Comparative effects of sulfhydryl reagents on membrane-bound and solubilized UDP-glucose:ceramide glucosyltransferase from Golgi membranes. Evidence for partial involvement of a thiol group in the nucleotide sugar binding site of the solubilized enzyme

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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Membrane‐bound calmodulin in Xenopus laevis oocytes as a novel binding site for melatonin

The FASEB Journal ◽

10.1096/fasebj.12.13.1401 ◽

1998 ◽

Vol 12 (13) ◽

pp. 1401-1408 ◽

Cited By ~ 39

Author(s):

M. Paz Romero ◽

Antonio García‐Pergañeda ◽

Juan M. Guerrero ◽

Carmen Osuna

Keyword(s):

Xenopus Laevis ◽

Binding Site ◽

Xenopus Laevis Oocytes ◽

Membrane Bound

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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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Characterization of the stilbenedisulphonate binding site on band 3 Memphis variant II (Pro-854→Leu)

Biochemical Journal ◽

10.1042/bj3170509 ◽

1996 ◽

Vol 317 (2) ◽

pp. 509-514 ◽

Cited By ~ 6

Author(s):

James M. SALHANY ◽

Renee L. SLOAN ◽

Lawrence M. SCHOPFER

Keyword(s):

Binding Site ◽

Conformational Changes ◽

Covalent Binding ◽

Band 3 ◽

Blood Group Antigen ◽

Cross Linking ◽

Anion Exchange Protein ◽

Membrane Bound ◽

And Control

Band 3 Memphis variant II is a mutant anion-exchange protein associated with the Diego a+ blood group antigen. There are two mutations in this transporter: Lys-56 → Glu within the cytoplasmic domain, and Pro-854 → Leu within the membrane-bound domain. The Pro-854 mutation, which is thought to give rise to the antigenicity, is located within the C-terminal subdomain of the membrane-bound domain. Yet, there is an apparent enhancement in the rate of covalent binding of H2DIDS (4,4´-di-isothiocyanatodihydro-2,2´-stilbenedisulphonate) to ‘lysine A’ (Lys-539) in the N-terminal subdomain, suggesting widespread conformational changes. In this report, we have used various kinetic assays which differentiate between conformational changes in the two subdomains, to characterize the stilbenedisulphonate site on band 3 Memphis variant II. We have found a significantly higher H2DIDS (a C-terminal-sensitive inhibitor) affinity for band 3 Memphis variant II, due to a lower H2DIDS ‘off’ rate constant, but no difference was found between mutant and control when DBDS (4,4´-dibenzamido-2,2´-stilbenedisulphonate) (a C-terminal-insensitive inhibitor) ‘off’ rates were measured. Furthermore, there were no differences in the rates of covalent binding to lysine A, for either DIDS (4,4´-di-isothiocyanato-2,2´-stilbenedisulphonate) or H2DIDS. However, the rate of covalent intrasubunit cross-linking of Lys-539 and Lys-851 by H2DIDS was abnormally low for band 3 Memphis variant II. These results suggest that the Pro-854 → Leu mutation causes a localized conformational change in the C-terminal subdomain of band 3.

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