In the first part of this work (paper 1, Xue, Y. et al. Biochemistry 2014, 53, 6473), we have studied the complex between the 10-residue peptide Sos and N-terminal SH3 domain from adaptor protein c-Crk. In the second part (this paper), we designed the double mutant of the c-Crk N-SH3 domain, W169F/Y186L, with the intention to eliminate the interactions responsible for tight peptide–protein binding, while retaining the interactions that create the initial electrostatic encounter complex. The resulting system was characterized experimentally by measuring the backbone and side-chain 15N relaxation rates, as well as binding shifts and 1HN temperature coefficients. In addition, it was also modeled via a series of ∼5 μs molecular dynamics (MD) simulations recorded in a large water box under an Amber ff99SB*-ILDN force field. Similar to paper 1, we have found that the strength of arginine-aspartate and arginine-glutamate salt bridges is overestimated in the original force field. To address this problem we have applied the empirical force-field correction described in paper 1. Specifically, the Lennard-Jones equilibrium distance for the nitrogen–oxygen pair across Arg-to-Asp/Glu salt bridges has been increased by 3%. This modification led to MD models in good agreement with the experimental data. The emerging picture is that of a fuzzy complex, where the peptide “dances” over the surface of the protein, making transient contacts via salt-bridge interactions. Every once in a while the peptide assumes a certain more stable binding pose, assisted by a number of adventitious polar and nonpolar contacts. On the other hand, occasionally Sos flies off the protein surface; it is then guided by electrostatic steering to quickly reconnect with the protein. The dynamic interaction between Sos and the double mutant of c-Crk N-SH3 gives rise to only small binding shifts. The peptide retains a high degree of conformational mobility, although it is appreciably slowed down due to its (loose) association with the protein. Note that spin relaxation data are indispensable in determining the dynamic status of the peptide. Such data can be properly modeled only on a basis of bona fide MD simulations, as shown in our study. We anticipate that in future the field will move away from the ensemble view of protein disorder and toward more sophisticated MD models. This will require further optimization of force fields, aimed specifically at disordered systems. Efforts in this direction have been recently initiated by several research groups; the empirical salt-bridge correction proposed in our work falls in the same category. MD models obtained with the help of suitably refined force fields and guided by experimental NMR data will provide a powerful insight into an intricate world of disordered biomolecules.

Intrinsically disordered proteins (IDPs) often rely on electrostatic interactions to bind their structured targets. To obtain insight into the mechanism of formation of the electrostatic encounter complex, we investigated the binding of the peptide Sos (PPPVPPRRRR), which serves as a minimal model for an IDP, to the c-Crk N-terminal SH3 domain. Initially, we measured 15N relaxation rates at two magnetic field strengths and determined the binding shifts for the complex of Sos with wild-type SH3. We have also recorded a 3 μs molecular dynamics (MD) trajectory of this complex using the Amber ff99SB*-ILDN force field. The comparison of the experimental and simulated data shows that MD simulation consistently overestimates the strength of salt bridge interactions at the binding interface. The series of simulations using other advanced force fields also failed to produce any satisfactory results. To address this issue, we have devised an empirical correction to the Amber ff99SB*-ILDN force field whereby the Lennard-Jones equilibrium distance for the nitrogen–oxygen pair across the Arg-to-Asp and Arg-to-Glu salt bridges has been increased by 3%. Implementing this correction resulted in a good agreement between the simulations and the experiment. Adjusting the strength of salt bridge interactions removed a certain amount of strain contained in the original MD model, thus improving the binding of the hydrophobic N-terminal portion of the peptide. The arginine-rich C-terminal portion of the peptide, freed from the effect of the overstabilized salt bridges, was found to interconvert more rapidly between its multiple conformational states. The modified MD protocol has also been successfully used to simulate the entire binding process. In doing so, the peptide was initially placed high above the protein surface. It then arrived at the correct bound pose within ∼2 Å of the crystallographic coordinates. This simulation allowed us to analyze the details of the dynamic binding intermediate, i.e., the electrostatic encounter complex. However, an experimental characterization of this transient, weakly populated state remains out of reach. To overcome this problem, we designed the double mutant of c-Crk N-SH3 in which mutations Y186L and W169F abrogate tight Sos binding and shift the equilibrium toward the intermediate state resembling the electrostatic encounter complex. The results of the combined NMR and MD study of this engineered system will be reported in the next part of this paper.

•We describe proton-decoupled CPMG sequence for measuring 15N R2 relaxation rates.•For disordered proteins, the new scheme achieves 3-fold improvement in precision.•The experiment is immune to various artefacts from amide solvent exchange.•15N offset correction is small and easy to calculate.•The experiment has also been adapted to measure 15Nε relaxation in Arg side chains.15N R2 relaxation is one of the most informative experiments for characterization of intrinsically disordered proteins (IDPs). Small changes in nitrogen R2 rates are often used to determine how IDPs respond to various biologically relevant perturbations such as point mutations, posttranslational modifications and weak ligand interactions. However collecting high-quality 15N relaxation data can be difficult. Of necessity, the samples of IDPs are often prepared with low protein concentration and the measurement time can be limited because of rapid sample degradation. Furthermore, due to hardware limitations standard experiments such as 15N spin-lock and CPMG can sample the relaxation decay only to ca. 150 ms. This is much shorter than 15N T2 times in disordered proteins at or near physiological temperature. As a result, the sampling of relaxation decay profiles in these experiments is suboptimal, which further lowers the precision of the measurements.Here we report a new implementation of the proton-decoupled (PD) CPMG experiment which allows one to sample 15N R2 relaxation decay up to ca. 0.5–1 s. The new experiment has been validated through comparison with the well-established spin-lock measurement. Using dilute samples of denatured ubiquitin, we have demonstrated that PD-CPMG produces up to 3-fold improvement in the precision of the data. It is expected that for intrinsically disordered proteins the gains may be even more substantial. We have also shown that this sequence has a number of favorable properties: (i) the spectra are recorded with narrow linewidth in nitrogen dimension; (ii) 15N offset correction is small and easy to calculate; (iii) the experiment is immune to various spurious effects arising from solvent exchange; (iv) the results are stable with respect to pulse miscalibration and rf field inhomogeneity; (v) with minimal change, the pulse sequence can also be used to measure R2 relaxation of 15Nε spins in arginine side chains. We anticipate that the new experiment will be a valuable addition to the NMR toolbox for studies of IDPs.Graphical abstractDownload high-res image (81KB)Download full-size image

With the advent of ultra-long MD simulations it becomes possible to model microsecond time-scale protein dynamics and, in particular, the exchange broadening effects (Rex) as probed by NMR relaxation dispersion measurements. This new approach allows one to identify the exchanging species, including the elusive “excited states”. It further helps to map out the exchange network, which is potentially far more complex than the commonly assumed 2- or 3-site schemes. Under fast exchange conditions, this method can be useful for separating the populations of exchanging species from their respective chemical shift differences, thus paving the way for structural analyses. In this study, recent millisecond-long MD trajectory of protein BPTI (Shaw et al. Science2010, 330, 341) is employed to simulate the time variation of amide 15N chemical shifts. The results are used to predict the exchange broadening of 15N lines and, more generally, the outcome of the relaxation dispersion measurements using Carr–Purcell–Meiboom–Gill sequence. The simulated Rex effect stems from the fast (∼10–100 μs) isomerization of the C14–C38 disulfide bond, in agreement with the prior experimental findings (Grey et al. J. Am. Chem. Soc.2003, 125, 14324).

Molecular dynamics (MD) simulations have been widely used to analyze dynamic conformational equilibria of folded proteins, especially in relation to NMR observables. However, this approach found little use in the studies of disordered proteins, where the sampling of vast conformational space presents a serious problem. In this paper, we demonstrate that the latest advances in computation technology make it possible to overcome this limitation. The experimentally validated (calibrated) MD models allow for new insights into structure/dynamics of disordered proteins. As a test system, we have chosen denatured ubiquitin in solution with 8 M urea at pH 2. High-temperature MD simulations in implicit solvent have been carried out for the wild-type ubiquitin as well as MTSL-tagged Q2C, D32C, and R74C mutants. To recalibrate the MD data (500 K) in relation to the experimental conditions (278 K, 8 M urea), the time axes of the MD trajectories were rescaled. The scaling factor was adjusted such as to maximize the agreement between the simulated and experimental 15N relaxation rates. The resulting effective length of the trajectories, 311 μs, ensures good convergence properties of the MD model. The constructed MD model was validated against the array of experimental data, including additional 15N relaxation parameters, multiple sets of paramagnetic relaxation enhancements (PREs), and the radius of gyration. In each case, a near-quantitative agreement has been obtained, suggesting that the model is successful. Of note, the MD-based approach rigorously predicts the quantities that are inherently dynamic, i.e., dependent on the motional correlation times. This cannot be accomplished, other than in empirical fashion, on the basis of static structural models (conformational ensembles). The MD model was further used to investigate the relative translational motion of the MTSL label and the individual HN atoms. The derived segmental diffusion coefficients proved to be nearly uniform along the peptide chain, averaging to D = 0.49–0.55 × 10–6 cm2/s. This result was verified by direct analysis of the experimental PRE data using the recently proposed Ullman-Podkorytov model. In this model, MTSL and HN moieties are treated as two tethered spheres undergoing mutual diffusion in a harmonic potential. The fitting of the experimental data involving D as a single adjustable parameter leads to D = 0.45 × 10–6 cm2/s, in good agreement with the MD-based analyses. This result can be compared with the range of estimates obtained from the resonance energy transfer experiments, D = 0.2–6.0 × 10–6 cm2/s.

Analyses of solution 15N relaxation data and solid-state 1HN−15N dipolar couplings from a small globular protein, α-spectrin SH3 domain, produce a surprisingly similar pattern of order parameters. This result suggests that there is little or no ns−μs dynamics throughout most of the sequence and, in particular, in the structured portion of the backbone. At the same time, evidence of ns−μs motions is found in the flexible loops and termini. These findings, corroborated by the MD simulations of α-spectrin SH3 in a hydrated crystalline environment and in solution, are consistent with the picture of protein dynamics that has recently emerged from the solution studies employing residual dipolar couplings.