Buttressing ligand-binding pockets in de novo designed NTF2-like domains

NTF2-like proteins are compact α + β fold domains with cone-shaped architectures and internal pockets, making them attractive scaffolds for the de novo design of small-molecule binders and enzymes. However, creating ligand-binding pockets often compromises folding stability, posing a key challenge i...

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Detalhes bibliográficos
Autores: Nadal, Marta, Albi-Puig, Joana, Castellví, Albert, Garcia-Franco, Paula M., Vega, Sonia, Velázquez-Campoy, Adrián, Marcos, Enrique
Formato: artículo
Estado:Versión publicada
Fecha de publicación:2025
País:España
Recursos:Consejo Superior de Investigaciones Científicas (CSIC)
Repositorio:DIGITAL.CSIC. Repositorio Institucional del CSIC
OAI Identifier:oai:digital.csic.es:10261/412124
Acesso em linha:http://hdl.handle.net/10261/412124
Access Level:acceso abierto
Palavra-chave:NTF2‐like
Protein stability
X‐ray crystallography
Protein design
Beta‐sheets
Molecular dynamics simulations
Ligand‐binding
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spelling Buttressing ligand-binding pockets in de novo designed NTF2-like domainsNadal, MartaAlbi-Puig, JoanaCastellví, AlbertGarcia-Franco, Paula M.Vega, SoniaVelázquez-Campoy, AdriánMarcos, EnriqueNTF2‐likeProtein stabilityX‐ray crystallographyProtein designBeta‐sheetsMolecular dynamics simulationsLigand‐bindingNTF2-like proteins are compact α + β fold domains with cone-shaped architectures and internal pockets, making them attractive scaffolds for the de novo design of small-molecule binders and enzymes. However, creating ligand-binding pockets often compromises folding stability, posing a key challenge in de novo protein design. Here, we introduce strategies to stabilize NTF2-like domains while preserving pocket geometry and accessibility. By expanding the hydrophobic core through computationally designed α-helical subdomains or homodimer interfaces buttressing the β-sheet's convex face, we enhance structural stability without blocking pocket access on the concave face. Biochemical, biophysical, and crystallographic analyses confirm that the designed buttressing elements maintain the intended fold and support diverse, well-formed hydrophobic ligand-binding pockets with increased preorganization. Our results demonstrate that structural stabilization and pocket optimization need not be mutually exclusive, providing a generalizable approach to create robust ligand-binding proteins. This framework addresses a major bottleneck in protein design and should fuel the development of NTF2-based scaffolds for applications in small-molecule biosensing and enzyme catalysis.This research was supported by grants from the Spanish Ministry of Science and Innovation (RYC2018-025295-I, EUR2020-112164, CNS2023-144990) and AGAUR (2021SGR00423) to E.M. J.A-P. was supported by a FPU predoctoral fellowship (under grant agreement FPU23/01041). A.C. was supported by STFC-UK/CCP4 “Agreement for the integration of methods into the CCP4 software distribution, ARCIMBOLDO_LOW”. This work was supported by the Ministerio de Ciencia e Innovación (MCIN/AEI/10.13039/501100011033/) and “ERDF A way of Making Europe” (PID2021-127296OB-I00) to A.V.C.Peer reviewedWiley-VCHAgencia Estatal de Investigación (España)Ministerio de Ciencia, Innovación y Universidades (España)Science and Technology Facilities Council (UK)Generalitat de CatalunyaEuropean CommissionConsejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]202620262025info:eu-repo/semantics/articlehttp://purl.org/coar/resource_type/c_6501Publisher's versioninfo:eu-repo/semantics/publishedVersionapplication/pdfhttp://hdl.handle.net/10261/412124reponame:DIGITAL.CSIC. Repositorio Institucional del CSICinstname:Consejo Superior de Investigaciones Científicas (CSIC)Inglés#PLACEHOLDER_PARENT_METADATA_VALUE##PLACEHOLDER_PARENT_METADATA_VALUE##PLACEHOLDER_PARENT_METADATA_VALUE##PLACEHOLDER_PARENT_METADATA_VALUE#info:eu-repo/grantAgreement/AEI/Plan Estatal de Investigación Científica y Técnica y de Innovación 2017-2020/RYC2018-025295-Iinfo:eu-repo/grantAgreement/AEI/Plan Estatal de Investigación Científica y Técnica y de Innovación 2017-2020/EUR2020-112164info:eu-repo/grantAgreement/AEI/Plan Estatal de Investigación Científica y Técnica y de Innovación 2021-2023/CNS2023-144990info:eu-repo/grantAgreement/AEI/Plan Estatal de Investigación Científica y Técnica y de Innovación 2021-2023/PID2021-127296OB-I00The underlying dataset has been published as supplementary material of the article in the publisher platform at DOI 10.1002/pro.70323Nadal, Marta; Albi-Puig, Joana; Marcos Enrique; 2025; Buttressing ligand-binding pockets in de novo designed NTF2-like domains [Dataset]; Zenodo; https://doi.org/10.5281/zenodo.17053179https://doi.org/10.1002/pro.70323Síinfo:eu-repo/semantics/openAccessoai:digital.csic.es:10261/4121242026-05-22T06:33:51Z
dc.title.none.fl_str_mv Buttressing ligand-binding pockets in de novo designed NTF2-like domains
title Buttressing ligand-binding pockets in de novo designed NTF2-like domains
spellingShingle Buttressing ligand-binding pockets in de novo designed NTF2-like domains
Nadal, Marta
NTF2‐like
Protein stability
X‐ray crystallography
Protein design
Beta‐sheets
Molecular dynamics simulations
Ligand‐binding
title_short Buttressing ligand-binding pockets in de novo designed NTF2-like domains
title_full Buttressing ligand-binding pockets in de novo designed NTF2-like domains
title_fullStr Buttressing ligand-binding pockets in de novo designed NTF2-like domains
title_full_unstemmed Buttressing ligand-binding pockets in de novo designed NTF2-like domains
title_sort Buttressing ligand-binding pockets in de novo designed NTF2-like domains
dc.creator.none.fl_str_mv Nadal, Marta
Albi-Puig, Joana
Castellví, Albert
Garcia-Franco, Paula M.
Vega, Sonia
Velázquez-Campoy, Adrián
Marcos, Enrique
author Nadal, Marta
author_facet Nadal, Marta
Albi-Puig, Joana
Castellví, Albert
Garcia-Franco, Paula M.
Vega, Sonia
Velázquez-Campoy, Adrián
Marcos, Enrique
author_role author
author2 Albi-Puig, Joana
Castellví, Albert
Garcia-Franco, Paula M.
Vega, Sonia
Velázquez-Campoy, Adrián
Marcos, Enrique
author2_role author
author
author
author
author
author
dc.contributor.none.fl_str_mv Agencia Estatal de Investigación (España)
Ministerio de Ciencia, Innovación y Universidades (España)
Science and Technology Facilities Council (UK)
Generalitat de Catalunya
European Commission
Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]
dc.subject.none.fl_str_mv NTF2‐like
Protein stability
X‐ray crystallography
Protein design
Beta‐sheets
Molecular dynamics simulations
Ligand‐binding
topic NTF2‐like
Protein stability
X‐ray crystallography
Protein design
Beta‐sheets
Molecular dynamics simulations
Ligand‐binding
description NTF2-like proteins are compact α + β fold domains with cone-shaped architectures and internal pockets, making them attractive scaffolds for the de novo design of small-molecule binders and enzymes. However, creating ligand-binding pockets often compromises folding stability, posing a key challenge in de novo protein design. Here, we introduce strategies to stabilize NTF2-like domains while preserving pocket geometry and accessibility. By expanding the hydrophobic core through computationally designed α-helical subdomains or homodimer interfaces buttressing the β-sheet's convex face, we enhance structural stability without blocking pocket access on the concave face. Biochemical, biophysical, and crystallographic analyses confirm that the designed buttressing elements maintain the intended fold and support diverse, well-formed hydrophobic ligand-binding pockets with increased preorganization. Our results demonstrate that structural stabilization and pocket optimization need not be mutually exclusive, providing a generalizable approach to create robust ligand-binding proteins. This framework addresses a major bottleneck in protein design and should fuel the development of NTF2-based scaffolds for applications in small-molecule biosensing and enzyme catalysis.
publishDate 2025
dc.date.none.fl_str_mv 2025
2026
2026
dc.type.none.fl_str_mv info:eu-repo/semantics/article
http://purl.org/coar/resource_type/c_6501
Publisher's version
info:eu-repo/semantics/publishedVersion
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/10261/412124
url http://hdl.handle.net/10261/412124
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.relation.none.fl_str_mv #PLACEHOLDER_PARENT_METADATA_VALUE#
#PLACEHOLDER_PARENT_METADATA_VALUE#
#PLACEHOLDER_PARENT_METADATA_VALUE#
#PLACEHOLDER_PARENT_METADATA_VALUE#
info:eu-repo/grantAgreement/AEI/Plan Estatal de Investigación Científica y Técnica y de Innovación 2017-2020/RYC2018-025295-I
info:eu-repo/grantAgreement/AEI/Plan Estatal de Investigación Científica y Técnica y de Innovación 2017-2020/EUR2020-112164
info:eu-repo/grantAgreement/AEI/Plan Estatal de Investigación Científica y Técnica y de Innovación 2021-2023/CNS2023-144990
info:eu-repo/grantAgreement/AEI/Plan Estatal de Investigación Científica y Técnica y de Innovación 2021-2023/PID2021-127296OB-I00
The underlying dataset has been published as supplementary material of the article in the publisher platform at DOI 10.1002/pro.70323
Nadal, Marta; Albi-Puig, Joana; Marcos Enrique; 2025; Buttressing ligand-binding pockets in de novo designed NTF2-like domains [Dataset]; Zenodo; https://doi.org/10.5281/zenodo.17053179
https://doi.org/10.1002/pro.70323
Sí
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
dc.publisher.none.fl_str_mv Wiley-VCH
publisher.none.fl_str_mv Wiley-VCH
dc.source.none.fl_str_mv reponame:DIGITAL.CSIC. Repositorio Institucional del CSIC
instname:Consejo Superior de Investigaciones Científicas (CSIC)
instname_str Consejo Superior de Investigaciones Científicas (CSIC)
reponame_str DIGITAL.CSIC. Repositorio Institucional del CSIC
collection DIGITAL.CSIC. Repositorio Institucional del CSIC
repository.name.fl_str_mv
repository.mail.fl_str_mv
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