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ABSTRACT: The Sec translocase mediates the post-translational translocation of a number of preproteins through the inner membrane in bacteria. In the initiatory translocation step, SecB targets the preprotein to the translocase by specific interaction with its receptor SecA. The latter is the ATPase of Sec translocase which mediates the post-translational translocation of preprotein through the protein-conducting channel SecYEG in the bacterial inner membrane. We examined the structures of Escherichia coli Sec intermediates in solution as visualized by negatively stained electron microscopy in order to probe the oligomeric states of SecA during this process. The symmetric interaction pattern between the SecA dimer and SecB becomes asymmetric in the presence of proOmpA, and one of the SecA protomers predominantly binds to SecB/proOmpA. Our results suggest that during preprotein translocation, the two SecA protomers are different in structure and may play different roles.
PLoS ONE 01/2011; 6(1):e16498. · 4.09 Impact Factor
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ABSTRACT: SecA is an obligatory component of the Escherichia coli general secretion pathway. However, the oligomeric structure of SecA and SecA conformational changes during translocation processes are still unclear. Here we obtained the three-dimensional structure of E. coli wild-type full-length SecA in solution by single particle cryo-electron microscopy and determined its oligomeric organization. In this structure, SecA occurs as a dimer in which the two protomers are arranged in an antiparallel mode, with a novel electrostatic interface, and both protomers are in closed conformation. The system developed here may provide a promising technique for studying dynamic structural changes in SecA.
Journal of Biological Chemistry 10/2008; 283(43):28783-7. · 4.77 Impact Factor
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ABSTRACT: Cells use molecular chaperones and proteases to implement the essential quality control mechanism of proteins. The DegP (HtrA) protein, essential for the survival of Escherichia coli cells at elevated temperatures with homologues found in almost all organisms uniquely has both functions. Here we report a mechanism for DegP to activate both functions via formation of large cage-like 12- and 24-mers after binding to substrate proteins. Cryo-electron microscopic and biochemical studies revealed that both oligomers are consistently assembled by blocks of DegP trimers, via pairwise PDZ1-PDZ2 interactions between neighboring trimers. Such interactions simultaneously eliminate the inhibitory effects of the PDZ2 domain. Additionally, both DegP oligomers were also observed in extracts of E. coli cells, strongly implicating their physiological importance.
Proceedings of the National Academy of Sciences 09/2008; 105(33):11939-44. · 9.68 Impact Factor
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ABSTRACT: SecA is a multifunctional protein involved in protein translocation in bacteria. The structure of SecA on membrane is dramatically altered compared with that in solution, accompanying with functional changes. We previously reported the formation of a novel ring-like structure of SecA on lipid layers, which may constitute part of the preprotein translocation channel. In the present work, two-dimensional crystallization of Escherichia coli SecA on lipid monolayers was performed to reveal the structural details of SecA on lipid layers and to investigate its function. The 2D crystals composed of ring-like structures were obtained by specific interaction between SecA and negatively charged lipid. The 2D projection map and 3D reconstruction from negative stained 2D crystals exhibited a distinct open channel-like structure of SecA, with an outer diameter of 7 nm and an inner diameter of 2 nm, providing the structural evidence for SecA importance in forming the part of the translocation channel. This pore structure is altered after transferring crystals to the SecB solution, indicating that the lipid-specific SecA structure has the SecB binding activity. The strategy developed here provides a promising technique for studying structure of SecA complex with its ligand on membrane.
Journal of Structural Biology 08/2007; 159(1):149-53. · 3.41 Impact Factor
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ABSTRACT: CorA is a primary Mg2+ transporter in bacteria, which also mediates influx of Ni2+ and Co2+. Topological studies suggested that it could be divided into a large soluble periplasmic domain (PPD) and three membrane-spanning alpha-helixes. In the present study, glutathione S-transferase (GST) fusion Escherichia coli CorA PPD was purified by GST affinity chromatography, and PPD was obtained by on-column thrombin digestion. Size-exclusion chromatography indicated that purified PPD exists as a homotetramer. Single particle electron microscopy analysis of PPD and two-dimensional crystals of GST-PPD indicated that E. coli CorA PPD is a pyramid-like homotetramer with a central cavity. Comparison of the CD spectra of full-length CorA and PPD also suggested that PPD has similar secondary structure to the full-length CorA. Dissociation constants for CorA and PPD with their substrates, determined by dose-dependent fluorescence quench of ligands, suggested that purified PPD retains its substrate binding ability as native CorA. The CorA PPD structure described here may provide structural information for the E. coli CorA functional oligomeric state.
Journal of Biological Chemistry 10/2006; 281(37):26813-20. · 4.77 Impact Factor
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ABSTRACT: The actin-binding protein p57 is a member of mammalian coronin-like proteins. The roles of this protein in phagocytic processes conceivably depend on its interactions with F-actin. Two regions, p57(1-34) and p57(111-204), were previously reported to be actin-binding sites. In this study, we found that the C-terminal region of p57, p57(297-461), also possessed F-actin binding activity. Furthermore, the leucine zipper domain at the C-terminus of p57(297-461) was essential for this actin-binding activity. The F-actin cross-linking assay revealed that the region contained in p57(297-461) was sufficient to cross-link actin filaments. Our results strongly suggested that there was a new actin-binding region at the C-terminus of p57.
Cell Research 02/2006; 16(1):106-12. · 8.19 Impact Factor
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ABSTRACT: As a member of small heat shock proteins, HSP16.3 was identified as the major membrane-bound protein of Mycobacterium tuberculosis during stationary phase. Previous studies revealed that HSP16.3 was in a nonameric form in solution. Here, two-dimensional crystal of HSP16.3 molecules on lipid monolayer was obtained for the first time. The crystal exhibited p422 symmetry with lattice parameters a=b=90A, gamma=90 degrees. The projection map of untilted crystals showed that the basic unit of the crystal was a rod-like structure with two high-density regions. The three-dimensional map at 2.2 nm resolution revealed a rod-like structure with a dimension of 56A x 32A x 25A, similar to the dimeric forms of M. jannaschii HSP16.5 and wheat HSP16.9. Cross-linking experiments confirmed that HSP16.3 nonamers dissociated into dimers upon interaction with the positively charged lipid layer. Surface plasmon resonance measurements revealed that both electrostatic and hydrophobic forces involved in the formation of the 2D crystal on the lipid monolayer. These results provide a basis for further investigation on the unique dimeric structure of HSP16.3 and its functions in vivo.
Biochemical and Biophysical Research Communications 11/2003; 310(2):360-6. · 2.48 Impact Factor
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ABSTRACT: SecA, an essential component of the general protein secretion pathway of bacteria, is present in Escherichia coli as soluble and membrane-integral forms. Here we show by electron microscopy that SecA assumes two characteristic forms in the presence of phospholipid monolayers: dumbbell-shaped elongated structures and ring-like pore structures. The ring-like pore structures with diameters of 8 nm and holes of 2 nm are found only in the presence of anionic phospholipids. These ring-like pore structures with larger 3- to 6-nm holes (without staining) were also observed by atomic force microscopic examination. They do not form in solution or in the presence of uncharged phosphatidylcholine. These ring-like phospholipid-induced pore-structures may form the core of bacterial protein-conducting channels through bacterial membranes.
Proceedings of the National Academy of Sciences 05/2003; 100(7):4221-6. · 9.68 Impact Factor
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ABSTRACT: C-reactive protein (CRP), as a classical member of acute phase reactants, plays a variety of physiological functions in vivo and in vitro. Here we show that CRP exists in different structural forms in solution and on phospholipid membranes. At least three types of CRP structures, i.e., the pentameric ring-like form, the small globulin-like form and the fibril-like structure, were observed by combination of size-exclusion chromatography and electron microscopy. The pentameric ring-like CRPs were discovered mostly on ligand containing membrane in a calcium-dependent manner. The globulin-like monomers found on negatively charged membrane in the absence of calcium exhibited structural stability. The fibril-like structures, that have not been reported before, were formed by face-to-face stacking of pentameric CRP in a number from several to hundreds. The freshly purified CRPs form short single-strand fibrils while those CRPs stored for more than several days form long and bundled fibrils. The different structural forms may convert to each other under certain conditions, which gives some hints as to the structural basis of multiple functions of CRP.
International Journal of Molecular Medicine 07/2002; 9(6):665-71. · 1.98 Impact Factor
