Different reactivity of two Brain sialyltransferases towards sulfhydryl reagents. Evidence for a thiol group involved in the nucleotide-sugar binding site of the NeuAc?2-3Gal?1-3GalNAc ?(2?6)sialyltransferase

The Na+-glucose cotransporter hSGLT1 is a member of a class of membrane proteins that harness Na+ electrochemical gradients to drive uphill solute transport. Although hSGLT1 belongs to one gene family (SLC5), recent structural studies of bacterial Na+ cotransporters have shown that Na+ transporters in different gene families have the same structural fold. We have constructed homology models of hSGLT1 in two conformations, the inward-facing occluded (based on vSGLT) and the outward open conformations (based on Mhp1), mutated in turn each of the conserved gates and ligand binding residues, expressed the SGLT1 mutants in Xenopus oocytes, and determined the functional consequences using biophysical and biochemical assays. The results establish that mutating the ligand binding residues produces profound changes in the ligand affinity (the half-saturation concentration, K0.5); e.g., mutating sugar binding residues increases the glucose K0.5 by up to three orders of magnitude. Mutation of the external gate residues increases the Na+ to sugar transport stoichiometry, demonstrating that these residues are critical for efficient cotransport. The changes in phlorizin inhibition constant ( Ki) are proportional to the changes in sugar K0.5, except in the case of F101C, where phlorizin Ki increases by orders of magnitude without a change in glucose K0.5. We conclude that glucose and phlorizin occupy the same binding site and that F101 is involved in binding to the phloretin group of the inhibitor. Substituted-cysteine accessibility methods show that the cysteine residues at the position of the gates and sugar binding site are largely accessible only to external hydrophilic methanethiosulfonate reagents in the presence of external Na+, demonstrating that the external sugar (and phlorizin) binding vestibule is opened by the presence of external Na+ and closes after the binding of sugar and phlorizin. Overall, the present results provide a bridge between kinetics and structural studies of cotransporters.

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Engineered occluded apo-intermediate of LacY

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1816267115 ◽

2018 ◽

Vol 115 (50) ◽

pp. 12716-12721 ◽

Cited By ~ 1

Author(s):

Irina Smirnova ◽

Vladimir Kasho ◽

H. Ronald Kaback

Keyword(s):

Binding Site ◽

Lactose Permease ◽

Binding Studies ◽

X Ray Crystallography ◽

Sugar Binding ◽

Transport Cycle ◽

Open Structures ◽

Unrestricted Access ◽

Tight Association ◽

Alternating Access

The lactose permease of Escherichia coli (LacY) utilizes an alternating access symport mechanism with multiple conformational intermediates, but only inward (cytoplasmic)- or outward (periplasmic)-open structures have been characterized by X-ray crystallography. It is demonstrated here with sugar-binding studies that cross-linking paired-Cys replacements across the closed cytoplasmic cavity stabilize an occluded conformer with an inaccessible sugar-binding site. In addition, a nanobody (Nb) that stabilizes a periplasmic-open conformer with an easily accessible sugar-binding site in WT LacY fails to cause the cytoplasmic cross-linked mutants to become accessible to galactoside, showing that the periplasmic cavity is closed. These results are consistent with tight association of the periplasmic ends in two pairs of helices containing clusters of small residues in the packing interface between N- and C-terminal six-helix bundles of the symporter. However, after reduction of the disulfide bond, the Nb markedly increases the rate of galactoside binding, indicating unrestricted access to the Nb epitope and the galactoside-binding site from the periplasm. The findings indicate that the cross-linked cytoplasmic double-Cys mutants resemble an occluded apo-intermediate in the transport cycle.

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Evaluation of the rate constants of sugar transport through maltoporin (LamB) of Escherichia coli from the sugar-induced current noise.

The Journal of General Physiology ◽

10.1085/jgp.105.3.385 ◽

1995 ◽

Vol 105 (3) ◽

pp. 385-401 ◽

Cited By ~ 53

Author(s):

C Andersen ◽

M Jordy ◽

R Benz

Keyword(s):

Escherichia Coli ◽

Binding Site ◽

Outer Membrane ◽

Rate Constants ◽

Sugar Transport ◽

Current Noise ◽

Induced Current ◽

Lipid Bilayer Membranes ◽

Sugar Binding ◽

Different Temperatures

LamB (maltoporin) of Escherichia coli outer membrane was reconstituted into artificial lipid bilayer membranes. The channel contains a binding site for sugars and is blocked for ions when the site is occupied by a sugar. The on and off reactions of sugar binding cause an increase of the noise of the current through the channel. The sugar-induced current noise of maltoporin was used for the evaluation of the sugar-binding kinetics for different sugars of the maltooligosaccharide series and for sucrose. The on rate constant for sugar binding was between 10(6) and 10(7) M-1.s-1 for the maltooligosaccharides and corresponds to the movement of the sugars from the aqueous phase to the central binding site. The off rate (corresponding to the release of the sugars from the channel) decreased with increasing number of glucose residues in the maltooligosaccharides from approximately 2,000 s-1 for maltotriose to 180 s-1 for maltoheptaose. The kinetics for sucrose movement was considerably slower. The activation energies of the stability constant and of the rate constants for sugar binding were evaluated from noise experiments at different temperatures. The role of LamB in the transport of maltooligosaccharides across the outer membrane is discussed.

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Comparison of human poly-N-acetyl-lactosamine synthase structure with GT-A fold glycosyltransferases supports a modular assembly of catalytic subsites

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.015305 ◽

2020 ◽

pp. jbc.RA120.015305

Author(s):

Renuka Kadirvelraj ◽

Jeong-Yeh Yang ◽

Hyun Woo Kim ◽

Justin H. Sanders ◽

Kelley W. Moremen ◽

...

Keyword(s):

Structural Analysis ◽

Binding Site ◽

Human Disease ◽

Catalytic Mechanism ◽

Substrate Recognition ◽

Nucleotide Sugar ◽

Modular Assembly ◽

Acceptor Specificity ◽

Donor Acceptor ◽

Prior Models

Poly-N-acetyl-lactosamine (poly-LacNAc) structures are composed of repeating [-Galβ(1,4)-GlcNAcβ(1,3)-]n glycan extensions. They are found on both N- and O-glycoproteins and glycolipids, and play an important role in development, immune function, and human disease. The majority of mammalian poly-LacNAc is synthesized by the alternating iterative action of β1,3-N-acetylglucosaminyltransferase 2 (B3GNT2) and β1,4-galactosyltransferases. B3GNT2 is in the largest mammalian glycosyltransferase family, GT31, but little is known about the structure, substrate recognition, or catalysis by family members. Here we report the structures of human B3GNT2 in complex with UDP:Mg2+, and in complex with both UDP:Mg2+ and a glycan acceptor, lacto-N-neotetraose. The B3GNT2 structure conserves the GT-A fold and the DxD motif that coordinates a Mg2+ ion for binding the UDP-GlcNAc sugar donor. The acceptor complex shows interactions with only the terminal Galβ(1,4)-GlcNAcβ(1,3)- disaccharide unit, which likely explains the specificity for both N- and O-glycan acceptors. Modeling of the UDP-GlcNAc donor supports a direct displacement inverting catalytic mechanism. Comparative structural analysis indicates that nucleotide sugar donors for GT-A fold glycosyltransferases bind in similar positions and conformations without conserving interacting residues, even for enzymes that use the same donor substrate. In contrast, the B3GNT2 acceptor binding site is consistent with prior models suggesting that the evolution of acceptor specificity involves loops inserted into the stable GT-A fold. These observations support the hypothesis that GT-A fold glycosyltransferases employ co-evolving donor, acceptor, and catalytic subsite modules as templates to achieve the complex diversity of glycan linkages in biological systems.

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A specific oligosaccharide-binding site in the alternansucrase catalytic domain mediates alternan elongation

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.013028 ◽

2020 ◽

Vol 295 (28) ◽

pp. 9474-9489 ◽

Cited By ~ 1

Author(s):

Manon Molina ◽

Claire Moulis ◽

Nelly Monties ◽

David Guieysse ◽

Sandrine Morel ◽

...

Keyword(s):

Binding Site ◽

Molar Mass ◽

Enzyme Stability ◽

Catalytic Site ◽

Glycoside Hydrolase Family ◽

Surface Binding ◽

High Molar Mass ◽

Sugar Binding ◽

Binding Pockets ◽

Domain V

Microbial α-glucans produced by GH70 (glycoside hydrolase family 70) glucansucrases are gaining importance because of the mild conditions for their synthesis from sucrose, their biodegradability, and their current and anticipated applications that largely depend on their molar mass. Focusing on the alternansucrase (ASR) from Leuconostoc citreum NRRL B-1355, a well-known glucansucrase catalyzing the synthesis of both high- and low-molar-mass alternans, we searched for structural traits in ASR that could be involved in the control of alternan elongation. The resolution of five crystal structures of a truncated ASR version (ASRΔ2) in complex with different gluco-oligosaccharides pinpointed key residues in binding sites located in the A and V domains of ASR. Biochemical characterization of three single mutants and three double mutants targeting the sugar-binding pockets identified in domain V revealed an involvement of this domain in alternan binding and elongation. More strikingly, we found an oligosaccharide-binding site at the surface of domain A, distant from the catalytic site and not previously identified in other glucansucrases. We named this site surface-binding site (SBS) A1. Among the residues lining the SBS-A1 site, two (Gln700 and Tyr717) promoted alternan elongation. Their substitution to alanine decreased high-molar-mass alternan yield by a third, without significantly impacting enzyme stability or specificity. We propose that the SBS-A1 site is unique to alternansucrase and appears to be designed to bind alternating structures, acting as a mediator between the catalytic site and the sugar-binding pockets of domain V and contributing to a processive elongation of alternan chains.

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