Cracking the allosteric code of NMR chemical shifts.

One of the broadest definitions of allostery is in terms of long-range couplings between remote sites within a molecular system (1–4). Allosteric couplings are critical to understand the molecular basis of physiological regulatory mechanisms as well as of pathological deregulation (1–4). Allostery is also opening new opportunities in drug design and diagnostics (1–5). Hence, there is considerable interest in elucidating allosteric mechanisms, which typically involve the modulation of functional conformational equilibria by allosteric effectors. One of the most effective means to monitor allosteric transitions is through NMR chemical shift changes resulting from a library of perturbations designed to interrogate a given allosteric system (6, 7). The perturbation library may include analogs of allosteric ligands and/or mutations designed to modulate allosteric conformational equilibria (7). Through statistical comparative analyses of the NMR chemical shift variations elicited by the selected perturbations it is possible to identify chemical shift patterns that serve as distinctive signatures for specific allosteric mechanisms (3, 7). However, the application of these methods to symmetric homooligomers, which are prototypical allosteric systems, has historically remained challenging due to difficulties in detecting and characterizing the unbound and bound protomers of elusive singly bound intermediates. In PNAS, Falk et al. (2) provide a brilliant solution to this problem by designing mutations that silence binding to a single protomer of a homodimeric enzyme. The method proposed by Falk et al. (2) reveals chemical shift patterns that cannot be fully rationalized through simple two-state exchange models, pointing to the need of more complex mechanisms that go beyond traditional allosteric paradigms. Allostery plays a central role in physiology, pathology, and pharmacology (1–5). The physiological role of allostery is primarily regulatory and is critical for cell homeostasis and signaling (1–4). In pathology, allostery is essential for the molecular rationalization of diseaserelated mutations and posttranslational modifications, whereas in pharmacology allosteric processes are of both therapeutic and diagnostic value (1–5). Targeting allosteric sites is therapeutically advantageous because it circumvents competition with endogenous ligands or substrates at orthosteric sites, thus enhancing potency. Furthermore, allosteric loci are typically less evolutionarily conserved than orthosteric sites, thus offering also a selectivity advantage. The diagnostic value of allostery stems from the notion that intrinsic allosteric transitions provide a means to effectively sense protein-bound biomarkers, which often remain elusive to traditional analytical methods originally developed for unbound metabolites (5). The allosteric phenomenon arises from the modulation of dynamic conformational equilibria by external Fig. 1. Relation between allosteric models and chemical shift patterns observed for the quadruplets proposed by Falk et al. (2). State equilibria for simplified versions of the MWC, KNF, and EAM (18, 19) are shown using the same color code as in the schematic HSQC spectra in the bottom panels. Each protomer is assumed to sample only two conformations (empty squares or circles) exchanging rapidly in the chemical shift NMR time scale. Filled squares denote allosteric ligands; lig0, lig1, and lig2 refer to the apo, singly bound, and doubly bound samples, respectively. The HSQC patterns in the bottom were generated under the assumption that chemical shifts sense primarily the conformation of the protomer for which they are measured, as opposed to ligand binding or the conformation of the adjacent protomer. This figure is based on refs. 2 and 18.