The binding versatility of human Serum Albumin and its allosteric modulation

Abstract

Human serum albumin (HSA), the most prominent protein in plasma, is best known for its exceptional ligand binding capacity. Seven binding sites for fatty acids (FA; FA1-FA7) have been found to be common for FAs ranging from C10:0 to C18:0; however, nine clefts can bind C10:0 FAs. Moreover, defatted HSA binds four thyroxine molecules at FA3-FA4, FA5, and FA7; on the other hand, the HSA-FA complex binds one thyroxine molecule at the II-III interdomain region (i.e., FA9). Furthermore, HSA binds the GA module of the bacterial protein PAB at domain II; this seems to add selective advantages to the bacterium in terms of growth, increasing its virulence by providing to the bacteria FAs and, possibly, other nutrients transported by HSA. Lastly, HSA displays three binding sites endowed with appropriate residues to coordinate metal ions. Remarkably, HSA participates in heme scavenging by binding the macrocycle at the FA1 site. In turn, heme endows HSA with globin-like reactivity and spectroscopic properties. Notably, ligand binding to HSA is competitively and allosterically modulated. To highlight the molecular bases of the allosteric and competitive binding properties of HSA, the following aspects have been investigated. (i) The reductive nitrosylation of ferric human serum hemealbumin (HSA-heme-Fe(III)) has been investigated from the kinetic and thermodynamic viewpoints. At pH = 5.5, HSA-hemeFe(III) binds NO reversibly, leading to the formation of nitrosylated HSA-heme-Fe(III) (HSA-heme-Fe(III)-NO). By contrast, at pH

-heme-Fe(III) leads to the transient formation of HSA-heme-Fe(III)-NO that precedes the formation of ferrous nitrosylated human serum heme-albumin (HSA-heme-Fe(II)-NO). The rate-limiting step for reductive nitrosylation of HSA-heme-Fe(III) is represented by the OH– -mediated reduction of HSA-heme-Fe(II)-NO+ to HSA-hemeFe(II). (ii) Carbonylation of ferrous heme-albumin (HSA-hemeFe(II)) has been studied from both kinetic and spectroscopic 1 viewpoints. The heme-Fe(II) atom is a mixture of a fourcoordinate intermediate-spin species (predominant at pH 5.8 and 7.0), a five-coordinate high-spin form (mainly at pH 7.0), and a six-coordinate low-spin species (predominant at pH 10.0). The acidic-to-alkaline reversible transition reflects conformational changes leading to the coordination of the heme-Fe(II) atom by the His146 residue. The presence of several HSA-heme-Fe(II) species accounts for the complex, multi-exponential kinetics observed, and reflects the very slow interconversion between the different species observed both for CO association and dissociation as a function of pH. (iii) HSA-heme-Fe(II) nitrosylation has been investigated from the kinetic viewpoint. Ibuprofen and warfarin binding to FA2 and FA7 sites, respectively, inhibit allosterically HSA-heme-Fe(II) nitrosylation. Remarkably, the affinity of ibuprofen and warfarin for HSAheme-Fe(II) is different from that for drug binding to HSA and HSA-heme-Fe(III), indicating that both heme binding and the redox state of the heme-Fe atom affect allosterically HSA(-hemeFe) properties. (iv -tetrahydrocannabinol (THC) and diazepam binding to HSA and HSA-heme-Fe(III) has been investigated from both thermodynamic and spectrophotometric viewpoints. THC binds to the FA2 and FA7 sites of HSA with very different affinity without affecting diazepam association. THC binding to the high-affinity FA2 site accounts for the low free fraction of the drug in plasma, and increases allosterically the heme-Fe(III) affinity for the FA1 site. Possibly, the HSA conformational transition(s) induced by THC binding could account for drug delivery to the liver through receptor-mediated endocytosis. As a whole, the present data highlight: (ii) the binding mode of drugs to HSA(-heme-Fe), (ii) the reactivity of HSA-heme-Fe(III) and HSA-heme-Fe(II), and (iii) the allosteric modulation mechanism(s) of HSA(-heme-Fe).