scholarly journals Asymmetry in catalysis by Thermotoga maritima membrane-bound pyrophosphatase demonstrated by a nonphosphorus allosteric inhibitor

2019 ◽  
Vol 5 (5) ◽  
pp. eaav7574 ◽  
Author(s):  
Keni Vidilaseris ◽  
Alexandros Kiriazis ◽  
Ainoleena Turku ◽  
Ayman Khattab ◽  
Niklas G. Johansson ◽  
...  
Keyword(s):  
Drug Target ◽  
Thermotoga Maritima ◽  
Thermophilic Bacterium ◽  
Integral Membrane Proteins ◽  
Sodium Ions ◽  
Homologous Proteins ◽  
Inhibitor Binding ◽  
Allosteric Inhibitor ◽  
Substrate Analog ◽  
Membrane Bound

Membrane-bound pyrophosphatases are homodimeric integral membrane proteins that hydrolyze pyrophosphate into orthophosphates, coupled to the active transport of protons or sodium ions across membranes. They are important in the life cycle of bacteria, archaea, plants, and parasitic protists, but no homologous proteins exist in vertebrates, making them a promising drug target. Here, we report the first nonphosphorus allosteric inhibitor of the thermophilic bacterium Thermotoga maritima membrane-bound pyrophosphatase and its bound structure together with the substrate analog imidodiphosphate. The unit cell contains two protein homodimers, each binding a single inhibitor dimer near the exit channel, creating a hydrophobic clamp that inhibits the movement of β-strand 1–2 during pumping, and thus prevents the hydrophobic gate from opening. This asymmetry of inhibitor binding with respect to each homodimer provides the first clear structural demonstration of asymmetry in the catalytic cycle of membrane-bound pyrophosphatases.

scholarly journals Asymmetry in catalysis by Thermotoga maritima membrane-bound pyrophosphatase demonstrated by a non-phosphorous allosteric inhibitor

2018 ◽  
Author(s):  
Keni Vidilaseris ◽  
Alexandros Kiriazis ◽  
Ainoleena Turku ◽  
Ayman Khattab ◽  
Niklas G. Johansson ◽  
...  
Keyword(s):  
Drug Target ◽  
Thermotoga Maritima ◽  
Thermophilic Bacterium ◽  
Integral Membrane Proteins ◽  
Sodium Ions ◽  
Exit Channel ◽  
Homologous Proteins ◽  
Inhibitor Binding ◽  
Allosteric Inhibitor ◽  
Membrane Bound

AbstractMembrane-bound pyrophosphatases are homodimeric integral membrane proteins that hydrolyse pyrophosphate into orthophosphates, coupled to the active transport of protons or sodium ions across membranes. They are important in the life cycle of bacteria, archaea, plants, and protist parasites, but no homologous proteins exist in vertebrates, making them a promising drug target. Here, we report the first non-phosphorous allosteric inhibitor (Ki of 1.8 ± 0.3 μM) of the thermophilic bacterium Thermotoga maritima membrane-bound pyrophosphatase and its bound structure at 3.7 Å resolution together with the substrate analogue imidodiphosphate. The unit cell contains two protein homodimers, each binding a single inhibitor dimer near the exit channel, creating a hydrophobic clamp that inhibits the movement of β-strand 1–2 during pumping, and thus preventing the hydrophobic gate from opening. This asymmetry of inhibitor binding with respect to each homodimer provide the first clear demonstration of asymmetry in the catalytic cycle of membrane-bound pyrophosphatases.

scholarly journals The Rnf complex is a Na+ coupled respiratory enzyme in a fermenting bacterium, Thermotoga maritima

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Martin Kuhns ◽  
Dragan Trifunović ◽  
Harald Huber ◽  
Volker Müller
Keyword(s):  
Electron Transport ◽  
Ion Transport ◽  
Cytoplasmic Membrane ◽  
Thermotoga Maritima ◽  
Physiological Role ◽  
Thermophilic Bacterium ◽  
Respiratory Enzyme ◽  
Membrane Bound ◽  
Reverse Electron Transport

Abstractrnf genes are widespread in bacteria and biochemical and genetic data are in line with the hypothesis that they encode a membrane-bound enzyme that oxidizes reduced ferredoxin and reduces NAD and vice versa, coupled to ion transport across the cytoplasmic membrane. The Rnf complex is of critical importance in many bacteria for energy conservation but also for reverse electron transport to drive ferredoxin reduction. However, the enzyme has never been purified and thus, ion transport could not be demonstrated yet. Here, we have purified the Rnf complex from the anaerobic, fermenting thermophilic bacterium Thermotoga maritima and show that is a primary Na+ pump. These studies provide the proof that the Rnf complex is indeed an ion (Na+) translocating, respiratory enzyme. Together with a Na+-F1FO ATP synthase it builds a simple, two-limb respiratory chain in T. maritima. The physiological role of electron transport phosphorylation in a fermenting bacterium is discussed.

scholarly journals A high-throughput method for orthophosphate determination of thermostable membrane-bound pyrophosphatase activity

2018 ◽  
Vol 10 (6) ◽  
pp. 646-651 ◽  
Author(s):  
Keni Vidilaseris ◽  
Juho Kellosalo ◽  
Adrian Goldman
Keyword(s):  
Membrane Proteins ◽  
Active Transport ◽  
High Throughput ◽  
Integral Membrane Proteins ◽  
Sodium Ions ◽  
High Throughput Method ◽  
Membrane Bound ◽  
Pyrophosphatase Activity

Membrane-bound pyrophosphatases (mPPases) are homodimeric integral membrane proteins that hydrolyse pyrophosphate into orthophosphates coupled to the active transport of protons or sodium ions across membranes.

Inhibitor Binding Sites in the Protein Tyrosine Phosphatase SHP-2

2020 ◽  
Vol 20 (11) ◽  
pp. 1017-1030
Author(s):  
Haonan Zhang ◽  
Zhengquan Gao ◽  
Chunxiao Meng ◽  
Xiangqian Li ◽  
Dayong Shi
Keyword(s):  
Small Molecule ◽  
Protein Tyrosine Phosphatase ◽  
Binding Sites ◽  
Drug Target ◽  
Tyrosine Phosphatase ◽  
Cancer Drug ◽  
Cellular Activity ◽  
Binding Modes ◽  
Inhibitor Binding ◽  
Protein Tyrosine

Protein tyrosine phosphatase 2 (SHP-2) has long been proposed as a cancer drug target. Several small-molecule compounds with different mechanisms of SHP-2 inhibition have been reported, but none are commercially available. Pool selectivity over protein tyrosine phosphatase 1 (SHP-1) and a lack of cellular activity have hindered the development of selective SHP-2 inhibitors. In this review, we describe the binding modes of existing inhibitors and SHP-2 binding sites, summarize the characteristics of the sites involved in selectivity, and identify the suitable groups for interaction with the binding sites.

scholarly journals Enhanced Co2+ activation and inhibitor binding of carboxypeptidase M at low pH. Similarity to carboxypeptidase H (enkephalin convertase)

1989 ◽  
Vol 261 (1) ◽  
pp. 289-291 ◽  
Author(s):  
P A Deddish ◽  
R A Skidgel ◽  
E G Erdös
Keyword(s):  
Plasma Membrane ◽  
Low Ph ◽  
Co2 Activation ◽  
Inhibitor Binding ◽  
Carboxypeptidase B ◽  
Acid Ph ◽  
Carboxypeptidase H ◽  
Membrane Bound ◽  
Carboxypeptidase N ◽  
Carboxypeptidase M

Carboxypeptidases H and M differ in their distribution and other properties, but both are activated by Co2+ and inhibited by guanidinoethylmercaptosuccinic acid. The higher degree of activation or inhibition of carboxypeptidase H by these agents at acid pH has been employed to identify this enzyme in tissues. We found that the activation or inhibition of both purified and plasma-membrane-bound human carboxy-peptidase M depends on the pH of the medium. CoCl2 activated over 6-fold at pH 5.5, but less than 2-fold at pH 7.5. Guanidinoethylmercaptosuccinic acid inhibited the membrane-bound carboxypeptidase M more effectively than the purified enzyme, and the IC50 was about 25-30 times lower at pH 5.5. As purified human plasma carboxypeptidase N and pancreatic carboxypeptidase B were also activated more at pH 5.5, we conclude that the increased activation by CoCl2 is due to the enhanced dissociation of Zn2+ below the pKa of the ligands that co-ordinate the cofactor in the protein. Thus increased activation or inhibition at acid pH would not differentiate basic carboxypeptidases.

The alternative oxidases: simple oxidoreductase proteins with complex functions

2013 ◽  
Vol 41 (5) ◽  
pp. 1305-1311 ◽  
Author(s):  
Luke Young ◽  
Tomoo Shiba ◽  
Shigeharu Harada ◽  
Kiyoshi Kita ◽  
Mary S. Albury ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Electron Transport ◽  
Alternative Oxidase ◽  
Structural Features ◽  
Transport Proteins ◽  
Integral Membrane Proteins ◽  
Complex Functions ◽  
Membrane Bound ◽  
Iron Carboxylate ◽  
Electron Transport Proteins

The alternative oxidases are membrane-bound monotopic terminal electron transport proteins found in all plants and in some agrochemically important fungi and parasites including Trypansoma brucei, which is the causative agent of trypanosomiasis. They are integral membrane proteins and reduce oxygen to water in a four electron process. The recent elucidation of the crystal structure of the trypanosomal alternative oxidase at 2.85 Å (1 Å=0.1 nm) has revealed salient structural features necessary for its function. In the present review we compare the primary and secondary ligation spheres of the alternative oxidases with other di-iron carboxylate proteins and propose a mechanism for the reduction of oxygen to water.

scholarly journals Screening for Thermotoga maritima Membrane-Bound Pyrophosphatase Inhibitors

10.3791/60619 ◽  
2019 ◽  
Author(s):  
Keni Vidilaseris ◽  
Niklas G. Johansson ◽  
Ainoleena Turku ◽  
Alexandros Kiriazis ◽  
Gustav Boije af Gennäs ◽  
...  
Keyword(s):  
Thermotoga Maritima ◽  
Membrane Bound

The structure ofAquifex aeolicusFtsH in the ADP-bound state reveals aC2-symmetric hexamer

2015 ◽  
Vol 71 (6) ◽  
pp. 1307-1318 ◽  
Author(s):  
Marina Vostrukhina ◽  
Alexander Popov ◽  
Elena Brunstein ◽  
Martin A. Lanz ◽  
Renato Baumgartner ◽  
...  
Keyword(s):  
Active Site ◽  
Thermotoga Maritima ◽  
Adenosine Diphosphate ◽  
Crystal Form ◽  
Bound State ◽  
Aquifex Aeolicus ◽  
Protein Activity ◽  
Quadruple Mutant ◽  
Membrane Bound ◽  
Open Issues

The crystal structure of a truncated, soluble quadruple mutant of FtsH fromAquifex aeolicuscomprising the AAA and protease domains has been determined at 2.96 Å resolution in space groupI222. The protein crystallizes as a hexamer, with the protease domain forming layers in theabplane. Contacts between these layers are mediated by the AAA domains. These are highly disordered in one crystal form, but are clearly visible in a related form with a shortercaxis. Here, adenosine diphosphate (ADP) is bound to each subunit and the AAA ring exhibits twofold symmetry. The arrangement is different from the ADP-bound state of an analogously truncated, soluble FtsH construct fromThermotoga maritima. The pore is completely closed and the phenylalanine residues in the pore line a contiguous path. The protease hexamer is very similar to those described for other FtsH structures. To resolve certain open issues regarding a conserved glycine in the linker between the AAA and protease domains, as well as the active-site switch β-strand, mutations have been introduced in the full-length membrane-bound protein. Activity analysis of these point mutants reveals the crucial importance of these residues for proteolytic activity and is in accord with previous interpretation of the active-site switch and the importance of the linker glycine residue.

Sign in / Sign up

Export Citation Format

Share Document