scholarly journals Active site voltage clamp fluorometry of the sodium glucose cotransporter hSGLT1

2017 ◽  
Vol 114 (46) ◽  
pp. E9980-E9988 ◽  
Author(s):  
Edurne Gorraitz ◽  
Bruce A. Hirayama ◽  
Aviv Paz ◽  
Ernest M. Wright ◽  
Donald D. F. Loo
Keyword(s):  
Binding Site ◽  
Conformational Changes ◽  
Side Chains ◽  
Mutant Proteins ◽  
Sugar Binding ◽  
Internal Surfaces ◽  
Sodium Glucose Cotransporter ◽  
Time Courses ◽  
Covalently Linked ◽  
Charge Movements

In the human sodium glucose cotransporter (hSGLT1) cycle, the protein undergoes conformational changes where the sugar-binding site alternatively faces the external and internal surfaces. Functional site-directed fluorometry was used to probe the conformational changes at the sugar-binding site. Residues (Y290, T287, H83, and N78) were mutated to cysteines. The mutants were expressed in Xenopus laevis oocytes and tagged with environmentally sensitive fluorescent rhodamines [e.g., tetramethylrhodamine (TMR)-thiols]. The fluorescence intensity was recorded as the mutants were driven into different conformations using voltage jumps. Sugar binding and transport by the fluorophore-tagged mutants were blocked, but Na+ binding and the voltage-dependent conformational transitions were unaffected. Structural models indicated that external Na+ binding opened a large aqueous vestibule (600 Å3) leading to the sugar-binding site. The fluorescence of TMR covalently linked to Y290C, T287C, and H83C decreased as the mutant proteins were driven from the inward to the outward open Na+-bound conformation. The time courses of fluorescence changes (milliseconds) were close to the SGLT1 capacitive charge movements. The quench in rhodamine fluorescence indicated that the environment of the chromophores became more polar with opening of the external gates as the protein transitioned from the inward to outward facing state. Structural analyses showed an increase in polar side chains and a decrease in hydrophobic side chains lining the vestibule, and this was reflected in solvation of the chromophore. The results demonstrate the opening and closing of external gates in real time, with the accompanying changes of polarity of the sugar vestibule.

scholarly journals Genetic Analysis of Phage Mu Mor Protein Amino Acids Involved in DNA Minor Groove Binding and Conformational Changes

2011 ◽  
Vol 286 (41) ◽  
pp. 35852-35862 ◽  
Author(s):  
Muthiah Kumaraswami ◽  
Lakshmi Avanigadda ◽  
Rajendra Rai ◽  
Hee-Won Park ◽  
Martha M. Howe
Keyword(s):  
Dna Binding ◽  
Rna Polymerase ◽  
Binding Site ◽  
Conformational Changes ◽  
Minor Groove ◽  
Minor Groove Binding ◽  
Groove Binding ◽  
Specific Chemical ◽  
Mutant Proteins ◽  
Dna Minor Groove

Gene expression during lytic development of bacteriophage Mu occurs in three phases: early, middle, and late. Transcription from the middle promoter, Pm, requires the phage-encoded activator protein Mor and the bacterial RNA polymerase. The middle promoter has a −10 hexamer, but no −35 hexamer. Instead Pm has a hyphenated inverted repeat that serves as the Mor binding site overlapping the position of the missing −35 element. Mor binds to this site as a dimer and activates transcription by recruiting RNA polymerase. The crystal structure of the His-Mor dimer revealed three structural elements: an N-terminal dimerization domain, a C-terminal helix-turn-helix DNA-binding domain, and a β-strand linker between the two domains. We predicted that the highly conserved residues in and flanking the β-strand would be essential for the conformational flexibility and DNA minor groove binding by Mor. To test this hypothesis, we carried out single codon-specific mutagenesis with degenerate oligonucleotides. The amino acid substitutions were identified by DNA sequencing. The mutant proteins were characterized for their overexpression, solubility, DNA binding, and transcription activation. This analysis revealed that the Gly-Gly motif formed by Gly-65 and Gly-66 and the β-strand side chain of Tyr-70 are crucial for DNA binding by His-tagged Mor. Mutant proteins with substitutions at Gly-74 retained partial activity. Treatment with the minor groove- and GC-specific chemical chromomycin A3 demonstrated that chromomycin prevented His-Mor binding but could not disrupt a pre-formed His-Mor·DNA complex, consistent with the prediction that Mor interacts with the minor groove of the GC-rich spacer in the Mor binding site.

Infrared spectra of N-acetyl-L-α-amino-acid N'-methylamides with aliphatic side chains in non-polar solvents

1981 ◽  
Vol 46 (3) ◽  
pp. 772-780 ◽  
Author(s):  
Jorga Smolíková ◽  
Jan Pospíšek ◽  
Karel Bláha
Keyword(s):  
Amino Acid ◽  
Conformational Changes ◽  
Infrared Spectra ◽  
Room Temperature ◽  
Side Chains ◽  
Polar Solvents

Infrared spectra of the L-alanine (I), L-leucine (II), L-valine (III) and L-tert-leucine (IV) N-acetyl N'-methylamides were measured. Amides I-IV are not self associated in tetrachlormethane in the concentration 2 . 10-5 mol l-1 at room temperature and in tetrachloroethylene in the concentration 1.5 . 10-4 mol l-1 at temperatures above 65° C. True conformational changes are observable only with the least flexible amide IV which exists at room temperature in a C5 conformation. This conformational type is also highly populated in the valine derivative III, but is less important in the alanine and leucine derivatives I and II in which the intramolecularly bonded C7 and the distorted hydrogen-nonbonded conformations contribute seriously.

scholarly journals The Inner Interhelix Loop 4–5 of the Melibiose Permease fromEscherichia coliTakes Part in Conformational Changes after Sugar Binding

2006 ◽  
Vol 281 (36) ◽  
pp. 25882-25892 ◽  
Author(s):  
Kerstin Meyer-Lipp ◽  
Natacha Séry ◽  
Constanta Ganea ◽  
Cécile Basquin ◽  
Klaus Fendler ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Sugar Binding

scholarly journals Function Trumps Form in Two Sugar Symporters, LacY and vSGLT

2021 ◽  
Vol 22 (7) ◽  
pp. 3572
Author(s):  
Jeff Abramson ◽  
Ernest M. Wright
Keyword(s):  
Binding Site ◽  
Sugar Transport ◽  
Electrochemical Potential ◽  
Inverted Repeats ◽  
Side Chains ◽  
Transmembrane Helices ◽  
Central Cavity ◽  
Potential Gradients ◽  
Sodium Binding ◽  
Major Facilitator

Active transport of sugars into bacteria occurs through symporters driven by ion gradients. LacY is the most well-studied proton sugar symporter, whereas vSGLT is the most characterized sodium sugar symporter. These are members of the major facilitator (MFS) and the amino acid-Polyamine organocation (APS) transporter superfamilies. While there is no structural homology between these transporters, they operate by a similar mechanism. They are nano-machines driven by their respective ion electrochemical potential gradients across the membrane. LacY has 12 transmembrane helices (TMs) organized in two 6-TM bundles, each containing two 3-helix TM repeats. vSGLT has a core structure of 10 TM helices organized in two inverted repeats (TM 1–5 and TM 6–10). In each case, a single sugar is bound in a central cavity and sugar selectivity is determined by hydrogen- and hydrophobic- bonding with side chains in the binding site. In vSGLT, the sodium-binding site is formed through coordination with carbonyl- and hydroxyl-oxygens from neighboring side chains, whereas in LacY the proton (H3O+) site is thought to be a single glutamate residue (Glu325). The remaining challenge for both transporters is to determine how ion electrochemical potential gradients drive uphill sugar transport.

scholarly journals Monoclonal antibodies identify residues 199–216 of the integrin α2 vWFA domain as a functionally important region within α2β1

2000 ◽  
Vol 350 (2) ◽  
pp. 485-493 ◽  
Author(s):  
Danny S. TUCKWELL ◽  
Lyndsay SMITH ◽  
Michelle KORDA ◽  
Janet A. ASKARI ◽  
Sentot SANTOSO ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Binding Site ◽  
Conformational Changes ◽  
Cation Binding ◽  
Inhibitory Antibody ◽  
Collagen Binding ◽  
Collagen Peptide ◽  
Binding Residue ◽  
Important Region ◽  
Domain Mapping

Integrin α2β1 is the major receptor for collagens in the human body, and the collagen-binding site on the α2 subunit von Willebrand factor A-type domain (vWFA domain) is now well defined. However, the biologically important conformational changes that are associated with collagen binding, and the means by which the vWFA domain is integrated into the whole integrin are not completely understood. We have raised monoclonal antibodies against recombinant α2 vWFA domain for use as probes of function. Three antibodies, JA202, JA215 and JA218, inhibited binding to collagen, collagen I C-propeptide and E-cadherin, demonstrating that their function is important for structurally diverse α2β1 ligands. Cross-blocking studies grouped the epitopes into two clusters: (I) JA202, the inhibitory antibody, Gi9, and a non-inhibitory antibody, JA208; (II) JA215 and JA218. Both clusters were sensitive to events at the collagen binding site, as binding of Gi9, JA202, JA215 and JA218 were inhibited by collagen peptide, JA208 binding was enhanced by collagen peptide, and binding of JA202 was decreased after mutagenesis of the cation-binding residue Thr221 to alanine. Binding of cluster I antibodies was inhibited by the anti-functional anti-β1 antibody Mab13, and binding of Gi9 and JA218 to α2β1 was inhibited by substituting Mn2+ for Mg2+, demonstrating that these antibodies were sensitive to changes initiated outside the vWFA domain. Mapping of epitopes showed that JA202 and Gi9 bound between residues 212–216, while JA208 bound between residues 199–216. We have therefore identified two epitope clusters with novel properties; i.e. they are intimately associated with the collagen-binding site, responsive to conformational changes at the collagen-binding site and sensitive to events initiated outside the vWFA domain.

scholarly journals Bridging the gap between structure and kinetics of human SGLT1

2012 ◽  
Vol 302 (9) ◽  
pp. C1293-C1305 ◽  
Author(s):  
Monica Sala-Rabanal ◽  
Bruce A. Hirayama ◽  
Donald D. F. Loo ◽  
Vincent Chaptal ◽  
Jeff Abramson ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Binding Site ◽  
Sugar Transport ◽  
Gene Families ◽  
Structural Studies ◽  
Sugar Binding ◽  
Substituted Cysteine Accessibility ◽  
Biochemical Assays ◽  
Functional Consequences ◽  
Binding Residues

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.

scholarly journals Engineered occluded apo-intermediate of LacY

2018 ◽  
Vol 115 (50) ◽  
pp. 12716-12721 ◽  
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.

scholarly journals Route, mechanism, and implications of proton import during Na+/K+ exchange by native Na+/K+-ATPase pumps

2014 ◽  
Vol 143 (4) ◽  
pp. 449-464 ◽  
Author(s):  
Natascia Vedovato ◽  
David C. Gadsby
Keyword(s):  
Binding Site ◽  
Single Molecule ◽  
Conformational Changes ◽  
Outward Current ◽  
Side Chain ◽  
Ion Binding ◽  
Inward Current ◽  
Na Release ◽  
K Transport ◽  
External K

A single Na+/K+-ATPase pumps three Na+ outwards and two K+ inwards by alternately exposing ion-binding sites to opposite sides of the membrane in a conformational sequence coupled to pump autophosphorylation from ATP and auto-dephosphorylation. The larger flow of Na+ than K+ generates outward current across the cell membrane. Less well understood is the ability of Na+/K+ pumps to generate an inward current of protons. Originally noted in pumps deprived of external K+ and Na+ ions, as inward current at negative membrane potentials that becomes amplified when external pH is lowered, this proton current is generally viewed as an artifact of those unnatural conditions. We demonstrate here that this inward current also flows at physiological K+ and Na+ concentrations. We show that protons exploit ready reversibility of conformational changes associated with extracellular Na+ release from phosphorylated Na+/K+ pumps. Reversal of a subset of these transitions allows an extracellular proton to bind an acidic side chain and to be subsequently released to the cytoplasm. This back-step of phosphorylated Na+/K+ pumps that enables proton import is not required for completion of the 3 Na+/2 K+ transport cycle. However, the back-step occurs readily during Na+/K+ transport when external K+ ion binding and occlusion are delayed, and it occurs more frequently when lowered extracellular pH raises the probability of protonation of the externally accessible carboxylate side chain. The proton route passes through the Na+-selective binding site III and is distinct from the principal pathway traversed by the majority of transported Na+ and K+ ions that passes through binding site II. The inferred occurrence of Na+/K+ exchange and H+ import during the same conformational cycle of a single molecule identifies the Na+/K+ pump as a hybrid transporter. Whether Na+/K+ pump–mediated proton inflow may have any physiological or pathophysiological significance remains to be clarified.

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