The TPR domain of PgaA is a multifunctional scaffold that binds PNAG and modulates PgaB-dependent polymer processing

Pfoh, Roland; Subramanian, Adithya S.; Huang, Jingjing; Little, Dustin J.; Forman, Adam; DiFrancesco, Benjamin R.; Balouchestani-Asli, Negar; Kitova, Elena N.; Klassen, John S.; Pomès, Régis; Nitz, Mark; Howell, P. Lynne

doi:10.1371/journal.ppat.1010750

Title: The TPR domain of PgaA is a multifunctional scaffold that binds PNAG and modulates PgaB-dependent polymer processing

Journal Article · Fri Aug 05 00:00:00 EDT 2022 · PLoS Pathogens

DOI:https://doi.org/10.1371/journal.ppat.1010750· OSTI ID:1903801

Pfoh, Roland ^[1]; Subramanian, Adithya S. ^[2]; Huang, Jingjing ^[2]; Little, Dustin J. ^[2]; Forman, Adam ^[3]; DiFrancesco, Benjamin R. ^[3]; Balouchestani-Asli, Negar ^[1]; Kitova, Elena N. ^[4]; Klassen, John S. ^[4]; Pomès, Régis ^[1]; Nitz, Mark ^[3];

^[2]

Hospital for Sick Children, Toronto (Canada)
Hospital for Sick Children, Toronto (Canada); University of Toronto, ON (Canada)
University of Toronto, ON (Canada)
University of Alberta, Edmonton, AB (Canada)

The synthesis of exopolysaccharides as biofilm matrix components by pathogens is a crucial factor for chronic infections and antibiotic resistance. Many periplasmic proteins involved in polymer processing and secretion in Gram-negative synthase dependent exopolysaccharide biosynthetic systems have been individually characterized. The operons responsible for the production of PNAG, alginate, cellulose and the Pel polysaccharide each contain a gene that encodes an outer membrane associated tetratricopeptide repeat (TPR) domain containing protein. While the TPR domain has been shown to bind other periplasmic proteins, the functional consequences of these interactions for the polymer remain poorly understood. Herein, we show that the C-terminal TPR region of PgaA interacts with the de-N-acetylase domain of PgaB, and increases its deacetylase activity. Additionally, we found that when the two proteins form a complex, the glycoside hydrolase activity of PgaB is also increased. To better understand structure-function relationships we determined the crystal structure of a stable TPR module, which has a conserved groove formed by three repeat motifs. Tryptophan quenching, mass spectrometry analysis and molecular dynamics simulation studies suggest that the crystallized TPR module can bind PNAG/dPNAG via its electronegative groove on the concave surface, and potentially guide the polymer through the periplasm towards the porin for export. Our results suggest a scaffolding role for the TPR domain that combines PNAG/dPNAG translocation with the modulation of its chemical structure by PgaB.

View Accepted Manuscript (DOE)

Cite

Export

Save

Research Organization:: Brookhaven National Lab. (BNL), Upton, NY (United States); Hospital for Sick Children, Toronto (Canada)

Sponsoring Organization:: USDOE; Canadian Institutes of Health Research (CIHR); Natural Sciences and Engineering Research Council of Canada (NSERC); Hospital for Sick Children Foundation Student Scholarship Program; University of Toronto; Canadian Foundation for Innovation; National Institutes of Health (NIH)

Grant/Contract Number:: MOP 43998; FDN154327

OSTI ID:: 1903801

Journal Information:: PLoS Pathogens, Vol. 18, Issue 8; ISSN 1553-7374

Publisher:: Public Library of ScienceCopyright Statement

Country of Publication:: United States

Language:: English

References (54)

Synthase-dependent exopolysaccharide secretion in Gram-negative bacteria Whitney, J. C.; Howell, P. L. Trends in Microbiology, Vol. 21, Issue 2 https://doi.org/10.1016/j.tim.2012.10.001	journal	February 2013
Combining in situ proteolysis and mass spectrometry to crystallize Escherichia coli PgaB Little, Dustin J.; Whitney, John C.; Robinson, Howard Acta Crystallographica Section F Structural Biology and Crystallization Communications, Vol. 68, Issue 7 https://doi.org/10.1107/S1744309112022075	journal	June 2012
Architecture of the Cellulose Synthase Outer Membrane Channel and Its Association with the Periplasmic TPR Domain Acheson, Justin F.; Derewenda, Zygmunt S.; Zimmer, Jochen Structure, Vol. 27, Issue 12 https://doi.org/10.1016/j.str.2019.09.008	journal	December 2019
Discovery and characterization of a Gram-positive Pel polysaccharide biosynthetic gene cluster Whitfield, Gregory B.; Marmont, Lindsey S.; Bundalovic-Torma, Cedoljub PLOS Pathogens, Vol. 16, Issue 4 https://doi.org/10.1371/journal.ppat.1008281	journal	April 2020
A Quantitative Chaperone Interaction Network Reveals the Architecture of Cellular Protein Homeostasis Pathways Taipale, Mikko; Tucker, George; Peng, Jian Cell, Vol. 158, Issue 2 https://doi.org/10.1016/j.cell.2014.05.039	journal	July 2014
Structural Basis for Translocation of a Biofilm-supporting Exopolysaccharide across the Bacterial Outer Membrane Wang, Yan; Andole Pannuri, Archana; Ni, Dongchun Journal of Biological Chemistry, Vol. 291, Issue 19 https://doi.org/10.1074/jbc.M115.711762	journal	March 2016
Crystal structure of the flexible tandem repeat domain of bacterial cellulose synthesis subunit C Nojima, Shingo; Fujishima, Ayumi; Kato, Koji Scientific Reports, Vol. 7, Issue 1 https://doi.org/10.1038/s41598-017-12530-0	journal	October 2017
Reliable Determinations of Protein–Ligand Interactions by Direct ESI-MS Measurements. Are We There Yet? Kitova, Elena N.; El-Hawiet, Amr; Schnier, Paul D. Journal of The American Society for Mass Spectrometry, Vol. 23, Issue 3 https://doi.org/10.1007/s13361-011-0311-9	journal	January 2012
Collaboration gets the most out of software Morin, Andrew; Eisenbraun, Ben; Key, Jason eLife, Vol. 2 https://doi.org/10.7554/eLife.01456	journal	September 2013
A Completely Reimplemented MPI Bioinformatics Toolkit with a New HHpred Server at its Core Zimmermann, Lukas; Stephens, Andrew; Nam, Seung-Zin Journal of Molecular Biology, Vol. 430, Issue 15 https://doi.org/10.1016/j.jmb.2017.12.007	journal	July 2018
The superhelical TPR-repeat domain of O-linked GlcNAc transferase exhibits structural similarities to importin α Jínek, Martin; Rehwinkel, Jan; Lazarus, Brooke D. Nature Structural & Molecular Biology, Vol. 11, Issue 10 https://doi.org/10.1038/nsmb833	journal	September 2004
P-LINCS: A Parallel Linear Constraint Solver for Molecular Simulation Hess, Berk Journal of Chemical Theory and Computation, Vol. 4, Issue 1 https://doi.org/10.1021/ct700200b	journal	December 2007
The Phyre2 web portal for protein modeling, prediction and analysis Kelley, Lawrence A.; Mezulis, Stefans; Yates, Christopher M. Nature Protocols, Vol. 10, Issue 6 https://doi.org/10.1038/nprot.2015.053	journal	May 2015
Analysis of HmsH and its role in plague biofilm formation Abu Khweek, Arwa; Fetherston, Jacqueline D.; Perry, Robert D. Microbiology, Vol. 156, Issue 5 https://doi.org/10.1099/mic.0.036640-0	journal	May 2010
Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions Krissinel, E.; Henrick, K. Acta Crystallographica Section D Biological Crystallography, Vol. 60, Issue 12 https://doi.org/10.1107/S0907444904026460	journal	November 2004
A Leap-frog Algorithm for Stochastic Dynamics Van Gunsteren, W. F.; Berendsen, H. J. C. Molecular Simulation, Vol. 1, Issue 3 https://doi.org/10.1080/08927028808080941	journal	March 1988
Alginate Polymerization and Modification Are Linked in Pseudomonas aeruginosa Moradali, M. Fata; Donati, Ivan; Sims, Ian M. mBio, Vol. 6, Issue 3 https://doi.org/10.1128/mBio.00453-15	journal	May 2015
AlphaFold Protein Structure Database: massively expanding the structural coverage of protein-sequence space with high-accuracy models Varadi, Mihaly; Anyango, Stephen; Deshpande, Mandar Nucleic Acids Research, Vol. 50, Issue D1 https://doi.org/10.1093/nar/gkab1061	journal	November 2021
GROMACS: High performance molecular simulations through multi-level parallelism from laptops to supercomputers Abraham, Mark James; Murtola, Teemu; Schulz, Roland SoftwareX, Vol. 1-2 https://doi.org/10.1016/j.softx.2015.06.001	journal	September 2015
Highly accurate protein structure prediction with AlphaFold Jumper, John; Evans, Richard; Pritzel, Alexander Nature https://doi.org/10.1038/s41586-021-03819-2	journal	July 2021
Apo- and Cellopentaose-bound Structures of the Bacterial Cellulose Synthase Subunit BcsZ Mazur, Olga; Zimmer, Jochen Journal of Biological Chemistry, Vol. 286, Issue 20 https://doi.org/10.1074/jbc.M111.227660	journal	March 2011
Comparison of multiple Amber force fields and development of improved protein backbone parameters Hornak, Viktor; Abel, Robert; Okur, Asim Proteins: Structure, Function, and Bioinformatics, Vol. 65, Issue 3 https://doi.org/10.1002/prot.21123	journal	November 2006
Sel1-like repeat proteins in signal transduction Mittl, Peer R. E.; Schneider-Brachert, Wulf Cellular Signalling, Vol. 19, Issue 1 https://doi.org/10.1016/j.cellsig.2006.05.034	journal	January 2007
Solution Properties of Hemicellulose Polysaccharides with Four Common Carbohydrate Force Fields Sauter, Jörg; Grafmüller, Andrea Journal of Chemical Theory and Computation, Vol. 11, Issue 4 https://doi.org/10.1021/ct500924f	journal	March 2015
The Pseudomonas aeruginosa homeostasis enzyme AlgL clears the periplasmic space of accumulated alginate during polymer biosynthesis Gheorghita, Andreea A.; Wolfram, Francis; Whitfield, Gregory B. Journal of Biological Chemistry, Vol. 298, Issue 2 https://doi.org/10.1016/j.jbc.2021.101560	journal	February 2022
Roles of pgaABCD Genes in Synthesis, Modification, and Export of the Escherichia coli Biofilm Adhesin Poly- -1,6-N-Acetyl-D-Glucosamine Itoh, Y.; Rice, J. D.; Goller, C. Journal of Bacteriology, Vol. 190, Issue 10 https://doi.org/10.1128/JB.01920-07	journal	March 2008
Crystal Structure and Pair Potentials: A Molecular-Dynamics Study Parrinello, M.; Rahman, A. Physical Review Letters, Vol. 45, Issue 14 https://doi.org/10.1103/PhysRevLett.45.1196	journal	October 1980
Scaffold Proteins: Hubs for Controlling the Flow of Cellular Information Good, M. C.; Zalatan, J. G.; Lim, W. A. Science, Vol. 332, Issue 6030 https://doi.org/10.1126/science.1198701	journal	May 2011
MDTraj: A Modern Open Library for the Analysis of Molecular Dynamics Trajectories McGibbon, Robert T.; Beauchamp, Kyle A.; Harrigan, Matthew P. Biophysical Journal, Vol. 109, Issue 8 https://doi.org/10.1016/j.bpj.2015.08.015	journal	October 2015
Gram-negative synthase-dependent exopolysaccharide biosynthetic machines Low, Kristin E.; Howell, P. Lynne Current Opinion in Structural Biology, Vol. 53 https://doi.org/10.1016/j.sbi.2018.05.001	journal	December 2018
Efficient synthesis and protein conjugation of β-(1→6)-d-N-acetylglucosamine oligosaccharides from the polysaccharide intercellular adhesin Leung, Carmen; Chibba, Anthony; Gómez-Biagi, Rodolfo F. Carbohydrate Research, Vol. 344, Issue 5 https://doi.org/10.1016/j.carres.2008.12.021	journal	March 2009
Toward the structural genomics of complexes: Crystal structure of a PE/PPE protein complex from Mycobacterium tuberculosis Strong, M.; Sawaya, M. R.; Wang, S. Proceedings of the National Academy of Sciences, Vol. 103, Issue 21 https://doi.org/10.1073/pnas.0602606103	journal	May 2006
GLYCAM06: A generalizable biomolecular force field. Carbohydrates: GLYCAM06 Kirschner, Karl N.; Yongye, Austin B.; Tschampel, Sarah M. Journal of Computational Chemistry, Vol. 29, Issue 4 https://doi.org/10.1002/jcc.20820	journal	September 2007
Synthesis of defined mono-de-N-acetylated β-(1→6)-N-acetyl-d-glucosamine oligosaccharides to characterize PgaB hydrolase activity Forman, Adam; Pfoh, Roland; Eddenden, Alexander Organic & Biomolecular Chemistry, Vol. 17, Issue 43 https://doi.org/10.1039/C9OB02079A	journal	January 2019
PgaB orthologues contain a glycoside hydrolase domain that cleaves deacetylated poly-β(1,6)-N-acetylglucosamine and can disrupt bacterial biofilms Little, Dustin J.; Pfoh, Roland; Le Mauff, François PLOS Pathogens, Vol. 14, Issue 4 https://doi.org/10.1371/journal.ppat.1006998	journal	April 2018
Modification and periplasmic translocation of the biofilm exopolysaccharide poly- -1,6-N-acetyl-D-glucosamine Little, D. J.; Li, G.; Ing, C. Proceedings of the National Academy of Sciences, Vol. 111, Issue 30 https://doi.org/10.1073/pnas.1406388111	journal	July 2014
Particle mesh Ewald: An N ⋅log( N ) method for Ewald sums in large systems Darden, Tom; York, Darrin; Pedersen, Lee The Journal of Chemical Physics, Vol. 98, Issue 12 https://doi.org/10.1063/1.464397	journal	June 1993
Fluorescamine: A Reagent for Assay of Amino Acids, Peptides, Proteins, and Primary Amines in the Picomole Range Udenfriend, S.; Stein, S.; Bohlen, P. Science, Vol. 178, Issue 4063 https://doi.org/10.1126/science.178.4063.871	journal	November 1972
Vectors for co-expression of an unrestricted number of proteins Scheich, C.; Kummel, D.; Soumailakakis, D. Nucleic Acids Research, Vol. 35, Issue 6 https://doi.org/10.1093/nar/gkm067	journal	March 2007
Coot model-building tools for molecular graphics Emsley, Paul; Cowtan, Kevin Acta Crystallographica Section D Biological Crystallography, Vol. 60, Issue 12, p. 2126-2132 https://doi.org/10.1107/S0907444904019158	journal	November 2004
Molecular dynamics with coupling to an external bath Berendsen, H. J. C.; Postma, J. P. M.; van Gunsteren, W. F. The Journal of Chemical Physics, Vol. 81, Issue 8 https://doi.org/10.1063/1.448118	journal	October 1984
Periplasmic space and the concept of the periplasm Graham, L. L.; Beveridge, T. J.; Nanninga, N. Trends in Biochemical Sciences, Vol. 16 https://doi.org/10.1016/0968-0004(91)90135-I	journal	January 1991
The Structure- and Metal-dependent Activity of Escherichia coli PgaB Provides Insight into the Partial De-N-acetylation of Poly-β-1,6-N-acetyl-d-glucosamine Little, Dustin J.; Poloczek, Joanna; Whitney, John C. Journal of Biological Chemistry, Vol. 287, Issue 37 https://doi.org/10.1074/jbc.M112.390005	journal	September 2012
Method for Distinguishing Specific from Nonspecific Protein−Ligand Complexes in Nanoelectrospray Ionization Mass Spectrometry Sun, Jiangxiao; Kitova, Elena N.; Wang, Weijie Analytical Chemistry, Vol. 78, Issue 9 https://doi.org/10.1021/ac0522005	journal	April 2006
Protein Sequence Analysis Using the MPI Bioinformatics Toolkit Gabler, Felix; Nam, Seung‐Zin; Till, Sebastian Current Protocols in Bioinformatics, Vol. 72, Issue 1 https://doi.org/10.1002/cpbi.108	journal	December 2020
An introduction to experimental phasing of macromolecules illustrated by SHELX ; new autotracing features Usón, Isabel; Sheldrick, George M. Acta Crystallographica Section D Structural Biology, Vol. 74, Issue 2 https://doi.org/10.1107/S2059798317015121	journal	February 2018
Domains, Motifs, and Scaffolds: The Role of Modular Interactions in the Evolution and Wiring of Cell Signaling Circuits Bhattacharyya, Roby P.; Reményi, Attila; Yeh, Brian J. Annual Review of Biochemistry, Vol. 75, Issue 1 https://doi.org/10.1146/annurev.biochem.75.103004.142710	journal	June 2006
Avogadro: an advanced semantic chemical editor, visualization, and analysis platform Hanwell, Marcus D.; Curtis, Donald E.; Lonie, David C. Journal of Cheminformatics, Vol. 4, Issue 1 https://doi.org/10.1186/1758-2946-4-17	journal	August 2012
A smooth particle mesh Ewald method Essmann, Ulrich; Perera, Lalith; Berkowitz, Max L. The Journal of Chemical Physics, Vol. 103, Issue 19 https://doi.org/10.1063/1.470117	journal	November 1995
ConSurf 2016: an improved methodology to estimate and visualize evolutionary conservation in macromolecules Ashkenazy, Haim; Abadi, Shiran; Martz, Eric Nucleic Acids Research, Vol. 44, Issue W1 https://doi.org/10.1093/nar/gkw408	journal	May 2016
PelA and PelB proteins form a modification and secretion complex essential for Pel polysaccharide-dependent biofilm formation in Pseudomonas aeruginosa Marmont, Lindsey S.; Whitfield, Gregory B.; Rich, Jacquelyn D. Journal of Biological Chemistry, Vol. 292, Issue 47 https://doi.org/10.1074/jbc.M117.812842	journal	November 2017
[20] Processing of X-ray diffraction data collected in oscillation mode Otwinowski, Zbyszek; Minor, Wladek Macromolecular Crystallography Part A, 307-326 https://doi.org/10.1016/S0076-6879(97)76066-X	book	January 1997
VMD: Visual molecular dynamics Humphrey, William; Dalke, Andrew; Schulten, Klaus Journal of Molecular Graphics, Vol. 14, Issue 1 https://doi.org/10.1016/0263-7855(96)00018-5	journal	February 1996
Activation Mechanism and Cellular Localization of Membrane-Anchored Alginate Polymerase in Pseudomonas aeruginosa Moradali, M. Fata; Ghods, Shirin; Rehm, Bernd H. A. Applied and Environmental Microbiology, Vol. 83, Issue 9 https://doi.org/10.1128/AEM.03499-16	journal	March 2017

Similar Records

AlgK is a TPR-containing Protein and the Periplasmic Component of a Novel Exopolysaccharide Secretin

Journal Article · Fri Jan 01 00:00:00 EST 2010 · Structure · OSTI ID:1903801

Keiski, C; Harwich, M; Jain, S; +6 more

Crystal Structures of the Tetratricopeptide Repeat Domains of Kinesin Light Chains: Insight into Cargo Recognition Mechanisms

Journal Article · Tue Oct 23 00:00:00 EDT 2012 · PLoS One · OSTI ID:1903801

Zhu, Haizhong; Lee, Han Youl; Tong, Yufeng; +7 more

Crystal structure of P58(IPK) TPR fragment reveals the mechanism for its molecular chaperone activity in UPR

Journal Article · Tue May 25 00:00:00 EDT 2010 · J. Mol. Biol. · OSTI ID:1903801

Tao, Jiahui; Petrova, Kseniya; Ron, David; +1 more

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
polymers
glycoside hydrolases
crystal structure
protein interactions
biochemical simulation
outer membrane protein
tryptophan
bacterial biofilms

Title: The TPR domain of PgaA is a multifunctional scaffold that binds PNAG and modulates PgaB-dependent polymer processing

Citation Formats

References (54)

Similar Records

Related Subjects