Structure of SAs

Although the three-dimensional structure of BSA has not been determined, the structure of HSA, with which BSA shares 75% sequence identity, has been well characterized for the apo protein as well as for a variety of complexes with a variety of long-chain fatty acids and other more compact hydrophobic molecules. The structure of HSA complexed with the anaesthetic halothane (C2F3Cl2Br) and myristic acid (CH3(CH2)12COOH) is shown in Figure 6.4.

The structure of HSA comprises three structurally homologous domains, each of just under 200 residues, denoted I-III (Curry et al., 1998) and involving residues 5-196, 197-383 and 384-582 respectively. Each domain has two sub-domains, each of —100 residues, denoted A and B. The structure lacks P-strands and is predominantly (68%) a-helical, with several lengthy loops connecting the A and B sub-domains. On ligand binding, there is substantial movement of the domains with respect to each other, but the tertiary structure of each domain undergoes only small changes (Curry et al., 1998). Medium- and long-chain fatty acids occupy five distinct

Figure 6.4 Structure of HSA complexed with halothane (slate/purple) partially occupying seven distinct sites and myristic acid (yellow/red) fully occupying five distinct sites (PDB code: 1e7c). Domain IA (residues 5-107) is shown in blue; domain IB (residues 108-196) is shown in light blue; domain IIA (residues 197-297) is shown in green; domain IIB (residues 297-383) is shown in light green; domain IIIA (residues 384-497) is shown in red; and domain IIIB (residues 498-582) is shown in light red. The single cysteine, Cys34, is labeled (Bhattacharya et al., 2000). The 17 disulfide bonds, which tie together individual sub-domains, are represented in stick format. Figure drawn with PyMOL (Delano, 2002) (see also Plate 6.4).

Figure 6.4 Structure of HSA complexed with halothane (slate/purple) partially occupying seven distinct sites and myristic acid (yellow/red) fully occupying five distinct sites (PDB code: 1e7c). Domain IA (residues 5-107) is shown in blue; domain IB (residues 108-196) is shown in light blue; domain IIA (residues 197-297) is shown in green; domain IIB (residues 297-383) is shown in light green; domain IIIA (residues 384-497) is shown in red; and domain IIIB (residues 498-582) is shown in light red. The single cysteine, Cys34, is labeled (Bhattacharya et al., 2000). The 17 disulfide bonds, which tie together individual sub-domains, are represented in stick format. Figure drawn with PyMOL (Delano, 2002) (see also Plate 6.4).

sites (dissociation constants 0.05-1 |iM) (Spector, 1975), one in domain I, a second between domains I and II and the remaining three in domain III, as characterized by X-ray techniques for HSA. In the case of halothane binding, two sites are located in domain I and five are located in domain II.

A comprehensive study of the binding of 17 distinct drugs to HSA, in the presence and absence of myristate, has been published recently (Ghuman et al, 2005). Whereas the binding of steroids to BSA is influenced by the binding of fatty acids, for HSA, there is much less influence (Watanabe and Sato, 1996). NMR titrations have shown that BSA, like HSA, binds five myristates; four of the five sites appear to be structurally homologous to those identified crystallographically for HSA (Hamilton et al., 1984, 1991; Cistola et al, 1987; Simard et al, 2005). The X-ray structure of equine serum albumin (ESA) is very similar to that of HSA, consistent with —75% sequence identity between these two proteins (Ho et al, 1993).

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