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1. A Single Amino Acid Substitution Changes the Self-Assembly Status of a Type IV Piliation Secretin

Author

Mohamed Chami, Anthony P. Pugsley, Nicholas N. Nickerson, Eduardo P. C. Rocha, Sophie S. Abby, Génétique Moléculaire, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Génomique évolutive des Microbes / Microbial Evolutionary Genomics, Center for Cellular Imaging and NanoAnalytics, Biozentrum [Basel, Suisse], University of Basel (Unibas)-University of Basel (Unibas), This work was supported in part by grants ANR-05-0307-01 and ANR-09-BLAN-0291. N.N.N. was supported by a postdoctoral fellowship from the Canadian Louis Pasteur Foundation during part of this work., ANR-09-BLAN-0291,Secretin(2009), and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Inovirus, medicine.disease_cause, digestive system, Microbiology, Insert (molecular biology), 03 medical and health sciences, fluids and secretions, Secretin, Extracellular, medicine, Cluster Analysis, Secretion, Molecular Biology, Phylogeny, 030304 developmental biology, 0303 health sciences, Mutation, [SDV.GEN.GPO]Life Sciences [q-bio]/Genetics/Populations and Evolution [q-bio.PE], Bacteria, Sequence Homology, Amino Acid, biology, 030306 microbiology, Articles, biology.organism_classification, [SDV.BIBS]Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], digestive system diseases, In vitro, Amino Acid Substitution, Filamentous bacteriophage, Biochemistry, Liposomes, Mutant Proteins, Fimbriae Proteins, Protein Multimerization, hormones, hormone substitutes, and hormone antagonists, Function (biology)
Abstract

Secretins form large multimeric complexes in the outer membranes of many Gram-negative bacteria, where they function as dedicated gateways that allow proteins to access the extracellular environment. Despite their overall relatedness, different secretins use different specific and general mechanisms for their targeting, assembly, and membrane insertion. We report that all tested secretins from several type II secretion systems and from the filamentous bacteriophage f1 can spontaneously multimerize and insert into liposomes in anin vitrotranscription-translation system. Phylogenetic analyses indicate that these secretins form a group distinct from the secretins of the type IV piliation and type III secretion systems, which do not autoassemblein vitro. A mutation causing a proline-to-leucine substitution allowed PilQ secretins from two different type IV piliation systems to assemblein vitro, albeit with very low efficiency, suggesting that autoassembly is an inherent property of all secretins.

Published
2012
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2. Pilotin-secretin recognition in the type II secretion system of Klebsiella oxytoca

Author

Tosi, Tommaso, Nickerson, Nicholas, Mollica, Luca, Jensen, Malene Ringkjøbing, Blackledge, Martin, Baron, Bruno, England, Patrick, Pugsley, Anthony, Dessen, Andréa, Institut de biologie structurale (IBS - UMR 5075 ), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Génétique Moléculaire, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Biophysique des Macromolécules et de leurs Interactions, Institut Pasteur [Paris] (IP), This work was supported by Grant ANR-09-BLAN-0291. N.N.N. was supported by a postdoctoral fellowship from the Canadian Louis Pasteur Foundation during part of this work. The co-ordinates of PulS have been deposited in the Protein Database under code 4A56., The authors wish to thank Ingrid Guilvout and Séverine Collin for their helpful discussions and members of the Molecular Genetics Unit at the Institut Pasteur for their support. We are also grateful to J. Marquez and the HTX Lab team (Partnership for Structural Biology, Grenoble, PSB) for access to and help with high throughput crystallization, Hassan Berlhali (EMBL Grenoble) for help with collection of PulS-Barium data and Carlos Contreras-Martel for his support in solving PulSV30 structure, the European Synchrotron Radiation Facility (ESRF, PSB) for access to beamlines, Bertrand Raynal (Centre of Biophysics of Macromolecules and their Interactions, Institut Pasteur) for hydrodynamic modelling calculations, and Jacques d'Alayer (Protein Microsequencing and Analysis Platform, Institut Pasteur) for N-terminal sequencing., and ANR-09-BLAN-0291,Secretin(2009)

Subjects
[SDV]Life Sciences [q-bio], MESH: Bacterial Outer Membrane Proteins, Klebsiella oxytoca, MESH: Protein Structure, Secondary, MESH: Klebsiella oxytoca, Protein Structure, Secondary, Protein Structure, Tertiary, MESH: Protein Structure, Tertiary, MESH: Protein Binding, MESH: Molecular Chaperones, MESH: Bacterial Secretion Systems, Bacterial Secretion Systems, Bacterial Outer Membrane Proteins, Molecular Chaperones, Protein Binding
Abstract

International audience; A crucial aspect of the functionality of bacterial type II secretion systems is the targeting and assembly of the outer membrane secretin. In the Klebsiella oxytoca type II secretion system, the lipoprotein PulS, a pilotin, targets secretin PulD monomers through the periplasm to the outer membrane. We present the crystal structure of PulS, an all-helical bundle that is structurally distinct from proteins with similar functions. Replacement of valine at position 42 in a charged groove of PulS abolished complex formation between a non-lipidated variant of PulS and a peptide corresponding to the unfolded region of PulD to which PulS binds (the S-domain), in vitro, as well as PulS function in vivo. Substitutions of other residues in the groove also diminished the interaction with the S-domain in vitro but exerted less marked effects in vivo. We propose that the interaction between PulS and the S-domain is maintained through a structural adaptation of the two proteins that could be influenced by cis factors such as the fatty acyl groups on PulS, as well as periplasmic transacting factors, which represents a possible paradigm for chaperonetarget protein interactions.

Published
2011
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3. Outer Membrane Targeting of Secretin PulD Protein Relies on Disordered Domain Recognition by a Dedicated Chaperone

Author

Bertrand Raynal, Bruno Baron, Andréa Dessen, Nicholas N. Nickerson, Tommasso Tosi, Patrick England, Anthony P. Pugsley, Génétique Moléculaire, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Institut de biologie structurale (IBS - UMR 5075 ), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Biophysique des Macromolécules et de leurs Interactions, This work was supported in part by French National Research Agency Grants ANR-05-0307-01 and ANR-09-BLAN-0291., ANR-05-BLAN-0307,SECRETIN,Structure and function of secretin in the bacterial type II protein secretion system(2005), and ANR-09-BLAN-0291,Secretin(2009)

Subjects
Protein Folding, MESH: Protein Structure, Quaternary, [SDV]Life Sciences [q-bio], MESH: Protein Structure, Secondary, MESH: Escherichia coli Proteins, Plasma protein binding, Biochemistry, Protein Structure, Secondary, MESH: Protein Structure, Tertiary, fluids and secretions, MESH: Nuclear Magnetic Resonance, Biomolecular, MESH: Bacterial Secretion Systems, Protein secondary structure, Bacterial Secretion Systems, MESH: Escherichia coli, Escherichia coli Proteins, Outer Membrane, MESH: Lipoproteins, Periplasm, Chaperone binding, Protein folding, MESH: Molecular Chaperones, Bacterial outer membrane, hormones, hormone substitutes, and hormone antagonists, Bacterial Outer Membrane Proteins, Protein Binding, Lipoproteins, MESH: Protein Folding, Biology, Intrinsically disordered proteins, digestive system, Microbiology, Escherichia coli, MESH: Protein Binding, Binding site, Chaperone Chaperonin, Protein Structure, Quaternary, Molecular Biology, Nuclear Magnetic Resonance, Biomolecular, Secretion, MESH: Bacterial Outer Membrane Proteins, Membrane Proteins, Cell Biology, digestive system diseases, Protein Structure, Tertiary, Intrinsically Disordered Proteins, Chaperone (protein), Biophysics, biology.protein, Molecular Chaperones
Abstract

International audience; Interaction of bacterial outer membrane secretin PulD with its dedicated lipoprotein chaperone PulS relies on a disorder-to-order transition of the chaperone binding (S) domain near the PulD C terminus. PulS interacts with purified S domain to form a 1:1 complex. Circular dichroism, one-dimensional NMR, and hydrodynamic measurements indicate that the S domain is elongated and intrinsically disordered but gains secondary structure upon binding to PulS. Limited proteolysis and mass spectrometry identified the 28 C-terminal residues of the S domain as a minimal binding site with low nanomolar affinity for PulS in vitro that is sufficient for outer membrane targeting of PulD in vivo. The region upstream of this binding site is not required for targeting or multimerization and does not interact with PulS, but it is required for secretin function in type II secretion. Although other secretin chaperones differ substantially from PulS in sequence and secondary structure, they have all adopted at least superficially similar mechanisms of interaction with their cognate secretins, suggesting that intrinsically disordered regions facilitate rapid interaction between secretins and their chaperones.

Published
2011
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