Renuka Kadirvelraj

Role of Packing Defects in the Evolution of Allostery and Induced Fit in Human UDP-Glucose Dehydrogenase.

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.

Hysteresis and negative cooperativity in human UDP-glucose dehydrogenase.

Human UDP-α-d-glucose 6-dehydrogenase (hUGDH) forms a hexamer that catalyzes the NAD(+)-dependent oxidation of UDP-α-d-glucose (UDG) to produce UDP-α-d-glucuronic acid. Mammalian UGDH displays hysteresis (observed as a lag in progress curves), indicating that the enzyme undergoes a slow transition from an inactive to an active state. Here we show that hUGDH is sensitive to product inhibition during the lag. The inhibition results in a systematic decrease in steady-state velocity and makes the lag appear to have a second-order dependence on enzyme concentration. Using transient-state kinetics, we confirm that the lag is in fact due to a substrate and cofactor-induced isomerization of the enzyme. We also show that the cofactor binds to the hUGDH:UDG complex with negative cooperativity. This suggests that the isomerization may be related to the formation of an asymmetric enzyme complex. We propose that the hysteresis in hUGDH is the consequence of a functional adaptation; by slowing the response of hUGDH to sudden increases in the flux of UDG, the other biochemical pathways that use this important metabolite (i.e., glycolysis) will have a competitive edge.

Cofactor Binding Triggers a Molecular Switch To Allosterically Activate Human UDP-α-d-glucose 6-Dehydrogenase

Human UDP-α-D-glucose dehydrogenase (hUGDH) catalyzes the NAD(+)-dependent oxidation of UDP-α-D-glucose (UDG) to produce UDP-α-D-glucuronic acid. The oligomeric structure of hUGDH is dynamic and can form two distinct hexameric complexes in solution. The active form of hUGDH consists of dimers that undergo a concentration-dependent association to form a hexamer with 32 symmetry. In the presence of the allosteric feedback inhibitor UDP-α-D-xylose (UDX), hUGDH changes shape to form an inactive, horseshoe-shaped complex. Previous studies have identified the UDX-induced allosteric mechanism that changes the hexameric structure to inhibit the enzyme. Here, we investigate the role of the 32 symmetry hexamer in the catalytic cycle. We engineered a stable hUGDH dimer by introducing a charge-switch substitution (K94E) in the hexamer-building interface (hUGDH(K94E)). The k(cat) of hUGDH(K94E) is ~160-fold lower than that of the wild-type enzyme, suggesting that the hexamer is the catalytically relevant state. We also show that cofactor binding triggers the formation of the 32 symmetry hexamer, but UDG is needed for the stability of the complex. The hUGDH(K94E) crystal structure at 2.08 Å resolution identifies loop(88-110) as the cofactor-responsive allosteric switch that drives hexamer formation; loop(88-110) directly links cofactor binding to the stability of the hexamer-building interface. In the interface, loop(88-110) packs against the Thr131-loop/α6 helix, the allosteric switch that responds to the feedback inhibitor UDX. We also identify a structural element (the S-loop) that explains the indirect stabilization of the hexamer by substrate and supports a sequential, ordered binding of the substrate and cofactor. These observations support a model in which (i) UDG binds to the dimer and stabilizes the S-loop to promote cofactor binding and (ii) cofactor binding orders loop(88-110) to induce formation of the catalytically active hexamer.

Conformational flexibility in the allosteric regulation of human UDP-α-D-glucose 6-dehydrogenase.

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.

Hysteresis in Human UDP-Glucose Dehydrogenase Is Due to a Restrained Hexameric Structure That Favors Feedback Inhibition

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.

HumanN-acetylglucosaminyltransferase II substrate recognition uses a modular architecture that includes a convergent exosite.

Significance Cell-surface and secreted glycoproteins are initially synthesized and glycosylated in the endoplasmic reticulum. Glycan structures are trimmed and remodeled as they transit the secretory pathway, resulting in multi-branched complex-type structures. The enzymes that remodel these structures have precise linkage and branch specificities, with the product of one reaction being specifically recognized as the substrate for the following reaction. These reactions include N-acetylglucosaminyltransferase II (MGAT2), an enzyme that initiates complex branch extension by precise recognition of its glycan substrate. The structural basis for MGAT2 substrate recognition is the subject of the present study. Structures of MGAT2-substrate complexes reveal both modular and convergent mechanisms for selective substrate recognition and catalysis and provide a generalized model for template-based synthesis of glycan structures by glycosyltransferases. Asn-linked oligosaccharides are extensively modified during transit through the secretory pathway, first by trimming of the nascent glycan chains and subsequently by initiating and extending multiple oligosaccharide branches from the trimannosyl glycan core. Trimming and branching pathway steps are highly ordered and hierarchal based on the precise substrate specificities of the individual biosynthetic enzymes. A key committed step in the synthesis of complex-type glycans is catalyzed by N-acetylglucosaminyltransferase II (MGAT2), an enzyme that generates the second GlcNAcβ1,2- branch from the trimannosyl glycan core using UDP-GlcNAc as the sugar donor. We determined the structure of human MGAT2 as a Mn2+-UDP donor analog complex and as a GlcNAcMan3GlcNAc2-Asn acceptor complex to reveal the structural basis for substrate recognition and catalysis. The enzyme exhibits a GT-A Rossmann-like fold that employs conserved divalent cation-dependent substrate interactions with the UDP-GlcNAc donor. MGAT2 interactions with the extended glycan acceptor are distinct from other related glycosyltransferases. These interactions are composed of a catalytic subsite that binds the Man-α1,6- monosaccharide acceptor and a distal exosite pocket that binds the GlcNAc-β1,2Man-α1,3Manβ- substrate “recognition arm.” Recognition arm interactions are similar to the enzyme–substrate interactions for Golgi α-mannosidase II, a glycoside hydrolase that acts just before MGAT2 in the Asn-linked glycan biosynthetic pathway. These data suggest that substrate binding by MGAT2 employs both conserved and convergent catalytic subsite modules to provide substrate selectivity and catalysis. More broadly, the MGAT2 active-site architecture demonstrates how glycosyltransferases create complementary modular templates for regiospecific extension of glycan structures in mammalian cells.

The role of intrinsic disorder in human UDP-glucose dehydrogenase

It is estimated that 33% of eukaryotic proteins contain at least one intrinsically disordered (ID) segment of 30 residues or longer. Many of these ID-peptides are believed to be important for enzyme function or regulation, but only a few examples have been examined experimentally. Here we show that the disordered C-terminus (ID-tail) of human UDP-α-D-glucose-6-dehydrogenase (hUGDH) contributes to the allosteric regulation of the enzyme. The crystal structures of hUGDH show that the ID-tail is disordered in both the active and allosterically inhibited conformations. Despite the disordered state, the deletion of the ID-tail (ΔC-term hUGDH) reduces the affinity for allosteric inhibitor UDP-xylose by more than an order of magnitude. The fact that the bound allosteric inhibitor is not solvent accessible suggests that any interaction between the ID-tail and the effector involves an indirect mechanism. Given that the hexameric assembly of hUGDH is important for the allosteric response, we examined the effect of the ID-tail on the structure of the enzyme. The crystal structures of ΔC-term and fulllength hUGDH reveal the same hexameric complex. However, sedimentation velocity analysis shows that the loss of the ID-tail weakens the hexamer in solution. To decouple the contribution of the hexameric structure and the ID-tail from the binding of UDP-Xyl, we used a stabilized hUGDH dimer (M11-hUGDH). The M11-hUGDH dimer binds UDP-Xylose with a greater affinity than the hexamer. As observed in the full-length enzyme, deletion of the ID-tail in the M11-hUGDH dimer reduces the affinity for allosteric effector (6.4-fold). Thus, we show that the mechanism by which the ID-tail favors allosteric inhibition is independent of its role in stabilizing the hUGDH hexamer.