Targeted removal of the FA2 site on human albumin prevents fatty acid-mediated inhibition of Zn2+-binding

Dongmei Wu, Stephen J. Hierons, Sirilata Polepalli, Michal Gucwa, Remi Fritzen, Michal Markiewicz, Juan Sabín, Wladek Minor, Krzysztof Murzyn, Claudia A. Blindauer, Alan J. Stewart*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review


Zinc is required for virtually all biological processes. In plasma, Zn2+ is predominantly transported by human serum albumin (HSA), which possesses two Zn2+-binding sites of differing affinities (sites A and B). Fatty acids (FAs) are also transported by HSA, with 7 structurally characterized FA-binding sites (named FA1-FA7) known. FA-binding inhibits Zn2+-HSA interactions, in a manner that can impact upon haemostasis and cellular zinc uptake, but the degree to which FA-binding at specific sites contributes this inhibition is unclear. Wildtype HSA and H9A, H67A, H247A, and Y150F/R257A/S287A (FA2-KO) mutant albumins were expressed in Pichia pastoris. ITC studies revealed that the Zn2+-binding capacity at the high-affinity Zn2+ site (site A) was reduced in H67A and H247A mutants, with site B less affected. The H9A mutation decreased Zn2+ binding at the lower-affinity site, establishing His9 as a site B ligand. Zn2+ binding to HSA and H9A was compromised by palmitate, consistent with FA-binding affecting site A. 13C-NMR experiments confirmed that the FA2-KO mutations prohibited FA-binding at site FA2. In contrast to wildtype HSA, Zn2+-binding to the FA2-KO mutant was unaffected by myristate, suggesting that FA-binding at FA2 is solely responsible for inhibition. Molecular dynamics studies identified the steric obstruction exerted by a FA bound in site FA2 that impedes the conformational change from open (FA-loaded) to closed (FA-free) states, required for Zn2+ 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 Zn2+ speciation in health and disease.
Original languageEnglish
JournalJournal of Lipid Research
VolumeIn Press
Early online date14 May 2024
Publication statusE-pub ahead of print - 14 May 2024


  • 13C-NMR
  • Allosteric interaction
  • Free fatty acids
  • Isothermal titration calorimetery
  • Serum albumin
  • Zinc


Dive into the research topics of 'Targeted removal of the FA2 site on human albumin prevents fatty acid-mediated inhibition of Zn2+-binding'. Together they form a unique fingerprint.

Cite this