Relationship of thermostability and binding affinity in metal-binding WW-domain minireceptors

• Verfasst von: Pham, Truc Lam [VerfasserIn]
• Verfasst von: Conde González, Marcos Rafael [VerfasserIn]
• Verfasst von: Fazliev, Sunnatullo [VerfasserIn]
• Verfasst von: Kishore, Agi [VerfasserIn]
• Verfasst von: Comba, Peter [VerfasserIn]
• Verfasst von: Thomas, Franziska [VerfasserIn]
• Titel: Relationship of thermostability and binding affinity in metal-binding WW-domain minireceptors
• Verf.angabe: Truc Lam Pham, Marcos R. Conde González, Sunnatullo Fazliev, Agi Kishore, Peter Comba, and Franziska Thomas
• E-Jahr: 2024
• Jahr: February 16, 2024
• Umfang: 8 S.
• Fussnoten: Zuerst veröffentlicht: 21. Dezember 2023 ; Illustrationen ; Gesehen am 12.02.2024
• Titel Quelle: Enthalten in: ChemBioChem
• Ort Quelle: Weinheim : Wiley-VCH, 2000
• Jahr Quelle: 2024
• Band/Heft Quelle: 25(2024), 4 vom: Feb., Artikel-ID e202300715, Seite 1-8
• ISSN Quelle: 1439-7633
• DOI: doi:10.1002/cbic.202300715
• Datenträger: Online-Ressource
• Sprache: eng
• Bibliogr. Hinweis: Forschungsdaten: Pham, Truc Lam, 1995 - : Relationship of thermostability and binding affinity in metal-binding WW-domain minireceptors [research data]
• Sach-SW: metal binding
• Sach-SW: miniprotein
• Sach-SW: protein engineering
• Sach-SW: WW domain
• Sach-SW: β-sheet peptide
• K10plus-PPN: 1880538970

Abstract

The design of metallo-miniproteins advances our understanding of the structural and functional roles of metals in proteins. We recently designed a metal-binding WW domain, WW-CA-Nle, which displays three histidine residues on its surface for coordination of divalent metals Ni(II), Zn(II) and Cu(II). However, WW-CA-Nle is a molten globule in the apo state and thus showed only moderate binding affinities with Kd values in the μM regime. In this report, we hypothesize that improved thermal stability of the apo state of the metal binding WW-domain scaffold should lead to improved preorganization of the metal-binding site and consequently to higher metal-binding affinities. By redesigning WW-CA-Nle, we obtained WW-CA variants, WW-CA-min and WW-CA-ANG, which were fully folded in the apo states and displayed moderate to excellent thermostabilities in the apo and holo states. We were able to show that the improved thermal stabilities led to improved metal binding, which was reflected in Kd values that were at least one order of magnitude lower compared to WW-CA-Nle. EPR spectroscopy and ITC measurements revealed a better defined and predisposed metal binding site in WW-CA-ANG.