Targeted removal of the FA2 site on human albumin prevents fatty acid-mediated inhibition of Zn 2+ binding.

Autor:	Wu D; School of Medicine, University of St. Andrews, St. Andrews, UK., Hierons SJ; School of Medicine, University of St. Andrews, St. Andrews, UK., Polepalli S; Department of Chemistry, University of Warwick, Coventry, UK., Gucwa M; Department of Computational Biophysics and Bioinformatics, Jagiellonian University, Krakow, Poland., Fritzen R; School of Medicine, University of St. Andrews, St. Andrews, UK., Markiewicz M; Department of Computational Biophysics and Bioinformatics, Jagiellonian University, Krakow, Poland., Sabín J; AFFINImeter Scientific Team, Software 4 Science Developments, Santiago de Compostela, Spain; Applied Physics Department, University of Santiago de Compostela, Santiago de Compostela, Spain., Minor W; Department of Molecular Physiology and Biological Physics, University of Virginia School of Medicine, Charlottesville, VA, USA., Murzyn K; Department of Computational Biophysics and Bioinformatics, Jagiellonian University, Krakow, Poland., Blindauer CA; Department of Chemistry, University of Warwick, Coventry, UK. Electronic address: C.Blindauer@warwick.ac.uk., Stewart AJ; School of Medicine, University of St. Andrews, St. Andrews, UK. Electronic address: ajs21@st-andrews.ac.uk.
Jazyk:	angličtina
Zdroj:	Journal of lipid research [J Lipid Res] 2024 Jun; Vol. 65 (6), pp. 100560. Date of Electronic Publication: 2024 May 14.
DOI:	10.1016/j.jlr.2024.100560
Abstrakt:	Zinc is required for virtually all biological processes. In plasma, Zn ²⁺ is predominantly transported by human serum albumin (HSA), which possesses two Zn ²⁺ -binding sites of differing affinities (sites A and B). Fatty acids (FAs) are also transported by HSA, with seven structurally characterized FA-binding sites (named FA1-FA7) known. FA binding inhibits Zn ²⁺ -HSA interactions, in a manner that can impact upon hemostasis and cellular zinc uptake, but the degree to which binding at specific FA sites contributes to this inhibition is unclear. Wild-type HSA and H9A, H67A, H247A, and Y150F/R257A/S287A (FA2-KO) mutant albumins were expressed in Pichia pastoris. Isothermal titration calorimetry studies revealed that the Zn ²⁺ -binding capacity at the high-affinity Zn ²⁺ site (site A) was reduced in H67A and H247A mutants, with site B less affected. The H9A mutation decreased Zn ²⁺ binding at the lower-affinity site, establishing His9 as a site B ligand. Zn ²⁺ binding to HSA and H9A was compromised by palmitate, consistent with FA binding affecting site A. ¹³ C-NMR experiments confirmed that the FA2-KO mutations prohibited FA binding at site FA2. Zn ²⁺ binding to the FA2-KO mutant was unaffected by myristate, suggesting binding at FA2 is solely responsible for inhibition. Molecular dynamics studies identified the steric obstruction exerted by bound FA in site FA2, which impedes the conformational change from open (FA-loaded) to closed (FA-free) states, required for Zn ²⁺ to bind at site A. The successful targeting of the FA2 site will aid functional studies exploring the interplay between circulating FA levels and plasma Zn ²⁺ speciation in health and disease. Competing Interests: Conflict of interest J. S. is a co-founder and CEO at AFFINImeter. All other authors declare that they have no conflicts of interest with the contents of this article. (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)
Databáze:	MEDLINE
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