Mixed, nonclassical behavior in a classic allosteric protein

Abstract

Allostery is a major driver of biological processes requiring coordination. Thus, it is one of the most fundamental and remarkable phenomena in nature, and there is motivation to understand and manipulate it to a multitude of ends. Today, it is often described in terms of two phenomenological models proposed more than a half-century ago involving only T(tense) or R(relaxed) conformations. Here, methyl-based NMR provides extensive detail on a dynamic T to R switch in the classical dimeric allosteric protein, yeast chorismate mutase (CM), that occurs in the absence of substrate, but only with the activator bound. Switching of individual subunits is uncoupled based on direct observation of mixed TR states in the dimer. This unique finding excludes both classic models and solves the paradox of a coexisting hyperbolic binding curve and highly skewed substrate-free T-R equilibrium. Surprisingly, structures of the activator-bound and effector-free forms of CM appear the same by NMR, providing another example of the need to account for dynamic ensembles. The apo enzyme, which has a sigmoidal activity profile, is shown to switch, not to R, but to a related high-energy state. Thus, the conformational repertoire of CM does not just change as a matter of degree depending on the allosteric input, be it effector and/or substrate. Rather, the allosteric model appears to completely change in different contexts, which is only consistent with modern ensemble-based frameworks.

Keywords: Allostery; MWC; NMR dynamics; ensemble allosteric model; relaxation dispersion.

Fig. 1.

CM structure and allosteric conformations. (A) CM with allosteric effector Trp bound (green sticks) and transition state analog inhibitor (TSI) bound in active sites (pink sticks), pdbID 3 csm. The effector-binding domain (EBR) is in gold. Individual helices are highlighted and T226 shown. (B) Superposition of T (2 csm) and R (1 csm) structures, with the superposition on the lightly colored protomers. Side chains are shown to indicate where larger conformational differences are observed.

Fig. 2.

ILV methyl HMQC reference spectra of CM conformational states. ApoCM is in the T-state by virtue of its near perfect overlay with inhibited TyrCM (A). The R-state has a distinct HMQC fingerprint as evidenced by significant differences between spectra of apoCM and the constitutively active T226I mutant bound to Trp (B). The R-form is similar to the tryptophan-TSI bound super-R state (C and SI Appendix, Fig. S1).

Fig. 3.

Activator-bound CM is also in the T-state. Near perfect ILV methyl HMQC overlay of apoCM (black) and TrpCM (green) (A). Locations of several R-state resonances are shown by gray arrows indicating R chemical shifts are not observed. TrpCM stands out as having significantly more conformational exchange on the µs–ms timescale (ΔR₂, see Methods) than the other CM states (B). Residues in the EBR are highlighted within dashed boxes.

Fig. 4.

µs–ms dynamics of apoCM and TrpCM. Relaxation dispersion curves are presented as two sets of two columns in which the first column is ¹H CPMG and the second ¹³C CEST for representative ILV residues. The first three rows are for methyl groups that were fit globally with a single k_ex and single population of high-energy, minor state (pB), albeit with different values for the two CM forms. Note the amplitudes of the ¹H CPMG curves and distance of the “dips” from the main peaks in the ¹³C CEST profiles are greater for TrpCM than apoCM, reflecting a higher population of the minor state and larger chemical shift changes between major and minor states for TrpCM. The last row gives representative probes from the EBR (I52) or the active site (V197) that were fit locally with different k_ex values.

Fig. 5.

T–R and non-T–R switching in TrpCM and apoCM. Correlation of ¹H CPMG (A) and ¹³C CEST (B) Δω values with chemical shift differences between TrpCM and TrpCM^T226I HMQC resonances. Data for TrpCM and apoCM are in blue and yellow, respectively. Adherence of TrpCM data to unit slope line indicates a large part of the molecule switches between the T and R states on the ms timescale in the absence of substrate. ApoCM undergoes dynamics but not a T–R switch. (C) Broad distribution of ILV probes undergoing T–R motion in TrpCM are shown in blue spheres, and deviant nuclei in other colors with rates according to the key. (D) Comparison of global sets of residues with ms motions in apoCM vs. TrpCM. (E) Nuclei with deviant, non-T–R motions in TrpCM cluster to the EBR and active site. (F) Zoom in on active site of the T-state with the TSI placed based on alignment with sRCM X-ray model describing non-T–R TrpCM motions that may be involved in substrate and/or product flux.

Fig. 6.

Direct observation of mixed T:R states in the CM dimer. (A) HMQC spectrum of TrpCM with 0.5 molar eq. of TSI in black showing a manifold of states for L104C^δ1. Reference spectra of the relevant symmetrical states are shown with single contours. Each of the two main peaks is comprised of two states described by dimer cartoons in which filled squares indicate TSI bound subunits. A third resonance of unknown origin(s) has the same chemical shift as the R-state. (B) ZZ-exchange spectroscopy of the same half-saturated sample shows exchange peaks connecting this third resonance to major states. The green dotted lines represent T to R interconversion of the unbound subunit of the single TSI-bound dimer, which is conclusive evidence of a mixed T:SR major state and independently switching subunits. (C) The exchange phenomenon outlined by the green dashed line panel B is shown.

Fig. 7.

CM does not adhere to a single 2-state allosteric model. In panels A, B, and C, circle = T, and square = R, and filled shape signifies substrate bound, and filled square is sRCM. The T state of the apo and Trp-bound states are shown in different colors because Trp modifies the T-ensemble in ways that include access to the R state (this work) and changes in conformational entropy (44). Observed substrate bound forms in panels A and C are depicted similarly based on similar k_cat (B) and HMQC spectra once TSI is bound (SI Appendix, Fig. S8). (A) States allowed under the MWC model are outlined in gray dashes, and the states observed in this work are outlined in green dashes. While TrpCM shows some behavior consistent with the MWC model, direct observation of mixed TR states (middle column) show other mechanisms are at play. Note uncoupling of the subunits and mixed TR states explain hyperbolic activity curve in panel B. (C) Removal of the activator changes both the ensemble and the allosteric mechanism to reveal a manifold of states more consistent with a KNF-type model; coupling of the subunits yields the sigmoidal activity curve in panel. Not shown in panel C is a high-energy state that is related to, but clearly not the R state (Results). Collectively, this behavior is only encompassed by an EAM.