scholarly journals Structural comparison of GLUT1 to GLUT3 reveal transport regulation mechanism in sugar porter family

2021 ◽  
Vol 4 (4) ◽  
pp. e202000858
Author(s):  
Tânia Filipa Custódio ◽  
Peter Aasted Paulsen ◽  
Kelly May Frain ◽  
Bjørn Panyella Pedersen
Keyword(s):  
Binding Site ◽  
Functional Data ◽  
Physiological Function ◽  
Regulation Mechanism ◽  
Sequence Motif ◽  
Open Conformation ◽  
Transport Regulation ◽  
Glucose Binding ◽  
The Difference ◽  
Kinetic Regulation

The human glucose transporters GLUT1 and GLUT3 have a central role in glucose uptake as canonical members of the Sugar Porter (SP) family. GLUT1 and GLUT3 share a fully conserved substrate-binding site with identical substrate coordination, but differ significantly in transport affinity in line with their physiological function. Here, we present a 2.4 Å crystal structure of GLUT1 in an inward open conformation and compare it with GLUT3 using both structural and functional data. Our work shows that interactions between a cytosolic “SP motif” and a conserved “A motif” stabilize the outward conformational state and increases substrate apparent affinity. Furthermore, we identify a previously undescribed Cl− ion site in GLUT1 and an endofacial lipid/glucose binding site which modulate GLUT kinetics. The results provide a possible explanation for the difference between GLUT1 and GLUT3 glucose affinity, imply a general model for the kinetic regulation in GLUTs and suggest a physiological function for the defining SP sequence motif in the SP family.

scholarly journals Structural comparison of GLUT1 to GLUT3 reveal transport regulation mechanism in Sugar Porter family

2020 ◽  
Author(s):  
Tânia Filipa Custódio ◽  
Peter Aasted Paulsen ◽  
Kelly May Frain ◽  
Bjørn Panyella Pedersen
Keyword(s):  
Binding Site ◽  
Functional Data ◽  
Physiological Function ◽  
Regulation Mechanism ◽  
Sequence Motif ◽  
Open Conformation ◽  
Glucose Binding ◽  
The Difference ◽  
Substrate Affinities ◽  
Kinetic Regulation

AbstractThe human glucose transporters GLUT1 and GLUT3 have a central role in glucose uptake as canonical members of the Sugar Porter (SP) family. GLUT1 and GLUT3 share a fully conserved substrate-binding site with identical substrate coordination, but differ significantly in transport affinity in line with their physiological function. Here we present a 2.4 Å crystal structure of GLUT1 in an inward open conformation and compare it with GLUT3 using both structural and functional data. Our work shows that interactions between a cytosolic “Sugar Porter motif” and a conserved “A motif” stabilize the outward conformational state and increases substrate apparent affinity. Furthermore, we identify a previously undescribed Cl- ion site in GLUT1 and an endofacial lipid/glucose binding site which modulate GLUT kinetics. The results provide a possible explanation for the difference between GLUT1 and GLUT3 glucose affinity, imply a general model for the kinetic regulation in GLUTs and suggest a physiological function for the defining SP sequence motif in the Sugar Porter family.Summary BlurbNew structure of GLUT1 compared to GLUT3 explain different substrate affinities. The result provide a functional rationale for key structural motifs that define the universal Sugar Porter family.

Exploring the Hexokinase Glucose Binding Site Through Correlation Analysis and Molecular Modeling of Glucosamine Inhibitors

1993 ◽  
Vol 6 (4) ◽  
pp. 271-282 ◽  
Author(s):  
Eugene A. Coats ◽  
Kenneth A. Skau ◽  
Carol A Caperelli ◽  
David Solomacha
Keyword(s):  
Molecular Modeling ◽  
Correlation Analysis ◽  
Binding Site ◽  
Glucose Binding

scholarly journals Crystal Structure of Circadian Clock Protein KaiA fromSynechococcus elongatus

2004 ◽  
Vol 279 (19) ◽  
pp. 20511-20518 ◽  
Author(s):  
Sheng Ye ◽  
Ioannis Vakonakis ◽  
Thomas R. Ioerger ◽  
Andy C. LiWang ◽  
James C. Sacchettini
Keyword(s):  
Crystal Structure ◽  
Circadian Clock ◽  
Binding Site ◽  
Regulation Mechanism ◽  
Response Regulators ◽  
Dimer Interface ◽  
Helix Bundle ◽  
Bacterial Response ◽  
Four Helix Bundle ◽  
Clock Protein

The circadian clock found inSynechococcus elongatus, the most ancient circadian clock, is regulated by the interaction of three proteins, KaiA, KaiB, and KaiC. While the precise function of these proteins remains unclear, KaiA has been shown to be a positive regulator of the expression of KaiB and KaiC. The 2.0-Å structure of KaiA ofS. elongatusreported here shows that the protein is composed of two independently folded domains connected by a linker. The NH2-terminalpseudo-receiver domain has a similar fold with that of bacterial response regulators, whereas the COOH-terminal four-helix bundle domain is novel and forms the interface of the 2-fold-related homodimer. The COOH-terminal four-helix bundle domain has been shown to contain the KaiC binding site. The structure suggests that the KaiB binding site is covered in the dimer interface of the KaiA “closed” conformation, observed in the crystal structure, which suggests an allosteric regulation mechanism.

scholarly journals Oversized galactosides as a probe for conformational dynamics in LacY

2018 ◽  
Vol 115 (16) ◽  
pp. 4146-4151 ◽  
Author(s):  
Irina Smirnova ◽  
Vladimir Kasho ◽  
Xiaoxu Jiang ◽  
Hong-Ming Chen ◽  
Stephen G. Withers ◽  
...  
Keyword(s):  
Binding Site ◽  
Conformational Dynamics ◽  
Dissociation Rate ◽  
Binding Kinetics ◽  
Lactose Permease ◽  
E Coli ◽  
Open Conformation ◽  
Dissociation Rate Constants ◽  
Kinetics Of ◽  
Micromolar Range

Binding kinetics of α-galactopyranoside homologs with fluorescent aglycones of different sizes and shapes were determined with the lactose permease (LacY) of Escherichia coli by FRET from Trp151 in the binding site of LacY to the fluorophores. Fast binding was observed with LacY stabilized in an outward-open conformation (kon = 4–20 μM−1·s−1), indicating unobstructed access to the binding site even for ligands that are much larger than lactose. Dissociation rate constants (koff) increase with the size of the aglycone so that Kd values also increase but remain in the micromolar range for each homolog. Phe27 (helix I) forms an apparent constriction in the pathway for sugar by protruding into the periplasmic cavity. However, replacement of Phe27 with a bulkier Trp does not create an obstacle in the pathway even for large ligands, since binding kinetics remain unchanged. High accessibility of the binding site is also observed in a LacY/nanobody complex with partially blocked periplasmic opening. Remarkably, E. coli expressing WT LacY catalyzes transport of α- or β-galactopyranosides with oversized aglycones such as bodipy or Aldol518, which may require an extra space within the occluded intermediate. The results confirm that LacY specificity is strictly directed toward the galactopyranoside ring and also clearly indicate that the opening on the periplasmic side is sufficiently wide to accommodate the large galactoside derivatives tested here. We conclude that the actual pathway for the substrate entering from the periplasmic side is wider than the pore diameter calculated in the periplasmic-open X-ray structures.

A mutation inactivating the distal SF1 binding site on the human anti-Müllerian hormone promoter causes persistent Müllerian duct syndrome

2019 ◽  
Vol 28 (19) ◽  
pp. 3211-3218 ◽  
Author(s):  
Helena F Schteingart ◽  
Jean-Yves Picard ◽  
Clara Valeri ◽  
Ian Marshall ◽  
Dominique Treton ◽  
...  
Keyword(s):  
Binding Site ◽  
Mullerian Duct ◽  
Mobility Shift ◽  
Müllerian Duct ◽  
Persistent Müllerian Duct Syndrome ◽  
First Case ◽  
Disorder Of Sexual Development ◽  
Persistent Mullerian Duct Syndrome ◽  
The Difference ◽  
Duct Syndrome

AbstractThe persistent Müllerian duct syndrome (PMDS) is a 46,XY disorder of sexual development characterized by the persistence of Müllerian duct derivatives, uterus and tubes, in otherwise normally masculinized males. The condition, transmitted as a recessive autosomal trait, is usually due to mutations in either the anti-Müllerian hormone (AMH) gene or its main receptor. Many variants of these genes have been described, all targeting the coding sequences. We report the first case of PMDS due to a regulatory mutation. The AMH promoter contains two binding sites for steroidogenic factor 1 (SF1), one at −102 and the other at −228. Our patient carries a single base deletion at −225, significantly decreasing its capacity for binding SF1, as measured by the electrophoresis mobility shift assay. Furthermore, by linking the AMH promoter to the luciferase gene, we show that the transactivation capacity of the promoter is significantly decreased by the mutation, in contrast to the disruption of the −102 binding site. To explain the difference in impact we hypothesize that SF1 could partially overcome the lack of binding to the −102 binding site by interacting with a GATA4 molecule linked to a nearby response element. We show that disruption of both the −102 SF1 and the −84 GATA response elements significantly decreases the transactivation capacity of the promoter. In conclusion, we suggest that the distance between mutated SF1 sites and potentially rescuing GATA binding motifs might play a role in the development of PMDS.

scholarly journals Histone-like Nucleoid-Structuring Protein (H-NS) Paralogue StpA Activates the Type I-E CRISPR-Cas System against Natural Transformation in Escherichia coli

2020 ◽  
Vol 86 (14) ◽  
Author(s):  
Dongchang Sun ◽  
Xudan Mao ◽  
Mingyue Fei ◽  
Ziyan Chen ◽  
Tingheng Zhu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Binding Site ◽  
Natural Transformation ◽  
Inducible Promoter ◽  
Regulation Mechanism ◽  
Type I ◽  
Content Type ◽  
E Coli ◽  
Double Stranded Dna ◽  
Dna Binding Site

ABSTRACT Working mechanisms of CRISPR-Cas systems have been intensively studied. However, far less is known about how they are regulated. The histone-like nucleoid-structuring protein H-NS binds the promoter of cas genes (Pcas) and suppresses the type I-E CRISPR-Cas system in Escherichia coli. Although the H-NS paralogue StpA also binds Pcas, its role in regulating the CRISPR-Cas system remains unidentified. Our previous work established that E. coli is able to take up double-stranded DNA during natural transformation. Here, we investigated the function of StpA in regulating the type I-E CRISPR-Cas system against natural transformation of E. coli. We first documented that although the activated type I-E CRISPR-Cas system, due to hns deletion, interfered with CRISPR-Cas-targeted plasmid transfer, stpA inactivation restored the level of natural transformation. Second, we showed that inactivating stpA reduced the transcriptional activity of Pcas. Third, by comparing transcriptional activities of the intact Pcas and the Pcas with a disrupted H-NS binding site in the hns and hns stpA null deletion mutants, we demonstrated that StpA activated transcription of cas genes by binding to the same site as H-NS in Pcas. Fourth, by expressing StpA with an arabinose-inducible promoter, we confirmed that StpA expressed at a low level stimulated the activity of Pcas. Finally, by quantifying the level of mature CRISPR RNA (crRNA), we demonstrated that StpA was able to promote the amount of crRNA. Taken together, our work establishes that StpA serves as a transcriptional activator in regulating the type I-E CRISPR-Cas system against natural transformation of E. coli. IMPORTANCE StpA is normally considered a molecular backup of the nucleoid-structuring protein H-NS, which was reported as a transcriptional repressor of the type I-E CRISPR-Cas system in Escherichia coli. However, the role of StpA in regulating the type I-E CRISPR-Cas system remains elusive. Our previous work uncovered a new route for double-stranded DNA (dsDNA) entry during natural transformation of E. coli. In this study, we show that StpA plays a role opposite to that of its paralogue H-NS in regulating the type I-E CRISPR-Cas system against natural transformation of E. coli. Our work not only expands our knowledge on CRISPR-Cas-mediated adaptive immunity against extracellular nucleic acids but also sheds new light on understanding the complex regulation mechanism of the CRISPR-Cas system. Moreover, the finding that paralogues StpA and H-NS share a DNA binding site but play opposite roles in transcriptional regulation indicates that higher-order compaction of bacterial chromatin by histone-like proteins could switch prokaryotic transcriptional modes.

Kinetic regulation mechanism of pbuE riboswitch

2015 ◽  
Vol 142 (1) ◽  
pp. 015103 ◽  
Author(s):  
Sha Gong ◽  
Yujie Wang ◽  
Wenbing Zhang
Keyword(s):  
Regulation Mechanism ◽  
Kinetic Regulation

scholarly journals Structure of the Fab Fragment of F105, a Broadly Reactive Anti-Human Immunodeficiency Virus (HIV) Antibody That Recognizes the CD4 Binding Site of HIV Type 1 gp120

2005 ◽  
Vol 79 (20) ◽  
pp. 13060-13069 ◽  
Author(s):  
Royce A. Wilkinson ◽  
Chayne Piscitelli ◽  
Martin Teintze ◽  
Lisa A. Cavacini ◽  
Marshall R. Posner ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Binding Site ◽  
Neutralizing Antibody ◽  
Binding Pocket ◽  
Human Antibody ◽  
Fab Fragment ◽  
Outer Domain ◽  
Immunodeficiency Virus ◽  
Cd4 Binding Site ◽  
The Difference

ABSTRACT We have determined the crystal structure of the Fab fragment from F105, a broadly reactive human antibody with limited potency that recognizes the CD4 binding site of gp120. The structure reveals an extended CDR H3 loop with a phenylalanine residue at the apex and shows a striking pattern of serine and tyrosine residues. Modeling the interaction between gp120 and F105 suggests that the phenylalanine may recognize the binding pocket of gp120 used by Phe43 of CD4 and that numerous tyrosine and serine residues form hydrogen bonds with the main chain atoms of gp120. A comparison of the F105 structure to that of immunoglobulin G1 b12, a much more potent and broadly neutralizing antibody with an overlapping epitope, suggests similarities that contribute to the broad recognition of human immunodeficiency virus by both antibodies. While the putative epitope for F105 shows significant overlap with that predicted for b12, it appears to differ from the b12 epitope in extending across the interface between the inner and outer domains of gp120. In contrast, the CDR loops of b12 appear to interact predominantly with the outer domain of gp120. The difference between the predicted epitopes for b12 and F105 suggests that the unique potency of b12 may arise from its ability to avoid the interface between the inner and outer domains of gp120.

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