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Enzymes can adapt to cold by wiggling regions far from their active site

Mutations introduced far from the active site of an enzyme can cause local unfolding that increases enzyme activity. This finding suggests how organisms that live in the cold can speed up biochemical reactions.

The biochemical sciences have tended to focus on processes that take place at ‘physiological’ temperatures of around 37 °C. But much of Earth’s surface is covered with ocean, ice or snow, and is replete with organisms that function at much lower temperatures. Life in these environments requires suitable biological adaptations, for example in the enzymes that maintain the chemical environment of the cell. In a paper in Nature, Saavedra et al.1 shed light on a biophysical mechanism for such low-temperature adaptation that operates at the molecular level. Their results show strikingly that protein modifications distant from an enzyme’s active site can modulate localized unfolding of the enzyme — effectively, wiggling of parts of the enzyme’s structure — that can control several facets of enzyme-reaction mechanisms.

Physical chemists have long known that the rate of chemical reactions depends on temperature, and that reaction rates generally decrease as temperatures drop. This temperature dependence also applies to enzyme-catalysed reactions, raising the intriguing question of how psychrophilic organisms (which live at low temperatures) can maintain their repertoire of enzyme-mediated functions. Related enzymes in psychrophilic organisms and in mesophilic organisms (which live at physiological temperatures) have similar activities — that is, the reactions they catalyse occur at similar rates2. For this to occur, the functional parameters of the cold-adapted enzymes must have been tuned to compensate for the lower temperatures.

A clue to how this tuning could occur came from previous observations3 that psychrophilic enzymes tend to have more surface glycine mutations (in which an amino-acid residue on the protein’s surface is replaced by a glycine residue) far from the active site than do similar enzymes in mesophilic organisms. However, the mechanistic details of this phenomenon were poorly understood. Saavedra et al. used an enzyme called adenylate kinase to test the mechanism by which such glycine mutations act from a distance to alter enzyme function.

Adenylate kinase catalyses reactions that help to maintain a balance of adenosine phosphates (molecules that act as the energy currency of cells). The authors chose this mesophilic enzyme because it has been used extensively as a model system for investigating enzyme biophysics, biochemistry and folding, including by researchers from the same laboratory as Saavedra and co-workers.

“By making these individual mutations we could either cold adapt the release of the product, or we could cold adapt the pick-up of the substrate.”

In the present work, the authors tested the previously discussed idea2 that tuning of entropy — a measure of disorder — is a major driving force in the adaptation of enzymes to low temperatures. They sought to probe the effect of surface glycine mutations, at locations far from the active site, that might change the ‘wiggling’ (an entropic effect) of the protein without changing its overall folded structure.

There are three domains in adenylate kinase: the CORE domain, which contains much of the active site, and LID and AMPbd, each of which contains part of the active site. The authors studied glycine mutations in both the LID and the AMPbd domains by using a combination of biophysical and structural techniques. These studies included measuring the stability of the enzyme variants and their binding affinity to a mimic of the enzyme’s substrate, and a more detailed characterization of the protein states and structural fluctuations by using nuclear magnetic resonance spectroscopy.

Taken together, Saavedra and colleagues’ results demonstrate that adenylate kinase exists in at least three different states, and that the mutations change the relative occupancy (stability) of these states. Compared with the wild-type protein, both the LID and the AMPbd mutants decrease the occupancy of the fully folded structure, but they increase the stability of two different states in which either the LID or the AMPbd domain is locally unfolded (Fig. 1). The increased stability of these locally unfolded states stems from the fact that the footprint of a glycine amino-acid residue is smaller than that of other amino-acid residues, which means that the protein chains in the glycine mutants are more flexible than those in the wild-type protein. Remarkably, these two types of local unfolding alter different aspects of the enzyme’s function: LID unfolding decreases its binding affinity, whereas AMPbd unfolding increases its activity.

Figure 1 | Entropic tuning of enzyme function allows cold adaptation. Saavedra et al.1 made mutants of the enzyme adenylate kinase, replacing non-glycine amino-acid residues with glycine residues at the surface of either the LID domain (red) or the AMPbd domain (blue). Both types of mutation caused local unfolding of the enzyme, increasing its disorder (entropy), and altered the enzyme’s functional behaviour, despite being distant from the active site. The LID mutations decreased the affinity of adenylate kinase for its substrates, whereas the AMPbd mutations increased the enzyme activity. Such entropic tuning of function might be an evolutionary mechanism that allows enzymes to cope with low temperatures, which usually slow enzymatic reactions.

A particularly interesting aspect of the authors’ work is that it helps us to understand how surface glycine mutations act at a distance to support cold adaptation. This phenomenon might seem mysterious at first glance, but it is encapsulated in the idea of allosteric regulation4,5 — a common form of enzyme regulation in which the binding of a molecular partner at a site distant from the active site affects enzyme activity. The conventional view of allosteric regulation has been that binding of the partner causes small structural changes that propagate through the protein to alter the structure of the active site. However, there is now evidence for mechanisms involving changes in the dynamics (entropy), rather than in the structure, of the unbound state for many cases of allosteric regulation68. The current work provides striking tests and examples of such entropic allosteric regulation for different enzyme properties.

Saavedra and colleagues’ results have substantial implications for the evolution of enzyme function. However, their mechanistic proposal for cold adaptation was tested for just one model enzyme, so its relevance for other cold-adapted enzymes requires further testing. Deeper insight into the biophysical mechanisms of cold adaptation will also benefit from more-detailed views of the structural fluctuations of enzymes afforded by single-molecule experiments9,10. Nevertheless, the findings open up new avenues for exploring allosteric control of multiple modes of protein function, both in natural evolution and in rational protein engineering11 for biotechnology.

Nature 558, 195-196 (2018)

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