Structure and Properties of a Natural Competence-Associated Pilin Suggest a Unique Pilus Tip-Associated DNA Receptor

ABSTRACT Natural competence is the term used to describe the uptake of “naked” extracellular DNA by bacteria; it plays a significant role in horizontal genetic exchange. It is associated with type IV pili, and specialized competence pili mediate DNA uptake. Here, we show that the crystal structure o...

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Autores principales:	Mohd Zulkifli Salleh, Vijaykumar Karuppiah, Matthew Snee, Angela Thistlethwaite, Colin W. Levy, David Knight, Jeremy P. Derrick
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2019
Materias:	X-ray crystallography genetic competence natural transformation systems pilus assembly surface receptor Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/659fe2932b8743b2a327295f116a2110
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spelling	oai:doaj.org-article:659fe2932b8743b2a327295f116a21102021-11-15T15:55:25ZStructure and Properties of a Natural Competence-Associated Pilin Suggest a Unique Pilus Tip-Associated DNA Receptor10.1128/mBio.00614-192150-7511https://doaj.org/article/659fe2932b8743b2a327295f116a21102019-06-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.00614-19https://doaj.org/toc/2150-7511ABSTRACT Natural competence is the term used to describe the uptake of “naked” extracellular DNA by bacteria; it plays a significant role in horizontal genetic exchange. It is associated with type IV pili, and specialized competence pili mediate DNA uptake. Here, we show that the crystal structure of a competence-associated protein from Thermus thermophilus, ComZ, consists of a type II secretion pseudopilin-like domain, with a large β-solenoid domain inserted into the β-sheet of the pilin-like fold. ComZ binds with high affinity to another competence-associated pilin, PilA2, which lies adjacent to the comZ gene in the genome. The crystal structure of PilA2 revealed a similar type II secretion pseudopilin-like fold, with a small subdomain; docking simulations predicted that PilA2 binds between the pseudopilin-like and β-solenoid domains of ComZ. Electrophoretic shift analysis and DNase protection studies were used to show that ComZ alone and the ComZ/PilA2 complex are able to bind DNA. Protection against reductive dimethylation was used in combination with mass spectrometry and site-directed mutagenesis to identify two lysine residues in ComZ which are involved in DNA binding. They are located between the two domains in ComZ, on the opposite side from the predicted PilA2 binding site. These results suggest a model in which PilA2 assists ComZ in forming the competence pilus tip and DNA binds to the side of the fiber. The results demonstrate how a type IV pilin can be adapted to a specific function by domain insertion and provide the first structural insights into a tip-located competence pilin. IMPORTANCE Thermus thermophilus is a thermophilic bacterium which is capable of natural transformation, the uptake of external DNA with high efficiency. DNA uptake is thought to be mediated by a competence-associated pilus, which binds the DNA substrate and mediates its transfer across the outer membrane and periplasm. Here, we describe the structural and functional analysis of two pilins which are known to be essential for DNA uptake, ComZ and PilA2. ComZ adopts an unusual structure, incorporating a large β-solenoid domain into the pilin structural framework. We argue on structural grounds that this structure cannot readily be accommodated into the competence pilus fiber unless it is at the tip. We also show that ComZ binds DNA and identify two lysine residues which appear to be important for DNA binding. These results suggest a model in which ComZ and PilA2 form a tip-associated DNA receptor which mediates DNA uptake.Mohd Zulkifli SallehVijaykumar KaruppiahMatthew SneeAngela ThistlethwaiteColin W. LevyDavid KnightJeremy P. DerrickAmerican Society for MicrobiologyarticleX-ray crystallographygenetic competencenatural transformation systemspilus assemblysurface receptorMicrobiologyQR1-502ENmBio, Vol 10, Iss 3 (2019)
institution	DOAJ
collection	DOAJ
language	EN
topic	X-ray crystallography genetic competence natural transformation systems pilus assembly surface receptor Microbiology QR1-502
spellingShingle	X-ray crystallography genetic competence natural transformation systems pilus assembly surface receptor Microbiology QR1-502 Mohd Zulkifli Salleh Vijaykumar Karuppiah Matthew Snee Angela Thistlethwaite Colin W. Levy David Knight Jeremy P. Derrick Structure and Properties of a Natural Competence-Associated Pilin Suggest a Unique Pilus Tip-Associated DNA Receptor
description	ABSTRACT Natural competence is the term used to describe the uptake of “naked” extracellular DNA by bacteria; it plays a significant role in horizontal genetic exchange. It is associated with type IV pili, and specialized competence pili mediate DNA uptake. Here, we show that the crystal structure of a competence-associated protein from Thermus thermophilus, ComZ, consists of a type II secretion pseudopilin-like domain, with a large β-solenoid domain inserted into the β-sheet of the pilin-like fold. ComZ binds with high affinity to another competence-associated pilin, PilA2, which lies adjacent to the comZ gene in the genome. The crystal structure of PilA2 revealed a similar type II secretion pseudopilin-like fold, with a small subdomain; docking simulations predicted that PilA2 binds between the pseudopilin-like and β-solenoid domains of ComZ. Electrophoretic shift analysis and DNase protection studies were used to show that ComZ alone and the ComZ/PilA2 complex are able to bind DNA. Protection against reductive dimethylation was used in combination with mass spectrometry and site-directed mutagenesis to identify two lysine residues in ComZ which are involved in DNA binding. They are located between the two domains in ComZ, on the opposite side from the predicted PilA2 binding site. These results suggest a model in which PilA2 assists ComZ in forming the competence pilus tip and DNA binds to the side of the fiber. The results demonstrate how a type IV pilin can be adapted to a specific function by domain insertion and provide the first structural insights into a tip-located competence pilin. IMPORTANCE Thermus thermophilus is a thermophilic bacterium which is capable of natural transformation, the uptake of external DNA with high efficiency. DNA uptake is thought to be mediated by a competence-associated pilus, which binds the DNA substrate and mediates its transfer across the outer membrane and periplasm. Here, we describe the structural and functional analysis of two pilins which are known to be essential for DNA uptake, ComZ and PilA2. ComZ adopts an unusual structure, incorporating a large β-solenoid domain into the pilin structural framework. We argue on structural grounds that this structure cannot readily be accommodated into the competence pilus fiber unless it is at the tip. We also show that ComZ binds DNA and identify two lysine residues which appear to be important for DNA binding. These results suggest a model in which ComZ and PilA2 form a tip-associated DNA receptor which mediates DNA uptake.
format	article
author	Mohd Zulkifli Salleh Vijaykumar Karuppiah Matthew Snee Angela Thistlethwaite Colin W. Levy David Knight Jeremy P. Derrick
author_facet	Mohd Zulkifli Salleh Vijaykumar Karuppiah Matthew Snee Angela Thistlethwaite Colin W. Levy David Knight Jeremy P. Derrick
author_sort	Mohd Zulkifli Salleh
title	Structure and Properties of a Natural Competence-Associated Pilin Suggest a Unique Pilus Tip-Associated DNA Receptor
title_short	Structure and Properties of a Natural Competence-Associated Pilin Suggest a Unique Pilus Tip-Associated DNA Receptor
title_full	Structure and Properties of a Natural Competence-Associated Pilin Suggest a Unique Pilus Tip-Associated DNA Receptor
title_fullStr	Structure and Properties of a Natural Competence-Associated Pilin Suggest a Unique Pilus Tip-Associated DNA Receptor
title_full_unstemmed	Structure and Properties of a Natural Competence-Associated Pilin Suggest a Unique Pilus Tip-Associated DNA Receptor
title_sort	structure and properties of a natural competence-associated pilin suggest a unique pilus tip-associated dna receptor
publisher	American Society for Microbiology
publishDate	2019
url	https://doaj.org/article/659fe2932b8743b2a327295f116a2110
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Structure and Properties of a Natural Competence-Associated Pilin Suggest a Unique Pilus Tip-Associated DNA Receptor

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