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1.
Nat Struct Mol Biol ; 2024 Apr 17.
Article in English | MEDLINE | ID: mdl-38632360

ABSTRACT

The Pyrococcus horikoshii amino acid transporter GltPh revealed, like other channels and transporters, activity mode switching, previously termed wanderlust kinetics. Unfortunately, to date, the basis of these activity fluctuations is not understood, probably due to a lack of experimental tools that directly access the structural features of transporters related to their instantaneous activity. Here, we take advantage of high-speed atomic force microscopy, unique in providing simultaneous structural and temporal resolution, to uncover the basis of kinetic mode switching in proteins. We developed membrane extension membrane protein reconstitution that allows the analysis of isolated molecules. Together with localization atomic force microscopy, principal component analysis and hidden Markov modeling, we could associate structural states to a functional timeline, allowing six structures to be solved from a single molecule, and an inward-facing state, IFSopen-1, to be determined as a kinetic dead-end in the conformational landscape. The approaches presented on GltPh are generally applicable and open possibilities for time-resolved dynamic single-molecule structural biology.

2.
Nat Commun ; 14(1): 2579, 2023 05 04.
Article in English | MEDLINE | ID: mdl-37142617

ABSTRACT

Excitatory amino acid transporters (EAATs) uptake glutamate into glial cells and neurons. EAATs achieve million-fold transmitter gradients by symporting it with three sodium ions and a proton, and countertransporting a potassium ion via an elevator mechanism. Despite the availability of structures, the symport and antiport mechanisms still need to be clarified. We report high-resolution cryo-EM structures of human EAAT3 bound to the neurotransmitter glutamate with symported ions, potassium ions, sodium ions alone, or without ligands. We show that an evolutionarily conserved occluded translocation intermediate has a dramatically higher affinity for the neurotransmitter and the countertransported potassium ion than outward- or inward-facing transporters and plays a crucial role in ion coupling. We propose a comprehensive ion coupling mechanism involving a choreographed interplay between bound solutes, conformations of conserved amino acid motifs, and movements of the gating hairpin and the substrate-binding domain.


Subject(s)
Amino Acid Transport System X-AG , Glutamic Acid , Humans , Amino Acid Transport System X-AG/metabolism , Ion Transport , Ions/metabolism , Glutamic Acid/metabolism , Sodium/metabolism , Potassium/metabolism
3.
J Am Chem Soc ; 145(15): 8583-8592, 2023 04 19.
Article in English | MEDLINE | ID: mdl-37023263

ABSTRACT

Limited chemical shift dispersion represents a significant barrier to studying multistate equilibria of large membrane proteins by 19F NMR. We describe a novel monofluoroethyl 19F probe that dramatically increases the chemical shift dispersion. The improved conformational sensitivity and line shape enable the detection of previously unresolved states in one-dimensional (1D) 19F NMR spectra of a 134 kDa membrane transporter. Changes in the populations of these states in response to ligand binding, mutations, and temperature correlate with population changes of distinct conformations in structural ensembles determined by single-particle cryo-electron microscopy (cryo-EM). Thus, 19F NMR can guide sample preparation to discover and visualize novel conformational states and facilitate image analysis and three-dimensional (3D) classification.


Subject(s)
Fluorine , Magnetic Resonance Imaging , Cryoelectron Microscopy/methods , Magnetic Resonance Spectroscopy , Protein Conformation
4.
Sci Adv ; 7(10)2021 03.
Article in English | MEDLINE | ID: mdl-33658209

ABSTRACT

Human excitatory amino acid transporter 3 (hEAAT3) mediates glutamate uptake in neurons, intestine, and kidney. Here, we report cryo-EM structures of hEAAT3 in several functional states where the transporter is empty, bound to coupled sodium ions only, or fully loaded with three sodium ions, a proton, and the substrate aspartate. The structures suggest that hEAAT3 operates by an elevator mechanism involving three functionally independent subunits. When the substrate-binding site is near the cytoplasm, it has a remarkably low affinity for the substrate, perhaps facilitating its release and allowing the rapid transport turnover. The mechanism of the coupled uptake of the sodium ions and the substrate is conserved across evolutionarily distant families and is augmented by coupling to protons in EAATs. The structures further suggest a mechanism by which a conserved glutamate residue mediates proton symport.


Subject(s)
Excitatory Amino Acid Transporter 3/chemistry , Protons , Binding Sites , Cryoelectron Microscopy , Excitatory Amino Acid Transporter 3/metabolism , Glutamic Acid/metabolism , Humans , Ions/metabolism , Sodium/chemistry
5.
Cell Res ; 28(6): 644-654, 2018 06.
Article in English | MEDLINE | ID: mdl-29588525

ABSTRACT

Acetate is an important metabolite in metabolism and cell signaling. Succinate-Acetate Permease (SatP) superfamily proteins are known to be responsible for acetate transport across membranes, but the nature of this transport remains unknown. Here, we show that the SatP homolog from Citrobacter koseri (SatP_Ck) is an anion channel that can unidirectionally translocate acetate at rates of the order of ~107 ions/s. Crystal structures of SatP_Ck in complex with multiple acetates at 1.8 Å reveal that the acetate pathway consists of four acetate-binding sites aligned in a single file that are interrupted by three hydrophobic constrictions. The bound acetates at the four sites are each orientated differently. The acetate at the cytoplasmic vestibule is partially dehydrated, whereas those in the main pore body are fully dehydrated. Aromatic residues within the substrate pathway may coordinate translocation of acetates via anion-π interactions. SatP_Ck reveals a new type of selective anion channel and provides a structural and functional template for understanding organic anion transport.


Subject(s)
Acetic Acid/metabolism , Bacterial Proteins/metabolism , Citrobacter koseri/metabolism , Monocarboxylic Acid Transporters/metabolism , Bacterial Proteins/chemistry , Binding Sites , Citrobacter koseri/chemistry , Crystallography, X-Ray , Enterobacteriaceae Infections/microbiology , Humans , Models, Molecular , Monocarboxylic Acid Transporters/chemistry , Protein Conformation , Succinates/metabolism
6.
Biochem Biophys Res Commun ; 446(1): 380-6, 2014 Mar 28.
Article in English | MEDLINE | ID: mdl-24613384

ABSTRACT

ADP-ribosylation factors (Arfs) play key roles in controlling membrane traffic and organelle structures. The activation of Arfs from GDP to GTP binding form is triggered by the guanine exchange factors (GEFs). There are six families of Arf-GEFs with a common guanine exchange catalytic domain (Sec7 domain) and various mechanisms of guanine exchange activity regulation. A loop region (loop>J motif) just following the helix J of Sec7 domain was found conserved and important for the catalytic activity regulation of Arf-GEFs. However, the molecular detail of the role the loop>J motif plays has been yet unclear. Here, we studied the catalytic domain of Sec7p, a yeast trans-Golgi network membrane localized Arf-GEFs, and found that the loop>J motif is indispensible for its GEF catalytic activity. Crystallographic, NMR spectrum and mutagenesis studies suggested that the loop>J motif with a key conserved residue Ile1010 modulates the fine conformation of Sec7 domain and thereby regulates its guanine exchange activity.


Subject(s)
Guanine Nucleotide Exchange Factors/chemistry , Guanine Nucleotide Exchange Factors/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Amino Acid Motifs , Amino Acid Sequence , Binding Sites/genetics , Conserved Sequence , Crystallography, X-Ray , Guanine Nucleotide Exchange Factors/genetics , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Nuclear Magnetic Resonance, Biomolecular , Protein Conformation , Protein Stability , Protein Structure, Secondary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Sequence Homology, Amino Acid , Static Electricity , Structural Homology, Protein
7.
J Hazard Mater ; 213-214: 184-92, 2012 Apr 30.
Article in English | MEDLINE | ID: mdl-22341981

ABSTRACT

Activated carbon (AC) supported manganese oxide sorbents were prepared by the supercritical water impregnation (SCWI) using two different precursor of Mn(NO(3))(2) (SCW(N)) and Mn(Ac)(2)·4H(2)O (SCW(A)). Their capacities of removing H(2)S from coal gas were evaluated and compared to the sorbents prepared by the pore volume impregnation (PVI) method. The structure and composition of different sorbents were characterized by XRD, SEM, TEM, XPS and XANES techniques. It is found that the precursor of active component plays the crucial role and SCW(N) sorbents show much better sulfidation performance than the SCW(A) sorbents. This is because the Mn(3)O(4) active phase of the SCW(N) sorbents are well dispersed on the AC support, while the Mn(2)SiO(4)-like species in the SCW(A) sorbent can be formed and seriously aggregated. The SCW(N) sorbents with 2.80% and 5.60% manganese are favorable for the sulfidation reaction, since the Mn species are better dispersed on the SCW(N) sorbents than those on the PV(N) sorbents and results in the better sulfidation performance of the SCW(N) sorbents. As the Mn content increases to 11.20%, the metal oxide particles on AC supports aggregate seriously, which leads to poorer sulfidation performance of the SCW(N)11.20% sorbents than that of the PV(N)11.20% sorbents.


Subject(s)
Carbon/chemistry , Coal/analysis , Hydrogen Sulfide/isolation & purification , Manganese/chemistry , Adsorption , Gases/chemistry , Hot Temperature , Manganese Compounds/chemistry , Microscopy, Electron, Scanning , Microscopy, Electron, Transmission , Nanoparticles , Oxides/chemistry , Photoelectron Spectroscopy , Porosity , Sulfides/chemistry , X-Ray Diffraction
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