Hydrogen exchange and ligand binding: Ligand‐dependent and ligand‐independent protection in the Src SH3 domain

Abstract

Amide hydrogen-deuterium exchange has proven to be a powerful tool for detecting and characterizing high-energy conformations in protein ensembles. Since interactions with ligands can modulate these highenergy conformations, hydrogen exchange appears to be an ideal experimental probe of the physical mechanisms underlying processes like allosteric regulation. The chemical mechanism of hydrogen exchange, however, can complicate such studies. Here, we examine hydrogen exchange rates in a simple model system, the c-Src SH3 domain interacting with a short peptide ligand. Addition of ligand slows the rates of hydrogen exchange at nearly every amide for which we can obtain data. Careful analysis, however, reveals that this slowing is due primarily to a reduction in the population of free protein in the system, and not to any specific property of the complex. We present a method to separate the contributions of free and bound protein to the exchange kinetics that has allowed us to identify the subset of amides where exchange arises directly from the complex. These results demonstrate that the slowing of hydrogen exchange induced by ligand interactions should be interpreted with caution, and more extensive experiments are required to correlate changes in hydrogen exchange with changes in structure or internal dynamics.Keywords: SH3; hydrogen exchange; ligand binding; protein dynamics Proteins are highly dynamic molecules. A variety of experimental techniques have revealed protein motions ranging from sidechain rotations to large domain rearrangements, on timescales from nanoseconds to years. Some of these motions can be thought of as transitions between the most populated, native conformation and less populated highenergy states. Under conditions that favor the native conformations, myriad high-energy forms are also sampled, ranging from small local fluctuations to complete unfolding. At equilibrium, the relative population of each conformation is determined by its energy according to a Boltzmann distribution.Although the existence of high-energy forms in the native state ensemble is well established, the functional significance of these conformations in many proteins remains unclear. In allosteric regulation, they may be extremely important. Models of allostery postulate an equilibrium between inactive, tense states and high-energy, active, relaxed states (Monod et al. 1965;Koshland et al. 1966). Positive effectors preferentially bind the active ensemble, shifting the equilibrium and activating the enzyme. This model is well recognized for large, multimeric proteins like hemoglobin and allosteric enzymes. In addition, phosphorylation of the bacterial signaling molecule NtrC has been shown to alter a preexisting equilibrium between low-and high-energy states, favoring active conformations (Volkman et al. 2001). Computer modeling has implicated a similar mechanism for allosteric control in the monomeric enzyme dihydrofolate reductase (Pan et al. 2000), and has suggested that this mechanism may be broadly applicabl...

Analysis of the Thermodynamics of Binding of an SH3 Domain to Proline-rich Peptides using a Chimeric Fusion Protein

“…15,16,[57][58][59] Long-range propagation of conformational tightening would therefore produce an amplification of the local energy changes of single-point mutations. Unfortunately, however, the comparative NMR relaxation study shown here for the SPCp41 chimera and the P7A mutant does not provide us with information about long-range effects in the SH3 domain brought about by the slow aggregation of the mutant chimera, which hindered a more thorough analysis of the data.…”

Fourier transform mass spectrometry to monitor hyaluronan–protein interactions: use of hydrogen/deuterium amide exchange

“…However, in the presence of HA 8 , the ligand can stabilize the folded (closed) form of the protein, thereby preventing additional deuterium uptake in the complex relative to the free protein. 46 It is also this phenomenon which allows binding affinities to be determined by PLIMSTEX studies. 47 Previous work has identified five critical amino acids necessary for Link_TSG6 HA-binding by site-directed mutagenesis (Lys11, Tyr12, Tyr59, Phe70 and Tyr78), and confirmed with NMR studies wherein eleven amino acids (Lys11, Tyr12, Val57, Tyr59, Pro60, Ile61, Phe70, Ill76, Tyr78, Arg81 and Trp88) were found to interact with the ligand.…”

Localizing Carbohydrate Binding Sites in Proteins Using Hydrogen/Deuterium Exchange Mass Spectrometry

“…These data suggest that changes in the structure or dynamics of the N-terminal region Interestingly, no such ligand-induced structural changes are evident from X-ray crystallography data [38]. It is also possible that ligand binding leads to a depopulation of some of the conformers, as this also can influence HDX rates [14].…”

“…When a ligand binds to a protein, some of the amide H's may become (more) protected against exchange (due to the formation of new or stronger inter-or intramolecular H-bonds or a reduction in solvent accessibility), resulting in decreased rates of exchange for the peptides containing these groups [10,14].…”

“…Consequently, the measured differences in peptide D-uptake may reflect the formation of intermolecular interactions or ligand binding-induced changes in protein conformation or dynamics, or a combination of these effects [9,10]. Additionally, ligand-induced changes in the population of different protein conformers may also affect the rate of D-uptake [14,29,30]. It has been proposed that docking simulations or sitedirected mutagenesis, in combination with HDX-MS data, may help to localize the ligand binding site [15,28].…”