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1. The c‐di‐ <scp>AMP</scp> ‐binding protein <scp>CbpB</scp> modulates the level of <scp>ppGpp</scp> alarmone in Streptococcus agalactiae

Author

Giovanni Covaleda‐Cortés, Ariel Mechaly, Terry Brissac, Heike Baehre, Laura Devaux, Patrick England, Bertrand Raynal, Sylviane Hoos, Myriam Gominet, Arnaud Firon, Patrick Trieu‐Cuot, Pierre Alexandre Kaminski, Biologie des Bactéries pathogènes à Gram-positif - Biology of Gram-Positive Pathogens, Université Paris Cité (UPCité)-Microbiologie Intégrative et Moléculaire (UMR6047), Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Cristallographie (Plateforme) - Crystallography (Platform), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Hannover Medical School [Hannover] (MHH), Biophysique Moléculaire (plateforme) - Molecular Biophysics (platform), This work was supported by grants from the French Government ‘Laboratory of Excellence - Integrative Biology of Emerging Infectious Diseases’ (LabEx IBEID, grant number ANR-10-LABX-62-IBEID to PTC), the Fondation pour la Recherche Médicale (FRM grant number DEQ20181039599 to PTC)., We acknowledge SOLEIL for provision of synchrotron radiation facilities, and we thank the staff of beamlines PROXIMA-1 and PROXIMA-2A for assistance. The authors are grateful to the Staff of the Crystallography platform at the Institut Pasteur for robot-driven crystallization screenings. We acknowledge Eduard Baquero Salazar for his help during the early stage of this work. We also acknowledge the help of Mariette Matondo and Thibault Chaze from the Proteomics facility of Institut Pasteur., and ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010)

Subjects
CbpB protein, Cyclic diadenosine monophosphate (c‐di‐AMP), [SDV]Life Sciences [q-bio], X‐ray crystallography & binding, Streptococcus, Cell Biology, Potassium homeostasis, CBS domain, Molecular Biology, Biochemistry
Abstract

International audience; Cyclic di-AMP is an essential signaling molecule in Gram-positive bacteria. This second messenger regulates the osmotic pressure of the cell by interacting directly with the regulatory domains, either RCK_C or CBS domains, of several potassium and osmolyte uptake membrane protein systems. Cyclic di-AMP also targets stand-alone CBS domain proteins such as DarB in Bacillus subtilis and CbpB in Listeria monocytogenes. We show here that the CbpB protein of Group B Streptococcus binds c-di-AMP with a very high affinity. Crystal structures of CbpB reveal the determinants of binding specificity and significant conformational changes occurring upon c-di-AMP binding. Deletion of the cbpB gene alters bacterial growth in low potassium conditions most likely due to a decrease in the amount of ppGpp caused by a loss of interaction between CbpB and Rel, the GTP/GDP pyrophosphokinase.

Published
2023

2. The crystal structure of PknI from Mycobacterium tuberculosis shows an inactive, pseudokinase-like conformation

Author

Tristan Wagner, Marco Bellinzoni, Nathalie Barilone, Matthieu Alexandre, Bertrand Raynal, Pedro M. Alzari, María-Natalia Lisa, Microbiologie structurale - Structural Microbiology (Microb. Struc. (UMR_3528 / U-Pasteur_5)), Institut Pasteur [Paris]-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Biophysique Moléculaire (plateforme) - Molecular Biophysics (platform), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), This work was partially supported by grants from the Institut Pasteur and the CNRS., We thank Ahmed Haouz and Patrick Weber (Institut Pasteur) for their help with robot‐driven crystallization screenings, Bruno Baron for the assistance during circular dichroism measurements, and Clément Moroldo for his work in optimizing cryoprotection of PknI crystals. We acknowledge the synchrotron sources Soleil (Saint‐Aubin, France) and ESRF (Grenoble, France) for granting access to their facilities, and their staff members for the helpful assistance., Institut Pasteur [Paris] (IP)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, Models, Molecular, Protein Conformation, alpha-Helical, MESH: Mycobacterium tuberculosis, MESH: Sequence Homology, Amino Acid, Gene Expression, MESH: Amino Acid Sequence, Crystallography, X-Ray, Biochemistry, SER/THR KINASE, purl.org/becyt/ford/1 [https], MESH: Recombinant Proteins, ACTIVATION SEGMENT, Cloning, Molecular, MESH: Bacterial Proteins, Conserved Sequence, MESH: Conserved Sequence, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Kinase, Chemistry, MESH: Escherichia coli, SIGNAL TRANSDUCTION, computer.file_format, Recombinant Proteins, 3. Good health, Cell biology, X-RAY CRYSTALLOGRAPHY, MESH: Protein Conformation, beta-Strand, Signal transduction, CIENCIAS NATURALES Y EXACTAS, signal transduction, MESH: Models, Molecular, Protein Binding, MESH: Gene Expression, Otras Ciencias Biológicas, [PHYS.PHYS.PHYS-BIO-PH]Physics [physics]/Physics [physics]/Biological Physics [physics.bio-ph], MESH: Sequence Alignment, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Protein Serine-Threonine Kinases, MESH: Protein-Serine-Threonine Kinases, Ciencias Biológicas, Mycobacterium tuberculosis, 03 medical and health sciences, Bacterial Proteins, Ser/Thr kinase, [CHIM.CRIS]Chemical Sciences/Cristallography, Escherichia coli, MESH: Protein Binding, MESH: Cloning, Molecular, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Protein Interaction Domains and Motifs, Amino Acid Sequence, Binding site, Kinase activity, purl.org/becyt/ford/1.6 [https], Molecular Biology, X-ray crystallography, MESH: Protein Conformation, alpha-Helical, MESH: Protein Interaction Domains and Motifs, Binding Sites, Sequence Homology, Amino Acid, Cell Biology, Atomic coordinates, Protein Data Bank, biology.organism_classification, MESH: Crystallography, X-Ray, 030104 developmental biology, activation segment, MESH: Binding Sites, Cytoplasm, Protein Conformation, beta-Strand, [INFO.INFO-BI]Computer Science [cs]/Bioinformatics [q-bio.QM], computer, Sequence Alignment
Abstract

Eukaryotic-like Ser/Thr protein kinases (ePKs) have been identified in many bacterial species, where they are known to mediate signalling mechanisms that share several features with their eukaryotic counterparts. In Mycobacterium tuberculosis, PknI is one of the 11 predicted ePKs and it has been related to bacterial virulence. In order to better understand the molecular basis of its role in mycobacterial signalling, we solved the crystal structure of the PknI cytoplasmic domain. We found that even though PknI possesses most conserved elements characteristic of Hanks-type kinases, it is degraded in several motifs that are essential for the ePKs catalytic activity. Most notably, PknI presents a remarkably short activation segment lacking a peptide–substrate binding site. Consistent with this observation and similar to earlier findings for eukaryotic pseudokinases, no kinase activity was detected for the catalytic domain of PknI, against different substrates and in various experimental conditions. Based on these results, we conclude that PknI may rely on unconventional mechanism(s) for kinase activity and/or it could play alternative role(s) in mycobacterial signalling. Database: Atomic coordinates and structure factors for the catalytic domain of M. tuberculosis PknI are in the Protein Data Bank under the accession codes 5M06 (wild-type PknI + ADP), 5M07 (PknI_C20A), 5M08 (PknI_C20A_R136A) and 5M09 (PknI_C20A_R136N). Fil: Lisa, María Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina. Universite de Paris V; Francia. Instituto Pasteur; Francia Fil: Wagner, Tristan. Instituto Pasteur; Francia. Universite de Paris V; Francia Fil: Alexandre, Matthieu. Instituto Pasteur; Francia. Universite de Paris V; Francia Fil: Barilone, Nathalie. Instituto Pasteur; Francia. Universite de Paris V; Francia Fil: Raynal, Bertrand. Instituto Pasteur; Francia. Universite de Paris V; Francia Fil: Alzari, Pedro M.. Instituto Pasteur; Francia. Universite de Paris V; Francia Fil: Bellinzoni, Marco. Instituto Pasteur; Francia. Universite de Paris V; Francia

Published
2016

3. Structural plasticity of the <scp>HHD</scp> 2 domain of whirlin

Author

Florence Cordier, Nicolas Wolff, Ahmed Haouz, Lucas Chataigner, Florent Delhommel, Frederick Saul, Récepteurs Canaux - Channel Receptors, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Collège doctoral [Sorbonne universités], Sorbonne Université (SU), Bioinformatique structurale - Structural Bioinformatics, Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], Cristallographie (Plateforme) - Crystallography (Platform), Financial support from the TGIR‐RMN‐THC Fr3050 CNRS for conducting the research is gratefully acknowledged. The authors acknowledge the Conseil Régional d'Ile de France for financial support through the SESAME 2014 NMRCHR program No. 14014526 (800 MHz spectrometer). This work was supported by the Programme Transversal de Recherche from Institut Pasteur (PTR grant no. 483 to NW), the Ministère de l'Enseignement Supérieur et de la Recherche (grant no. 883/2013 to FD), and the Comité Berthe Fouassier – Maladies de l'oeil de la Fondation de France (no. 00071779 to FD)., We acknowledge synchrotrons SOLEIL for provision of synchrotron radiation facilities and we would like to thank the staff of beamlines PROXIMA 1 and SWING for assistance. The authors are grateful to the staff of the Crystallographic platform at Institut Pasteur for robot‐driven crystallization screening. We thank Bertrand Raynal (Biophysics platform, Institut Pasteur) for SAXS expertise and helpful discussions and Amel Bahloul and Christine Petit (Génétique et Physiologie de l'Audition, Institut Pasteur) for helpful discussions., Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), and Collège Doctoral

Subjects
Models, Molecular, 0301 basic medicine, Gene isoform, Scaffold protein, Protein Conformation, [SDV]Life Sciences [q-bio], Usher syndrome, Sequence Homology, Protomer, Whirlin, 030105 genetics & heredity, Crystallography, X-Ray, Biochemistry, 03 medical and health sciences, Protein Domains, Scattering, Small Angle, otorhinolaryngologic diseases, medicine, Humans, [CHIM]Chemical Sciences, Amino Acid Sequence, Nonsyndromic deafness, Molecular Biology, Chemistry, Cilium, Stereocilia, Membrane Proteins, Cell Biology, medicine.disease, 030104 developmental biology, swapped dimer, Biophysics, Protein Multimerization, Harmonin Homology Domain, Function (biology)
Abstract

International audience; Whirlin is a protein essential to sensory neurons. Its defects are responsible for nonsyndromic deafness or for the Usher syndrome, a condition associating congenital deafness and progressive blindness. This large multidomain scaffolding protein is expressed in three isoforms with different functions and localizations in stereocilia bundles of hearing hair cells or in the connecting cilia of photoreceptor cells. The HHD2 domain of whirlin is the only domain shared by all isoforms, but its function remains unknown. In this article, we report its crystal structure in two distinct conformations, a monomeric five‐helix bundle, similar to the known structure of other HHD domains, and a three‐helix bundle organized as a swapped dimer. Most of the hydrophobic contacts and electrostatic interactions that maintain the globular monomeric form are conserved at the protomer interface of the dimer. NMR experiments revealed that the five‐helix conformation is predominant in solution, but exhibits increased dynamics on one face encompassing the hinge loops. Using NMR and SAXS, we also show that HHD2 does not interact with its preceding domains. Our findings suggest that structural plasticity might play a role in the function of the HHD2 domain

Published
2018

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