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Protein structures are dynamic and can explore a large conformational landscape.sup.1,2. Only some of these structural substates are important for protein function (such as ligand binding, catalysis and regulation).sup.3-5. How evolution shapes the structural ensemble to optimize a specific function is poorly understood.sup.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 peptide segments greater than 30 residues in length.sup.6, the majority of which have no known function.sup.7-9. Here we show that the entropic force produced by an intrinsically disordered carboxy terminus (ID-tail) shifts the conformational ensemble of human UDP-[alpha]-d-glucose-6-dehydrogenase (UGDH) towards 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 intrinsically disordered segment, and is consistent with the entropic force generated by an unstructured peptide attached to the protein surface.sup.10-13. Our data show that the unfolded state of the ID-tail rectifies the dynamics and structure of UGDH to favour 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 intrinsic disorder in the proteome. The carboxy terminus of human UDP-[alpha]-d-glucose-6-dehydrogenase is structurally disordered, but has sequence-independent effects on the conformation of the enzyme and binding of an allosteric inhibitor, suggesting a reason for the persistence of intrinsically disordered peptide segments in the proteome., Author(s): Nicholas D. Keul [sup.1] , Krishnadev Oruganty [sup.2] , Elizabeth T. Schaper Bergman [sup.3] , Nathaniel R. Beattie [sup.1] , Weston E. McDonald [sup.1] , Renuka Kadirvelraj [sup.1] , [...]

The role of conserved water in the complexation of the legume lectin concanavalin A (Con A) with a synthetic analog of the natural trisaccharide, in which a hydroxyethyl side chain has replaced the hydroxyl group at the C-2 position in the central mannosyl residue, is studied. The crystal structure of Con A bound to the modified trimannoside has disclosed the irrefutable presence of the conserved water in the lectin binding site and has enabled molecular dynamics (MD) and thermodynamic integration (TI) calculations to assess the affect of the water on ligand affinity.

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 υ through the protein core, changing packing interactions and rotating a helix (α6136-144) 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 υ 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. [ABSTRACT FROM AUTHOR]

Mammalian Asn-linked glycans are extensively processed as they transit the secretory pathway to generate diverse glycans on cell surface and secreted glycoproteins. Additional modification of the glycan core by α-1,6-fucose addition to the innermost GlcNAc residue (core fucosylation) is catalyzed by an α-1,6-fucosyltransferase (FUT8). The importance of core fucosylation can be seen in the complex pathological phenotypes of FUT8 null mice, which display defects in cellular signaling, development, and subsequent neonatal lethality. Elevated core fucosylation has also been identified in several human cancers. However, the structural basis for FUT8 substrate specificity remains unknown.Here, using various crystal structures of FUT8 in complex with a donor substrate analog, and with four distinct glycan acceptors, we identify the molecular basis for FUT8 specificity and activity. The ordering of three active site loops corresponds to an increased occupancy for bound GDP, suggesting an induced-fit folding of the donor-binding subsite. Structures of the various acceptor complexes were compared with kinetic data on FUT8 active site mutants and with specificity data from a library of glycan acceptors to reveal how binding site complementarity and steric hindrance can tune substrate affinity. The FUT8 structure was also compared with other known fucosyltransferases to identify conserved and divergent structural features for donor and acceptor recognition and catalysis. These data provide insights into the evolution of modular templates for donor and acceptor recognition among GT-B fold glycosyltransferases in the synthesis of diverse glycan structures in biological systems., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Boruah et al.)