scholarly journals Structural capture of an intermediate transport state of a CLC CI-/H+ antiporter

2018 ◽  
Author(s):  
Kunwoong Park ◽  
Hyun-Ho Lim
Keyword(s):  
Crystal Structures ◽  
Conformational Heterogeneity ◽  
Conformational States ◽  
Cellular Processes ◽  
Chloride Homeostasis ◽  
Transport Cycle ◽  
Multiple Conformations ◽  
Structural Levels

AbstractThe CLC family proteins are involved in a variety of cellular processes, where chloride homeostasis needs to be controlled. Two distinct classes of CLC proteins, Cl- channels and Cl-/H+ antiporters, have been functionally and structurally investigated over the last several decades. Recent studies have revealed that the conformational heterogeneity of the critical glutamate residue, Gluex could explain the transport cycle of CLC-type Cl-/H+ antiporters. However, the presence of multiple conformations of the Gluex has been suggested from combined structural snapshots of two different CLC antiporters. Thus, we aimed to investigate the presence of these three intermediate conformations in CLC-ec1, the most deeply studied CLC at both functional and structural levels. By comparing crystal structures of E148D, E148A mutant and wildtype CLC-ec1 with varying anion concentrations, we suggest that the Gluex indeed take at least three distinct conformational states in a single CLC antiporter, CLC-ec1.

scholarly journals Mutation of external glutamate residue reveals a new intermediate transport state and anion binding site in a CLC Cl−/H+ antiporter

2019 ◽  
Vol 116 (35) ◽  
pp. 17345-17354 ◽  
Author(s):  
Kunwoong Park ◽  
Byoung-Cheol Lee ◽  
Hyun-Ho Lim
Keyword(s):  
Binding Site ◽  
Chloride Concentration ◽  
Anion Binding ◽  
Cyanidioschyzon Merolae ◽  
Conformational Heterogeneity ◽  
Wild Type ◽  
Physiological Processes ◽  
Transport Cycle ◽  
Multiple Conformations ◽  
Structural Levels

The CLC family of proteins are involved in a variety of physiological processes to control cellular chloride concentration. Two distinct classes of CLC proteins, Cl− channels and Cl−/H+ antiporters, have been functionally and structurally investigated over the last several decades. Previous studies have suggested that the conformational heterogeneity of the critical glutamate residue, Gluex, could explain the transport cycle of CLC-type Cl−/H+ antiporters. However, the presence of multiple conformations (Up, Middle, and Down) of the Gluex has been suggested from combined structural snapshots of 2 different CLC antiporters: CLC-ec1 from Escherichia coli and cmCLC from a thermophilic red alga, Cyanidioschyzon merolae. Thus, we aimed to investigate further the heterogeneity of Gluex-conformations in CLC-ec1, the most deeply studied CLC antiporter, at both functional and structural levels. Here, we show that the crystal structures of the Gluex mutant E148D and wild-type CLC-ec1 with varying anion concentrations suggest a structural intermediate, the “Midlow” conformation. We also found that an extra anion can be located above the external Cl−-binding site in the E148D mutant when the anion concentration is high. Moreover, we observed that a carboxylate in solution can occupy either the external or central Cl−-binding site in the ungated E148A mutant using an anomalously detectable short carboxylic acid, bromoacetate. These results lend credibility to the idea that the Gluex can take at least 3 distinct conformational states during the transport cycle of a single CLC antiporter.

scholarly journals Crystal structures of an E1–E2–ubiquitin thioester mimetic reveal molecular mechanisms of transthioesterification

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Lingmin Yuan ◽  
Zongyang Lv ◽  
Melanie J. Adams ◽  
Shaun K. Olsen
Keyword(s):  
Complex Formation ◽  
Crystal Structures ◽  
Molecular Mechanisms ◽  
Conformational State ◽  
Biochemical Data ◽  
Conformational States ◽  
Product Release ◽  
Closed Conformation ◽  
Open Conformation ◽  
Conjugating Enzymes

AbstractE1 enzymes function as gatekeepers of ubiquitin (Ub) signaling by catalyzing activation and transfer of Ub to tens of cognate E2 conjugating enzymes in a process called E1–E2 transthioesterification. The molecular mechanisms of transthioesterification and the overall architecture of the E1–E2–Ub complex during catalysis are unknown. Here, we determine the structure of a covalently trapped E1–E2–ubiquitin thioester mimetic. Two distinct architectures of the complex are observed, one in which the Ub thioester (Ub(t)) contacts E1 in an open conformation and another in which Ub(t) instead contacts E2 in a drastically different, closed conformation. Altogether our structural and biochemical data suggest that these two conformational states represent snapshots of the E1–E2–Ub complex pre- and post-thioester transfer, and are consistent with a model in which catalysis is enhanced by a Ub(t)-mediated affinity switch that drives the reaction forward by promoting productive complex formation or product release depending on the conformational state.

scholarly journals High-resolution structures of inhibitor complexes of human indoleamine 2,3-dioxygenase 1 in a new crystal form

2018 ◽  
Vol 74 (11) ◽  
pp. 717-724 ◽  
Author(s):  
Shukun Luo ◽  
Ke Xu ◽  
Shaoyun Xiang ◽  
Jie Chen ◽  
Chunyun Chen ◽  
...  
Keyword(s):  
Drug Discovery ◽  
High Resolution ◽  
Crystal Structures ◽  
Active Site ◽  
Crystal Form ◽  
Crystal Forms ◽  
Cellular Processes ◽  
Potential Target ◽  
Indoleamine 2,3 Dioxygenase

Human indoleamine 2,3-dioxygenase 1 (IDO1) is a heme-dependent enzyme with important roles in many cellular processes and is a potential target for drug discovery against cancer and other diseases. Crystal structures of IDO1 in complex with various inhibitors have been reported. Many of these crystals belong to the same crystal form and most of the reported structures have resolutions in the range 3.2–2.3 Å. Here, three new crystal forms of human IDO1 obtained by introducing a surface mutation, K116A/K117A, distant from the active site are reported. One of these crystal forms diffracted to 1.5 Å resolution and can be readily used for soaking experiments to determine high-resolution structures of IDO1 in complex with the substrate tryptophan or inhibitors that coordinate the heme. In addition, this mutant was used to produce crystals of a complex with an inhibitor that targets the apo form of the enzyme under the same conditions; the structure of this complex was determined at 1.7 Å resolution. Overall, this mutant represents a robust platform for determining the structures of inhibitor and substrate complexes of IDO1 at high resolution.

scholarly journals Crystal Structures of HLA-A*0201 Complexed with Melan-A/MART-126(27L)-35Peptidomimetics Reveal Conformational Heterogeneity and Highlight Degeneracy of T Cell Recognition

2010 ◽  
Vol 53 (19) ◽  
pp. 7061-7066 ◽  
Author(s):  
Céline Douat-Casassus ◽  
Oleg Borbulevych ◽  
Marion Tarbe ◽  
Nadine Gervois ◽  
Francine Jotereau ◽  
...  
Keyword(s):  
T Cell ◽  
Crystal Structures ◽  
Conformational Heterogeneity ◽  
Cell Recognition ◽  
T Cell Recognition

scholarly journals Crystal structures of hFPPS in complex with novel anticancer drug leads

2014 ◽  
Vol 70 (a1) ◽  
pp. C712-C712
Author(s):  
Jaeok Park ◽  
Chun Leung ◽  
Yih-Shyan Lin ◽  
Joris De Schutter ◽  
Youla Tsantrizos ◽  
...  
Keyword(s):  
Physicochemical Properties ◽  
Crystal Structures ◽  
Anticancer Agents ◽  
Active Site ◽  
Farnesyl Pyrophosphate ◽  
Allosteric Site ◽  
Cellular Processes ◽  
Inhibitory Site ◽  
Active Site Cavity

Human farnesyl pyrophosphate synthase (hFPPS) produces farnesyl pyrophosphate, an isoprenoid required for a variety of essential cellular processes. Inhibition of hFPPS has been well established as the mechanism of action of the nitrogen-containing bisphosphonate (N-BP) drugs, currently best known for their anti-bone resorptive effects. Recent investigations indicate that hFPPS inhibition also produces potent anticancer effects both in vitro and vivo: N-BPs inhibit proliferation, motility, and viability of tumor cells, and act in synergy with other anticancer agents [1,2]. However, the physicochemical properties of the current N-BP drugs seriously compromise their full anticancer potential in non-skeletal tissues. They show poor membrane permeability and extreme affinity to bone, due mainly to their highly charged bisphosphonate moiety, which mimics the pyrophosphate of the substrates of hFPPS. Both the substrates and N-BPs bind to hFPPS via Mg ion-mediated interactions between their pyrophosphate/bisphosphonate moiety and two aspartate-rich surfaces of the enzyme's active site cavity. Recently, we took a structure-guided approach to develop bisphosphonates with higher lipophilicity for enhanced uptake into non-skeletal tissues. Surprisingly, some of the new compounds were found to bind to hFPPS even in the absence of Mg ions. Crystal structures of hFPPS in complex with a representative compound revealed that this bisphosphonate binds to the enzyme's active site in the presence of Mg ions, but also to a nearby allosteric inhibitory site in their absence. Furthermore, removal of a phosphonate group from the bisphosphonate moiety of this compound resulted in an inhibitor that binds exclusively to the allosteric site. Based on the crystal structures with these lead compounds, we generated of a novel class of non-bisphosphonate, allosteric inhibitors of hFPPS with superior physicochemical properties than those of the current N-BP drugs for broader tissue distribution.

scholarly journals Structures of the substrate-engaged 26S proteasome reveal the mechanisms for ATP hydrolysis-driven translocation

2018 ◽  
Author(s):  
Andres H. de la Peña ◽  
Ellen A. Goodall ◽  
Stephanie N. Gates ◽  
Gabriel C. Lander ◽  
Andreas Martin
Keyword(s):  
Conformational Changes ◽  
Atp Hydrolysis ◽  
26S Proteasome ◽  
Atp Binding ◽  
Conformational States ◽  
Phosphate Release ◽  
Protein Substrates ◽  
Cellular Processes ◽  
Hexameric Atpase ◽  
Central Pore

AbstractThe 26S proteasome is the primary eukaryotic degradation machine and thus critically involved in numerous cellular processes. The hetero-hexameric ATPase motor of the proteasome unfolds and translocates targeted protein substrates into the open gate of a proteolytic core, while a proteasomal deubiquitinase concomitantly removes substrate-attached ubiquitin chains. However, the mechanisms by which ATP hydrolysis drives the conformational changes responsible for these processes have remained elusive. Here we present the cryo-EM structures of four distinct conformational states of the actively ATP-hydrolyzing, substrate-engaged 26S proteasome. These structures reveal how mechanical substrate translocation accelerates deubiquitination, and how ATP-binding, hydrolysis, and phosphate-release events are coordinated within the AAA+ motor to induce conformational changes and propel the substrate through the central pore.

Why are reactions of 2- and 8-thioquinoline derivatives with iodine different?

2018 ◽  
Vol 74 (8) ◽  
pp. 974-980
Author(s):  
Boris V. Rudakov ◽  
Adam I. Stash ◽  
Gennady I. Makarov ◽  
Yury V. Matveychuk ◽  
Dmitry A. Zherebtsov ◽  
...  
Keyword(s):  
Comparative Analysis ◽  
Crystal Structures ◽  
Free Rotation ◽  
Conformational States ◽  
Relative Stabilities ◽  
Reaction Proceeding ◽  
Reaction Centres

The crystal structures of 1,2-dihydro-1,1′-bi[thiazolo[3,2-a]quinoline]-10a,10a′-diium diiodide hemihydrate, C22H16N2S2 2+·2I−·0.5H2O, and 1,2-dihydro-1,1′-bi[thiazolo[3,2-a]quinoline]-10a,10a′-diium iodide triiodide, C22H16N2S2 2+·I−·I3 −, obtained during the reaction of 1,4-bis(quinolin-2-ylsulfanyl)but-2-yne (2TQB) with iodine, have been determined at 120 K. The crystalline products contain the dication as a result of the reaction proceeding along the iodocyclization pathway. This is fundamentally different from the previously observed reaction of 1,4-bis(quinolin-8-ylsulfanyl)but-2-yne (8TQB) with iodine under similar conditions. A comparative analysis of the possible conformational states indicates differences in the relative stabilities and free rotation for the 2- and 8-thioquinoline derivatives which lead to a disparity in the convergence of the potential reaction centres for 2TQB and 8TQB.

Watching conformational dynamics of ABC transporters with single-molecule tools

2015 ◽  
Vol 43 (5) ◽  
pp. 1041-1047 ◽  
Author(s):  
Florence Husada ◽  
Giorgos Gouridis ◽  
Ruslan Vietrov ◽  
Gea K. Schuurman-Wolters ◽  
Evelyn Ploetz ◽  
...  
Keyword(s):  
Single Molecule ◽  
Abc Transporters ◽  
Fluorescent Dyes ◽  
Substrate Binding ◽  
Conformational Dynamics ◽  
Cell Volume Regulation ◽  
Size Exclusion ◽  
Conformational States ◽  
Functional Interpretation ◽  
Transport Cycle

ATP-binding cassette (ABC) transporters play crucial roles in cellular processes, such as nutrient uptake, drug resistance, cell-volume regulation and others. Despite their importance, all proposed molecular models for transport are based on indirect evidence, i.e. functional interpretation of static crystal structures and ensemble measurements of function and structure. Thus, classical biophysical and biochemical techniques do not readily visualize dynamic structural changes. We recently started to use single-molecule fluorescence techniques to study conformational states and changes of ABC transporters in vitro, in order to observe directly how the different steps during transport are coordinated. This review summarizes our scientific strategy and some of the key experimental advances that allowed the substrate-binding mechanism of prokaryotic ABC importers and the transport cycle to be explored. The conformational states and transitions of ABC-associated substrate-binding domains (SBDs) were visualized with single-molecule FRET, permitting a direct correlation of structural and kinetic information of SBDs. We also delineated the different steps of the transport cycle. Since information in such assays are restricted by proper labelling of proteins with fluorescent dyes, we present a simple approach to increase the amount of protein with FRET information based on non-specific interactions between a dye and the size-exclusion chromatography (SEC) column material used for final purification.

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