Visualization of transient encounter complexes in protein–protein association

Abstract

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. Furthermore, rate enhancement through non-specific binding may occur by either a reduction in dimensionality or the presence of a short-range, non-specific attractive potential. Here, using paramagnetic relaxation enhancement, 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 amino-terminal domain of enzyme I and the phosphocarrier protein HPr. Neither the stereospecific complex alone nor any single alternative conformation can account fully for the intermolecular paramagnetic relaxation enhancement data. Restrained rigid-body simulated annealing refinement against the paramagnetic relaxation enhancement data enables us to obtain an atomic probability distribution map of the non-specific encounter complex ensemble that qualitatively correlates with the electrostatic surface potentials on the interacting proteins. Qualitatively similar results are presented for two other protein-protein complexes.

Practical aspects of 1H transverse paramagnetic relaxation enhancement measurements on macromolecules

“…An isotropic g-tensor for the unpaired electrons of Mn 2+ in EDTA-Mn 2+ conjugated to either DNA or protein was also found in previous studies, including systems ranging from 20 to 50 kDa (10,19,(23)(24)(25)(26)(27)(28).…”

“…Expression and purification of HPr (E32C) and the conjugation reaction with N-[S-(2-pyridylthio)cysteaminyl]ethylene-diamine-N,N,N′N′-teatracetic acid (Toronto Research Chemicals) were carried out as described (28). The final conjugated HPr(E32C)-EDTAMn 2+ or -EDTA-Ca 2+ samples were further purified by Mono-Q anion-exchange chromatography.…”

“…The PRE can also provide information relating to large-scale dynamics that accompany changes of paramagnetic center -1 H distances, for example in non-specific protein-DNA interactions (24,25) and interdomain motions (26). A recent major advance in the field is the finding that in the fast exchange regime the intermolecular PRE can provide a powerful probe to detect and characterize transient, lowly populated intermediates in macromolecular binding events, thereby providing structural information on encounter complexes that cannot be obtained by any other biophysical technique (27,28).…”

Protein dynamics and function from solution state NMR spectroscopy

“…In such cases, minor states can be identified on the basis of a mismatch between measured PREs and PREs calculated on the basis of a structure of the major state a. An ensemble approach can then be used to fit the PRE data more accurately, revealing the presence of the minor state or states (Tang et al 2006). …”

“…Another ubiquitin ensemble that was constructed by refinement against RDCs and is sensitive to motions on a ps-to-ms time scale is described in some detail in Section 4 (Lange et al 2008). Ensemble approaches have also been used to visualize transient encounter complexes (Bashir et al 2010;Tang et al 2006), quantify enzyme dynamics , describe the solution structures of intrinsically disordered proteins (Jensen et al 2013(Jensen et al , 2014Silvestre-Ryan et al 2013), and probe correlated motions in proteins (Bouvignies et al 2005;Bryn Fenwick et al 2014;Clore & Schwieters, 2004a;Fenwick et al 2011).…”