Structural basis of binding of fluorescent, site-specific dansylated amino acids to human serum albumin

Abstract

Human serum albumin (HSA) has two primary binding sites for drug molecules. These sites selectively bind different dansylated amino acid compounds, which-due to their intrinsic fluorescence-have long been used as specific markers for the drug pockets on HSA. We present here the co-crystal structures of HSA in complex with six dansylated amino acids that are specific for either drug site 1 (dansyl-l-asparagine, dansyl-l-arginine, dansyl-l-glutamate) or drug site 2 (dansyl-l-norvaline, dansyl-l-phenylalanine, dansyl-l-sarcosine). Our results explain the structural basis of the site-specificity of different dansylated amino acids. They also show that fatty acid binding has only a modest effect on binding of dansylated amino acids to drug site 1 and identify the location of secondary binding sites.

Fig.1

Location of binding sites for dansylated amino acids determined by X-ray crystallography. (a) Simulated annealing F_o − F_c omit map (contoured at 3σ) showing DanF bound to drug site 2 in sub-domain IIIA. DanF is shown in a stick representation with atoms coloured by atom-type: C – purple; O – red; and N – blue. Sub-domain IIIA is shown in a ribbon representation (blue). Selected protein side-chains are shown as sticks, with carbon atoms coloured grey. (b) Overview of DanN bound to drug sites 1 and 2 in HSA. The ligands are shown in a stick representation with a semi-transparent molecular surface. The protein secondary structure is shown coloured by sub-domain. This colour scheme is maintained throughout. (c) Overview of DanN bound to sub-domains IIA and IB in HSA–myristate. The seven fatty acid binding sites on the protein are labelled FA1–FA7. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig.2

Binding interactions made by dansylated amino acids (DanN, DanE, DanR) in drug site 1 (sub-domain IIA) of HSA in the absence and presence of fatty acid. (a) Binding of DanN (coloured by atom type as in Fig. 1b). Hydrogen bonds are shown as dotted cyan lines. (b) Comparison of the binding of DanN and warfarin (Petitpas et al., 2001). The view is rotated slightly to the left compared to panel a; the polypeptide containing Y150 has been removed for clarity. (c) Binding of DanN to HSA in the presence of myristate (shown as CPK spheres, coloured by atom type: carbon – grey and oxygen – red). (d) Comparison of the binding of DanN, DanE and DanR to HSA–myristate. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig.3

Binding interactions made by dansylated amino acids (DanSRC, DanNV, DanF and DanN) in drug site 2 (sub-domain IIIA) of HSA. (a) Binding of DanF (coloured by atom type as in Fig. 1a). Hydrogen bonds are shown as dotted cyan lines. (b) Comparison of the binding of DanF and indoxyl sulphate (IDS) Ghuman et al., 2005; carbon atoms in IDS are coloured dark yellow. (c) Comparison of the binding of DanF, DanNV and DanSRC to drug site 2 of HSA. (d) Comparison of the binding of DanF and DanN to drug site 2 of HSA. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)