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human serum albumin


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Structural Biology
Journal of Structural Biology 162 (2008) 40–49 www.elsevier.com/locate/yjsbi

A new drug binding subsite on human serum albumin and drug–drug interaction studied by X-ray crystallography
Lili Zhu a,b, Feng Yang a,b, Liqing Chen c, Edward J. Meehan c, Mingdong Huang a,b,*
a

State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, The Chinese Academy of Sciences, 155 Yang Qiao Xi Lu, Fuzhou, Fujian 350002, China b Graduate School of Chinese Academy of Sciences, The Chinese Academy of Sciences, Beijing 10039, China c Laboratory for Structural Biology, Department of Chemistry, Graduate Programs of Biotechnology, Chemistry and Materials Science, University of Alabama in Huntsville, Huntsville, AL 35899, USA Received 17 August 2007; received in revised form 15 November 2007; accepted 12 December 2007 Available online 28 December 2007

Abstract 30 -Azido-30 -deoxythymidine (AZT) is the ?rst clinically e?ective drug for the treatment of human immunode?ciency virus infection. The drug interaction with human serum albumin (HSA) has been an important component in understanding its mechanism of action, especially in drug distribution and in drug–drug interaction on HSA in the case of multi-drug therapy. We present here crystal structures of a ternary HSA–Myr–AZT complex and a quaternary HSA–Myr–AZT–SAL complex (Myr, myristate; SAL, salicylic acid). From this study, a new drug binding subsite on HSA Sudlow site 1 was identi?ed. The presence of fatty acid is needed for the creation of this subsite due to fatty acid induced conformational changes of HSA. Thus, the Sudlow site 1 of HSA can be divided into three non-overlapped subsites: a SAL subsite, an indomethacin subsite and an AZT subsite. Binding of a drug to HSA often in?uences simultaneous binding of other drugs. From the HSA–Myr–AZT–SAL complex structure, we observed the coexistence of two drugs (AZT and SAL) in Sudlow site 1 and the competition between these two drugs in subdomain IB. These results provide new structural information on HSA–drug interaction and drug–drug interaction on HSA. ? 2007 Elsevier Inc. All rights reserved.
Keywords: Human serum albumin; 30 -Azido-30 -deoxythymidine; Salicylic acid; Drug–albumin interaction; Drug–drug interaction; Drug binding site; Xray crystallography

1. Introduction Human serum albumin (HSA) is the most abundant protein constituent of blood plasma and serves as a major protein storage component for endogenous and external compounds. HSA has three homologous domains (named I, II, and III); each domain is made up of two separate sub-

Corresponding author. Address: State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, The Chinese Academy of Sciences, 155 Yang Qiao Xi Lu, Fuzhou, Fujian 350002, China. E-mail addresses: zhulili@fjirsm.ac.cn (L. Zhu), fyang@fjirsm.ac.cn (F. Yang), chenlq@uah.edu (L. Chen), meehane@uah.edu (E.J. Meehan), mhuang@fjirsm.ac.cn (M. Huang). 1047-8477/$ - see front matter ? 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.jsb.2007.12.004

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domains (named A and B) connected by a random coil (He and Carter, 1992). Extensive biochemical studies in the 1970s resulted in the widely accepted proposal of two main drug-binding sites in HSA (Sudlow et al., 1975), denoted as Sudlow site 1 and Sudlow site 2. Sudlow site 1 (located in subdomain IIA) was shown to prefer large heterocyclic and negatively charged compounds, while Sudlow site 2 (located in subdomain IIIA) was the preferred site for small aromatic carboxylic acids (Sudlow et al., 1975, 1976). In addition, there are a number of minor binding sites (Bhattacharya et al., 2000a; Ghuman et al., 2005; Sengupta and Hage, 1999; Zunszain et al., 2003) that allow multiple drug molecules to be bound simultaneously onto HSA and lead to higher drug binding capacity of HSA. HSA plays a central role in drug pharmacokinetics (Herve et al., 1994;

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Vorum, 1999), and thus a?ects therapeutic drug dosage. Out of the four aspects of pharmacokinetics (absorption, distribution, metabolism, excretion), distribution is the one that this protein controls, because most drugs travel in plasma and reach the target tissues bound to this protein (Colmenarejo, 2003; Herve et al., 1994).

30 -Azido-30 -deoxythymidine (AZT, structure 1) (Ho?man et al., 1985), is the ?rst clinically e?ective drug used for the treatment of human immunode?ciency virus infection. It is a synthetic pyrimidinic analogue that di?ers from natural nucleoside thymidine (dThd) in having an azido substituent in place of the hydroxyl group at the 30 -position of the deoxyribose ring. As a variant of dThd, AZT typically has the same reactivity as dThd and follows the same metabolic pathways in the cell although with radically altered kinetic parameters (Samuels, 2006). AZT itself is a pro-drug and needs to be converted into the pharmaceutically active form 30 -azido-30 -deoxythymidine-50 -triphosphate (AZTTP). AZTTP can then interact with DNA polymerases and be incorporated into a replicating DNA strand in the place of thymidine triphosphate (dTTP). The azido group prevents further replication of the DNA molecule, leading to chain termination (Bradshaw et al., 2005; Kedar et al., 1990; Reardon and Miller, 1990). In today’s anti-HIV therapy regiment, AZT is administrated at 600 mg total per day (either 200 mg three times per day or 300 mg twice per day), resulting in a steady-state serum AZT concentration of $0.8 mM (Fletcher et al., 2002). Such high dosage is needed because of the short half-life time of AZT in plasma (%1.1 h) (DiPiro, 1997). The AZT binding to plasma proteins, especially albumin, controls the concentration of the free drug, and hence a?ects the drug’s pharmacokinetics, storage, toxicity, transportability to the tissue and through cell membranes. It is known that AZT exhibits poor binding to human serum albumin (HSA) (Quevedo et al., 2001). Such characteristic was believed to be one of the main reasons for its short half-life time (Luzier and Morse, 1993; Quevedo et al., 2001). However, the structure basis of such characteristic has not been reported in details.

Interaction of AZT with HSA has been studied by capillary electrophoresis, FTIR and CD spectroscopic methods (Gaudreau et al., 2002). Capillary electrophoresis and spectroscopic results showed two major binding sites of AZT on HSA with binding constants K1 of 1.9 ? 106 M?1 and K2 of 2.1 ? 104 M?1. AZT exhibits a higher a?nity to therapeutic HSA than to puri?ed HSA (Quevedo et al., 2001). A crystallographic study in 1992 con?rmed AZT binding with HSA (He and Carter, 1992), but did not give details on the binding interactions because of the limited resolution of the data. Therefore, a higher resolution crystal structure of the HSA–AZT complexes is useful to show the detailed drug binding modes and to identify residues of HSA that are key determinants of binding speci?city. Drug–drug interaction on HSA often occurred in multidrug therapy (Aubry et al., 1995; Brenner et al., 2002; Mubashar et al., 2002; Schmit et al., 1996; Schwarzenbach, 2002). HSA is a ?exible protein and has a number of drug binding sites (He and Carter, 1992; Sengupta and Hage, 1999; Sudlow et al., 1975, 1976). Binding of a drug can in?uence simultaneous binding of other drugs (Bertucci and Domenici, 2002). This can lead to (1) the displacement in the binding site of the drug by the one that has a higher a?nity to the transporting albumin, and/or (2) the alteration of the albumin structure, resulting in the change (increase or decrease) of the protein a?nity toward the drugs (Kragh-Hansen et al., 2002; Sulkowska et al., 2006). The competition of two drugs usually decreases the amount of drug bound to the albumin (Onks et al., 1991). Alterations caused by drug–drug interactions in the HSA binding of drugs may alter the volume of distribution, clearance, and elimination of a drug and may modulate its therapeutic e?ect. It is useful to classify drugbinding sites so that the risk of drug interactions can be evaluated. Therefore, identifying coexistence or competition of drugs on HSA is of great importance. The mechanism of cooperative binding of both cytarabine (araC) and ?uorouracil, used in combination therapy, to BSA has been investigated using UV and NMR spectroscopy (Sulkowska et al., 2004). The study showed the competition of these two drugs and the removal of ?uorouracil by araC from the common binding site. Study on the competition between araC and aspirin binding with BSA suggested that araC reduced the a?nity of albumin toward aspirin. On the other hand, the interaction between araC and BSA was weaker in the presence of aspirin (Sulkowska et al., 2006). Over the last decade, several techniques have been developed and applied to study the drug–drug interaction on binding to serum albumin (HSA or bovine serum albumin, BSA) (Bai et al., 2005; Cui et al., 2004; Kuchimanchi et al., 2001; Sowell et al., 2001). However, there are not many detailed structural studies of such drug–drug interaction (Ghuman et al., 2005). In this study, we use salicylic acid (SAL) as a competitive drug to determine the extent and nature of AZT binding to HSA by X-ray crystallography. The interactions of

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SAL with HSA have been extensively investigated for decades (He and Carter, 1992; Honma et al., 1991; KraghHansen et al., 1990; Lee et al., 1995; Ozer and Tacal, 2001; Parsons and Sathe, 1991; Yang et al., 2007). This study provides new structural information on HSA–drug interaction and drug–drug interaction on HSA.

2. Materials and methods 2.1. Protein puri?cation, complex formation and crystallization Fatty acid free HSA was purchased from Sigma Inc. (catalogue number A3782) and further puri?ed to remove HSA dimer, according to the published protocols (Curry et al., 1998; Yang et al., 2007). The protein was dissolved in a 20 mM potassium phosphate bu?er (pH 7.5) to about 100 mg/ml and stored in a ?80 °C refrigerator before use. Sodium myristate, 30 -azido-30 -deoxythymidine, and salicylic acid were purchased from Sigma Inc. Sodium myristate was dissolved into ethanol, and then was diluted to 2.5 mM using 20 mM potassium phosphate, pH 7.5, and stored at 4 °C in refrigerator. Sodium myristate will precipitate upon cooling. To make HSA–Myr complexes, the 2.5 mM sodium myristate solution was heated to facilitate dispersion of the fatty acid and let cool brie?y. Then 720 ll of 2.5 mM myristate solution was mixed with 100 ll of 100 mg/ml HSA and this mixture was incubated at room temperature for 2 h. Excessive undissolved myristate was found in the mixture and was removed by centrifugation at 12,000g for 4 min. The HSA–Myr complexes were concentrated to 100 mg/ml using a 10,000 Da molecular mass cut-o? centrifugal concentrator (Millipore). To study the crystal structures of HSA in complex with drugs, drug molecules can be soaked into preformed HSA crystals, or HSA–drug complex is formed ?rst and then is used to generate crystals of HSA–drug complex (co-crystallization). In our case, we did not obtain the crystals of HSA–Myr–AZT complex by co-crystallization, but succeeded in soaking AZT into HSA–Myr crystals. This soaking approach has been shown to be useful in studying other HSA–drug complexes (Curry et al., 1998; Petitpas et al., 2001; Yang et al., 2007). Crystallization was carried out by sitting drop vapour di?usion at room temperature. The HSA–Myr complex was centrifuged at 12,000g for 4 min before setting up crystallization experiments. HSA– Myr complexes (2.0 ll) were mixed with equal volumes of the reservoir solution, consisting of 31–34% (w/v) polyethylene glycol 3350 and 50 mM potassium phosphate (pH 7.5). Crystals grew spontaneously as clusters of rods after three days. Streak-seeding was used to improve crystal quality (Yang et al., 2007). In all cases, crystals were obtained by streak seeding into drops that had been equilibrated for overnight. AZT and salicylic acid were dissolved into alcohol and diluted to 20 mM with the

solution consisting of 32% (w/v) polyethylene glycol 3350, 50 mM potassium phosphate pH 7.5. HSA–Myr crystals were washed by the reservoir solution, and then placed into 50 ll solution containing 20 mM AZT or comprising 10 mM AZT and 10 mM SAL solution (50 ll) for about 24 h, respectively. These ?nal drugs concentrations were identi?ed by testing a series of concentrations to ensure enough drugs binding on HSA and at the same time not to etch the HSA–Myr crystals after soaking. 2.2. Data collection and structure determination X-ray di?raction data collection was carried out at 100 K on the APS SER-CAT beam line 22-ID, Chicago, USA. The di?raction data were indexed and processed using the HKL2000 program. Crystals of HSA–Myr– AZT complex and HSA–Myr–AZT–SAL complex were both isomorphous with the HSA–myristate complex (PDB code 1E7G) (Bhattacharya et al., 2000b). A HSA model (PDB entry 1E7G, stripped of its myristate) was used as a molecular replacement model for phasing of the X-ray data. The positioned model was initially re?ned using a rigid body protocol in CNS (Brunger et al., 1998) with the model splitting into its six subdomains and then subjected to cycles of positional and B-factor re?nement. To minimize model bias, composite and r-weighted 2Fo ? Fc omit maps (Brunger et al., 1998) were used throughout this work. These maps were used to guide the positions of ligands and to make manual adjustments by program O (Jones et al., 1991) to the protein model prior to further cycles of re?nement. Then we put ligands molecules to the extra empty electron densities, respectively. Final re?nement statistics were summarized in Table 1. Figures depicting the structure were prepared by PyMOL (DeLano, 2002). The coordinates of HSA–Myr–AZT and HSA–Myr–AZT–SAL are deposited in the Protein Data Bank (PDB code 3B9L and 3B9M). 3. Results 3.1. Structure of the HSA–Myr–AZT complex and model reliability The crystal structure of HSA–AZT in the absence of myristate was previously reported (He and Carter, 1992). Without fatty acids, this HSA–AZT crystal di?racted only ? to a resolution of 4 A, limiting the detailed analysis of molecular interaction between HSA and AZT. In this study, we crystallized HSA in the presence of sodium myristate, which very often facilitates the crystal formation (Yang et al., 2007). Under physiological conditions, 0.1–2 molecules of fatty acid (FA) are bound to each HSA molecule, but under certain disease states the molar ratio of FA/HSA can rise to much higher levels (Bahr et al., 1991; Brodersen et al., 1990; Simard et al., 2006; Spector, 1986). The crystals of HSA–Myr–AZT complex were

L. Zhu et al. / Journal of Structural Biology 162 (2008) 40–49 Table 1 Data collection and model re?nement statistics HSA–Myr–drug Space group ? Cell parameters a, b, c (A), b (°) ? Resolution range (A) Data redundancy Completeness (%) I/rIa Rmerge (%)b Model re?nement Rmodel (%)c Rfree (%)d rmsd from ideal bond ? lengths (A) rmsd from ideal angles (°)
a b

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HSA–Myr–AZT C2 183.90, 38.70, 95.87, 104.35 46.44–2.6 3.3 87.5 11.4 (2.2) 6.3 (38) 22.3 29.2 0.010 1.5

HSA–Myr– AZT–SAL C2 181.54, 38.46, 95.37, 104.92 46.08–2.7 6.0 87.9 29.2 (4.3) 6.5 (32) 24.2 31.6 0.011 1.5

tory stereochemistry: 86.1% of residues in the most favourable regions and 12.8% in the additional allowed region in the Ramachandran plot. The root mean square deviations ? (rmsd) of bond length and bond angles are 0.011 A and 1.5°, respectively (Table 1). The composite 2Fo ? Fc rAweighted map from this model clearly gave an unambiguous indication of positions and orientations for two SAL molecules and one AZT molecule. One SAL molecule was in Sudlow site 1 together with one AZT molecule (Fig. 2A), whereas the other SAL was found in subdomain IB (Fig. 2B). Interestingly, no AZT was observed in subdomain IB, suggesting that SAL can replace AZT but not the Myr1 at the subdomain IB. 4. Discussion 4.1. A new drug binding subsite on HSA Comparison of our HSA–Myr–AZT structure with other HSA–Myr–drug complexes reveals a new drug binding subsite on HSA in the presence of myristate (Fig. 3). The existence of this new subsite was also observed, and thus further con?rmed by another crystal structure (HSA–Myr–AZT–SAL) in this study. In general, most drugs studied are found to bind in the central portion of Sudlow site 1 that is within the core of subdomain IIA. For example, SAL occupies the predominantly apolar compartment of Sudlow site 1 (Yang et al., 2007) (Fig. 3). In the crystal structure of HSA saturated with myristates (Bhattacharya et al., 2000b), this same compartment was occupied by a myristate molecule (Myr7). The binding of Myr7 at this subsite is believed to be quite weak (Bhattacharya et al., 2000b; Cui et al., 2004; Hamilton, 2004), because most drugs can displace Myr7 and occupy in this pocket (e.g., warfarin (Petitpas et al., 2001), phenylbutazone (Ghuman et al., 2005), azapropazone (Ghuman et al., 2005), aspirin and SAL (Yang et al., 2007)). Indomethacin is an exception in that it binds exclusively to the front, lower sub-compartment of Sudlow site 1 and does not displace the seventh myristate (Myr7) (Ghuman et al., 2005) (Fig. 3). This subsite is largely delineated by L198, F206, A210, F211, W214 from subdomain IIA, L481 from subdomain IIIA and residues V343 and L347 from IB. Di?erent from these two above-mentioned subsites, AZT binds at a new and distinct subsite (Fig. 3). In our HSA–Myr–AZT structure, this new AZT subsite is close to subdomain IB and is besieged by hydrophilic and polar amino acids which include E153, S192, L195, Q196 from subdomain IB and L199, H242, R257, E292 from Sudlow site 1 (Table 2). Similar to indomethacin, AZT does not displace Myr7 but coexist with Myr7. The carboxylate group OE1 of Q196 forms electrostatic interactions with the oxygen atom of tetrahydrofuran ring of AZT ? (3.22 A), and the carboxylate group of Myr7 makes direct ? interaction with the azido group of AZT (3.47 A). The OE1 atom of E153 forms a hydrogen bond with the hydroxyl ? group of deoxythymidine of AZT (2.65 A) and interacts

Values for the outermost resolution shell are given in parentheses. PP PP Rmerge ? 100 ? j j jIhj ? Ihj= j j Ihj , where Ih is the weighted mean intensity of the symmetry-related re?ections Ihj. P P c Rmodel ? 100 ? hkljF obs ? F calc j= hklF obs , where Fobs and Fcalc are the observed and calculated structure factors, respectively. d Rfree is calculated using a randomly selected 5% sample of re?ection data omitted from the re?nement.

obtained using HSA–Myr crystals soaked with 20 mM AZT for 24 h (see Section 2). These crystals di?racted up ? to 2.6 A. After crystallographic re?nement with drugs in the right positions and orientations, the structure was re?ned to a R value of 0.223 and Rfree of 0.292 with overall ? coordinate error of 0.38 A (Table 1). The Ramachandran plot showed 87% of the residues are in the most favoured region and 11.7% in the additional allowed region. The ?nal model of HSA–Myr–AZT had satisfactory stereochemistry with root mean square deviations (rmsd) of bond ? length and bond angles of 0.007 A and 1.5°, respectively. The composite 2Fo ? Fc rA-weighted map clearly showed the electron density for seven myristate and two AZT molecules (Fig. 1A). One molecule of AZT bound to subdomain IIA of HSA (also named Sudlow site 1) and the other AZT bound to subdomain IB of HSA. Both AZT molecules are coexisting with a myristate molecule. In Sudlow site 1, AZT bound to HSA in the presence of the seventh myristate (Myr7) (Fig. 1B). In subdomain IB, AZT bound to HSA in the presence of the ?rst myristate (Myr1) (Fig. 1C). 3.2. Structure of the HSA–Myr–AZT–SAL complex and model reliability The HSA–Myr–AZT–SAL crystals were obtained using HSA–Myr crystals soaked in solution comprising 10 mM AZT and 10 mM salicylic acid for 24 h and di?racted up ? to 2.7 A. After crystallographic re?nement with drugs in the right positions and orientations, the structure was re?ned with R dropping from 0.452 to 0.242, Rfree from ? 0.397 to 0.316 and an overall coordinate error of 0.43 A (Table 1). The ?nal model is well-re?ned and has a satisfac-

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Fig. 1. Crystal structure of the HSA–Myr–AZT complex (A) Overall structure of the HSA–Myr–AZT complex. The protein secondary structure is shown as ribbon with the sub-domains colour-coded as follows: IA, red; IB, warmpink; IIA, green; IIB, splitpea; IIIA, blue; IIIB, skyblue. This colour scheme is maintained throughout the paper. Ligands are shown in a space-?lling representation, coloured by atom type: carbon—grey; nitrogen—blue; oxygen— red. (B) Stereo view of r-weighted 2Fo ? Fc omit map of HSA–Myr–AZT structure (blue, contoured at 1r) shows the AZT binding in Sudlow site 1. (C) Stereo view of r-weighted 2Fo ? Fc omit map (blue, contoured at 1r) for AZT binding in IB in the HSA–Myr–AZT structure. (For interpretation of the references to colour in this ?gure legend, the reader is referred to the web version of this article.)

? with the oxygen atom of tetrahydrofuran of AZT (3.81 A). The OE2 of E153 interacts with the hydroxyl group of ? deoxythymidine of AZT (3.54 A) and the oxygen atom of

? tetrahydrofuran of AZT (3.99 A). The hydroxyl group of ? from the 20 -carboxylate of deoxyS192 is positioned 3.25 A thymide of AZT. The azido group of AZT provides electro-

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Fig. 2. Crystal structure of the HSA–Myr–AZT–SAL complex in stereo view (A) AZT and SAL binding in Sudlow site 1. (B) SAL binding in subdomain IB. r-weighted 2Fo ? Fc omit maps (blue, contoured at 1r) are also shown. (For interpretation of the references to colour in this ?gure legend, the reader is referred to the web version of this article.)

? static interactions with the NE2 of H242 (3.45 A) and the ? NH1 of R257 (3.40 A). By all appearances, this AZT subsite is hydrophilic and electrostatic interactions are important for AZT binding to this subsite. Based on these results, it can be seen that the traditional Sudlow site 1 at the subdomain IIA core can be further divided into three subsites: a SAL binding subsite, an indomethacin binding subsite and an AZT binding subsite. There are no overlaps between these three subsites. The AZT subsite locates at the entrance of Sudlow site 1 and is close to subdomain IB. 4.2. E?ect of myristate on AZT binding Structure of the complex between AZT and defatted HSA that does not contain fatty acid was reported in ? 1992 at 4.0 A (He and Carter, 1992). Only one AZT was found within subdomain IIIA (also named Sudlow site 2) in this structure (He and Carter, 1992). No AZT was observed in Sudlow site 1 and subdomain IB in that HSA–AZT structure in the absence of fatty acid. In the presence of fatty acid (myristate), we observed that the

existence of AZT in Sudlow site 1 and subdomain IB. Thus, fatty acid seems facilitating the AZT binding on HSA. Detailed analysis suggested that the existence of AZT subsite in Sudlow site 1 depends on the presence of myristate. In the absence of myristate (PDB code 1BM0), the side chain of Y150 projects into the Sudlow site 1 and overlaps with the AZT binding subsite (Fig. 4). Myristate binding to HSA induces a substantial conformational change in HSA involving rotations of domains I and III relative to domain II. A myristate molecule (Myr2) forms a hydrogen bond with Y150 side chain, leading to the reorientation of the side chain of Y150 outside of Sudlow site 1 binding site, thus generating the AZT subsite (Fig. 4). The myristate-induced overall conformational changes of HSA and the side chain reorientation of Y150 were not observed in the crystal structure of HSA in complex with a shorter fatty acid (decanoic acid, PDB code 1TF0) (Lejon et al., 2004). Only three decanoic acids were identi?ed in 1TF0 structure, in contrast to 5–7 myristates typically observed in HSA–Myr structures. It seems that there is not enough decanoic acid occupancy in 1TF0 struc-

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4.3. Coexistence of two drugs in Sudlow site 1 of HSA As shown above, the Sudlow site 1 is a large drug binding site and contains three subsites: a SAL subsite, an indomathacin subsite, and an AZT subsite in the presence of fatty acid. There is no overlap among these three subsites. Does this Sudlow site 1 accommodate more than one drug? We tried to answer this question by soaking the HSA–Myr crystals with two drugs simultaneously: AZT and salicylic acid, each at a concentration of 10 mM. Salicylic acid is a widely used non-steroidal anti-coagulant and anti-in?ammatory drug and is known to bind at the Sudlow site 1 (Yang et al., 2007). The structure of this ternary complex indeed revealed the electron density for both AZT and SAL in the Sudlow site 1 (Fig. 2A). The structure of the quaternary complex HSA–Myr– AZT–SAL shows that AZT is surrounded by polar residues Y150, E153, K195, K199, C245, H242, and R257 (Table 2). This location of AZT in Sudlow site 1 is similar to that of AZT in HSA–Myr–AZT complex, further con?rming the presence of AZT at this subsite. SAL in this quaternary complex is located at the Myr7 subsite, replacing the Myr7 of HSA–Myr–AZT structure. This con?rms our previous observation that SAL binds stronger on HSA compared with myristate at this site (Yang et al., 2007). In the HSA–Myr–AZT–SAL structure, the close dis? tance between SAL and AZT is 6.1 A, suggesting no direct interaction between the SAL and AZT at Sudlow site 1. The presence of two drug molecules in Sudlow site 1 was observed in another structural study of HSA with indomathacin/azapropazone and indomathacin/phenulbutazone pairs (Ghuman et al., 2005), which also proved the Sudlow site 1 could accommodate more than one drugs. Coexistence of two drugs on HSA was also observed using techniques other than X-ray crystallography. Cosalane binding to HSA was determined in the presence of salicylic acid by a gel ?ltration technique, which showed that cosalane and SAL could bind to HSA simultaneously (Kuchimanchi et al., 2001). Using nuclear magnetic resonance (NMR) relaxation measurements, ibuprofen (IBP)

Fig. 3. Sudlow site 1 can be divided into three subsites: AZT subsite (this study, lightblue), indomethacin subsite (imn, PDB code 2bxm, orange), and SAL subsite (SAL, PDB code 2i30, pink). Drugs are shown in a stick representation with a semi-transparent van der Waals surface. Selected side chains that surrounded AZT sites are also shown. (For interpretation of the references to colour in this ?gure legend, the reader is referred to the web version of this article.)

ture to induce HSA conformational changes. The key fatty acid site (Myr2) that causes HSA conformational changes was not occupied in 1TF0 structure. Another possibility is that decanoic acid is a fatty acid with chain length too short to cause HSA conformational changes. The e?ect of fatty acid on AZT binding to HSA was previously analysed by gel ?ltration method (Quevedo et al., 2001). It was found that the bound fractions of AZT to commercial fatty acid-free HSA, puri?ed HSA (all fatty acids removed by charcoal treatment) and therapeutic use HSA (containing fatty acid) were 12.9 ± 0.6%, 11.07 ± 1.4% and 20.28 ± 3.0%, respectively (Quevedo et al., 2001). This result also suggested a considerably increased binding of AZT to HSA in the presence of fatty acid.

Table 2 Interactions between drugs and ligands of HSA Drugs in di?erent subdomain of HSA–Myr complex AZT (in subdomain IIA of HSA– Myr–AZT complex) AZT (in subdomain IB of HSA– Myr–AZT complex) AZT (in subdomain IIA of HSA– Myr–AZT–SAL complex) SAL (in subdomain IIA of HSA– Myr–AZT–SAL complex) SAL (in subdomain IB of HSA– Myr–AZT–SAL complex) ? ? H146(3.01 A), myr1(3.05 A) Hydrogen bond ? ? ? E153(3.54 A), S192(3.25 A), Q196(3.22 A), ? ? ? H242(3.45 A), R257(3.40 A), myr7(3.47 A) ? ? Y161(3.61 A), myr1(3.68 A) ? ? E153(3.48 A), R257(3.45 A) Salt bridge ? E153(2.65 A) ? Y161(2.76 A) Surrounding ligands of the drug pocket Y150, E153, S192, K195, Q196, K199, V241, H242, C245, R257, A291, E292, myr7 L115, Y138, I142, H146, F149, L154, F157, Y161, L185, R186, G189, K190, S193 Y148,Y150, E153, K195, Q196, K199, C200,V241, H242, C245, R257, A291, SAL L219, R222, F223, L238, H242, R257, L260, I264, S287, I290, A291 L115, I142, H146, F149, L154, F157, Y161, L185, R186, G189, K190

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Fig. 4. E?ect of myristate on AZT binding: interaction between Myr2 and Y150 leads to the reorientation of the side chain of Y150 and the creation of the AZT binding subsite. The structure of HSA–Myr–AZT (this study, opaque) and HSA without fatty acid (PDB code 1BM0, semi-transparent) were superposed by aligning the positions of residues belonging to domain II (197–383). Ligands coloured by atom type: nitrogen—blue; oxygen— red; carbon—grey in HSA–AZT–myristate and carbon—cygan in defatted HSA. (For interpretation of the references to colour in this ?gure legend, the reader is referred to the web version of this article.)

binding by one order of magnitude (Kuchimanchi et al., 2001). The competitive binding of ibuprofen (IBP) and SAL to HSA was also studied by using nuclear magnetic resonance (NMR) relaxation measurements. The results from this study revealed that when the concentration of one ligand (IBP or SAL) was increased in the solution, the free fraction of another one was increased because of the competitive binding (Cui et al., 2004). All these results support that the competitive binding of drug–drug to HSA does exist. In summary, we identify a new AZT binding subsite in Sudlow site 1. The presence of this binding site is depends on the presence of fatty acid (myristates). We also use salicylic acid as a competitive drug to determine the extent and nature of AZT binding to HSA by X-ray crystallography. Such structural information might facilitate the further characterization of drug–HSA interactions. Due to drug–drug interaction on HSA in multi-drug therapy, one drug binding with HSA may alter the active fraction of the other drugs. Therefore, study of drug binding sites and drug–drug interaction on HSA provide a basis to understand drug transport in plasma and to determine drug dosages. Acknowledgments This work was supported by Grants from the Natural Science Foundation of China (30430190, 30625011) and 973 (2007CB914304) to M.H., and NSF-EPSCoR of USA. Use of the Advanced Photon Source was supported by the US Department of Energy, O?ce of Science, O?ce of Basic Energy Sciences, under contract No. W-31-109Eng-38. We thank the sta?s of the APS SER-CAT beamline 22ID for help with data collection. References
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and SAL was shown to share one identical high-a?nity binding site and certain low-a?nity binding sites on HSA molecule (Cui et al., 2004). All these results indicate two drugs can interact with HSA simultaneously. 4.4. Competition of two drugs on HSA The HSA–Myr–AZT–SAL structure not only shows the co-existence of two drugs (AZT and SAL) at one large binding site (Sudlow site 1), but also reveal the competition of two drugs. In subdomain IB of the structure of quaternary HSA–Myr–AZT–SAL complex, SAL is bound within the binding pocket formed by I142, F149, L154, F157, Y161, L185 and G189 (Table 2). The carboxylate group ? of SAL is positioned 3.78 A from Y161 and forms hydro? gen bonds with the ?rst myristate (Myr1) (3.05 A). Moreover, the hydroxyl group of SAL forms hydrogen bonds ? with H146 (3.01 A) (Fig. 2B). In subdomain IB of the structure of ternary HSA–Myr–AZT complex, AZT locates below the ?rst myristate (Myr1) and the azido group of AZT provides electrostatic interactions with the ? carboxylate group of Myr1 (3.68 A) (Fig. 1C).Through the comparison between HSA–Myr–AZT structure and HSA–Myr–AZT–SAL structure, it can be seen that, in subdomain IB, AZT was replaced by SAL when both drugs were soaked at equal concentrations (10 mM). This suggests that SAL binds stronger than AZT at subdomain IB. Competition of two drugs binding on HSA was widely recognized. The competition binding between SAL and cosalane to HSA was studied by a gel ?ltration technique, the results of this work showed that SAL reduced cosalane

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