Structural and biochemical characterisation of Co2+-binding sites on serum albumins and their interplay with fatty acids

Dongmei Wu, Michal Gucwa, Mateusz P. Czub, David R. Cooper, Ivan G. Shabalin, Remi Fritzen, Swati Arya, Ulrich Schwarz-Linek, Claudia A. Blindauer, Wladek Minor*, Alan J. Stewart*

*Corresponding author for this work

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Serum albumin–Co2+ interactions are of clinical importance. They play a role in mediating the physiological effects associated with cobalt toxicity and are central to the albumin cobalt binding (ACB) assay for diagnosis of myocardial ischemia. To further understand these processes, a deeper understanding of albumin–Co2+ interactions is required. Here, we present the first crystallographic structures of human serum albumin (HSA; three structures) and equine serum albumin (ESA; one structure) in complex with Co2+. Amongst a total of sixteen sites bearing a cobalt ion across the structures, two locations were prominent, and they relate to metal-binding sites A and B. Site-directed mutagenesis and isothermal titration calorimetry (ITC) were employed to characterise sites on HSA. The results indicate that His9 and His67 contribute to the primary (putatively corresponding to site B) and secondary Co2+-binding sites (site A), respectively. The presence of additional multiple weak-affinity Co2+ binding sites on HSA was also supported by ITC studies. Furthermore, addition of 5 molar equivalents of the non-esterified fatty acid palmitate (C16:0) reduced the Co2+-binding affinity at both sites A and B. The presence of bound myristate (C14:0) in the HSA crystal structures provided insight into the fatty acid-mediated structural changes that diminish the affinity of the protein toward Co2+. Together, these data provide further support for the idea that ischemia-modified albumin corresponds to albumin with excessive fatty-acid loading. Collectively, our findings provide a comprehensive understanding of the molecular underpinnings governing Co2+ binding to serum albumin.
Original languageEnglish
Pages (from-to)6244-6258
Number of pages15
JournalChemical Science
Issue number23
Early online date23 May 2023
Publication statusPublished - 21 Jun 2023


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