Crystallographic analysis reveals a unique lidocaine binding site on human serum albumin
Introduction
Human serum albumin (HSA) is the most abundant plasma protein (∼0.6 mM) and comprises 50–60% of the total plasma protein in humans. An even larger pool of HSA is found in the extravascular spaces although at a lower concentration (Peters, 1996). As one of its main functions, HSA serves as a depot and transport protein for numerous endogenous and exogenous compounds. Among the exogenous compounds is a wide range of drugs, and binding to HSA affects their absorption, distribution, metabolism and elimination. Because HSA has a limited number of high-affinity binding sites, detailed molecular information about these sites is very helpful in the assessment of cooperative effects of binding of other drugs or endogenous ligands. However, the distribution of compounds will also be affected by low-affinity binding to HSA due to its high concentration. In addition, structural information of both high and low-affinity binding sites is useful when designing new drugs whether the aim is to avoid binding to HSA, or to make use of its depot function.
HSA is an α-helical protein (67% α-helix) consisting of a single polypeptide chain of 585 amino acids that form a heart-shaped protein with three homologous domains (I–III) (Carter and Ho, 1994, Sugio et al., 1999). Each of the domains is composed of two subdomains (A and B) with distinct helical folding patterns connected by flexible loops. Small-angle X-ray scattering studies of HSA in solution show general agreement with the crystal structure (Olivieri and Craievich, 1995). Also, a combined phosphorescence depolarisation–hydrodynamic modelling study has proposed that the overall conformation of HSA in neutral solution is very similar to that observed in crystal structures (Ferrer et al., 2001).
The drugs binding to HSA are mainly lipophilic compounds with anionic or electronegative features, and crystal structures of many HSA complexes have been determined (Carter and Ho, 1994, Bhattacharya et al., 2000b, Petitpas et al., 2001, Ghuman et al., 2005). In plasma, cationic drugs usually bind to α1-acid glycoprotein (orosomucoid) (Schönfeld et al., 2008) however, some of them bind to HSA (Kragh-Hansen, 1981). No structural information about such binding sites is available. Therefore, in the present work we have performed a crystallographic analysis of HSA complexed with lidocaine, a Na+ channel blocker widely used as a local anesthetic and antiarrhythmic drug. The structural work was supplemented with intrinsic fluorescence data, equilibrium dialysis and isothermal titration calorimetry. The study revealed a new crystal form of a HSA-ligand that has not been observed before and the existence of a unique binding site located between domains I and III. Furthermore we compare our results to a 2.3 Å resolution crystal structure of HSA that we have obtained with bound sulphate.
Section snippets
Materials
HSA (A-7223, 95% pure), expressed in Pichia pastoris, was obtained from Sigma–Aldrich (St. Louis, MO, USA). For removing hydrophobic and hydrophilic ligands and additives, the protein was treated with charcoal at pH 3 as described by Chen (1967), dialysed extensively against deionised water, freeze-dried and then stored at −20 °C until use. HSA (A-1887, >96% pure and essentially fatty acid free) was also supplied by Sigma–Aldrich.
Lidocaine was from Sigma–Aldrich, and [carbonyl-14C]lidocaine
Structure of the HSA–lidocaine complex and of HSA with sulphate
Lidocaine is a tertiary amine compound, and its protonated form is shown in Fig. 1. The drug and HSA were dissolved in phosphate buffer, pH 7.5, at a 20:1 M ratio of ligand to protein and co-crystallised as a dimer in the unusual space group I41 (Fig. 2a). The dimer consists of one HSA molecule without ligand and one HSA molecule with a single, bound lidocaine (an asymmetric dimer). A combination of crystal packing and low-affinity binding of lidocaine may explain why an asymmetric 2:1
Discussion
HSA binding of cationic organic ligands has not been studied much, and only a few and fairly simple studies have dealt with lidocaine binding. Thus, Sawinski and Rapp have reported binding to two sites with the same K-value (Sawinski and Rapp, 1963), whereas other studies have suggested low-affinity binding to only one site (Krauss et al., 1986, Bailey and Briggs, 2004). The present results are in accordance with the latter binding model. The equilibrium dialysis results propose binding to one
Acknowledgments
This work was supported by the A.P. Møller Foundation for the Advancement of Medical Science and by a Hallas–Møller stipend of the Novo-Nordisk Foundation.
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