A conformational equilibrium controls the Vav DH domain

One emerging view of allostery, and protein behavior generally, describes function in terms of pre-existing equilibria. In this view, proteins are not like switches that get turned on and off, but rather are like dials that are turned "more on" or "more off" depending on the conditions. In this view, regulatory modifications such as phosphorylation do not enforce an active conformation so much as promote it. Because some relaxation measurements are sensitive to conformational exchange, NMR is well-suited to examine systems with this behavior. In the most recent edition of Nature Structural and Molecular Biology, a group from Michael Rosen's lab discover that this kind of equilibrium is governing the behavior of the DH domain from the Vav protein.

Vav activates Rho GTPases by inducing the exchange of GDP for GTP, making it a Guanine nucleotide Exchange Factor (GEF). This activity is performed by the DH domain, and is inhibited by a small neighboring element called the acidic region (Ac). As you can see from the figure of the combined Ac and DH domains (AD) at right, Ac (red) inhibits the DH domain (blue) by forming a helix that binds to the active site (explore this structure at the PDB, noting that the numbering is off by 167). Phosphorylation of Y174 (red side chain) unfolds the helix and exposes the active site, which would be a nice model except for two things. First, as you can see, Y174 is pretty well buried in this structure, which would make it difficult to phosphorylate. Second, mutation of Y174 to phenylalanine, a residue that cannot be phosphorylated, activates DH domain activity. How can Y174 get phosphorylated? What is phosphorylation actually doing?

One possible answer is that the existing structure doesn't tell the whole story. A protein, after all, doesn't have just a single structure, but rather an ensemble of structures across a population or time. While AD may spend most most of its time in this inhibited state, it's possible that sometimes it adopts an alternate conformation that allows Y174 phosphorylation. Li et al. set out to assess this possibility using measurements of R₂ relaxation dispersion. Residues that have a large field-dependence of transverse relaxation (ΔR₂) are undergoing some sort of conformational exchange process that changes their chemical shift between two or more states.

Using CPMG experiments on methyl-bearing side chains, Li et al. identified two groups of residues engaged in conformational exchange processes, shown at left. The first group (orange side chains) have a large ΔR₂ that vanishes once Y174 is phosphorylated. The second group (green side chains) have high ΔR₂ in both the phosphorylated and unphosphorylated states, but the rates are slightly higher in the former. Residues that were observed to have low ΔR₂ are shown with gray side chains. All of this indicates that there are two dynamic processes occuring on the microsecond-millisecond timescale. The first encompasses some change in the chemical shift of the acidic helix, while the second involves some unknown process. However, because the Group 2 residues react to the phosphorylation state, these processes are likely linked in some way. I notice that the Group 2 residues are clustered around loops and joints in the upper half of the domain (in this view), while residues not adjacent to loops or joints do not appear to have significant ΔR₂. It is possible that the observed dispersion represents some flexing of the domain around these loops, and that the rate of this motion increases slightly when the binding site is unoccupied.

That's all very interesting, but it's also bad news for the analysis, because it means the observed relaxation dispersion would have to be fit to a four-state model in order to obtain populations and kinetic parameters. Previous analysis by several groups has shown this to be a dubious proposition, so Li et al. take an alternative approach. Rather than try to fit out populations from the dispersion data, they make a series of mutations to AD to push the populations of the two states in one direction or the other. They find a number of states where the methyl peaks lie on a line between the open (phosphorylated) and bound (unmodified) states. The Y174F mutation lies very close to the phosphorylated state, interestingly enough, implying that the phosphate group itself is not a significant determinant of chemical shifts in the open state. Using a combination of HSQC peak positions and ΔR₂ measurements, Li et al. determine for each mutant or modification what population of the ensemble is in the open state. They find that this NMR-assigned population correlates with the rate constant (k_cat/K_M) for phosphorylation.

This implies a model in which regulation of DH by Ac involves an equilibrium between the bound and open states. In the bound state, Ac forms a helix in the binding site, an effect strongly dependent on a hydrogen bond to the OH of the Y174 (R332 may be the partner here). However, in this state Ac samples the open state about 10% of the time. While in the unbound state, Ac can be phosphorylated, a modification that prevents helix formation or binding; probably by steric interference in the binding site. This stabilization of the open state dramatically increases the chances that the Vav DH domain will be in an active state when it encounters a target. Thus, DH regulation depends on a population shift of an underlying equilibrium, not a singular on/off switch. This model has the advantage of accounting for how Y174 gets phosphorylated and why a mutation that prevents phosphorylation nonetheless leads to a constitutively activated state.

In vivo, Vav consists of many other domains in addition to the AD construct used here. These domains are known to contribute to the inhibition of DH; given these results it is probable that they do so by stabilizing the bound state. Because Ac binding causes the formation of a negatively-charged surface on one side of the helix, charge stabilization is a likely mechanism. Further research will hopefully identify these mechanisms, as well as the origin of the second conformational exchange process revealed by the experiments in this paper. This study is a good example of scientists making the best use of limited data to describe an instance of this important, but difficult to characterize, regulatory mechanism.

1. Li, P., Martins, I.R., Amarasinghe, G.K., Rosen, M.K. (2008). Internal dynamics control activation and activity of the autoinhibited Vav DH domain. Nature Structural & Molecular Biology, 15(6), 613-618. DOI: 10.1038/nsmb.1428