German Edition: DOI: 10.1002/ange.201503698 Protein Structure International Edition: DOI: 10.1002/anie.201503698 A Structural Ensemble for the Enzyme Cyclophilin Reveals an Orchestrated Mode of Action at Atomic Resolution CelestineN. Chi, Beat Vçgeli, Stefan Bibow, Dean Strotz, Julien Orts, Peter Güntert, and Roland Riek* Abstract: For enzyme activity, an exact structural and motional orchestration of the active site and its surroundings is believed to be key. In order to reveal such possible phenomena at atomic resolution on the basis of experimental evidence, an experimental restraint driven two-state ensemble of the prototypical enzyme cyclophilin was determined by using a recently introduced exact NOE approach. The ensemble description reveals the presence of an open and a closed state of cyclophilin, which is indicative of large-scale correlated motion. In the open state, the catalytic site is preorganized for catalysis, thus suggesting the mechanism of action to be conformational sampling, while the ligand-binding loop appears to act through an induced fit mechanism. This finding is supported by affinity measurements of a cyclophilin designed to be more open. Overall, more than 60–70 % of the side-chain conformations of cyclophilin appear to be corre- lated. The catalytic mechanisms of enzymes are believed to rely on a dynamic interplay between well-arranged structural states. [1] The magnitude of the conformational change may cover a large range in both space and time. The most relevant time scale for protein action is believed to be in the ms–ms range. Evidence of such dynamics has been found, for example, for the well-studied human cyclophilin A, [1a–c, 2] a peptidylprolyl cis–trans isomerase. Peptidylprolyl cis–trans isomerases cata- lyze interconversion between the cis and trans isomers of the peptide bond of proline residue within a substrate. [3] For cyclophilin A, NMR relaxation experiments revealed ms motions both during catalysis and in the apo state that can be interpreted as a two-state interconversion process. [2c–e, 4] In combination with room temperature X-ray crystallography [2d] and mutagenesis studies, [5] it has been suggested that the presence of a dynamic network encompassing the active site and its close neighborhood is key for activity. This finding has been complemented by a proposed mode of action of cyclophilin A derived from molecular dynamics simulation restricted by NMR restraints in combination with density functional theory calculations. [6] The calculations indicate that cyclophilin A acts through an electrostatic handle mechanism at the carbonyl of the residue preceding the proline in the substrate. The traditional approaches dedicated to elucidating such slow conformational dynamics are R11 NMR relaxation measurements and fluorescence-based techniques. [7] How- ever, it is difficult to represent the spatial sampling of these slow motions. [1d] New methodologies combining NMR probes with molecular dynamics simulations are being advanced to unravel this problem,. [1d, 6] Recently, we introduced another concept that makes use of exact Nuclear Overhauser Effect (NOE)-derived distance restraints. [8] In order to obtain a plausible description of the various substates of cyclo- philin A at atomic resolution, we employed an ensemble structure calculation with the use of eNOEs and residual dipolar couplings (RDCs). Following an established protocol [8] with the software packages eNORA [9] and CYANA, [10] ensemble structure calculations were performed with a total of 3629 eNOE- based distance restraints, 396 H-N RDCs derived from four alignment media, 279 scalar couplings, and 128 angle restraints from 13 Ca chemical shifts (Table S1 in the Support- ing Information). As a measure of the quality of the calculated structures, the CYANA target function, which is a weighted sum of all squared violations of the experimental restraints, is used. It drops significantly from one state to two states and levels off after three states (Figure 1c). This observation indicates that, in contrast to the single-state structure, multistate ensembles describe the experimental data well (Figure 1c and Table S1). In order to test for self- consistency of the experimental data, a cross validation test was performed with a jackknife procedure that repeats structure calculation ten times with 10 % of the experimental input data randomly deleted such that each distance restraint is omitted exactly once. The back-calculated target function of the omitted data then represents the entire data set. The decrease in this target function for higher-state ensembles (Figure 1 c) indicates again that the experimental data are well described by two or more states. Similar cross-validations were also done with the RDCs and the 3 J HNHA couplings (Figure 1 d and 1 e). Again, a significant and a moderate drop in the target function values for the 3 J HNHA couplings and the RDCs is observed when increasing the number of states from one to two. As a representative for the following discussion, the two- state ensemble described by a structural bundle of 2  20 conformers (PDB ID: 2n0t, Figure 1 a, b) is used in order to [*] Dr. C.N. Chi, Dr. B. Vçgeli, Dr. S. Bibow, D. Strotz, Dr. J. Orts, Prof. Dr. P. Güntert, Prof. Dr. R. Riek Laboratory of Physical Chemistry, ETH Zurich ETH-Hçnggerberg, 8093 Zürich (Switzerland) E-mail: roland.riek@phys.chem.ethz.ch Prof. Dr. P. Güntert Institute of Biophysical Chemistry, Center for Biomolecular Magnetic Resonance, Goethe University Frankfurt am Main Max-von-Laue-Strasse 9, 60438 Frankfurt am Main (Germany) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/anie.201503698. Angewandte Chemie 11657 Angew. Chem. Int. Ed. 2015, 54, 11657 –11661 # 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim