Interdomain zinc site on human albumin

Abstract

Albumin is the major transport protein in blood for Zn 2؉ , a metal ion required for physiological processes and recruited by various drugs and toxins. However, the Zn 2؉ -binding site(s) on albumin is ill-defined. We have analyzed the 18 x-ray crystal structures of human albumin in the PDB and identified a potential five-coordinate Zn site at the interface of domains I and II consisting of N ligands from His-67 and His-247 and O ligands from Asn-99, Asp-249, and H 2O, which are the same amino acid ligands as those in the zinc enzymes calcineurin, endonucleotidase, and purple acid phosphatase. The site is preformed in unliganded apo-albumin and highly conserved in mammalian albumins. We have used 111 Cd NMR as a probe for Zn 2؉ binding to recombinant human albumin. We show that His-67 3 Ala (His67Ala) mutation strongly perturbs Cd 2؉ binding, whereas the mutations Cys34Ala, or His39Leu and Tyr84Phe (residues which may H-bond to Cys-34) have no effect. Weak Cl ؊ binding to the fifth coordination site of Cd 2؉ was demonstrated. Cd 2؉ binding was dramatically affected by high fatty acid loading of albumin. Analysis of the x-ray structures suggests that fatty acid binding to site 2 triggers a spring-lock mechanism, which disengages the upper (His-67͞Asn-99) and lower (His-247͞Asp-249) halves of the metal site. These findings provide a possible mechanism whereby fatty acids (and perhaps other small molecules) could influence the transport and delivery of zinc in blood.

Deciphering metal-induced oxidative damages on glycated albumin structure and function

“…Regarding copper ions, serum albumin has one strong binding site in the N-terminal tripeptide (Asp-Ala-His) for Cu(II), but histidine residues are also suggested as a major ligand, similarly to cysteine [35]. Moreover a previous study showed that both metals could be bound to albumin via histidine-imidazole ring in the MBS/site A [17,60], or in N-terminal site for copper [65]. Indeed, both nitrogen atoms of the 17 histidine residues present in bovine albumin are potential donors for transition metal ions.…”

“…Another metal binding site, called B, whose location remains unknown was characterized for some metals including Zn(II) [59]. In the metal-binding site A, the imidazole nitrogens of His-67 in domain I and His-247 in domain II could be identified as main ligands for Zn(II) [60]. If site A is the preferential site for Zn(II), it is also a secondary binding site for Cu(II) [61,62].…”

Structure, Properties, and Engineering of the Major Zinc Binding Site on Human Albumin

“…On the basis of 111 Cd NMR studies, sitedirected mutagenesis, and molecular modeling, we have proposed that the major zinc site on albumin, termed site A, is located at the interface of domains I and II and is formed by the side chains of His 67 and Asn 99 from domain I and by His 247 and Asp 249 from domain II (Fig. 1B) (17,18). Here, we report the first direct structural characterization of a zinc site on albumin, using Zn K-edge x-ray absorption fine structure (EXAFS) 2 spectroscopy.…”

“…Model Building and Refinement-The EXAFS data were processed and analyzed as described previously (19) using full (three-dimensional) multiple scattering. We used our model of the zinc site (17), based on a published crystal structure at 2.5 Å of ligand-free (apo)albumin (Protein Data Bank code 1AO6 (9)), as a starting point for EXAFS data refinement. The model included the side chains of His 67 , Asn 99 , and Asp 249 , as well as the side chain and backbone of His 247 and all atoms of Gly 248 , including the peptide bond to Asp 249 (30 atoms).…”

“…The starting models of the mutants were generated by analogy to the wild-type starting model (17), using Sybyl (version 6.8), employing a customized TRIPOS force field incorporating zinc-specific parameters. Molecular graphics representations were generated using MOLMOL (version 2000 K) (22) and Weblab Viewer (Accelrys).…”