Hidden alternative structures of proline isomerase essential for catalysis

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Abstract
X-ray crystallography and NMR spectroscopy are two powerful tools used by structural biologists to determine the three-dimensional structures and characterize the dynamic properties of proteins. In this paper, the authors used both of these methods to identify and characterize a 'hidden' high-energy substate of human cyclophilin A, a proline isomerase. This was made possible by engineering a modified form of the enzyme incorporating a mutation at a distance from the active site that could stabilize this previously hidden conformation by inverting the equilibrium between the various substates and reducing the conformational interconversion rates and the catalytic rate. This approach should be broadly applicable to many other proteins and could lead to the reinterpretation of crystal structures determined previously. X-ray crystallography and NMR spectroscopy are two powerful tools to determine the three-dimensional structures and characterize the dynamic properties of proteins. The two methods are now combined to structurally unravel interconverting substrates of a human proline isomerase. Crystallographic approaches are used to define minor protein conformations and, combined with NMR analysis, to show how collective motions contribute to the catalytic power of an enzyme. A long-standing challenge is to understand at the atomic level how protein dynamics contribute to enzyme catalysis. X-ray crystallography can provide snapshots of conformational substates sampled during enzymatic reactions1, while NMR relaxation methods reveal the rates of interconversion between substates and the corresponding relative populations1,2. However, these current methods cannot simultaneously reveal the detailed atomic structures of the rare states and rationalize the finding that intrinsic motions in the free enzyme occur on a timescale similar to the catalytic turnover rate. Here we introduce dual strategies of ambient-temperature X-ray crystallographic data collection and automated electron-density sampling to structurally unravel interconverting substates of the human proline isomerase, cyclophilin A (CYPA, also known as PPIA). A conservative mutation outside the active site was designed to stabilize features of the previously hidden minor conformation. This mutation not only inverts the equilibrium between the substates, but also causes large, parallel reductions in the conformational interconversion rates and the catalytic rate. These studies introduce crystallographic approaches to define functional minor protein conformations and, in combination with NMR analysis of the enzyme dynamics in solution, show how collective motions directly contribute to the catalytic power of an enzyme.