Dynamics in the Transient Complex of Plastocyanin−Cytochrome f from Prochlorothrix hollandica

Abstract

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.

Replica exchange simulations of transient encounter complexes in protein–protein association

“…protein binding ͉ transient nonspecific encounter complexes ͉ paramagnetic relaxation enhancement ͉ bacterial phosphotransferase system E xperimental and theoretical studies have provided evidence that transient nonspecific encounter complexes play an important role in protein binding and function (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17). The formation of weakly bound nonspecific complexes, often dominated by long-range electrostatic interactions, enhances the on-rate of binding by increasing the interaction cross-section and reducing the conformational space to be searched on the path to the specific complex.…”

“…PRE measurements have unequivocally demonstrated the existence of transient encounter complexes in protein-DNA (19,20) and protein-protein (4)(5)(6)(7)(8)21) interactions. These experiments rely on three key observations: (i) the observed intermolecular PRE relaxation rates in the fast-exchange regime are population averages of all complexes present in solution; (ii) the magnitude of the PRE is proportional to ͗r Ϫ6 ͘, where r is the distance between a proton and a covalently attached paramagnetic label; and (iii) PRE effects are strong owing to the large magnetic moment of an unpaired electron.…”

Protein Recognition

“…Indeed, ultra-weak complexes might, in at least some instances, resemble the encounter complexes predicted for strong interactions, insofar as they comprise a dynamic ensemble of orientations rather than a single well-defined state [46]. NMR data collected on the transient complex of plastocyanin-cytochrome f can not be satisfied by a single structure, but only by a family of structures displaying significant variability, indicating that the complex is rather dynamic [46].…”

Paramagnetic Tools in Protein NMR

“…Various approaches have been proposed for visualisation of dynamic complexes characterised with paramagnetism, mostly based on a minimum set of structures that can describe the data [20,72,88,[125][126][127]. This area is still under development and is hindered not only by the fact that NMR provides only averages over all the populated conformers, but also by the fact that the mean of observables depends nonlinearly on the fractional populations due to the high powers in the distance dependence of PCS and PRE.…”