Allosteric feedback inhibition is the mechanism by which metabolic end products regulate their own biosynthesis by binding to an upstream enzyme. Despite its importance in controlling metabolism, there are relatively few allosteric mechanisms understood in detail. This is because allostery does not have an identifiable structural motif, making the discovery of new allosteric enzymes a difficult process. The lack of a conserved motif implies that the evolution of each allosteric mechanism is unique. Here we describe an atypical allosteric mechanism in human UDP-α-d-glucose 6-dehydrogenase (hUGDH) based on an easily acquired and identifiable structural attribute: packing defects in the protein core. In contrast to classic allostery, the active and allosteric sites in hUGDH are present as a single, bifunctional site. Using two new crystal structures, we show that binding of the feedback inhibitor, UDP-α-d-xylose, elicits a distinct induced-fit response; a buried loop translates ∼4 Å along and rotates ∼180° about the main chain axis, requiring surrounding side chains to repack. This allosteric transition is facilitated by packing defects, which negate the steric conformational restraints normally imposed by the protein core. Sedimentation velocity studies show that this repacking favors the formation of an inactive hexameric complex with unusual symmetry. We present evidence that hUGDH and the unrelated enzyme dCTP deaminase have converged to very similar atypical allosteric mechanisms using the same adaptive strategy, the selection for packing defects. Thus, the selection for packing defects is a robust mechanism for the evolution of allostery and induced fit.
Protein structures are dynamic and can explore a large conformational landscape1,2. Only some of these structural substates are important for protein function (i.e. ligand binding, catalysis and regulation)3–5. How evolution shapes the structural ensemble to optimize a specific function is poorly understood>3,4. One of the constraints on the evolution of proteins is the stability of the folded ‘native’ state. Despite this, 44% of the human proteome contains intrinsically disordered (ID) peptide segments >30 residues in length6, the majority of which have no known function7–9. Here we show that the entropic force produced by an ID carboxy-terminus (ID-tail) shifts the conformational ensemble of human UDP-α-D-glucose-6-dehydrogenase (hUGDH) toward a substate with a high affinity for an allosteric inhibitor. The function of the ID-tail does not depend on its sequence or chemical composition. Instead, the affinity enhancement can be accurately predicted based on the length of the ID segment and is consistent with the entropic force generated by an unstructured peptide attached to the protein surface10–13. Our data show that the unfolded state of the ID-tail rectifies the dynamics and structure of hUGDH to favor inhibitor binding. Because this entropic rectifier does not have any sequence or structural constraints, it is an easily acquired adaptation. This model implies that evolution selects for disordered segments to tune the energy landscape of proteins, which may explain the persistence of ID in the proteome.
Human UDP-α-d-glucose-6-dehydrogenase (hUGDH) displays hysteresis because of a slow isomerization from an inactive state (E*) to an active state (E). Here we show that the structure of E* constrains hUGDH in a conformation that favors feedback inhibition at physiological pH. The feedback inhibitor UDP-α-d-xylose (UDP-Xyl) competes with the substrate UDP-α-d-glucose for the active site. Upon binding, UDP-Xyl triggers an allosteric switch that changes the structure and affinity of the intersubunit interface to form a stable but inactive horseshoe-shaped hexamer. Using sedimentation velocity studies and a new crystal structure, we show that E* represents a stable conformational intermediate between the active and feedback-inhibited conformations. Because the allosteric switch occludes the cofactor and substrate binding sites in the inactive hexamer, the intermediate conformation observed in the crystal structure is consistent with the E* transient observed in relaxation studies. Steady-state analysis shows that the E* conformation enhances the affinity of hUGDH for the allosteric inhibitor UDP-Xyl by 8.6-fold (Ki = 810 nM). We present a model in which the constrained quaternary structure permits a small effector molecule to leverage a disproportionately large allosteric response.
UDP-α-D-xylose (UDX) acts as a feedback inhibitor of human UDP-α-D-glucose 6-dehydrogenase (hUGDH) by activating an unusual allosteric switch, the Thr131 loop. UDX binding induces the Thr131 loop to translate ~5 Å through the protein core, changing packing interactions and rotating a helix (α6(136-144)) to favor the formation of an inactive hexameric complex. But how does to conformational change occur given the steric packing constraints of the protein core? To answer this question, we deleted Val132 from the Thr131 loop to approximate an intermediate state in the allosteric transition. The 2.3 Å resolution crystal structure of the deletion construct (Δ132) reveals an open conformation that relaxes steric constraints and facilitates repacking of the protein core. Sedimentation velocity studies show that the open conformation stabilizes the Δ132 construct as a hexamer with point group symmetry 32, similar to that of the active complex. In contrast, the UDX-inhibited enzyme forms a lower-symmetry, horseshoe-shaped hexameric complex. We show that the Δ132 and UDX-inhibited structures have similar hexamer-building interfaces, suggesting that the hinge-bending motion represents a path for the allosteric transition between the different hexameric states. On the basis of (i) main chain flexibility and (ii) a model of the conformational change, we propose that hinge bending can occur as a concerted motion between adjacent subunits in the high-symmetry hexamer. We combine these results in a structurally detailed model for allosteric feedback inhibition and substrate--product exchange during the catalytic cycle.
The different tendencies of dinuclear azacryptates of the m-CH 2 C 6 H 4 CH 2 and 2,5-furano-spaced hosts L 1 and L 2 to catalyse CO 2 uptake-reactions within these sterically-protected host cavities are examined. Bridging methylcarbonates are generated catalytically upon exposure of methanol solutions of L 1 , but not L 2 , di-transition cation cryptates to atmospheric CO 2 . X-Ray crystallographic structures of homodinuclear µ-carbonato cryptates of both ligands and µ-methylcarbonato cryptates of L 1 with later first transition series cations are reported. ESI-MS spectra show loss of H 2 CO 3 from µ-carbonato cryptates in collision activation experiments.The oxo-anion, carbonate, currently attracts attention in diverse areas of chemistry. Transformations involving this species, its reaction precursors and products are of considerable biological 1 significance; particularly important here are the carboanhydrases which play an essential role in processes such as photosynthesis, respiration, calcification and pH control. In the light of increasing concern about CO 2 build-up from fossil fuel consumption and potential resulting greenhouse effects, improved understanding of the biological handling of carbonate-related species acquires increasing urgency. Any potential new application of CO 2 as feedstock in chemical processes, will for this reason, attract much interest. In addition, the many and varied coordination modes of the carbonate oxo-anion are of spectroscopic interest as is their capacity to transmit magnetic interaction. 2-4 The carbonate system is also significant in the context of anion coordination chemistry as its behaviour within small molecule hosts may help to elucidate details of transport and location of carbonate or carboxylate anions in enzyme processes.The frequently used strategy of anion coordination via protonated amine, 5,6 or other acidic host, can be problematic in such pH-sensitive systems, so we have adopted an alternative strategy: the oxoanions here are retained via their bridging coordination of cations held within a cryptand cavity. We have already used the "cryptate as host" strategy to coordinate pseudo-halide anions such as azide and cyanate, and studied the spectroscopy and magnetochemistry 7,8 resulting from the consequent (and on first observation unprecedented) colinear M-NXY-M bridging geometry.The secondary coordination of anionic or other bridges between cations themselves coordinated by a cryptand host molecule was quite some time ago termed cascade coordination by Jean-Marie Lehn, 9 in the implicit expectation that the bridging groups might be activated, by reason of their dicoordination, toward further and possibly useful chemical reaction. However, such outcome was not apparent in our pseudo-halide † Electronic supplementary information (ESI) available: magnetic data. See http://www.rsc.org/suppdata/dt/b1/b110449g/
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