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 Structure of an ancestral ADP-dependent kinase with fructose-6P reveals key residues for binding, catalysis, and ligand-induced conformational changes

2020 ◽  
pp. jbc.RA120.015376
Author(s):  
Sebastián M. Muñoz ◽  
Victor Castro-Fernandez ◽  
Victoria Guixe
Keyword(s):  
Conformational Changes ◽  
Active Site ◽  
Substrate Specificities ◽  
Closed Conformation ◽  
Sugar Binding ◽  
Bifunctional Enzymes ◽  
Glucose Binding ◽  
Critical Residue ◽  
Key Residues ◽  
Human And Mouse

ADP-dependent kinases were first described in archaea, although their presence has also been reported in bacteria and eukaryotes (human and mouse). This enzyme family comprises three substrate specificities; specific phosphofructokinases (ADP-PFK), specific glucokinases (ADP-GK), and bifunctional enzymes (ADP-PFK/GK). Although many structures are available for members of this family, no one exhibits fructose-6P at the active site. Employing an ancestral enzyme, we obtain the first structure of an ADP-dependent kinase (AncMsPFK) with fructose-6P at its active site. Key residues for sugar-binding and catalysis were identified by alanine scanning, being D36 a critical residue for F6P binding and catalysis. However, this residue hinders glucose binding since its mutation to alanine converts the AncMsPFK enzyme into a specific ADP-GK. Residue K179 is critical for F6P binding, while residues N181 and R212 are also important for this sugar-binding, but to a lesser extent. This structure also provides evidence for the requirement of both substrates (sugar and nucleotide) to accomplish the conformational change leading to a closed conformation. This suggests that AncMsPFK mainly populates two states (open and closed) during the catalytic cycle, as reported for specific ADP-PFK. This situation differs from that described for specific ADP-GK enzymes, where each substrate independently causes a sequential domain closure, resulting in three conformational states (open, semi-closed and closed).

scholarly journals Sugar Binding and Protein Conformational Changes in Lactose Permease

2006 ◽  
Vol 91 (11) ◽  
pp. 3972-3985 ◽  
Author(s):  
Ying Yin ◽  
Morten Ø. Jensen ◽  
Emad Tajkhorshid ◽  
Klaus Schulten
Keyword(s):  
Conformational Changes ◽  
Lactose Permease ◽  
Sugar Binding ◽  
Protein Conformational Changes

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 Molecular Basis of ChvE Function in Sugar Binding, Sugar Utilization, and Virulence in Agrobacterium tumefaciens

2009 ◽  
Vol 191 (18) ◽  
pp. 5802-5813 ◽  
Author(s):  
Fanglian He ◽  
Gauri R. Nair ◽  
Cinque S. Soto ◽  
Yehchung Chang ◽  
Lillian Hsu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Agrobacterium Tumefaciens ◽  
Conformational Changes ◽  
Binding Protein ◽  
Sugar Transport ◽  
Homology Model ◽  
Maximal Effect ◽  
Sugar Utilization ◽  
Sugar Binding ◽  
Binding Cleft

ABSTRACT ChvE is a chromosomally encoded protein in Agrobacterium tumefaciens that mediates a sugar-induced increase in virulence (vir) gene expression through the activities of the VirA/VirG two-component system and has also been suggested to be involved in sugar utilization. The ChvE protein has homology to several bacterial periplasmic sugar-binding proteins, such as the ribose-binding protein and the galactose/glucose-binding protein of Escherichia coli. In this study, we provide direct evidence that ChvE specifically binds the vir gene-inducing sugar d-glucose with high affinity. Furthermore, ChvE mutations resulting in altered vir gene expression phenotypes have been isolated and characterized. Three distinct categories of mutants have been identified. Strains expressing the first class are defective in both virulence and d-glucose utilization as a result of mutations to residues lining the sugar-binding cleft. Strains expressing a second class of mutants are not adversely affected in sugar binding but are defective in virulence, presumably due to impaired interactions with the sensor kinase VirA. A subset of this second class of mutants includes variants of ChvE that also result in defective sugar utilization. We propose that these mutations affect not only interactions with VirA but also interactions with a sugar transport system. Examination of a homology model of ChvE shows that the mutated residues associated with the latter two phenotypes lie in two overlapping solvent-exposed sites adjacent to the sugar-binding cleft where conformational changes associated with the binding of sugar might have a maximal effect on ChvE's interactions with its distinct protein partners.

scholarly journals Structure of an EIIC sugar transporter trapped in an inward-facing conformation

2018 ◽  
Vol 115 (23) ◽  
pp. 5962-5967 ◽  
Author(s):  
Zhenning Ren ◽  
Jumin Lee ◽  
Mahdi Muhammad Moosa ◽  
Yin Nian ◽  
Liya Hu ◽  
...  
Keyword(s):  
Rigid Body ◽  
Single Molecule ◽  
Conformational Changes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Rigid Body Motion ◽  
Body Motion ◽  
Sugar Uptake ◽  
Phosphotransferase System ◽  
Sugar Binding

The phosphoenolpyruvate-dependent phosphotransferase system (PTS) transports sugar into bacteria and phosphorylates the sugar for metabolic consumption. The PTS is important for the survival of bacteria and thus a potential target for antibiotics, but its mechanism of sugar uptake and phosphorylation remains unclear. The PTS is composed of multiple proteins, and the membrane-embedded Enzyme IIC (EIIC) component transports sugars across the membrane. Crystal structures of two members of the glucose superfamily of EIICs, bcChbC and bcMalT, were solved in the inward-facing and outward-facing conformations, and the structures suggest that sugar translocation could be achieved by movement of a structured domain that contains the sugar-binding site. However, different conformations have not been captured on the same transporter to allow precise description of the conformational changes. Here we present a crystal structure of bcMalT trapped in an inward-facing conformation by a mercury ion that bridges two strategically placed cysteine residues. The structure allows direct comparison of the outward- and inward-facing conformations and reveals a large rigid-body motion of the sugar-binding domain and other conformational changes that accompany the rigid-body motion. All-atom molecular dynamics simulations show that the inward-facing structure is stable with or without the cross-linking. The conformational changes were further validated by single-molecule Föster resonance energy transfer (smFRET). Combined, these results establish the elevator-type mechanism of transport in the glucose superfamily of EIIC transporters.

Influence of Calcium Ions on the Plant Cell Surface: Membrane Fusions and Conformational Changes

1974 ◽  
Vol 32 ◽  
pp. 154-155
Author(s):  
D. James Morré ◽  
Charles E. Bracker ◽  
William J. VanDerWoude
Keyword(s):  
Cell Wall ◽  
Cell Surface ◽  
Conformational Changes ◽  
Calcium Ions ◽  
Surface Membrane ◽  
Carboxyl Groups ◽  
Cross Linking ◽  
Ultrastructural Evidence ◽  
Glycine Max L ◽  
Wall Extensibility

Calcium ions in the concentration range 5-100 mM inhibit auxin-induced cell elongation and wall extensibility of plant stems. Inhibition of wall extensibility requires that the tissue be living; growth inhibition cannot be explained on the basis of cross-linking of carboxyl groups of cell wall uronides by calcium ions. In this study, ultrastructural evidence was sought for an interaction of calcium ions with some component other than the wall at the cell surface of soybean (Glycine max (L.) Merr.) hypocotyls.

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