Nuclear magnetic resonance spectroscopy has been used to establish a vital role for protein motion in the formation of a protein–DNA complex. The finding potentially opens up fresh approaches for modifying protein function. See Letter p.236
The continuing development of the tools of structural biology and their increasingly sophisticated application to studies of a wide range of biological molecules are some of the most noteworthy accomplishments of biophysics. The structures obtained have been used to explain molecular function, to design and modify proteins so as to engineer new biological properties, and in the rational generation of pharmaceuticals. Yet despite the well-documented successes, there have also been many cases in which the beautiful, high-resolution pictures produced leave many questions unanswered. Part of the reason is that the biological function of a molecule is driven by free energy changes that depend on contributions from both static (enthalpic) and dynamic (entropic) interactions1. Although static structures can provide atomic-resolution information about enthalpy, describing entropy at a similar level of detail has been far more difficult.
On page 236 of this issue, Tzeng and Kalodimos2 quantify the role of dynamics in their study of the catabolite activator protein, CAP. They have used nuclear magnetic resonance (NMR) spectroscopy to demonstrate that the binding activity of CAP can be regulated by conformational entropy — the entropy component associated with the number of conformational states that the protein samples.
CAP acts as a transcriptional activator — it binds to DNA to increase gene transcription. It is also an allosteric protein: binding of a ligand molecule at one site can introduce changes in both the structure3,4 and the dynamics5 of distal sites. More specifically, binding of a small nucleotide molecule, cyclic AMP (cAMP), to the cAMP-binding domain of CAP leads to substantial structural rearrangements in distal DNA-binding domains, priming the protein for DNA binding (Fig. 1a). Tzeng and Kalodimos studied CAP in the unbound and DNA-bound states.
The authors exploited CAP's allostery by engineering mutations in the protein at sites remote from the DNA-binding interface, but which nevertheless modulate DNA binding. By using NMR to study the derived mutants, as well as different nucleotide-bound forms of the protein, the authors established that the protein interconverts between inactive states that cannot bind DNA and active states that can, and that, for the mutants examined, the relative populations of these states can be very different.
In the simplest of binding models for CAP, the affinity of the protein for its target DNA is directly proportional to the fraction of molecules in the active conformation, as has been seen previously in different contexts for other systems6,7,8,9. But Tzeng and Kalodimos observed little such correlation. Indeed, the affinities of some of the CAP mutants for DNA are 50-fold greater, and others are 25-fold lower, than would have been predicted on the basis of the numbers of molecules populating the active state. This clearly indicates that static structures alone cannot explain CAP's behaviour.
The authors therefore went on to measure the enthalpic and entropic contributions to the CAP mutants' DNA-binding affinities using a calorimetric technique. Although the resulting data are informative, they are not at atomic resolution and they lump together contributions from a variety of terms. Of these contributions, one of the most useful to evaluate is conformational entropy, which counts the number of states adopted by bonds in CAP's 'backbone' and amino-acid side chains.
To gain more insight into the conformational entropy of the CAP mutants, Tzeng and Kalodimos carried out NMR experiments to quantify the amplitudes of motion of methyl groups in side chains, at the picosecond-to-nanosecond timescale (one picosecond is 10−12 seconds). They found that, on DNA binding, some of the mutants undergo very large net changes in conformational entropy that significantly increase the strength of association (Fig. 1b). In other cases, they observed that enthalpy changes drive binding, and that entropy changes oppose it.
Notably, the authors obtained an atomic-level description of how the picosecond–nanosecond dynamics of CAP respond to formation of the CAP–DNA complex from which the conformational entropy change for CAP–DNA binding was established. The pattern that emerges is not simple. For example, large changes in amplitudes of motion might have been expected only at regions close to the binding interface, but such changes extend much farther away, involving methyl groups more than 50 ångströms from the interface. Remarkably, the authors found a strong linear correlation between NMR-derived measures of conformational entropy change and the total change in entropy measured by calorimetry. Such a correlation has previously been observed10 in the binding of the calmodulin protein to its target peptides.
Tzeng and Kalodimos's findings show that, although entropy changes are crucial for CAP's function, there are other ways of modulating the strength of DNA binding to the protein. Even though the correlation between the fractional population of CAP molecules in the active conformation and DNA-binding affinity is poor, increasing the population that is in the active state still remains one avenue for increasing affinity. The authors' NMR experiments revealed that, of the nine CAP mutants that occupy predominantly inactive structures, two interchange with active conformations on the millisecond timescale; approximately 2% of the molecules are in the active conformation for one of these mutants, and 7% for the other. Unlike the other seven variants, for which active populations could not be detected, these two mutants bound DNA.
Most interestingly, Tzeng and Kalodimos's work suggests an approach for manipulating protein function through allostery. For example, one could imagine targeting a drug to a site in an enzyme that is far removed from the active site, in order to modify the enzyme's function. By combining allosteric drugs of this sort with more traditional pharmaceuticals that bind to active sites, it may be possible to moderate the drug resistance that so often plagues conventional therapies. Understanding the fundamental roles of dynamics in protein function will also facilitate new ways of exploiting proteins and of modifying their activities. Tzeng and Kalodimos's work takes an important step in this direction.
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