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Protein-Nucleic Acid Interaction: Inhibition Through Allostery

SBKB [doi:10.1038/sbkb.2012.151]
Technical Highlight - July 2013
Short description: NMR studies highlight the importance of investigating transitional species to fully understand protein function.

Energy landscape of mutant CAP showing the two states, inactive (I) and active (A), and their fractional populations. DNA binding selects the active conformation in a population shift mechanism. 1

Small molecule inhibitors often compete directly with native enzyme substrates or binding partners. However, because these sites are frequently conserved among similar proteins, allosteric inhibitors, which exert their activity by binding at a site distal to the active or partner interaction site, may offer increased specificity.

Kalodimos and Tzeng investigated the mechanism behind the allosteric inhibition of catabolite activator protein (CAP) by cyclic guanosine monophosphate (cGMP). Normally, the binding of cyclic adenosine monophosphate (cAMP) to the cyclic nucleotide-binding domain (CBD) of CAP induces an allosteric change in the DNA-binding domain (DBD) and promotes DNA binding. The authors used a previously identified mutant of CAP in which the ability of the DBD to bind to DNA is reduced upon binding of cGMP to the CBD. NMR analyses did not reveal major structural differences between the wild-type and mutant CAP DBDs, indicating that both should adopt the inactive conformation. However, the mutant CAP DBD was able to bind DNA, whereas the wild-type DBD was not.

Speculating that the mutant DBD may be able to transiently adopt an active conformation not accessible to the wild-type species, the authors used an NMR technique called relaxation dispersion, which can provide information on lowly populated species. In fact, NMR is unique in that it can provide information on both the ground state, or most populated species, as well as higher-energy intermediate states that are transient and less populated. It is these higher-energy states that are often responsible for protein activity.

The relaxation dispersion results revealed a transient intermediate state in the mutant CAP that was not detected in the wild-type protein. While this transient state is a minor species, accounting for 7% of the total protein population, it is the conformation that is able to bind DNA.

While binding of cGMP did not alter the structure of the DBD, it did prevent the formation of the intermediate state in the mutant CAP. Further structural analysis of the mutant CAP revealed that the mutation promotes a coil-to-helix transition, which is disrupted upon cGMP binding. These results demonstrate the utility of investigating lowly populated, transitional species to better understand, and perhaps better manipulate, protein function.

Jennifer Cable


  1. S.-R. Tzeng and C.G. Kalodimos. Allosteric inhibition through suppression of transient conformational states.
    Nat Chem Bio. (5 May 2013). doi:10.1038/nchembio.1250

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