measurements to obtain ifferent families of enzymes, often referred to as “peptidyl- information about interproton distances (i.e., intermolecular prolyl isomerases” (PPIases), catalyze proline isomerization, NOE restraints) between the enzyme and the substrate; there- D cis a process that involves the interconversion between the cis and fore NOEs were measured as averages over the -bound and trans trans isomers of the N-terminal amide bond of the amino acid the -bound conformations during the isomerization reaction SI Text proline (1–3). This isomerization process is an intrinsically slow ( ). We then performed molecular dynamics simulations reaction, typically occurring on the time scale of several minutes under physiological conditions. Hence it often represents a rate- Significance limiting step in biochemical reactions and indeed is used ubiq- uitously as a molecular switch in regulation (1–7). One of the most widespread molecular switches in biochemical The possible mechanisms by which PPIases speed up this pathways is based on the isomerization of the amino acid reaction have been the subject of intense scrutiny (8–16), although proline, a process that normally is facilitated by enzymes consensus descriptions of such mechanisms have not yet emerged. known as “proline isomerases.” We show that cyclophilin A, A question of particular relevance is the specific manner in which one of the most common proline isomerases, acts by a simple the electrostatic field in the catalytic site may facilitate the isom- mechanism, which we describe as an “electrostatic handle.” In erization reaction. To investigate this problem, we considered the this mechanism, the enzyme creates an electrostatic environ- case of cyclophilin A, a member of the cyclophilin family of ment in its catalytic site that rotates a peptide bond in the PPIases (17–20). Previous studies have suggested that con- substrate by pulling the electric dipole associated with the formations resembling those typical of the cis-bound and the carbonyl group preceding the peptide bond itself. Our results trans-bound states are populated through conformational fluctu- thus identify a specific mechanism by which electrostatics is exploited in enzyme catalysis. ations in the free state of the enzyme and therefore functional BIOPHYSICS AND insights into its mechanism of action might be obtained from the – Author contributions: C.C., A.B.S., E.Z.E., and M.V. designed research; C.C., A.B.S., M.J.H., COMPUTATIONAL BIOLOGY study of the free state (21 23). E.Z.E., and M.V. performed research; C.C., N.G.I., F.Z., E.Z.E., and M.V. contributed new The approach that we followed in studying the mechanism of reagents/analytic tools; C.C., A.B.S., M.J.H., E.Z.E., and M.V. analyzed data; and C.C., action of cyclophilin A is based on the simultaneous de- M.J.H., E.Z.E., and M.V. wrote the paper. termination of the structures of the cis-bound and trans-bound The authors declare no conflict of interest. states of the complex between the enzyme and its substrate as the This article is a PNAS Direct Submission. catalytic process takes place. Our results reveal that the mech- 1To whom correspondence should be addressed. Email: [email protected]. anism of the reaction involves the presence of an electrostatic This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. field that acts on the N-terminal peptide bond of the proline 1073/pnas.1404220111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1404220111 PNAS | July 15, 2014 | vol. 111 | no. 28 | 10203–10208 Downloaded by guest on October 2, 2021 AB C Fig. 1. Ensembles of structures representing the conformational fluctuations of cyclophilin A in the trans-bound (A), the cis-bound (B), and the free (C) states. The ensembles have been determined using backbone chemical shifts as replica-averaged restraints (free state) and backbone chemical shifts and interchain NOEs replica-averaged restraints (bound state). The simulations were performed with a modified version of GROMACS, using the Amber99SB*-ILDN force- field and applying the CamShift and NOE restraints over two replicas (37). More details are provided in SI Text. with replica-averaged chemical shifts (37) and intermolecular in the function of cyclophilin A (13–15). More specifically, density NOE restraints (29), a technique that enables the information functional theory (DFT) calculations (SI Text) of the electrostatic provided by NMR measurements to be incorporated in the field acting on the glycine–proline peptide bond were carried out structural determination procedure in a manner consistent with for the cis-bound and the trans-bound ensembles (Figs. S1–S3). the maximum entropy principle (41–43). We used two replicas Our results indicate that the z component, defined as the normal of the system; the initial structures were chosen with the proline to the ring plane defined by the N, Cα, and Cγ atoms of the in the model peptide in the cis conformation in the first replica proline residue, is approximately the same for the cis-bound and and in the trans conformation in the second replica (SI Text). the trans-bound states (Fig. 4). These calculations resulted in two (cis-bound and trans-bound) Having determined the electrostatic field present in the active conformational ensembles (Fig. 1) with corresponding free- site of cyclophilin A during the catalytic process, we investigated energy landscapes (Fig. 2). The agreement between experimen- its specific effect on the proline isomerization process. To obtain tal and calculated intermolecular NOEs and chemical shifts was an initial insight into this effect, we performed DFT calculations excellent (Table S1 and Fig. 3). For comparison, we also carried (SI Text) on a model system, the N-acetyl-L-prolyl-N-methyl- out similar calculations for the free state of the enzyme (Fig. 2; see amide (Ace-Pro-Nme) proline dipeptide, in vacuo and compared also Conformational Fluctuations in the Free State). the potential energy surface of this system in the presence and absence of an electrostatic field corresponding to that found in A Possible Electrostatic Handle Mechanism of Catalysis. To formulate the active site of cyclophilin A. The potential energy surfaces for a hypothesis about the mechanism of catalysis, we analyzed the the isomerization process as a function of the ω and ψ angles ensemble of conformations representing the bound state of (Fig. 4) indicate that in the absence of electrostatic fields the cyclophilin A and its substrate. This ensemble can be divided into trans isomer is about 20 kJ/mol more stable than the cis isomer, cis-bound and trans-bound subensembles.