scholarly journals Cryo-EM structures of Escherichia coli cytochrome bo3 reveal bound phospholipids and ubiquinone-8 in a dynamic substrate binding site

2021 ◽  
Vol 118 (34) ◽  
pp. e2106750118 ◽  
Author(s):  
Jiao Li ◽  
Long Han ◽  
Francesca Vallese ◽  
Ziqiao Ding ◽  
Sylvia K. Choi ◽  
...  
Keyword(s):  
Hydrogen Bond ◽  
Binding Site ◽  
Substrate Binding ◽  
Transmembrane Helix ◽  
Proton Pumping ◽  
Side Chain ◽  
Substrate Binding Site ◽  
Cytochrome Bo3 ◽  
Redox Active ◽  
Quinol Oxidases

Two independent structures of the proton-pumping, respiratory cytochrome bo3 ubiquinol oxidase (cyt bo3) have been determined by cryogenic electron microscopy (cryo-EM) in styrene–maleic acid (SMA) copolymer nanodiscs and in membrane scaffold protein (MSP) nanodiscs to 2.55- and 2.19-Å resolution, respectively. The structures include the metal redox centers (heme b, heme o3, and CuB), the redox-active cross-linked histidine–tyrosine cofactor, and the internal water molecules in the proton-conducting D channel. Each structure also contains one equivalent of ubiquinone-8 (UQ8) in the substrate binding site as well as several phospholipid molecules. The isoprene side chain of UQ8 is clamped within a hydrophobic groove in subunit I by transmembrane helix TM0, which is only present in quinol oxidases and not in the closely related cytochrome c oxidases. Both structures show carbonyl O1 of the UQ8 headgroup hydrogen bonded to D75I and R71I. In both structures, residue H98I occupies two conformations. In conformation 1, H98I forms a hydrogen bond with carbonyl O4 of the UQ8 headgroup, but in conformation 2, the imidazole side chain of H98I has flipped to form a hydrogen bond with E14I at the N-terminal end of TM0. We propose that H98I dynamics facilitate proton transfer from ubiquinol to the periplasmic aqueous phase during oxidation of the substrate. Computational studies show that TM0 creates a channel, allowing access of water to the ubiquinol headgroup and to H98I.

scholarly journals Crystal Structure of Tetrameric Homoisocitrate Dehydrogenase from an Extreme Thermophile, Thermus thermophilus: Involvement of Hydrophobic Dimer-Dimer Interaction in Extremely High Thermotolerance

2005 ◽  
Vol 187 (19) ◽  
pp. 6779-6788 ◽  
Author(s):  
Junichi Miyazaki ◽  
Kuniko Asada ◽  
Shinya Fushinobu ◽  
Tomohisa Kuzuyama ◽  
Makoto Nishiyama
Keyword(s):  
Crystal Structure ◽  
Binding Site ◽  
Thermal Inactivation ◽  
Hydrophobic Interactions ◽  
Substrate Binding ◽  
Thermus Thermophilus ◽  
Lysine Biosynthesis ◽  
Side Chain ◽  
Substrate Binding Site ◽  
Tetramer Formation

ABSTRACT The crystal structure of homoisocitrate dehydrogenase involved in lysine biosynthesis from Thermus thermophilus (TtHICDH) was determined at 1.85-Å resolution. Arg85, which was shown to be a determinant for substrate specificity in our previous study, is positioned close to the putative substrate binding site and interacts with Glu122. Glu122 is highly conserved in the equivalent position in the primary sequence of ICDH and archaeal 3-isopropylmalate dehydrogenase (IPMDH) but interacts with main- and side-chain atoms in the same domain in those paralogs. In addition, a conserved Tyr residue (Tyr125 in TtHICDH) which extends its side chain toward a substrate and thus has a catalytic function in the related β-decarboxylating dehydrogenases, is flipped out of the substrate-binding site. These results suggest the possibility that the conformation of the region containing Glu122-Tyr125 is changed upon substrate binding in TtHICDH. The crystal structure of TtHICDH also reveals that the arm region is involved in tetramer formation via hydrophobic interactions and might be responsible for the high thermotolerance. Mutation of Val135, located in the dimer-dimer interface and involved in the hydrophobic interaction, to Met alters the enzyme to a dimer (probably due to steric perturbation) and markedly decreases the thermal inactivation temperature. Both the crystal structure and the mutation analysis indicate that tetramer formation is involved in the extremely high thermotolerance of TtHICDH.

scholarly journals Structure of the cytochrome aa3-600 heme-copper menaquinol oxidase bound to inhibitor HQNO shows TM0 is part of the quinol binding site

2019 ◽  
Vol 117 (2) ◽  
pp. 872-876 ◽  
Author(s):  
Jingjing Xu ◽  
Ziqiao Ding ◽  
Bing Liu ◽  
Sophia M. Yi ◽  
Jiao Li ◽  
...  
Keyword(s):  
Binding Site ◽  
Transmembrane Helix ◽  
Proton Pumping ◽  
Spectroscopic Methods ◽  
Amino Acid Residues ◽  
X Ray ◽  
Membrane Bound ◽  
Quinol Binding ◽  
Quinol Oxidases ◽  
Single Binding Site

Virtually all proton-pumping terminal respiratory oxygen reductases are members of the heme-copper oxidoreductase superfamily. Most of these enzymes use reduced cytochrome c as a source of electrons, but a group of enzymes have evolved to directly oxidize membrane-bound quinols, usually menaquinol or ubiquinol. All of the quinol oxidases have an additional transmembrane helix (TM0) in subunit I that is not present in the related cytochrome c oxidases. The current work reports the 3.6-Å-resolution X-ray structure of the cytochrome aa3-600 menaquinol oxidase from Bacillus subtilis containing 1 equivalent of menaquinone. The structure shows that TM0 forms part of a cleft to accommodate the menaquinol-7 substrate. Crystals which have been soaked with the quinol-analog inhibitor HQNO (N-oxo-2-heptyl-4-hydroxyquinoline) or 3-iodo-HQNO reveal a single binding site where the inhibitor forms hydrogen bonds to amino acid residues shown previously by spectroscopic methods to interact with the semiquinone state of menaquinone, a catalytic intermediate.

scholarly journals X-ray crystallographic analysis of a MATE multidrug transporter from V. cholerae

2014 ◽  
Vol 70 (a1) ◽  
pp. C709-C709
Author(s):  
Tsukasa Kusakizako ◽  
Yoshiki Tanaka ◽  
Andrés Maturana ◽  
Christopher Hipolito ◽  
Teruo Kuroda ◽  
...  
Keyword(s):  
High Resolution ◽  
Binding Site ◽  
Crystal Structures ◽  
Multiple Drug Resistance ◽  
Substrate Binding ◽  
Transmembrane Helix ◽  
Common Mechanism ◽  
Multiple Drug ◽  
Coupling Mechanism ◽  
Substrate Binding Site

MATE (Multidrug And Toxic compound Extrusion) family transporters are highly conserved from Bacteria to Eukarya including human, and export a broad range of xenobiotics using either a proton or a sodium ion gradient across the membrane. Especially in bacterial pathogens, MATE transporters contribute to their multiple drug resistance (MDR). To understand how MATE transporters export various substrates such as drugs and thus how pathogens acquire MDR, structural analyses are essential. The crystal structures of several MATE transporters from pathogens have been reported. However, because of the limited resolution and the lack of drug-MATE transporters complex structures, the recognition mechanism of various substrates and the coupling mechanism of the cation influx and the drug efflux have been poorly understood. Although the high-resolution structures of MATE transporters from non-pathogenic archaeal P. furiosus (PfMATE) have been reported, PfMATE shares low sequence identity with MATE transporters from pathogens such as V. cholerae. Therefore, further findings of the structural mechanism of MDR caused by MATE transporters from pathogens have been needed. To understand the substrate recognition and transport mechanism of MATE transporters from pathogens, we determined the crystal structures of one of MATE transporters from V. cholerae (VcMATE) at 2.5-2.7 Å resolutions using in meso crystallization method. The high-resolution structures of VcMATE show two distinct conformations, as observed in the structures of PfMATE, and reveal the large movement of transmembrane helix 1 and the putative substrate-binding site. The structures suggest that the bending of transmembrane helix 1 and the sequential collapse of the putative substrate-binding site induce the release of the bound substrate. This conformational change during the substrate transport may be a common mechanism among MATE transporters from pathogens to non-pathogens.

Comprehensive in silico Study of GLUT10: Prediction of Possible Substrate Binding Sites and Interacting Molecules

2020 ◽  
Vol 21 (2) ◽  
pp. 117-130 ◽  
Author(s):  
Mohammad J. Hosen ◽  
Mahmudul Hasan ◽  
Sourav Chakraborty ◽  
Ruhshan A. Abir ◽  
Abdullah Zubaer ◽  
...  
Keyword(s):  
Virtual Screening ◽  
Binding Site ◽  
Glucose Transporter ◽  
Substrate Binding ◽  
Mutation Screening ◽  
Homology Model ◽  
Connective Tissue Disorder ◽  
Crystallographic Structure ◽  
Substrate Binding Site

Objectives: The Arterial Tortuosity Syndrome (ATS) is an autosomal recessive connective tissue disorder, mainly characterized by tortuosity and stenosis of the arteries with a propensity towards aneurysm formation and dissection. It is caused by mutations in the SLC2A10 gene that encodes the facilitative glucose transporter GLUT10. The molecules transported by and interacting with GLUT10 have still not been unambiguously identified. Hence, the study attempts to identify both the substrate binding site of GLUT10 and the molecules interacting with this site. Methods: As High-resolution X-ray crystallographic structure of GLUT10 was not available, 3D homology model of GLUT10 in open conformation was constructed. Further, molecular docking and bioinformatics investigation were employed. Results and Discussion: Blind docking of nine reported potential in vitro substrates with this 3D homology model revealed that substrate binding site is possibly made with PRO531, GLU507, GLU437, TRP432, ALA506, LEU519, LEU505, LEU433, GLN525, GLN510, LYS372, LYS373, SER520, SER124, SER533, SER504, SER436 amino acid residues. Virtual screening of all metabolites from the Human Serum Metabolome Database and muscle metabolites from Human Metabolite Database (HMDB) against the GLUT10 revealed possible substrates and interacting molecules for GLUT10, which were found to be involved directly or partially in ATS progression or different arterial disorders. Reported mutation screening revealed that a highly emergent point mutation (c. 1309G>A, p. Glu437Lys) is located in the predicted substrate binding site region. Conclusion: Virtual screening expands the possibility to explore more compounds that can interact with GLUT10 and may aid in understanding the mechanisms leading to ATS.

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