Structural and dynamic insights revealing how lipase binding domain MD1 of Pseudomonas aeruginosa foldase affects lipase activation

Abstract Folding and cellular localization of many proteins of Gram-negative bacteria rely on a network of chaperones and secretion systems. Among them is the lipase-specific foldase Lif, a membrane-bound steric chaperone that tightly binds (K D = 29 nM) and mediates folding of the lipase LipA, a vi...

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Autores principales:	Aldino Viegas, Peter Dollinger, Neha Verma, Jakub Kubiak, Thibault Viennet, Claus A. M. Seidel, Holger Gohlke, Manuel Etzkorn, Filip Kovacic, Karl-Erich Jaeger
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2020
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/1f6b74cffdfc456e8750adee00c92bc3
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spelling	oai:doaj.org-article:1f6b74cffdfc456e8750adee00c92bc32021-12-02T15:54:10ZStructural and dynamic insights revealing how lipase binding domain MD1 of Pseudomonas aeruginosa foldase affects lipase activation10.1038/s41598-020-60093-42045-2322https://doaj.org/article/1f6b74cffdfc456e8750adee00c92bc32020-02-01T00:00:00Zhttps://doi.org/10.1038/s41598-020-60093-4https://doaj.org/toc/2045-2322Abstract Folding and cellular localization of many proteins of Gram-negative bacteria rely on a network of chaperones and secretion systems. Among them is the lipase-specific foldase Lif, a membrane-bound steric chaperone that tightly binds (K D = 29 nM) and mediates folding of the lipase LipA, a virulence factor of the pathogenic bacterium P. aeruginosa. Lif consists of five-domains, including a mini domain MD1 essential for LipA folding. However, the molecular mechanism of Lif-assisted LipA folding remains elusive. Here, we show in in vitro experiments using a soluble form of Lif (sLif) that isolated MD1 inhibits sLif-assisted LipA activation. Furthermore, the ability to activate LipA is lost in the variant sLifY99A, in which the evolutionary conserved amino acid Y99 from helix α1 of MD1 is mutated to alanine. This coincides with an approximately three-fold reduced affinity of the variant to LipA together with increased flexibility of sLifY99A in the complex as determined by polarization-resolved fluorescence spectroscopy. We have solved the NMR solution structures of P. aeruginosa MD1 and variant MD1Y99A revealing a similar fold indicating that a structural modification is likely not the reason for the impaired activity of variant sLifY99A. Molecular dynamics simulations of the sLif:LipA complex in connection with rigidity analyses suggest a long-range network of interactions spanning from Y99 of sLif to the active site of LipA, which might be essential for LipA activation. These findings provide important details about the putative mechanism for LipA activation and point to a general mechanism of protein folding by multi-domain steric chaperones.Aldino ViegasPeter DollingerNeha VermaJakub KubiakThibault ViennetClaus A. M. SeidelHolger GohlkeManuel EtzkornFilip KovacicKarl-Erich JaegerNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 10, Iss 1, Pp 1-15 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Aldino Viegas Peter Dollinger Neha Verma Jakub Kubiak Thibault Viennet Claus A. M. Seidel Holger Gohlke Manuel Etzkorn Filip Kovacic Karl-Erich Jaeger Structural and dynamic insights revealing how lipase binding domain MD1 of Pseudomonas aeruginosa foldase affects lipase activation
description	Abstract Folding and cellular localization of many proteins of Gram-negative bacteria rely on a network of chaperones and secretion systems. Among them is the lipase-specific foldase Lif, a membrane-bound steric chaperone that tightly binds (K D = 29 nM) and mediates folding of the lipase LipA, a virulence factor of the pathogenic bacterium P. aeruginosa. Lif consists of five-domains, including a mini domain MD1 essential for LipA folding. However, the molecular mechanism of Lif-assisted LipA folding remains elusive. Here, we show in in vitro experiments using a soluble form of Lif (sLif) that isolated MD1 inhibits sLif-assisted LipA activation. Furthermore, the ability to activate LipA is lost in the variant sLifY99A, in which the evolutionary conserved amino acid Y99 from helix α1 of MD1 is mutated to alanine. This coincides with an approximately three-fold reduced affinity of the variant to LipA together with increased flexibility of sLifY99A in the complex as determined by polarization-resolved fluorescence spectroscopy. We have solved the NMR solution structures of P. aeruginosa MD1 and variant MD1Y99A revealing a similar fold indicating that a structural modification is likely not the reason for the impaired activity of variant sLifY99A. Molecular dynamics simulations of the sLif:LipA complex in connection with rigidity analyses suggest a long-range network of interactions spanning from Y99 of sLif to the active site of LipA, which might be essential for LipA activation. These findings provide important details about the putative mechanism for LipA activation and point to a general mechanism of protein folding by multi-domain steric chaperones.
format	article
author	Aldino Viegas Peter Dollinger Neha Verma Jakub Kubiak Thibault Viennet Claus A. M. Seidel Holger Gohlke Manuel Etzkorn Filip Kovacic Karl-Erich Jaeger
author_facet	Aldino Viegas Peter Dollinger Neha Verma Jakub Kubiak Thibault Viennet Claus A. M. Seidel Holger Gohlke Manuel Etzkorn Filip Kovacic Karl-Erich Jaeger
author_sort	Aldino Viegas
title	Structural and dynamic insights revealing how lipase binding domain MD1 of Pseudomonas aeruginosa foldase affects lipase activation
title_short	Structural and dynamic insights revealing how lipase binding domain MD1 of Pseudomonas aeruginosa foldase affects lipase activation
title_full	Structural and dynamic insights revealing how lipase binding domain MD1 of Pseudomonas aeruginosa foldase affects lipase activation
title_fullStr	Structural and dynamic insights revealing how lipase binding domain MD1 of Pseudomonas aeruginosa foldase affects lipase activation
title_full_unstemmed	Structural and dynamic insights revealing how lipase binding domain MD1 of Pseudomonas aeruginosa foldase affects lipase activation
title_sort	structural and dynamic insights revealing how lipase binding domain md1 of pseudomonas aeruginosa foldase affects lipase activation
publisher	Nature Portfolio
publishDate	2020
url	https://doaj.org/article/1f6b74cffdfc456e8750adee00c92bc3
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_version_	1718385437417930752

Structural and dynamic insights revealing how lipase binding domain MD1 of Pseudomonas aeruginosa foldase affects lipase activation

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