Mechanistic details of a protein–protein association pathway revealed by paramagnetic relaxation enhancement titration measurements

Abstract

Protein-protein association generally proceeds via the intermediary of a transient, lowly populated, encounter complex ensemble. The mechanism whereby the interacting molecules in this ensemble locate their final stereospecific structure is poorly understood. Further, a fundamental question is whether the encounter complex ensemble is an effectively homogeneous population of nonspecific complexes or whether it comprises a set of distinct structural and thermodynamic states. Here we use intermolecular paramagnetic relaxation enhancement (PRE), a technique that is exquisitely sensitive to lowly populated states in the fast exchange regime, to characterize the mechanistic details of the transient encounter complex interactions between the N-terminal domain of Enzyme I (EIN) and the histidine-containing phosphocarrier protein (HPr), two major bacterial signaling proteins. Experiments were conducted at an ionic strength of 150 mM NaCl to eliminate any spurious nonspecific associations not relevant under physiological conditions. By monitoring the dependence of the intermolecular transverse PRE (Γ 2 ) rates measured on 15 N-labeled EIN on the concentration of paramagnetically labeled HPr, two distinct types of encounter complex configurations along the association pathway are identified and dissected. The first class, which is in equilibrium with and sterically occluded by the specific complex, probably involves rigid body rotations and small translations near or at the active site. In contrast, the second class of encounter complex configurations can coexist with the specific complex to form a ternary complex ensemble, which may help EIN compete with other HPr binding partners in vivo by increasing the effective local concentration of HPr even when the active site of EIN is occupied.enzyme I-histidine containing phosphocarrier protein complex | encounter complex | lowly populated states | NMR | phosphotransferase system S pecific protein-protein interactions underlie virtually every process in the cell. In general, specific protein-protein recognition proceeds via a two-step process (1-3): weak association via diffusion-controlled intermolecular collisions results in the formation of an ensemble of short-lived, encounter complexes located in multiple local free energy minima of a two-dimensional funnel-like energy landscape on the protein surface (4); subsequent rearrangement along the energy landscape, involving translations and rotations of the two partner proteins, permits the global free energy minimum to be located, resulting in the formation of a well-defined specific complex stabilized by a defined set of electrostatic and van der Waals interactions. From a functional perspective, encounter complex ensembles are thought to play a critical role in fine tuning reaction fluxes inside the cell (5), enhancing association on-rates by increasing the interaction crosssection and reducing the conformational search space on the path to the specific complex (6-11). Despite the importance of encounter complex ensembles in...

Interplay between Minor and Major Groove-binding Transcription Factors Sox2 and Oct1 in Translocation on DNA Studied by Paramagnetic and Diamagnetic NMR

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NMR and Bioinformatics Discovery of Exosites That Tune Metalloelastase Specificity for Solubilized Elastin and Collagen Triple Helices

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