Formation of a Secretion-Competent Protein Complex by a Dynamic Wrap-around Binding Mechanism |
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Authors: | Arun A. Gupta Ines Reinartz Gogulan Karunanithy Alessandro Spilotros Venkateswara Rao Jonna Anders Hofer Dmitri I. Svergun Andrew J. Baldwin Alexander Schug Magnus Wolf-Watz |
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Affiliation: | 1. Department of Chemistry, Umeå University, SE-901 87 Umeå, Sweden;2. Steinbuch Centre for Computing, Karlsruhe Institute of Technology, 76131 Karlsruhe, Germany;3. Department of Physics, Karlsruhe Institute of Technology, 76131 Karlsruhe, Germany;4. Department of Chemistry, Physical and Theoretical Chemistry Laboratory, University of Oxford, South Parks Road, Oxford, OX13QZ, United Kingdom;5. European Molecular Biological Laboratory, Hamburg Outstation, Notkestrasse 85, Hamburg 22603, Germany;6. Department of Medical Biochemistry and Biophysics, Umeå University, SE-901 87 Umeå, Sweden;7. John von Neumann Institute for Computing, Jülich Supercomputer Centre, Forschungszentrum Jüllich, 52428 Jülich, Germany |
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Abstract: | Bacterial virulence is typically initiated by translocation of effector or toxic proteins across host cell membranes. A class of gram-negative pathogenic bacteria including Yersinia pseudotuberculosis and Yersinia pestis accomplishes this objective with a protein assembly called the type III secretion system. Yersinia effector proteins (Yop) are presented to the translocation apparatus through formation of specific complexes with their cognate chaperones (Syc). In the complexes where the structure is available, the Yops are extended and wrap around their cognate chaperone. This structural architecture enables secretion of the Yop from the bacterium in early stages of translocation. It has been shown previously that the chaperone-binding domain of YopE is disordered in its isolation but becomes substantially more ordered in its wrap-around complex with its chaperone SycE. Here, by means of NMR spectroscopy, small-angle X-ray scattering and molecular modeling, we demonstrate that while the free chaperone-binding domain of YopH (YopHCBD) adopts a fully ordered and globular fold, it populates an elongated, wrap-around conformation when it engages in a specific complex with its chaperone SycH2. Hence, in contrast to YopE that is unstructured in its free state, YopH transits from a globular free state to an elongated chaperone-bound state. We demonstrate that a sparsely populated YopHCBD state has an elevated affinity for SycH2 and represents an intermediate in the formation of the protein complex. Our results suggest that Yersinia has evolved a binding mechanism where SycH2 passively stimulates an elongated YopH conformation that is presented to the type III secretion system in a secretion-competent conformation. |
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Keywords: | T3SS type III secretion system CBDs chaperone-binding domains SAXS small-angle X-ray scattering GEMMA Gas-phase Electrophoretic Mobility Macromolecule Analysis HSQC heteronuclear single quantum coherence ITC isothermal titration calorimetry SBMs structure-based models CPMG Carr–Purcell–Meiboom–Gill NMR spectroscopy protein complex binding mechanism |
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