On the Dynamic Nature of the Transition State for Protein–Protein Association as Determined by Double-mutant Cycle Analysis and Simulation

Harel, Michal; Cohen, Marion C.; Schreiber, Gideon

doi:10.1016/j.jmb.2007.05.032

Cited by 38 publications

(66 citation statements)

References 61 publications

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“…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)(7)(8)(9)(10)(11). Despite the importance of encounter complex ensembles in protein-protein association, little is known of their structures and configurations because their populations are low, their lifetimes are short, and they are difficult to trap, rendering them essentially invisible to conventional structural and biophysical methods.…”

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confidence: 99%

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

Fawzi

Doucleff

Suh

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

113

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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...

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mentioning

confidence: 99%

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

Fawzi

Doucleff

Suh

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

113

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“…Detailed examination of kinetic constants and thermodynamic driving forces, however, has been performed for relatively few protein-protein interaction complexes. Although significant progress has been made in the understanding and prediction of association rate constants, the determinants of dissociation rates remain poorly understood (1)(2)(3)(4)(5). The ability to predict the kinetic constants would be a significant contribution to the understanding of interaction networks in the systems biology era (6).…”

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confidence: 99%

Analysis of the Binding Forces Driving the Tight Interactions between β-Lactamase Inhibitory Protein-II (BLIP-II) and Class A β-Lactamases

Brown¹,

Chow²,

Sankaran

et al. 2011

Journal of Biological Chemistry

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␤-Lactamases hydrolyze ␤-lactam antibiotics to provide drug resistance to bacteria. ␤-Lactamase inhibitory protein-II (BLIP-II) is a potent proteinaceous inhibitor that exhibits low picomolar affinity for class A ␤-lactamases. This study examines the driving forces for binding between BLIP-II and ␤-lactamases using a combination of presteady state kinetics, isothermal titration calorimetry, and x-ray crystallography. The measured dissociation rate constants for BLIP-II and various ␤-lactamases ranged from 10 ؊4 to 10 ؊7 s ؊1 and are comparable with those found in some of the tightest known protein-protein interactions. The crystal structures of BLIP-II alone and in complex with Bacillus anthracis Bla1 ␤-lactamase revealed no significant side-chain movement in BLIP-II in the complex versus the monomer. The structural rigidity of BLIP-II minimizes the loss of the entropy upon complex formation and, as indicated by thermodynamics experiments, may be a key determinant of the observed potent inhibition of ␤-lactamases.Protein-protein interactions govern many cellular processes, and an understanding of the determinants of molecular recognition would facilitate the rationale design of interactions for therapeutic purposes. Detailed examination of kinetic constants and thermodynamic driving forces, however, has been performed for relatively few protein-protein interaction complexes. Although significant progress has been made in the understanding and prediction of association rate constants, the determinants of dissociation rates remain poorly understood (1-5). The ability to predict the kinetic constants would be a significant contribution to the understanding of interaction networks in the systems biology era (6). Several model systems have been developed to analyze the principles of protein-protein interactions, and one such model is the interaction between ␤-lactamase enzymes and a set of ␤-lactamase inhibitory proteins (BLIPs) 3 (7, 8). ␤-Lactamases act by hydrolyzing the four-membered ring of ␤-lactam antibiotics, e.g. penicillins and cephalosporins, rendering the drugs inactive (9, 10). There are four classes of ␤-lactamases (A-D) based on primary amino acid sequences (11,12). Class B ␤-lactamases are metalloenzymes that use a zinc-coordinated catalytic water to hydrolyze the ␤-lactam ring whereas classes A, C, and D are serine hydrolyases (13-15). Class A ␤-lactamases are widespread in both Gram-positive and Gramnegative bacteria and exhibit broad substrate hydrolysis profiles that include penicillins, cephalosporins, and for a few enzymes, carbapenems (12,14).The catalytic mechanism of serine ␤-lactamases is divided into two stages, acylation and deacylation (16). In class A enzymes, the catalytic Ser-70 residue nucleophilically attacks the carbonyl of the ␤-lactam and forms an acyl-intermediate (17,18). An essential Glu-166 residue activates a highly coordinated water for deacylation (18,19). Substitutions at Glu-166 result in an acylated, inactive enzyme (20 -22).Clinically available mechanism-based inhibitor...

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“…In comparison, the partitioning of binding energy within large, weakly charged interfaces appears to be more complex because hydrophobic interfaces are more malleable (8). Studies based on weakly charged interactions by Schreiber and coworkers (3,9) indicate that individual interface side chains have little or no effect on association rates or energetic consequence on forming the TS. However, a number of fundamental questions about the thermodynamic and structural linkages that actually define the TS in weakly charged interactions are yet to be answered.…”

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Principal determinants leading to transition state formation of a protein–protein complex, orientation trumps side-chain interactions

Horn¹,

Sosnick

Kossiakoff³

2009

Proc. Natl. Acad. Sci. U.S.A.

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The binding transition state (TS) is the rate-limiting step for transient molecular interactions. This important step in the molecular recognition process, however, is largely understood only at a qualitative level. To establish a more quantitative picture of the TS structure, we exploit a set of biophysical techniques that have provided major insights in protein folding applications. As a model system representing the large class of ''weakly charged'' proteinprotein interactions, we examine the binding of a variety of human growth hormone (hGH) variants to the human growth hormone receptor (hGHR) and the human prolactin receptor (hPRLR). hGH variants were chosen to probe different features of the TS structure, based on their highly reengineered interfaces. Both Eyring and urea (m value) analyses suggest that the majority of binding surface burial occurs after TS. A comprehensive analysis showed that individual hGH interface residues do not contribute energetically to the stability of the TS, but there is a TS ''hot spot'' in the receptor. Zinc dependence studies that take advantage of an endogenous tetracoordinated interfacial metal binding demonstrate that surfaces of the molecules have attained a high orientational complementarity by the time the TS is reached. The model that best fits these data are that a ''knobs-into-holes'' process precisely aligns the two molecular interfaces in forming the TS structure. Surprisingly, most of the thermodynamic character of the binding reaction is focused in the fine-tuning process occurring after TS. equilibrium thermodynamics ͉ hot spot ͉ protein recognition ͉ transition state thermodynamics ͉ binding pathways O rganized networks of transient protein-protein interactions play fundamental roles in initiating many types of biological processes. In most cases, proper function depends on the timing and duration of the interaction, which is coordinated with a finely tuned affinity between the binding partners. The energy landscape describing the binding event between two molecules consists of a number of intermediates whose magnitude and location depend on the specific interaction. Nevertheless, highly simplified models of the reaction pathway consist of three principal states (1): Step 1, the unbound reactants; Step 2, the transition state (TS); and Step 3, the bound complex. The molecular details of two of the states, Steps 1 and 3, have been captured in numerous high-resolution structure determinations. However, the molecular recognition processes that connect Steps 1 and 3 go through a poorly understood TS intermediate step, which is characterized by a protein-protein interface that is still partially solvated, containing unoptimized side-chain interactions (2-7).There are two major classes of protein-protein interactions that describe most protein-protein association processes. One class is regulated primarily by electrostatic forces that provide both a long-range steering function and dominate the overall binding energy. The second class is more predominant and involve...

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On the Dynamic Nature of the Transition State for Protein–Protein Association as Determined by Double-mutant Cycle Analysis and Simulation

Cited by 38 publications

References 61 publications

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

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

Analysis of the Binding Forces Driving the Tight Interactions between β-Lactamase Inhibitory Protein-II (BLIP-II) and Class A β-Lactamases

Principal determinants leading to transition state formation of a protein–protein complex, orientation trumps side-chain interactions

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