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Research Article

Structural and Functional Insights into Peptidoglycan Access for the Lytic Amidase LytA of Streptococcus pneumoniae

Peter Mellroth, Tatyana Sandalova, Alexey Kikhney, Francisco Vilaplana, Dusan Hesek, Mijoon Lee, Shahriar Mobashery, Staffan Normark, Dmitri Svergun, Birgitta Henriques-Normark, Adnane Achour
Rino Rappuoli, Editor
Peter Mellroth
aDepartment of Microbiology, Tumor and Cell Biology (MTC), Karolinska Institutet, Stockholm, Sweden
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Tatyana Sandalova
bScience for Life Laboratory, Center for Infectious Medicine (CIM), Department of Medicine, Karolinska University Hospital Huddinge, Karolinska Institutet, Stockholm, Sweden
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Alexey Kikhney
cEuropean Molecular Biology Laboratory (EMBL), Hamburg Outstation, Hamburg, Germany
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Francisco Vilaplana
dDivision of Glycoscience, School of Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Centre, Stockholm, Sweden
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Dusan Hesek
eDepartments of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana, USA
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Mijoon Lee
eDepartments of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana, USA
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Shahriar Mobashery
eDepartments of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana, USA
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Staffan Normark
aDepartment of Microbiology, Tumor and Cell Biology (MTC), Karolinska Institutet, Stockholm, Sweden
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Dmitri Svergun
cEuropean Molecular Biology Laboratory (EMBL), Hamburg Outstation, Hamburg, Germany
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Birgitta Henriques-Normark
aDepartment of Microbiology, Tumor and Cell Biology (MTC), Karolinska Institutet, Stockholm, Sweden
fDepartment of Laboratory Medicine, Division of Clinical Microbiology, Karolinska University Hospital, Stockholm, Sweden
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Adnane Achour
bScience for Life Laboratory, Center for Infectious Medicine (CIM), Department of Medicine, Karolinska University Hospital Huddinge, Karolinska Institutet, Stockholm, Sweden
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Rino Rappuoli
Novartis Vaccines and Diagnostics
Roles: Editor
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DOI: 10.1128/mBio.01120-13
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ABSTRACT

The cytosolic N-acetylmuramoyl-l-alanine amidase LytA protein of Streptococcus pneumoniae, which is released by bacterial lysis, associates with the cell wall via its choline-binding motif. During exponential growth, LytA accesses its peptidoglycan substrate to cause lysis only when nascent peptidoglycan synthesis is stalled by nutrient starvation or β-lactam antibiotics. Here we present three-dimensional structures of LytA and establish the requirements for substrate binding and catalytic activity. The solution structure of the full-length LytA dimer reveals a peculiar fold, with the choline-binding domains forming a rigid V-shaped scaffold and the relatively more flexible amidase domains attached in a trans position. The 1.05-Å crystal structure of the amidase domain reveals a prominent Y-shaped binding crevice composed of three contiguous subregions, with a zinc-containing active site localized at the bottom of the branch point. Site-directed mutagenesis was employed to identify catalytic residues and to investigate the relative impact of potential substrate-interacting residues lining the binding crevice for the lytic activity of LytA. In vitro activity assays using defined muropeptide substrates reveal that LytA utilizes a large substrate recognition interface and requires large muropeptide substrates with several connected saccharides that interact with all subregions of the binding crevice for catalysis. We hypothesize that the substrate requirements restrict LytA to the sites on the cell wall where nascent peptidoglycan synthesis occurs.

IMPORTANCE Streptococcus pneumoniae is a human respiratory tract pathogen responsible for millions of deaths annually. Its major pneumococcal autolysin, LytA, is required for autolysis and fratricidal lysis and functions as a virulence factor that facilitates the spread of toxins and factors involved in immune evasion. LytA is also activated by penicillin and vancomycin and is responsible for the lysis induced by these antibiotics. The factors that regulate the lytic activity of LytA are unclear, but it was recently demonstrated that control is at the level of substrate recognition and that LytA required access to the nascent peptidoglycan. The present study was undertaken to structurally and functionally investigate LytA and its substrate-interacting interface and to determine the requirements for substrate recognition and catalysis. Our results reveal that the amidase domain comprises a complex substrate-binding crevice and needs to interact with a large-motif epitope of peptidoglycan for catalysis.

  • Copyright © 2014 Mellroth et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Structural and Functional Insights into Peptidoglycan Access for the Lytic Amidase LytA of Streptococcus pneumoniae
Peter Mellroth, Tatyana Sandalova, Alexey Kikhney, Francisco Vilaplana, Dusan Hesek, Mijoon Lee, Shahriar Mobashery, Staffan Normark, Dmitri Svergun, Birgitta Henriques-Normark, Adnane Achour
mBio Feb 2014, 5 (1) e01120-13; DOI: 10.1128/mBio.01120-13

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Structural and Functional Insights into Peptidoglycan Access for the Lytic Amidase LytA of Streptococcus pneumoniae
Peter Mellroth, Tatyana Sandalova, Alexey Kikhney, Francisco Vilaplana, Dusan Hesek, Mijoon Lee, Shahriar Mobashery, Staffan Normark, Dmitri Svergun, Birgitta Henriques-Normark, Adnane Achour
mBio Feb 2014, 5 (1) e01120-13; DOI: 10.1128/mBio.01120-13
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