Kinetic data on a number of protein-protein associations have provided evidence for the initial formation of a pre-equilibrium encounter complex that subsequently relaxes to the final stereospecific complex. Site-directed mutagenesis and Brownian dynamics simulations have suggested that the rate of association can be modulated by perturbations in charge distribution outside the direct interaction surfaces. Further, rate enhancement through non-specific binding may occur by a reduction of dimensionality or the presence of a short-range, non-specific attractive potential. Here, using paramagnetic relaxation enhancement (PRE) we directly demonstrate the existence and visualize the distribution of an ensemble of transient, non-specific encounter complexes under equilibrium conditions for a relatively weak protein-protein complex between the N-terminal domain of enzyme I (EIN) and the phosphocarrier protein HPr. Neither the stereospecific complex alone nor any single alternative conformation can account fully for the intermolecular PRE data. Restrained rigid body simulated annealing refinement against the PRE data permits one to obtain an atomic probability distribution map that depicts the non-specific encounter complex ensemble and suggests an important role for electrostatic interactions. Qualitatively similar results were demonstrated with two other complexes between HPr and IIamannose and IIAMannitol. Another example of large scale motions involves the E. coli mannitol transporter (IIMtl) that comprises three domains connected by flexible linkers: a transmembrane domain (C) and two cytoplasmic domains (A and B). IIMtl catalyzes three successive phosphoryl transfer reactions: one intermolecular (from HPr to the A domain) and two intramolecular (from the A to the B domain, and from the B domain to the incoming sugar bound to the C domain). A key functional requirement of IIMtl is that the A and B cytoplasmic domains be able to rapidly associate and dissociate while maintaining reasonably high occupancy of an active stereospecific AB complex to ensure effective phosphoryl transfer along the pathway. We have investigated the rate of intramolecular domain-domain association and dissociation in IIBAMtl using 1H relaxation dispersion spectroscopy in the rotating frame. The open, dissociated state (comprising an ensemble of states) and the closed, associated state (comprising the stereospecific complex) are approximately equally populated. The first order rate constants for intramolecular association and dissociation are 1.7(0.3)x104 and 1.8(0.4)x104 s-1, respectively. These values compare to rate constants of 500 s-1 for phosphoryl transfer, derived from qualitative lineshape analysis of 1H-15N correlation spectra taken during the course of active catalysis. Thus, on average 80 association/dissociation events are required to effect a single phosphoryl transfer reaction. We can therefore conclude that intramolecular phosphoryl transfer between the A and B domains of IIMtl is rate limited by chemistry and not the rate of formation or dissociation of a stereospecific complex in which the active sites are optimally apposed.
Showing the most recent 10 out of 13 publications
