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      Structural Characterization of the Complex of SecB and Metallothionein-Labeled proOmpA by Cryo-Electron Microscopy

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      Author(s): 1 , 1 , 2 , 1 , *
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      Journal: PLoS ONE
      Publisher: Public Library of Science

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          Abstract

          ProOmpA is a preprotein that is translocated across the plasma membrane by the general secretory pathway in Escherichia coli. The molecular chaperon SecB in Sec pathway can recognize and bind proOmpA for its translocation. However, the structure of the SecB/proOmpA complex remains unknown. Here, we constructed an uncleavable proOmpA fused with metallothionein at its C-terminus and labeled it with metals in vitro for the study of cryo-electron microscopy. Using single particle cryo-electron microscopy, we reconstructed 3D structure of the stable SecB/proOmpA complex. The structure shows that the major portion of preprotein locates on one side of SecB tetramer, resulting in an asymmetric binding pattern. This work also provides a possible approach to the structure determination of small protein complexes by cryo-electron microscopy.

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          The binding cascade of SecB to SecA to SecY/E mediates preprotein targeting to the E. coli plasma membrane.

          David J Hendrick, W Wickner, F. Ulrich Hartl … (1990)
          The export of many E. coli proteins such as proOmpA requires the cytosolic chaperone SecB and the membrane-bound preprotein translocase. Translocase is a multisubunit enzyme with the SecA protein as its peripheral membrane domain and the SecY/E protein as its integral domain. SecB, by binding to proOmpA in the cytosol, prevents its aggregation or association with membranes at nonproductive sites. The SecA receptor binds the proOmpA-SecB complex (Kd approximately 6 x 10(-8) M) through direct recognition of both the SecB (Kd approximately 2 x 10(-7) M) as well as the leader and mature domains of the precursor protein. SecB has a dual function in stabilizing the precursor and in passing it on to membrane-bound SecA, the next step in the pathway. SecA itself is bound to the membrane by its affinity (Kd approximately 4 x 10(-8) M) for SecY/E and for acidic lipids. The functions of SecB and SecA as a two-stage receptor system are linked by their affinity for each other.
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            E. coli mutant pleiotropically defective in the export of secreted proteins.

            Donald B. Oliver, Jon Beckwith (1981)
            A hybrid beta-galactosidase molecule containing a substantial portion of the amino-terminal sequence of the maltose-binding protein is inserted in the cytoplasmic membrane of E. coli; in this location, the protein has very low enzymatic activity. The strain producing it is, therefore, Lac-. Selection for derivatives of the fusion strain that are able to grow on lactose yields mutants in which the hybrid protein has become cytoplasmic, and thus has higher enzymatic activity. Among such derivatives, we have isolated a temperature-sensitive conditional lethal mutant that accumulates the precursor of the maltose-binding protein in the cytoplasm, and also accumulates precursors of alkaline phosphatase, lambda receptor protein and the ompF gene gene product. A number of periplasmic proteins are, however, properly localized at the nonpermissive temperature. The temperature-sensitive lesion has been genetically mapped to 2.5 min on the E. coli map, within or near a cluster of genes responsible for cell division and septation. The principle behind the genetic selection employed here should be useful in obtaining other secretion mutants to characterize the cell's secretion machinery.
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              Direct observation of chaperone-induced changes in a protein folding pathway.

              Philipp Bechtluft, Ruud G H van Leeuwen, Matthew Tyreman … (2007)
              How chaperone interactions affect protein folding pathways is a central problem in biology. With the use of optical tweezers and all-atom molecular dynamics simulations, we studied the effect of chaperone SecB on the folding and unfolding pathways of maltose binding protein (MBP) at the single-molecule level. In the absence of SecB, we find that the MBP polypeptide first collapses into a molten globulelike compacted state and then folds into a stable core structure onto which several alpha helices are finally wrapped. Interactions with SecB completely prevent stable tertiary contacts in the core structure but have no detectable effect on the folding of the external alpha helices. It appears that SecB only binds to the extended or molten globulelike structure and retains MBP in this latter state. Thus during MBP translocation, no energy is required to disrupt stable tertiary interactions.
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                Author and article information

                Contributors
                Vladimir N. Uversky: Role: Editor
                Journal
                Journal ID (nlm-ta): PLoS One
                Journal ID (iso-abbrev): PLoS ONE
                Journal ID (publisher-id): plos
                Journal ID (pmc): plosone
                Title: PLoS ONE
                Publisher: Public Library of Science (San Francisco, USA )
                ISSN (Electronic): 1932-6203
                Publication date Collection: 2012
                Publication date (Electronic): 4 October 2012
                Volume: 7
                Issue: 10
                Electronic Location Identifier: e47015
                Affiliations
                [1 ]State Key Laboratory of Biomembrane and Membrane Biotechnology, Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing, China
                [2 ]Department of Biology, Georgia State University, Atlanta, Georgia, United States of America
                University of South Florida College of Medicine, United States of America
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: QZ SFS. Performed the experiments: QZ. Analyzed the data: QZ SS SFS. Contributed reagents/materials/analysis tools: QZ. Wrote the paper: QZ SS PT SFS.

                Article
                Publisher ID: PONE-D-12-18611
                DOI: 10.1371/journal.pone.0047015
                PMC ID: 3464278
                PubMed ID: 23056562
                SO-VID: 3b9c57b8-360a-4c94-94fd-0c82e1759a8d
                Copyright statement: Copyright @ 2012
                License:

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                Date received : 22 June 2012
                Date accepted : 11 September 2012
                Page count
                Pages: 10
                Funding
                This work was supported by the National Basic Research program of China (2011CB910500/2010CB833706/2010CB912400) and the National Natural Science Foundation of China (30830028/30970591). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Subject: Research Article
                Subject: Biology
                Subject: Biochemistry
                Subject: Proteins
                Subject: Chaperone Proteins
                Subject: Protein Interactions
                Subject: Protein Structure
                Subject: Transmembrane Transport Proteins
                Subject: Macromolecular Assemblies
                Subject: Biophysics
                Subject: Microbiology
                Subject: Bacterial Pathogens
                Subject: Escherichia Coli
                Subject: Bacteriology

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                ScienceOpen disciplines: Uncategorized

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