Over the last two decades, multiple kinds of NMR experiments have repeatedly shown that protein structures are quite variable, frequently shifting to minor conformations. The most striking evidence in this line has come from hydrogen-exchange experiments, which have demonstrated that virtually all proteins undergo excursions to partially-folded states at equilibrium. As R2 relaxation-dispersion experiments have become more widely used, excursions to alternative folded states have repeatedly been detected. The challenge now is to find ways to characterize these low-population states. Advanced crystallographic techniques have proven useful in determining some of these alternative structures. However, proteins are not always amenable to crystallography, and the minor state in the crystal may not correspond exactly to the minor state in solution. Therefore there is an ongoing effort to define these states by NMR. Lewis Kay’s group in Toronto is in the forefront of this effort, and recently reported the solution structure of a minor state of a T4 lysozyme mutant (1).
Lysozyme is an extremely common enzyme because it has the useful property of degrading the peptidoglycan that makes up bacterial cell walls. This makes it a natural antibiotic against gram-positive bacteria, and as a result it is found in many secretions and fluids, including saliva and egg whites. Because it is plentiful it has been widely studied, with many mutants made and characterized for their activity and stability. Lysozyme also crystallizes easily — doing this was actually part of my biochemistry lab class back in college. So, many structures of the enzyme and its mutants are available.
One lysozyme mutant that has interesting properties is the L99A mutant of the lysozyme from the T4 bacteriophage. This mutation creates a cavity in the upper part of the protein that is known to bind hydrophobic ligands such as benzene (right, benzene in purple, PDB code 3DMX). However, crystal structures show this binding pocket to be completely buried, even when empty. This poses the question of how the ligand gets in. Although the structure of L99A is very similar to WT, the Kay lab noticed that the NMR spectra of the mutant contained broadened peaks, indicating the presence of an exchange process between two conformations. Therefore, the Kay lab used R2 relaxation-dispersion to show that the protein sampled a minor state that accounted for 3% of the total protein, with a lifetime of about 1 ms (2). This conformation was presumed to be the binding-competent form of the protein. However, without a structure of this state, they could not confirm that the pocket was accessible. This led to their present attempts to characterize this low-population state using NMR.
As I have mentioned before, R2 relaxation-dispersion experiments can provide three important pieces of information: the populations of the two conformational states (pG, pE for ‘Ground’ and ‘Excited’), the rate of exchange between them (kEX = kGE + kEG), and the difference in chemical shift between the two states at each nucleus (|Δω|). Because the chemical shift is determined by the protein conformation, and because additional experiments can determine the sign of Δω, it should be possible to figure out the structure of the alternate state, given enough relaxation-dispersion data. Therefore, the Kay lab performed a large number of experiments to determine Δω for nearly all of the backbone 15N, 13C, and 1H atoms, as well as many side-chain methyl groups. They then fed this data to the CS-ROSETTA protocol, which can determine a protein structure using chemical shifts alone. While holding the majority of the protein in a single conformation, they allowed CS-ROSETTA to remodel the part of the mutant where they had detected conformational fluctuations.
Using this method, they were able to produce a structure of the transiently-populated minor state of the mutant protein, which I show to the left in comparison to the major conformation (PDB codes 2LCB and 3DMV, respectively, aligned using residues 10-100, 150-160). The most dramatic change is that two of the helices have been fused into one. As you can see, the new helix clashes with the usual position of phenylalanine 114 (pale green, because of the overlap it’s hard to see), which has in turn shifted so that it occupies part of the cavity where benzene binds (pale blue). This suggests, contra the Kay group’s earlier work, that the minor state is also incapable of binding to benzene.
This is a difficult prediction to test in the L99A system because the minor state (E) lives for such a short time that it’s difficult to tell whether anything binds to it or not. Therefore, Bouvignies et al. made a double-mutant protein with the L99A mutation and an additional G113A mutation that was predicted to stabilize the long helix observed in the minor form. This turned out to be the case: the E structure was enriched in the double mutant. In addition, the interconversion rate was slow enough that at low temperature distinct peaks could be observed for each conformation, as well as cross-peaks indicating exchange between them (I discussed this kind of experiment in my previous posts about cyclophilin). Under these conditions, the minor form is sufficiently populous and long-lived to determine whether ligands bind to it.
The Kay group did this by adding an equimolar amount of benzene to the reaction and observing whether there were exchange peaks. If you examine their figure 3c, it’s clear that exchange occurs between all three possible states: (G)round, (E)xcited, and (B)ound. This might seem to contradict their hypothesis. However, the E→B exchange peaks have very low intensity and take significantly longer to reach a maximum than the other exchange peaks. Therefore, this exchange peak may represent a low-frequency E→G→B event rather than direct exchange between the E and B states. Fits of the exchange curves seem to substantiate this interpretation, as the fit tended towards a value of zero for kEB and the χ2 jumped up significantly when kEB was fixed to a very low number.
My only concern with this result is that the kEG rate changes from ~31 to ~36 s-1 when benzene is added (kGE remains the same). It’s possible that the presence of benzene really does accelerate this process, or that the errors are underestimated. The model might also be janky in some hidden way, but my back-of-the-envelope check of the parameters suggests that the results are consistent with what is known about benzene binding to the L99A mutant, e.g. various ways of calculating the KD from these data produce a value of approximately 1 mM, matching earlier results.
If the E state does not represent a binding-competent state, that means the protein must be exchanging to yet another, still-undetected state. According to Bouviginies et al., the E structure they determined can account for all of the observed chemical exchange. If the alternative state that is capable of admitting benzene to the hydrophobic pocket cannot be detected by relaxation-dispersion experiments, it must constitute a very small fraction of the overall protein population (< 1%) and undergo very fast exchange. In principle, the existence of such a process can be detected using experiments designed to measure the intrinsic R2 of a residue, and also should be detectable using 1H experiments directed towards the methyl groups (the side chains likely represent the best bet for explaining the phenomenon). It does not appear that those experiments have been done yet, but I’m certain they’re underway.
Bouvignies et al. made a third construct incorporating the R119P mutation to stabilize the E state even further. This succeeded, producing a protein that spent most of its time in the E state and occasionally sampled the G state. The paper contains no data as to whether benzene detectably binds this mutant, although that strikes me as an obvious experiment to try. Presumably the obligate route through a high-energy intermediate would slow the kinetics of binding relative to the single mutant. If the penalty for adopting the G fold in this mutant is high enough, it might also significantly reduce the affinity.
The findings in this paper are not of any immediate practical use. The L99A mutant is a biophysical curiosity, not a disease target, and most of these techniques have been presented before, at least individually. However, this does serve as a very nice example of the advanced NMR methods that allow the determination of minor states, and of the surprising findings that can be derived from them. This paper should serve as a model approach to this sort of question, which may find broad applicability in the study of signaling, ligand binding, and protein evolution.
Disclaimer: I am currently collaborating with David Baker’s lab on a research project using ROSETTA.
1) Bouvignies G, Vallurupalli P, Hansen D, Correia B, Lange O, Bah A, Vernon R, Dahlquist FW, Baker D, & Kay LE (2011). Solution structure of a minor and transiently formed state of a T4 lysozyme mutant Nature, 477 (7362), 111-114 DOI: 10.1038/nature10349
2) Mulder FA, Mittermaier A, Hon B, Dahlquist FW, & Kay LE (2001). Studying excited states of proteins by NMR spectroscopy. Nature structural biology, 8 (11), 932-5 PMID: 11685237