A point mutation remodels a binding interface

Decades of studies involving extensive mutagenesis of proteins and protein domains have impressed on us the idea that the folded tertiary structures of proteins are fairly resilient. While a particular mutation may abolish function by directly ablating a key chemical group, it is rare for a single mutation, or even a group of several mutations, to significantly change the overall conformation of a folded polypeptide chain. When a major change does result, it often takes the form of complete denaturation. Because of this, it may seem that protein folds occupy stable islands in sequence space, surrounded by a sea of sequences that form random coils or molten globules. However, there is some evidence that this view is mistaken, that substantially divergent structures may have very similar sequences. A paper recently published in PNAS adds to this view by describing a major change in the structure of a PAS domain resulting from 1-3 mutations.

PAS is a large family of protein-protein interaction domains contained in many signaling proteins. Although many of them have cofactors that modulate their binding, some PAS domains are constitutively active, which is the case for the domain under study here, the PAS-B domain from one of the founding members of the family, the aryl hydrocarbon receptor nuclear transporter (ARNT). The PAS-B domain was believed to dimerize with PAS domains from other proteins through one side of its β-sheet, and as a consequence Evans et al. decided to make several mutations on the outer surface of the sheet and monitor their effects on dimer formation.

One of these mutations, Y456T, had a strange effect on the domain's NMR spectrum: about 30 new peaks showed up in the ¹H-¹⁵N HSQC. Because this spectrum should show a single peak for each chemically unique proton-nitrogen pair, this result suggests that the pure proteins in the magnet exist in two distinct conformations. By lowering the temperature and repurifying the protein, Evans et al. were able to mostly separate these two conformations from each other. However, over time these conformationally purified samples became heterogeneous again, which means that the conformations can freely interconvert. This process was very slow, however — too slow to be detected using NMR relaxation techniques. From monitoring the HSQCs the authors concluded that the time constant for interconversion in the mutant is on the order of 16 hours.

Intrigued by what they were seeing, Evans et al. made additional mutations to ARNT PAS-B and found that the proportion of protein in each structure can be adjusted within a wide range by mutation. Using a triple-mutant system they were able to drive about 99% of the proteins into the new conformation. Using this mutant the authors were able to assign the resonances in the HSQC spectrum and solve the structure using NOEs. They learned that the chemical shift changes in the mutant are quite widespread, as you can see in the figure I have shamelessly stolen (left) for your benefit. In this figure the chemical shift changes are mapped onto their structure of the new conformation using color, ranging from green (very little or no change) to red (significant changes) — the sites of the three mutations are shown. As you can see, the chemical shift effect is widespread, covering almost the whole β-sheet of the protein and reaching to the helix on the opposite side.

Closer analysis of the structure shows why this is so. The strand of the β-sheet on which Y456 is situated has shifted its register by 3 spots. This has two effects. The first is that all of the hydrogen bonds involving that strand must be broken, at a substantial energetic cost. This is probably the reason the interconversion process is so slow. Moreover, because the register shifts by an odd number of residues, the strand must flip over, exposing to solvent the residues that were buried in the original structure, while burying the residues that were previously solvent-exposed. Although the backbone and side chain orientations in the strand overlay reasonably well between the two structures, the chemical groups available for interactions are completely different. Unsurprisingly, this alternate structure has very low affinity for its natural targets — titration experiments showed that the alternate conformation bound to its partner from hypoxia inducible factor at least 100x worse than the native structure.

Of course, in living cells with a wide variety of surfaces to bind to, the mutant PAS-B might find an alternative partner for which it has high affinity. Studies that attempt to understand protein-protein interfaces from an engineering or evolutionary perspective typically adopt the assumption that point mutations have little effect beyond adding a particular functional group here or there. This study indicates that this attitude underestimates the ability of point mutations to radically remodel interface surfaces. While a Y→T mutation may not seem particularly conservative, a Y→S mutation has similar effects and requires only a single nucleotide base change. It is not inconceivable that this alternate conformation could be reached in vivo, potentially giving rise to completely novel protein-protein interactions.

One might well wonder how common rearrangements of this kind are likely to be. As the authors point out, structural plasticity in the β-sheet is likely to be a common feature of PAS domains, making it difficult to assess whether this kind of mutational effect is widespread in other folds. However, the authors cite several examples of β-strand register shifts in other proteins. In addition, our decades of alanine-scanning mutagenesis have little to tell us about how common these kinds of rearrangements are, for two main reasons. First, the structural effects strongly depend on what a given residue is mutated to (see Table 1); had Evans et al. been content to leave things at alanine mutations they would never have detected this effect. Second, widely-applicable techniques for sensitively detecting small populations of alternate conformations have not been available until recently.

While the conformational transition induced by the Y456T mutation preserves the protein's overall fold and stability, it rearranges the hydrogen bonding contacts of the main β-sheet and shortens a loop. Obviously this is not as dramatic a change as found in lymphotactin. However, this alternate structure has significant consequences for PAS-B function. Unexpectedly, this single point mutation can radically remodel the PAS-B binding surface. Moreover, this result adds to the evidence that new structures (even in the context of known folds) may be accessed with only a few changes in amino acid sequence, without any need to detour through molten-globule intermediates.

M. R. Evans, P. B. Card, K. H. Gardner (2009). ARNT PAS-B has a fragile native state structure with an alternative β-sheet register nearby in sequence space Proceedings of the National Academy of Sciences, 106 (8), 2617-2622 DOI: 10.1073/pnas.0808270106