The ATPase domain of SecA can form a tetramer in solution 1 1Edited by I. B. Holland

Dempsey, Brian R.; Economou, Anastassios; Dunn, Stanley D.; Shilton, Brian H.

doi:10.1006/jmbi.2001.5279

Cited by 29 publications

(48 citation statements)

References 55 publications

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“…We found a sedimentation coefficient (s 20,w ) of 4.05, which is similar to the sedimentation coefficient of 4.07 for the 68-kDa monomeric fragment of SecA (44). However, the frictional coefficient ratio (f/f 0 ) was found equal to 1.95, which indicates that the TIM10 complex is a significantly extended particle (for comparison, BSA with a molecular mass of 65 kDa and hemoglobin with a molecular mass of 68 kDa have frictional coefficient ratios of 1.33 and 1.28, respectively).…”

Section: Tim9 and Tim10 Stabilize Each Other And Assemble Into The Tisupporting

confidence: 79%

Assembly of Tim9 and Tim10 into a Functional Chaperone

Vial¹,

Lu²,

Allen³

et al. 2002

Journal of Biological Chemistry

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The TIM10 complex is localized in the mitochondrial intermembrane space and mediates insertion of hydrophobic proteins at the inner membrane. We have characterized TIM10 assembly and analyzed the structural properties of its subunits, Tim9 and Tim10. Both proteins are ␣-helical with a protease-resistant central domain, and each self-associates to form mainly dimers and trimers in solution. Tim9 and Tim10 bound to one another with submicromolar affinity in equimolar amounts and assembled in a stable, significantly extended complex that was indistinguishable from the native mitochondrial TIM10 complex. Importantly, the reconstituted TIM10 complex is functional because it bound to the physiological substrate ADP/ATP carrier and displayed chaperone activity in refolding the model substrate firefly luciferase. These data demonstrate that the individual subunits can exist as independent, dynamically self-associating proteins. Assembly into the thermodynamically stable hexameric complex is necessary for the TIM10 chaperone function.Almost all mitochondrial proteins are synthesized in the cytosol and then imported into the organelle in a process that is dictated by each protein's sequence and that is ensured by the function of specialized translocation machineries in the organelle (1-4). Most import and intramitochondrial sorting pathways are variants of the general "matrix pathway," first described in detail for matrix-targeted proteins (1-4). In this pathway, a mitochondrial precursor, which is usually synthesized with an N-terminal, positively charged, amphiphilic presequence, first interacts with cytosolic chaperones. It is then bound by a hetero-oligomeric receptor system on the surface of mitochondria in a process that requires ATP hydrolysis in the cytosol. The polypeptide chain is then transported across two hetero-oligomeric protein import channels, the TOM complex in the outer membrane and the TIM23 complex in the inner membrane. Translocation is completed by the electrophoretic function of the electrochemical potential across the inner membrane and the ATP-powered import motor attached to the inner side of the TIM23 complex. In this pathway, targeting of the polypeptide chain from one complex to the other appears to be directed by increasing avidity of the positive presequence for a series of acidic receptor domains ("acid chain hypothesis") (5-9).The structural basis of this mechanism is becoming increasingly clear through advances in understanding the structural characteristics of key components at different steps of this pathway. First, the solution structure (determined by NMR) of the cytosolic part of the Tom20 receptor in complex with a synthetic presequence has been solved (10, 11): this showed that the presequence is in ␣-helical conformation and that binding between the receptor and the presequence involves hydrophobic stretches. Second, the existence of a hydrophilic channel of TOM40 as the outer membrane import pore and its dynamic behavior have been established (12-16). Third, Tim23 was shown to f...

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Section: Tim9 and Tim10 Stabilize Each Other And Assemble Into The Tisupporting

confidence: 79%

Assembly of Tim9 and Tim10 into a Functional Chaperone

Vial¹,

Lu²,

Allen³

et al. 2002

Journal of Biological Chemistry

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“…Although we cannot rule out the possibility that SecA functions as a monomer or even a tetramer, as suggested by previous studies (35,36), the structure of the A-B dimer suggests that this is the active form. The two subunits of the A-B dimer are related by a nearly perfect twofold axis of symmetry perpendicular to a plane defined by an elliptical pore found in the center of the dimer (Fig.…”

Section: Resultsmentioning

confidence: 59%

Crystal structure of Mycobacterium tuberculosis SecA, a preprotein translocating ATPase

Sharma

Arockiasamy

Ronning

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

176

219

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In bacteria, the majority of exported proteins are translocated by the Sec system, which recognizes the signal sequence of a preprotein and uses ATP and the proton motive force to mediate protein translocation across the cytoplasmic membrane. SecA is an essential protein component of this system, containing the molecular motor that facilitates translocation. Here we report the threedimensional structure of the SecA protein of Mycobacterium tuberculosis. Each subunit of the homodimer contains a ''motor'' domain and a translocation domain. The structure predicts that SecA can interact with the SecYEG pore and function as a molecular ratchet that uses ATP hydrolysis for physical movement of the preprotein. Knowledge of this structure provides a framework for further elucidation of the translocation process.

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“…Measurements of MBP-WT and MBP-DM proteins were made at BioCAT (beamline 18ID) of the Advanced Photon Source. Details of SAXS measurements and data processing have been described previously (19,20).…”

Section: Methodsmentioning

confidence: 99%

Insights into the Conformational Equilibria of Maltose-binding Protein by Analysis of High Affinity Mutants

Telmer

Shilton

2003

Journal of Biological Chemistry

110

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ATP binding cassette transporters couple ATP hydrolysis to the transmembrane transport of a diverse range of compounds. Members of the ATP binding cassette transporter superfamily are characterized by two membrane-integral domains that each contain 6 or more membrane spanning helices, but are otherwise poorly conserved, and two peripheral ATP binding cassette domains that display sequence conservation across the entire superfamily (1). In addition to the membrane complex, ATP binding cassette systems that catalyze nutrient uptake have primary receptors (binding proteins) that serve two functions: they provide a high affinity binding site for the transported molecule and they regulate the ATPase activity of the integral membrane complex.We are interested in the function of the primary receptors in the transport process. As a group, these proteins have been intensively studied by x-ray crystallography and other biophysical techniques (for a review, see Ref.2). They typically contain two domains separated by a hinge region; the substrate binds in the cleft between the two domains, and the protein undergoes a large conformational change, leading to closure of the cleft. With respect to the maltose transport system, domain closure in the binding protein is thought to be the first step toward molecular shape recognition by the membrane complex (3, 4), although it has been shown that both substrate-loaded and substrate-free binding proteins have a role in the transport cycle (5-8). The association and dissociation of the substrate, and attendant conformational changes in the binding protein (MBP), 1 may have direct effects on transport kinetics and regulation of ATP hydrolysis by MalFGK 2 . To investigate the role of binding protein affinity on the transport process, our goal was to engineer MBP molecules with greater affinity for maltose, without changing residues in either the maltose binding site or in regions thought to interact with MalFGK 2 .Crystal structures of MBP in both the closed and open conformations have been solved (9, 10), and they show that binding of maltose results in a large conformational change of the protein, bringing the two domains together such that the substrate is buried inside the cleft. In solution, unliganded MBP is in the open conformation (11); however, there is no obvious energetic barrier to closure of the ligand binding cleft, either in the hinge or in the interface surrounding the ligand binding site. Rather, an interface on the opposite side of the hinge from the ligand binding site appears to maintain the protein in an * This work was supported by Natural Sciences and Engineering Research Council Grant 21749-1999 (to B. H. S.); the macromolecular x-ray facility at the University of Western Ontario was financed with grants from the Canada Foundation for Innovation, Ontario Challenge Fund, and Western's Academic Development Fund; fluorescence and SPR studies were carried out at the University of Western Ontario Biomolecular Interactions and Conformations Facility, supported by a Mu...

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The ATPase domain of SecA can form a tetramer in solution 1 1Edited by I. B. Holland

Cited by 29 publications

References 55 publications

Assembly of Tim9 and Tim10 into a Functional Chaperone

Assembly of Tim9 and Tim10 into a Functional Chaperone

Crystal structure of Mycobacterium tuberculosis SecA, a preprotein translocating ATPase

Insights into the Conformational Equilibria of Maltose-binding Protein by Analysis of High Affinity Mutants

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