Role of hydrophobic interactions in the encounter complex formation of the plastocyanin and cytochrome f complex revealed by paramagnetic NMR spectroscopy.

Protein complex formation is thought to be at least a two-step process, in which the active complex is preceded by the formation of an encounter complex. The interactions in the encounter complex are usually dominated by electrostatic forces, whereas the active complex is also stabilized by noncovalent short-range forces. Here, the complex of cytochrome f and plastocyanin, electron-transfer proteins involved in photosynthesis, was studied using paramagnetic relaxation NMR spectroscopy. Spin labels were attached to cytochrome f, and the relaxation enhancements of plastocyanin nuclei were measured, demonstrating that a large part of the cytochrome f surface area is sampled by plastocyanin. In contrast, plastocyanin is always oriented with its hydrophobic patch toward cytochrome f. The complex was visualized using ensemble docking, showing that the encounter complex is stabilized by hydrophobic as well as electrostatic interactions. The results suggest a model of electrostatic preorientation before the proteins make contact, followed by the formation of an encounter complex that rapidly leads to electron-transfer active conformations by gradual increase of the overlap of nonpolar surface areas on cytochrome f and plastocyanin. In this model the distinction between the encounter and active complexes vanishes, at least in the case of electron-transfer complexes, which do not require a high degree of specificity.

Dynamics in the transient complex of plastocyanin-cytochrome f from Prochlorothrix hollandica.

The nature of transient protein complexes can range from a highly dynamic ensemble of orientations to a single well-defined state. This represents variation in the equilibrium between the encounter and final, functional state. The transient complex between plastocyanin (Pc) and cytochrome f (cytf) of the cyanobacterium Prochlorothrix hollandica was characterized by NMR spectroscopy. Intermolecular pseudocontact shifts and chemical shift perturbations were used as restraints in docking calculations to determine the structure of the wild-type Pc-cytf complex. The orientation of Pc is similar to orientations found in Pc-cytf complexes from other sources. Electrostatics seems to play a modest role in complex formation. A large variability in the ensemble of lowest energy structures indicates a dynamic nature of the complex. Two unusual hydrophobic patch residues in Pc have been mutated to the residues found in other plastocyanins (Y12G/P14L). The binding constants are similar for the complexes of cytf with wild-type Pc and mutant Pc, but the chemical shift perturbations are smaller for the complex with mutant Pc. Docking calculations for the Y12G/P14L Pc-cytf complex did not produce a converged ensemble of structures. Simulations of the dynamics were performed using the observed averaged NMR parameters as input. The results indicate a surprisingly large amplitude of mobility of Y12G/P14L Pc within the complex. It is concluded that the double mutation shifts the complex further from the well-defined toward the encounter state.