Structural basis for the binding of naproxen to human serum albumin in the presence of fatty acids and the GA module

Abstract

The previously determined crystal structure of the bacterial albumin‐binding GA module in complex with human serum albumin (HSA) suggested the possibility of utilizing the complex in the study of ligand binding to HSA. As a continuation of these studies, the crystal structure of the HSA–GA complex with the drug molecule naproxen and the fatty acid decanoate bound to HSA has been determined to a resolution of 2.5 Å. In terms of drug binding, the structure suggests that the binding of decanoate to the albumin molecule may play a role in making the haemin site in subdomain IB of the albumin molecule available for the binding of naproxen. In addition, structure comparisons with solved structures of HSA and of the HSA–GA complex show that the GA module is capable of binding to different conformations of HSA. The HSA–GA complex therefore emerges as a possible platform for the crystallographic study of specific HSA–drug interactions and of the influence exerted by the presence of fatty acids.

Lessons from the Crystallographic Analysis of Small Molecule Binding to Human Serum Albumin

“…In our experience cooling to 100K usually resulted in significant degradation of the order of the crystal lattice that increased mosaic spread and reduced the useful resolution of the data. Progress in this area has been made elsewhere and there are now several reports of HSA structures determined using data collected from cryo-cooled crystals, 12,28,30,31) though unfortunately some published reports lack an account of how cryo-cooling was achieved. 12,28) In contrast, Lejon and colleagues describe the use of either mineral oil or addition of 20% glycerol to the crystal harvest buffer to achieve cryo-cooling that permitted the determination of high-resolution structures (2.5-2.7°A) of complexes of HSA with a small domain from a bacterial surface protein.…”

“…12,28) In contrast, Lejon and colleagues describe the use of either mineral oil or addition of 20% glycerol to the crystal harvest buffer to achieve cryo-cooling that permitted the determination of high-resolution structures (2.5-2.7°A) of complexes of HSA with a small domain from a bacterial surface protein. 30,31) Elsewhere, 10-15% ethylene glycol has also been used as a cryo-protectant for crystals of defatted HSA grown from PEG 6000 (Kim L. Hein, Jesper V. Møller, Ulrich Kragh-Hansen, and Poul Nissen, unpublished data). In both cases the crystals were flash cooled in liquid nitrogen after incubation in the cryo-buffer.…”

“…In the structure of the HSA-myrisate-hemin complex reported by Carter and colleagues, 28) the position of the carboxylate of the fatty acid bound in site 4 (sub-domain IIIA) is altered by about 2.8°A, so that it can no longer hydrogen bond to Ser 489 23) but instead appears close enough to interact with the side-chain or Asn 391. In addition, in the ternary complex of HSA, decanoic acid and the bacterial albumin-binding GA domain, 30) there is no fatty acid bound to site 5 (subdomain IIIB), even though this has been identified as a possible high-affinity site for this medium chain lipid. 42) Crystallographic analysis of HSA complexes with other endogenous ligands: Fatty acids are the primary cargo of HSA and have the most binding sites of any endogenous ligand.…”

A fluorescent fatty acid probe, DAUDA, selectively displaces two myristates bound in human serum albumin

“…Protein crystals are usually frozen with mother liquor containing some cryoprotectants, typically 20% glycerol or ethylene glycol. 18,19 However, these typical cryoprotectant cannot be used for HSA. We have discovered a proper cryoprotectant (5-10% DMSO) to cryo-cool HSA crystals.…”

Crystallographic analysis reveals a unique lidocaine binding site on human serum albumin

“…A combination of crystal packing and low-affinity binding of lidocaine may explain why an asymmetric 2:1 HSA:lidocaine complex has occurred. An examination of the literature (Carter and Ho, 1994;Sugio et al, 1999;Ghuman et al, 2005;Lejon et al, 2008;Zunszain et al, 2008;Zhu et al, 2008) revealed that so far no other HSA crystal has been reported to belong to this type of space group. The structure was determined by molecular replacement and refined at 3.3 Å resolution.…”