scholarly journals Regulatory coiled-coil domains promote head-to-head assemblies of AAA+ chaperones essential for tunable activity control

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Marta Carroni ◽  
Kamila B Franke ◽  
Michael Maurer ◽  
Jasmin Jäger ◽  
Ingo Hantke ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Stress Resistance ◽  
Ground State ◽  
Resting State ◽  
Coiled Coil ◽  
Ring Structure ◽  
Cellular Toxicity ◽  
Constitutive Activation ◽  
Interaction Sites ◽  
Bacterial Protease

Ring-forming AAA+ chaperones exert ATP-fueled substrate unfolding by threading through a central pore. This activity is potentially harmful requiring mechanisms for tight repression and substrate-specific activation. The AAA+ chaperone ClpC with the peptidase ClpP forms a bacterial protease essential to virulence and stress resistance. The adaptor MecA activates ClpC by targeting substrates and stimulating ClpC ATPase activity. We show how ClpC is repressed in its ground state by determining ClpC cryo-EM structures with and without MecA. ClpC forms large two-helical assemblies that associate via head-to-head contacts between coiled-coil middle domains (MDs). MecA converts this resting state to an active planar ring structure by binding to MD interaction sites. Loss of ClpC repression in MD mutants causes constitutive activation and severe cellular toxicity. These findings unravel an unexpected regulatory concept executed by coiled-coil MDs to tightly control AAA+ chaperone activity.

scholarly journals Regulatory coiled-coil domains promote head-to-head assemblies of AAA+ chaperones essential for tunable activity control

2017 ◽  
Author(s):  
Marta Carroni ◽  
Kamila B. Franke ◽  
Michael Maurer ◽  
Jasmin Jäger ◽  
Ingo Hantke ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Stress Resistance ◽  
Ground State ◽  
Resting State ◽  
Coiled Coil ◽  
Ring Structure ◽  
Cellular Toxicity ◽  
Constitutive Activation ◽  
Interaction Sites ◽  
Bacterial Protease

AbstractRing-forming AAA+ chaperones exert ATP-fueled substrate unfolding by threading through a central pore. This activity is potentially harmful requiring mechanisms for tight repression and substrate-specific activation. The AAA+ chaperone ClpC with the peptidase ClpP forms a bacterial protease essential to virulence and stress resistance. The adaptor MecA activates ClpC by targeting substrates and stimulating ClpC ATPase activity. We show how ClpC is repressed in its ground state by determining ClpC cryo-EM structures with and without MecA. ClpC forms large two-helical assemblies that associate via head-to-head contacts between coiled-coil middle domains (MDs). MecA converts this resting state to an active planar ring structure by binding to MD interaction sites. Loss of ClpC repression in MD mutants causes constitutive activation and severe cellular toxicity. These findings unravel an unexpected regulatory concept executed by coiled-coil MDs to tightly control AAA+ chaperone activity.

scholarly journals Location of smooth-muscle myosin and tropomyosin binding sites in the C-terminal 288 residues of human caldesmon

1995 ◽  
Vol 312 (2) ◽  
pp. 617-625 ◽  
Author(s):  
P A J Huber ◽  
I D C Fraser ◽  
S B Marston
Keyword(s):  
Smooth Muscle ◽  
Coiled Coil ◽  
Protein Fragments ◽  
Chicken Gizzard ◽  
Interaction Sites ◽  
Extended Region ◽  
Muscle Tropomyosin ◽  
Ionic Nature ◽  
Domain 3 ◽  
Recombinant Fragments

We have produced nine recombinant fragments, H1 to H9, from a human cDNA that codes for the C-terminal 288 residues of caldesmon. The fragment H1, encompassing the 288 residues, is equivalent to domains 3 and 4 of caldesmon (amino acids 506-793 in human, 476-737 in the chicken gizzard sequence). It has been shown [Huber, Redwood, Avent, Tanner and Marston (1993) J. Muscle Res. Cell Motil. 14, 385-391] to bind to actin, Ca(2+)-calmodulin, tropomyosin and myosin. The fragments, H2 to H9, differ in length between 60 and 176 residues and cover the whole of domains 3 and 4 with many of the fragments overlapping. We have characterized the myosin and tropomyosin binding of these fragments. The binding of both tropomyosin and myosin is highly dependent on salt concentration, indicating the ionic nature of these interactions. The location of the myosin binding is an extended region encompassing the junction of domains 3/4 and domain 4a (residues 622-714, human; 566-657, chicken gizzard). Tropomyosin binds in a smaller region within domain 4a of caldesmon (residues 663-714, human; 606-657 chicken gizzard). We confirmed predictions based on sequence similarities of a tropomyosin binding site in domain 3 of caldesmon; however, this site bound to skeletal-muscle tropomyosin and had little affinity for the smooth-muscle tropomyosin isoform. None of the protein fragments H2-H9 retained the affinity of the parent fragment H1 for either myosin or tropomyosin. This indicates the need for several interaction sites scattered over an extended region to attain higher affinity. The regions interacting with caldesmon in both tropomyosin and myosin are coiled-coil structures. This is probably the reason for their shared interaction sites on caldesmon and their similar natures of binding.

scholarly journals Structure-activity relationships of retinoids as inhibitors of calmodulin-dependent human erythrocyte Ca2+-ATPase activity and calmodulin binding to membranes

1991 ◽  
Vol 277 (3) ◽  
pp. 603-606 ◽  
Author(s):  
F B Davis ◽  
T J Smith ◽  
P J Davis ◽  
S D Blas
Keyword(s):  
Enzyme Activity ◽  
Retinoic Acid ◽  
Atpase Activity ◽  
Human Erythrocyte ◽  
Enzyme Inhibitors ◽  
Ring Structure ◽  
Erythrocyte Membranes ◽  
Calmodulin Binding ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid

All-trans retinoic acid displaces the binding of radiolabelled calmodulin to human erythrocyte membranes, and inhibits the activity of plasma membrane Ca(2+)-stimulated, Mg(2+)-dependent ATPase (Ca(2+)-ATPase; EC 3.6.1.3). This enzyme is dependent upon the action of calmodulin. In this study we explored the structural attributes of the retinoids which confer this ability to inhibit enzyme activity and calmodulin binding. With respect to the fatty acid side-chain, a clear requirement for inhibition is a trans-configuration of the polar end-group. The importance of the ring structure is indicated by the ineffectiveness of polyprenoic acid and a benzene ring retinoid analogue as inhibitors of enzyme activity and calmodulin binding. There was good correlation between the relative potencies of the analogues as enzyme inhibitors and as inhibitors of calmodulin binding. The ability of selected retinoid analogues, at physiological concentrations with respect to all-trans retinoic acid, to inhibit erythrocyte Ca(2+)-ATPase activity and membrane binding of calmodulin underscores the structurally specific effects of these compounds on the interaction of calmodulin with the membrane-bound enzyme.

scholarly journals A higher-order configuration of the heterodimeric DOT1L–AF10 coiled-coil domains potentiates their leukemogenenic activity

2019 ◽  
Vol 116 (40) ◽  
pp. 19917-19923 ◽  
Author(s):  
Xiaosheng Song ◽  
Liuliu Yang ◽  
Mingzhu Wang ◽  
Yue Gu ◽  
Buqing Ye ◽  
...  
Keyword(s):  
Leucine Zipper ◽  
Coiled Coil ◽  
Binding Mode ◽  
Chromosomal Translocations ◽  
Chimeric Proteins ◽  
Binary Complex ◽  
Combined Approach ◽  
Terminal Portion ◽  
Interaction Sites ◽  
Functional Analyses

Chromosomal translocations of MLL1 (Mixed Lineage Leukemia 1) yield oncogenic chimeric proteins containing the N-terminal portion of MLL1 fused with distinct partners. The MLL1–AF10 fusion causes leukemia through recruiting the H3K79 histone methyltransferase DOT1L via AF10’s octapeptide and leucine zipper (OM-LZ) motifs. Yet, the precise interaction sites in DOT1L, detailed interaction modes between AF10 and DOT1L, and the functional configuration of MLL1–AF10 in leukeomogenesis remain unknown. Through a combined approach of structural and functional analyses, we found that the LZ domain of AF10 interacts with the coiled-coil domains of DOT1L through a conserved binding mode and discovered that the C-terminal end of the LZ domain and the OM domain of AF10 mediate the formation of a DOT1L–AF10 octamer via tetramerization of the binary complex. We reveal that the oligomerization ability of the DOT1L–AF10 complex is essential for MLL1–AF10’s leukemogenic function. These findings provide insights into the molecular basis of pathogenesis by MLL1 rearrangements.

Stability of the two wings of the coiled-coil domain of ClpB chaperone is critical for its disaggregation activity

2009 ◽  
Vol 421 (1) ◽  
pp. 71-77 ◽  
Author(s):  
Yo-hei Watanabe ◽  
Yosuke Nakazaki ◽  
Ryoji Suno ◽  
Masasuke Yoshida
Keyword(s):  
Atpase Activity ◽  
Leucine Zipper ◽  
Coiled Coil ◽  
Chaperone Activity ◽  
Aaa Atpase ◽  
Coiled Coil Domain ◽  
Central Pore ◽  
Substrate Proteins ◽  
Fold Stimulation ◽  
Stimulation Of

The ClpB chaperone forms a hexamer ring and rescues aggregated proteins in co-operation with the DnaK system. Each subunit of ClpB has two nucleotide-binding modules, AAA (ATPase associated with various cellular activities)-1 and AAA-2, and an 85-Å (1 Å=0.1 nm)-long coiled-coil. The coiled-coil consists of two halves: wing-1, leaning toward AAA-1, and wing-2, leaning away from all the domains. The coiled-coil is stabilized by leucine zipper-like interactions between leucine and isoleucine residues of two amphipathic α-helices that twist around each other to form each wing. To destabilize the two wings, we developed a series of mutants by replacing these residues with alanine. As the number of replaced residues increased, the chaperone activity was lost and the hexamer became unstable. The mutants, which had a stable hexameric structure but lost the chaperone activities, were able to exert the threading of soluble denatured proteins through their central pore. The destabilization of wing-1, but not wing-2, resulted in a several-fold stimulation of ATPase activity. These results indicate that stability of both wings of the coiled-coil is critical for full functioning of ClpB, but not for the central-pore threading of substrate proteins, and that wing-1 is involved in the communication between AAA-1 and AAA-2.

scholarly journals Domain mapping on the human metastasis regulator protein h-Prune reveals a C-terminal dimerization domain

2007 ◽  
Vol 407 (2) ◽  
pp. 199-205 ◽  
Author(s):  
Sabine Middelhaufe ◽  
Livia Garzia ◽  
Uta-Maria Ohndorf ◽  
Barbara Kachholz ◽  
Massimo Zollo ◽  
...  
Keyword(s):  
Active Sites ◽  
Catalytic Domain ◽  
Sequence Similarity ◽  
Limited Proteolysis ◽  
Coiled Coil ◽  
Metastasis Suppressor ◽  
Dimerization Domain ◽  
Interaction Sites ◽  
Domain Mapping ◽  
Human Orthologue

The human orthologue of the Drosophila prune protein (h-Prune) is an interaction partner and regulator of the metastasis suppressor protein NM23-H1 (non-metastatic protein 23). Studies on a cellular breast-cancer model showed that inhibition of the cAMP-specific PDE (phosphodiesterase) activity of h-Prune lowered the incidence of metastasis formation, suggesting that inhibition of h-Prune could be a therapeutic approach towards metastatic tumours. H-Prune shows no sequence similarity with known mammalian PDEs, but instead appears to belong to the DHH (Asp-His-His) superfamily of phosphoesterases. In order to investigate the structure and molecular function of h-Prune, we expressed recombinant h-Prune in a bacterial system. Through sequence analysis and limited proteolysis, we identified domain boundaries and a potential coiled-coil region in a C-terminal cortexillin homology domain. We found that this C-terminal domain mediated h-Prune homodimerization, as well as its interaction with NM23-H1. The PDE catalytic domain of h-Prune was mapped to the N-terminus and shown to be active, even when present in a monomeric form. Our findings indicate that h-Prune is composed of two independent active sites and two interaction sites for the assembly of oligomeric signalling complexes.

scholarly journals X-ray crystal structure of voltage-gated proton channel

2014 ◽  
Vol 70 (a1) ◽  
pp. C1501-C1501
Author(s):  
Kohei Takeshita ◽  
Souhei Sakata ◽  
Eiki Yamashita ◽  
Yuichiro Fujiwara ◽  
Yasushi Okamura ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Resting State ◽  
Coiled Coil ◽  
Proton Channel ◽  
Functional Coupling ◽  
Voltage Sensing ◽  
Voltage Gated ◽  
Extracellular Region ◽  
Hydrophobic Barrier ◽  
Activated State

The voltage-gated proton channel, Hv1 (VSOP) has a voltage-sensor domain (VSD) but lacks an authentic pore domain, and the VSD of Hv1 plays dual roles of voltage sensing and proton permeation. Hv1 is required for high-level superoxide production by phagocytes through its tight functional coupling with NADPH oxidase to eliminate pathogens. Hv1 is also expressed in human sperm and has been suggested to regulate motility through activating pH-sensitive calcium channels. The activities of Hv1 also have pathological implications, such as exacerbation of ischemic brain damage and progression of cancer. In this study, our crystal structure of mouse Hv1 (mHv1) showed a "closed umbrella" shape with a long helix consisting of the cytoplasmic coiled-coil and the voltage-sensing helix, S4, and featured a wide inner-accessible vestibule. We also found a Zn2+ion at the extracellular region of mHv1 protomer. The binding of Zn2+strongly suggested that the crystal structure of mHv1 represents the resting state, since Zn2+specifically inhibits activities of voltage-gated proton channels. Actually, two out of three arginines as sensor residues (R204 and R207) were located lower than the conserved phenylalanine, F146, on the S2 in a charge transfer center. This makes contrast with previous structures of other VSDs in the activated state where many positive residues of S4 were located upper than the conserved phenylalanine. Additionally, the crystal structure of mHv1 highlighted two hydrophobic barriers. Aspartic acid (D108), which is critical for proton selective permeation, was located facing intracellular vestibule below the inner hydrophobic barrier, thereby being accessible to water from the cytoplasm. Another hydrophobic layer of extracellular side probably ensures interruption of the proton pathway of mHv1 in resting state. These findings provide a novel platform for understanding the general principles of voltage sensing and proton permeation.

scholarly journals Conformational changes during the reaction cycle of Plasma Membrane Ca2+-ATPase in the autoinhibited and activated states

2021 ◽  
Author(s):  
Nicolás A Saffioti ◽  
Marilina de Sautu ◽  
Ana Sol Riesco ◽  
Mariela Soledad Ferreira-Gomes ◽  
Juan Pablo F.C. Rossi ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Atpase Activity ◽  
Binding Site ◽  
Conformational Changes ◽  
Ground State ◽  
Binding Pocket ◽  
Product State ◽  
Extracellular Medium ◽  
Reaction Cycle ◽  
Calmodulin Binding

Plasma membrane Ca2+-ATPase (PMCA) transports Ca2+ by a reaction cycle including phosphorylated intermediates. Calmodulin binding to the C-terminal tail disrupts autoinhibitory interactions, activating the pump. To assess the conformational changes during the reaction cycle, we studied the structure of different PMCA states using a fluorescent probe, hydrophobic photolabeling, controlled proteolysis and Ca2+-ATPase activity.  Our results show that calmodulin binds to E2P-like states, and during dephosphorylation, the hydrophobicity in the nucleotide-binding pocket decreases and the Ca2+ binding site becomes inaccessible to the extracellular medium. Autoinhibitory interactions are disrupted in E1Ca and in the E2P ground state whereas they are stabilized in the E2∙Pi product state. Finally, we propose a model that describes the conformational changes during the Ca2+ transport of PMCA.

scholarly journals Autoinhibition regulates the motility of the C. elegans intraflagellar transport motor OSM-3

2006 ◽  
Vol 174 (7) ◽  
pp. 931-937 ◽  
Author(s):  
Miki Imanishi ◽  
Nicholas F. Endres ◽  
Arne Gennerich ◽  
Ronald D. Vale
Keyword(s):  
Atpase Activity ◽  
Single Molecule ◽  
Intramolecular Interaction ◽  
Optical Trap ◽  
Coiled Coil ◽  
Intraflagellar Transport ◽  
Wild Type ◽  
C Elegans ◽  
Sensory Cilia

OSM-3 is a Kinesin-2 family member from Caenorhabditis elegans that is involved in intraflagellar transport (IFT), a process essential for the construction and maintenance of sensory cilia. In this study, using a single-molecule fluorescence assay, we show that bacterially expressed OSM-3 in solution does not move processively (multiple steps along a microtubule without dissociation) and displays low microtubule-stimulated adenosine triphosphatase (ATPase) activity. However, a point mutation (G444E) in a predicted hinge region of OSM-3's coiled-coil stalk as well as a deletion of that hinge activate ATPase activity and induce robust processive movement. These hinge mutations also cause a conformational change in OSM-3, causing it to adopt a more extended conformation. The motility of wild-type OSM-3 also can be activated by attaching the motor to beads in an optical trap, a situation that may mimic attachment to IFT cargo. Our results suggest that OSM-3 motility is repressed by an intramolecular interaction that involves folding about a central hinge and that IFT cargo binding relieves this autoinhibition in vivo. Interestingly, the G444E allele in C. elegans produces similar ciliary defects to an osm-3–null mutation, suggesting that autoinhibition is important for OSM-3's biological function.

scholarly journals Coupling of ATPase activity, microtubule binding and mechanics in the dynein motor domain

2018 ◽  
Author(s):  
Stefan Niekamp ◽  
Nicolas Coudray ◽  
Nan Zhang ◽  
Ronald D. Vale ◽  
Gira Bhabha
Keyword(s):  
Atpase Activity ◽  
Conformational Change ◽  
Conformational Changes ◽  
Large Scale ◽  
Atp Hydrolysis ◽  
Molecular Motor ◽  
Coiled Coil ◽  
Microtubule Binding ◽  
Dynein Motor ◽  
In The Wild

The movement of a molecular motor protein along a cytoskeletal track requires communication between enzymatic, polymer-binding, and mechanical elements. Such communication is particularly complex and not well understood in the dynein motor, an ATPase that is comprised of a ring of six AAA domains, a large mechanical element (linker) spanning over the ring, and a microtubule-binding domain (MTBD) that is separated from the AAA ring by a ~135 Å coiled-coil stalk. We identified mutations in the stalk that disrupt directional motion, have microtubule-independent hyperactive ATPase activity, and nucleotide-independent low affinity for microtubules. Cryo-electron microscopy structures of a mutant that uncouples ATPase activity from directional movement reveal that nucleotide-dependent conformational changes occur normally in one half of the AAA ring, but are disrupted in the other half. The large-scale linker conformational change observed in the wild-type protein is also inhibited, revealing that this conformational change is not required for ATP hydrolysis. These results demonstrate an essential role of the stalk in regulating motor activity and coupling conformational changes across the two halves of the AAA ring.

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