Keep a lid on it: A troika in kinase allostery

The malaria parasite Plasmodium falciparum encodes a cGMP-dependent protein kinase G (PfPKG) that is critical for its life cycle. Specific cGMP analogs are able to act as partial agonists of PfPKG. Using the exquisite diagnostic power of NMR chemical shifts, Byun et al. demonstrate that the extent of agonism by these cGMP derivatives relates to the degree of stabilization of a unique inactive conformation that shares structural features with both the ligand-free, inactive and the cGMP-bound, active states. The observation of this third state helps to generalize a novel paradigm for the allosteric activation of kinase function and may open opportunities for the development of novel therapeutics.

The malaria parasite Plasmodium falciparum encodes a cGMP-dependent protein kinase G (PfPKG) that is critical for its life cycle. Specific cGMP analogs are able to act as partial agonists of PfPKG. Using the exquisite diagnostic power of NMR chemical shifts, Byun et al. demonstrate that the extent of agonism by these cGMP derivatives relates to the degree of stabilization of a unique inactive conformation that shares structural features with both the ligand-free, inactive and the cGMP-bound, active states. The observation of this third state helps to generalize a novel paradigm for the allosteric activation of kinase function and may open opportunities for the development of novel therapeutics.
The protozoan Plasmodium falciparum, the principal causative agent for malaria-related deaths, encodes more than 5 dozen proteins with sequence signatures characteristic of eukaryotic protein kinases (1). These include a cyclic guanosine 39,59-monophosphate (cGMP)-dependent protein kinase G (PfPKG) that has been shown to regulate multiple stages of the parasite's life cycle (2). As a result, PfPKG has been explored as a target for the development of antimalarials; the promise shown by a series of imidazopyridine inhibitors validates that this is a functionally relevant strategy (3). PfPKG is a multidomain enzyme that, like its mammalian counterparts, is allosterically activated by cGMP. Targeting this allosteric site could enable the discovery of inhibitors with improved selectivity, although a rational exploitation of this strategy requires a more complete understanding of the mechanism of activation by cGMP and its derivatives. A new study from Byun et al. (4) delves into the details of this mechanistic question. Their studies reveal an unexpected inactive conformation that has broad implications for the allosteric activation of PfPKG.
The structural organization of PfPKG is more elaborate than in mammalian PKGs, in that it encodes four, rather than two, cyclic nucleotide-binding domains (CBD-A through D) attached to the N terminus of a catalytic kinase domain (KD). CBD-D plays a central role in the cGMP-induced activation of the enzyme, making numerous contacts with the KD in stabilizing the auto-inhibited inactive state (5). A key conformational change in CBD-D, related to activation, is induced by cGMP binding and involves the displacement and rotation of a C-terminal helix (aC). This conformational change results in a key aC residue, Arg 528 , swapping partners, from forming a salt bridge with Asp 597 (on the N-lobe of the KD) in the auto-inhibited state (5) to participating in an intradomain hydrogen bond with Tyr 480 in the cGMP-bound state (6). The cGMP-bound state of CBD-D is further stabilized by a unique "capping triad" that deviates from its human equivalent (6) and involves the aC residues, Gln 532 and Asp 533 , that stack against the guanine moiety of cGMP, forming intradomain hydrogen bonds with Arg 484 on the so-called phosphate-binding cassette. These various CBD-D residues may be considered diagnostic of the activation state of PfPKG. Byun et al. (4) exploit these features to great effect in a series of sophisticated NMR experiments on the isolated CBD-D, providing convincing evidence for the role of a hidden inactive state (or states), induced by cGMP analogs, that has a profound influence on enzymatic activity.
Byun et al. (4) analyzed the effects of the cGMP analogs, 8-NBD-cGMP (C8 of the imidazole ring modified by a bulky 2-[7-nitro-4-benzofurazanyl]aminoethylthio moiety) and 8-pCPT-cGMP (C8 modified by a less bulky 4-chlorophenylthio moiety) on the catalytic activity of a stripped down construct of PfPKG comprising CBD-D and the KD. They found that the degree of activation of this minimal enzyme, relative to that induced by cGMP, depended on the size of the C8 modification, with the bulkier 8-NBD modification leading to reduced activation. To probe the origins of this effect, the authors analyzed the chemical shift perturbations induced in CBD-D by these analogs relative to the absence (apo) or the presence of the natural activator, cGMP (holo). Making a reasonable assumption that the chemical shifts of the apo and holo states were indicative of the inactive (I 1 ) and active (A) conformations, respectively (Fig. 1), the authors performed a procedure termed chemical shift projection analysis (CHESPA) (7) using correlation spectra of CBD-D in the presence of the analogs. CHESPA provides a measure of the angle between two sets of two-dimensional vectors, one connecting the peak positions of corresponding residues in the spectra of the apo and holo states (reference) and a second that connects the peak in the holo spectrum to that in the presence of, in this case, a modified cGMP analog (perturbation). When an analog induces an active (or active-like) conformation, the angle between these two vectors is close to 180°. However, the authors found that several vectors corresponding to residues outside the binding site deviated from the expected antiparallel behavior, suggesting the presence of additional "hidden" states, represented by a single (third) conformation for simplicity. The authors then calculated the fractional shifts (i.e. the normalized projections of the perturbation vector onto the reference for each residue). These fractional shifts provide a measure of the relative population of activelike (close to 0 or positive) or inactive-like (negative) conformations represented by this third state. The fact that these projections were almost uniformly negative suggested that this third conformation (I 2 ) was inactive-like. Further, the values of these projections in specific regions showed some very interesting trends.
This was especially true for the so-called "pre-lid" and "lid" regions encompassing aC that populated inactive-like conformations to different extents in the presence of the two C8-modified cGMP analogs; the degree of kinase activation induced by these analogs could be reliably predicted from the NMR-determined populations of the A, I 1 , and I 2 states. Finally, the authors used molecular dynamics simulations to probe the structural features characteristic of I 2 , finding that the presence of a bulky substituent at the guanine C8 impairs formation of the key Tyr 480 -Arg 528 hydrogen bond and results in nonideal interactions within the Arg 484 /Gln 532 /Asp 533 capping triad. This results in the incomplete engagement of aC through intradomain contacts that characterize the A state. The authors concluded that the extent of agonism by a given cGMP analog depended on its ability to promote an I 2 state that is characterized by an engaged pre-lid but a disengaged lid; both regions are fully engaged in A and disengaged in I 1 . Indeed, the influence of I 2 on the overall activity is somewhat reminiscent of the cAMP-induced partial agonism on PKG 1b (8). This partial agonism by related ligands through apparently similar mechanisms (i.e. through additional hidden conformations) could represent a general regulatory feature for this class of enzymes.
A question for the future is whether these observations can be exploited to design novel PfPKG-centric antimalarial therapies. Targeting this bound-but-inactive state, I 2 , discovered here by Byun et al. (4), could represent a viable strategy for allosteric inhibitor development. This possibility is buoyed by the observation that 8-NBD-cGMP engages PfPKG with the same affinity as its natural activator, cGMP, suggesting that I 2 can indeed be stabi-lized without weakened binding. Nevertheless, additional fundamental work is needed toward achieving this goal, not least of which is to investigate whether the simple picture presented here, in terms of A, I 1 , and I 2 , is muddied in full-length PfPKG by the presence of the other CBDs and additional regulatory elements (5).
Acknowledgments-I thank Dr. Sebastien Alphonse for useful discussions and for help in preparing Fig. 1.
Funding and additional information-Work on eukaryotic kinases in the Ghose laboratory is supported by National Institutes of Health Grant GM123252 and National Science Foundation Grant PHY1811770. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Conflict of interest-The author declares that he has no conflicts of interest with the contents of this article..