scholarly journals Function of the Vibrio PomB plug region based on the stator rotation model for bacterial flagellar motor

2021 ◽  
Author(s):  
Michio Homma ◽  
Hiroyuki Terashima ◽  
Hiroaki Koiwa ◽  
Seiji Kojima
Keyword(s):  
Cross Linking ◽  
Ion Flow ◽  
Bacterial Flagella ◽  
B Subunit ◽  
Loop Region ◽  
Flagellar Motors ◽  
Flagellar Filament ◽  
A Subunit ◽  
Biological World ◽  
Protein Motor

AbstractBacterial flagella are the only real rotational motor organs in the biological world. The spiral-shaped flagellar filaments that extend from the cell surface rotate like a screw to create a propulsive force. The base of the flagellar filament has a protein motor consisting of a stator and a rotor embedded in the membrane. The motor part has stators composed of two types of membrane subunits, PomA(MotA) and PomB(MotB), which are energy converters coupled to the ion flow that assemble around the rotor. Recently, structures of the stator, in which two molecules of MotB stuck in the center of the MotA ring made of five molecules, were reported and a model in which the MotA ring rotates with respect to MotB, which is coupled to the influx of ions, was proposed. We focused on the Vibrio PomB plug region, which has been reported to control the activation of flagellar motors. We searched for the plug region, which is the interacting region, through site-directed photo-cross-linking and disulfide cross-linking experiments. Our results demonstrated that it interacts with the extracellular short loop region of PomA, which is between transmembrane 3 and 4. Although the motor halted following cross-linking, its function was recovered with a reducing reagent that disrupted the disulfide bond. Our results support the hypothesis, which has been inferred from the stator structure, that the plug region terminates the ion inflow by stopping the rotation of the rotor.ImportanceThe flagellar biological motor resembles a mechanical motor, which is composed of stator and rotor and where the rotational force is transmitted by gear-like movements. We hypothesized that the flagellar the rotation of stator that the pentamer of A subunits revolves around the axis of the B subunit dimer with ion flow. The plug region of the B subunit has been shown to regulate the ion flow. Herein, we demonstrated that the ion flow was terminated by the crosslinking between the plug region and the A subunit. These finding support the rotation hypothesis and explain the role of the plug region in terminating the rotation.

scholarly journals Putative spanner function of the Vibrio PomB plug region in the stator rotation model for flagellar motor

2021 ◽  
Author(s):  
Michio Homma ◽  
Hiroyuki Terashima ◽  
Hiroaki Koiwa ◽  
Seiji Kojima
Keyword(s):  
Flagellar Motor ◽  
Disulfide Crosslinking ◽  
Ion Flow ◽  
Bacterial Flagella ◽  
B Subunit ◽  
Loop Region ◽  
Flagellar Filament ◽  
Biological World ◽  
Ion Influx ◽  
Protein Motor

Bacterial flagella are the best-known rotational organelles in the biological world. The spiral-shaped flagellar filaments that extending from the cell surface rotate like a screw to create a propulsive force. At the base of the flagellar filament lies a protein motor that consists of a stator and a rotor embedded in the membrane. The stator is composed of two types of membrane subunits, PomA(MotA) and PomB(MotB), which are energy converters that assemble around the rotor to couple rotation with the ion flow. Recently, stator structures, where two MotB molecules are inserted into the center of a ring made of five MotA molecules, were reported. This structure inspired a model in which the MotA ring rotates around the MotB dimer in response to ion influx. Here, we focus on the Vibrio PomB plug region, which is involved in flagellar motor activation. We investigated the plug region using site-directed photo-crosslinking and disulfide crosslinking experiments. Our results demonstrated that the plug interacts with the extracellular short loop region of PomA, which is located between transmembrane helices 3 and 4. Although the motor stopped rotating after crosslinking, its function recovered after treatment with a reducing reagent that disrupted the disulfide bond. Our results support the hypothesis, which has been inferred from the stator structure, that the plug region terminates the ion influx by blocking the rotation of the rotor as a spanner. Importance The biological flagellar motor resembles a mechanical motor. It is composed of a stator and a rotor. The force is transmitted to the rotor by the gear-like stator movements. It has been proposed that the pentamer of MotA subunits revolves around the axis of the B subunit dimer in response to ion flow. The plug region of the B subunit regulates the ion flow. Here, we demonstrated that the ion flow was terminated by crosslinking the plug region of PomB with PomA. These findings support the rotation hypothesis and explain the role of the plug region in blocking the rotation of the stator unit.

Platelet Calmodulin Mediates The Calcium Dependent Activation Of Factor XIII

1981 ◽  
Author(s):  
D Kahn ◽  
N Crawford ◽  
I Cohen
Keyword(s):  
Factor Xiii ◽  
Sulfhydryl Group ◽  
Cross Linking ◽  
Platelet Factor ◽  
Subunit A ◽  
Calcium Dependent ◽  
B Subunit ◽  
A Subunit ◽  
Subunit B

Transglutaminases are ubiquitous in cells and require calcium for their activation. The factor XIII zymogen, present in plasma and in the platelet cytosol, requires for its activation both a limited proteolytic activity on the catalytic subunit,“a”, and, in the use of the plasma enzyme, calcium for dissociating subunit“a” from the carrier subunit“b”. Calcium is also necessary for exposing the reactive sulfhydryl group. We have recently suggested a role for the platelet factor XIII in the generation of calcium-dependent cross-linking processes in platelets. Since calmodulin is present in considerable amounts in the platelet cytosol and is known to regulate the activity of various calcium-dependent enzymes and cellular reactions, we have investigated its possible role in factor XIII activation. Since the“a” subunit of platelet factor XIII is identical to its plasma counterpart, the more easily purified plasma zymogen was used. The effect of calmodulin on the two calcium-dependent steps of factor XIII activation was investigated following thrombin-stimulated hydrolysis of the“a” subunit. Platelet calmodulin was found to enhance by at least 3 fold the calcium-dependent unmasking of the reactive sulfhydryl groups which were titrated with 14C-iodoacetamide. Calmodulin also enhanced by at least 4 fold the calcium-dependent dissociation of the b subunit from its complex with the thrombin-hydrolyzed“a” subunit. The calmodulin mediation of the calcium-dependent steps of factor XIII activation may be important for regulating the factor XIII-dependent cross-linking reactions in platelets and is reminiscent of the calcium-related regulatory role of fibrinogen on factor XIII activation which could prevail in plasma. An investigation of the possible role of calmodulin on other tissue transglutaminases is warranted.

Abstract 1653: The Role of the Fibrinogen Alpha Chain in Controlling the Rate of Factor XIII Activation: Implications for Coronary Artery Thrombus Formation

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Kerrie A Smith ◽  
Penelope J Adamson ◽  
Robert A Ariens ◽  
Helen Philippou ◽  
Peter J Grant
Keyword(s):  
Factor Xiii ◽  
Plaque Rupture ◽  
Thrombus Formation ◽  
Coagulation Factor ◽  
Cross Linking ◽  
Active Site Cysteine ◽  
B Subunit ◽  
Thrombin Cleavage ◽  
A Subunit ◽  
Α Chain

Background: The development of an obstructive arterial thrombus is dependent on the generation of a platelet rich fibrin mesh secondary to plaque rupture. Fibrin itself is formed by the cleavage of fibrinogen by thrombin with subsequent fibrin cross-linking by activated Factor XIII. Critical to these processes, fibrinogen α-chain residues 242– 424 have a major regulatory role in the dissociation of coagulation Factor XIII A subunit (FXIIIA) from the Factor XIII B subunit (FXIIIB), however the mechanisms for this enhancing effect have not been determined. Methods: α-Chain residues 242– 424 were expressed as a GST-fusion protein in E .coli, along with six further truncations; αC fragments α242– 402, α242–387, α242–374, α242–341, α242–289 and α242–265. FXIII-A and an inactive double thrombin cleavage mutant (R37A/K513A) were also expressed as GST-fusion proteins in E . coli . Results: Using surface plasmon resonance (SPR) and ELISA we can confirm the presence of at least two binding sites on the αC for activated FXIII-A within residues α388 – 424 and α242–265 with an overall KD of 4.9 ± 2.29 μM. This interaction was specific for activated FXIII-A, as the inactive FXIII-A double thrombin cleavage mutant did not bind to the αC as indicated by SPR and ELISA. We demonstrated that the binding of the αC is dependent on a conformational change which occurs during activation of FXIII-A with calcium and thrombin. FXIII-A activated with thrombin alone did not bind αC as determined by SPR and ELISA. Competition studies showed that the αC competitively inhibits binding with a K I of 1.2 μM confirming the specificity of this interaction. Iodoacetamide blocking of FXIII-A active site cysteine did not prevent binding of the αC suggesting this interaction is independent of FXIII-A cross-linking. Furthermore we have identified a novel interaction between FXIIIB and α-chain by ELISA. Utilising the αC fragments we have localised two binding regions between α290–341 and α242–265 with a K D of 123nM ± 11 and 286nM ± 24.7 respectively. Conclusions: The binding of both FXIII A- and B-subunits to this area of the fibrinogen α-chain suggests a crucial role in controlling the amount of activated FXIII generated for clot stabilisation and presents a potential target for therapeutic intervention.

Changed properties of the A subunit in DNA gyrase with a B subunit mutation

1982 ◽  
Vol 186 (4) ◽  
pp. 572-574 ◽  
Author(s):  
E. S. Bogdanova ◽  
S. M. Mirkin ◽  
Zh. G. Shmerling
Keyword(s):  
Dna Gyrase ◽  
B Subunit ◽  
A Subunit

Control of protein phosphatase 2A by simian virus 40 small-t antigen

1991 ◽  
Vol 11 (4) ◽  
pp. 1988-1995
Author(s):  
S I Yang ◽  
R L Lickteig ◽  
R Estes ◽  
K Rundell ◽  
G Walter ◽  
...  
Keyword(s):  
Protein Phosphatase ◽  
Simian Virus 40 ◽  
Histone H1 ◽  
Simian Virus ◽  
T Antigen ◽  
Light Chains ◽  
B Subunit ◽  
Phosphatase 2A ◽  
A Subunit ◽  
Small T Antigen

Soluble, monomeric simian virus 40 (SV40) small-t antigen (small-t) was purified from bacteria and assayed for its ability to form complexes with protein phosphatase 2A (PP2A) and to modify its catalytic activity. Different forms of purified PP2A, composed of combinations of regulatory subunits (A and B) with a common catalytic subunit (C), were used. The forms used included free A and C subunits and AC and ABC complexes. Small-t associated with both the free A subunit and the AC form of PP2A, resulting in a shift in mobility during nondenaturing polyacrylamide gel electrophoresis. Small-t did not interact with the free C subunit or the ABC form. These data demonstrate that the primary interaction is between small-t and the A subunit and that the B subunit of PP2A blocks interaction of small-t with the AC form. The effect of small-t on phosphatase activity was determined by using several exogenous substrates, including myosin light chains phosphorylated by myosin light-chain kinase, myelin basic protein phosphorylated by microtubule-associated protein 2 kinase/ERK1, and histone H1 phosphorylated by protein kinase C. With the exception of histone H1, small-t inhibited the dephosphorylation of these substrates by the AC complex. With histone H1, a small stimulation of dephosphorylation by AC was observed. Small-t had no effect on the activities of free C or the ABC complex. A maximum of 50 to 75% inhibition was obtained, with half-maximal inhibition occurring at 10 to 20 nM small-t. The specific activity of the small-t/AC complex was similar to that of the ABC form of PP2A with myosin light chains or histone H1 as the substrate. These results suggested that small-t and the B subunit have similar qualitative and quantitative effects on PP2A enzyme activity. These data show that SV40 small-antigen binds to purified PP2A in vitro, through interaction with the A subunit, and that this interaction inhibits enzyme activity.

scholarly journals Identification of Potential Novel Interacting Partners for Coagulation Factor XIII B (FXIII-B) Subunit, a Protein Associated with a Rare Bleeding Disorder

2019 ◽  
Vol 20 (11) ◽  
pp. 2682 ◽  
Author(s):  
Sneha Singh ◽  
Mohammad Suhail Akhter ◽  
Johannes Dodt ◽  
Peter Volkers ◽  
Andreas Reuter ◽  
...  
Keyword(s):  
Factor Xiii ◽  
Regulatory Protein ◽  
Coagulation Factor ◽  
Factor H ◽  
Complement C3 ◽  
Complement Factor ◽  
B Subunit ◽  
Coagulation Factor Xiii ◽  
A Subunit ◽  
Complement C1q

Coagulation factor XIII (FXIII) is a plasma-circulating heterotetrameric pro-transglutaminase complex that is composed of two catalytic FXIII-A and two protective/regulatory FXIII-B subunits. FXIII acts by forming covalent cross-links within a preformed fibrin clots to prevent its premature fibrinolysis. The FXIII-A subunit is known to have pleiotropic roles outside coagulation, but the FXIII-B subunit is a relatively unexplored entity, both structurally as well as functionally. Its discovered roles so far are limited to that of the carrier/regulatory protein of its partner FXIII-A subunit. In the present study, we have explored the co-presence of protein excipients in commercial FXIII plasma concentrate FibrogamminP by combination of protein purification and mass spectrometry-based verification. Complement factor H was one of the co-excipients observed in this analysis. This was followed by performing pull down assays from plasma in order to detect the putative novel interacting partners for the FXIII-B subunit. Complement system proteins, like complement C3 and complement C1q, were amongst the proteins that were pulled down. The only protein that was observed in both experimental set ups was alpha-2-macroglobulin, which might therefore be a putative interacting partner of the FXIII/FXIII-B subunit. Future functional investigations will be needed to understand the physiological significance of this association.

scholarly journals Protection against Lethal Leptospirosis after Vaccination with LipL32 Coupled or Coadministered with the B Subunit of Escherichia coli Heat-Labile Enterotoxin

2012 ◽  
Vol 19 (5) ◽  
pp. 740-745 ◽  
Author(s):  
André A. Grassmann ◽  
Samuel R. Félix ◽  
Carolina Ximendes dos Santos ◽  
Marta G. Amaral ◽  
Amilton C. P. Seixas Neto ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Immune Response ◽  
Subunit Vaccine ◽  
Lethal Dose ◽  
Control Group ◽  
Hamster Model ◽  
Content Type ◽  
B Subunit ◽  
Heat Labile ◽  
A Subunit

ABSTRACTLeptospirosis, a worldwide zoonosis, lacks an effective, safe, and cross-protective vaccine. LipL32, the most abundant, immunogenic, and conserved surface lipoprotein present in all pathogenic species ofLeptospira, is a promising antigen candidate for a recombinant vaccine. However, several studies have reported a lack of protection when this protein is used as a subunit vaccine. In an attempt to enhance the immune response, we used LipL32 coupled to or coadministered with the B subunit of theEscherichia coliheat-labile enterotoxin (LTB) in a hamster model of leptospirosis. After homologous challenge with 5× the 50% lethal dose (LD50) ofLeptospira interrogans, animals vaccinated with LipL32 coadministered with LTB and LTB::LipL32 had significantly higher survival rates (P< 0.05) than animals from the control group. This is the first report of a protective immune response afforded by a subunit vaccine using LipL32 and represents an important contribution toward the development of improved leptospirosis vaccines.

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