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Binding of the General Anesthetics Propofol and Halothane to Human Serum Albumin

2000; Elsevier BV; Volume: 275; Issue: 49 Linguagem: Inglês

10.1074/jbc.m005460200

1083-351X

Ananyo A. Bhattacharya, Stephen Curry, Nicholas P. Franks,

Mass Spectrometry Techniques and Applications

Human serum albumin (HSA) is one of the most abundant proteins in the circulatory system and plays a key role in the transport of fatty acids, metabolites, and drugs. For many drugs, binding to serum albumin is a critical determinant of their distribution and pharmacokinetics; however, there have as yet been no high resolution crystal structures published of drug-albumin complexes. Here we describe high resolution crystal structures of HSA with two of the most widely used general anesthetics, propofol and halothane. In addition, we describe a crystal structure of HSA complexed with both halothane and the fatty acid, myristate. We show that the intravenous anesthetic propofol binds at two discrete sites on HSA in preformed pockets that have been shown to accommodate fatty acids. Similarly we show that the inhalational agent halothane binds (at concentrations in the pharmacologically relevant range) at three sites that are also fatty acid binding loci. At much higher halothane concentrations, we have identified additional sites that are occupied. All of the higher affinity anesthetic binding sites are amphiphilic in nature, with both polar and apolar parts, and anesthetic binding causes only minor changes in local structure. Human serum albumin (HSA) is one of the most abundant proteins in the circulatory system and plays a key role in the transport of fatty acids, metabolites, and drugs. For many drugs, binding to serum albumin is a critical determinant of their distribution and pharmacokinetics; however, there have as yet been no high resolution crystal structures published of drug-albumin complexes. Here we describe high resolution crystal structures of HSA with two of the most widely used general anesthetics, propofol and halothane. In addition, we describe a crystal structure of HSA complexed with both halothane and the fatty acid, myristate. We show that the intravenous anesthetic propofol binds at two discrete sites on HSA in preformed pockets that have been shown to accommodate fatty acids. Similarly we show that the inhalational agent halothane binds (at concentrations in the pharmacologically relevant range) at three sites that are also fatty acid binding loci. At much higher halothane concentrations, we have identified additional sites that are occupied. All of the higher affinity anesthetic binding sites are amphiphilic in nature, with both polar and apolar parts, and anesthetic binding causes only minor changes in local structure. human serum albumin How general anesthetics exert their effects in the central nervous system has remained a puzzle for more than 150 years, but there is now a growing consensus that they act by binding directly to protein targets (1Franks N.P. Lieb W.R. Nature. 1994; 367: 607-614Crossref PubMed Scopus (1626) Google Scholar). The identity of these targets, however, remains uncertain, although a large body of evidence is accumulating on the functional effects of general anesthetics on a variety of possible candidates (1Franks N.P. Lieb W.R. Nature. 1994; 367: 607-614Crossref PubMed Scopus (1626) Google Scholar, 2Krasowski M.D. Harrison N.L. Cell. Mol. Life Sci. 1999; 55: 1278-1303Crossref PubMed Scopus (338) Google Scholar). Most of these data come from electrophysiological measurements, coupled more recently with the techniques of molecular genetics. Although these approaches are crucial in understanding the actions of general anesthetics, they give at best only indirect information on the forces that are involved in anesthetic-protein interactions and virtually no information on the molecular architectures of anesthetic binding sites. The lack of direct structural information is due at least in part to the fact that the most likely targets for general anesthetics are thought to be neuronal ion channels. These are, of course, integral membrane proteins and have proven to be exceptionally difficult to crystallize in a form that is suitable for high resolution x-ray diffraction analysis. However, there are several soluble proteins to which anesthetics are known to bind, and studies with these proteins have provided valuable information on the nature of anesthetic binding sites. Most of this work has been done with serum proteins and luciferase enzymes, but so far the only example of an anesthetic-sensitive protein for which there is also high resolution structural data is firefly luciferase (3Franks N.P. Jenkins A. Conti E. Lieb W.R. Brick P. Biophys. J. 1998; 75: 2205-2211Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). Perhaps the most extensively studied anesthetic binding protein is serum albumin, and there have been numerous attempts to characterize the binding sites involved (4Eckenhoff R.G. Shuman H. Anesthesiology. 1993; 79: 96-106Crossref PubMed Scopus (76) Google Scholar, 5Dubois B.W. Cherian S.F. Evers A.S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6478-6482Crossref PubMed Scopus (60) Google Scholar, 6Dubois B.W. Evers A.S. Biochemistry. 1992; 31: 7069-7076Crossref PubMed Scopus (85) Google Scholar, 7Eckenhoff R.G. J. Biol. Chem. 1996; 271: 15521-15526Crossref PubMed Scopus (64) Google Scholar, 8Johansson J.S. Eckenhoff R.G. Dutton P.L. Anesthesiology. 1995; 83: 316-324Crossref PubMed Scopus (117) Google Scholar), none of them, however, using direct structural techniques. This protein is not only amenable to high resolution structural analysis but, more importantly, is known to play a key role in the pharmacological actions of several general anesthetics. The importance of serum albumin in anesthetic pharmacology derives from its high concentration in the circulatory system (approximately 0.6 mm in plasma) and from its ability to bind an extraordinarily diverse range of drugs (including most anesthetics), metabolites, and fatty acids (for reviews, see Refs. 9Kragh-Hansen U. Dan. Med. Bull. 1990; 37: 57-84PubMed Google Scholar, 10Carter D.C. Ho J.X. Adv. Protein Chem. 1994; 45: 152-203Google Scholar, 11Peters T. All about Albumin: Biochemistry, Genetics, and Medical Applications. Academic Press, Inc., San Diego, CA1996Google Scholar). In several cases more than 50% of a clinically administered general anesthetic will be bound to serum albumin, and in some cases, such as the intravenous agent propofol, approximately 80% is bound (12Altmayer P. Buch U. Buch H.P. Arzneim.-Forsch. 1995; 45: 1053-1056PubMed Google Scholar). Consequently, any changes in the interactions between an anesthetic and serum albumin, either by fatty acids or other drugs competing for binding or by genetic modifications in the protein itself, could result in significant changes in the pharmacologically active concentration of the anesthetic. Although a high resolution structure of human serum albumin was published some years ago (13He X.M. Carter D.C. Nature. 1992; 358: 209-215Crossref PubMed Scopus (3452) Google Scholar), the unavailability of the three-dimensional coordinates did not encourage others to extend this work. Curry et al. (14Curry S. Mandelkow H. Brick P. Franks N. Nat. Struct. Biol. 1998; 5: 827-835Crossref PubMed Scopus (1171) Google Scholar) subsequently published a high resolution structure of the protein that identified the principal fatty acid binding sites, and this was followed by the publication of an independent determination of the native structure (15Sugio S. Kashima A. Mochizuki S. Noda M. Kobayashi K. Protein Eng. 1999; 12: 439-446Crossref PubMed Scopus (1481) Google Scholar). The protein is heart-shaped and contains 585 amino acids. It is organized into three homologous domains (labeled I-III), and each domain consists of two sub-domains (A and B) that share common structural elements (Fig. 1). In this paper we have used x-ray crystallography to provide high resolution information on the nature and locations of the principal binding sites for two of the most widely used general anesthetics, the intravenous agent propofol and the inhalational agent halothane (see Structures I and II). Figure 1The structure of HSA and the locations of fatty acid binding sites. The native structure of HSA (A) and the structure of HSA in the presence of myristate (B), showing the locations of eight fatty acid binding sites. Fatty acids FA4 and FA8 are shown in a darker shade ofgray for clarity of presentation. Further details on the fatty acid binding sites have been published elsewhere (14Curry S. Mandelkow H. Brick P. Franks N. Nat. Struct. Biol. 1998; 5: 827-835Crossref PubMed Scopus (1171) Google Scholar, 23Curry S. Brick P. Franks N.P. Biochim. Biophys. Acta. 1999; 1441: 131-140Crossref PubMed Scopus (450) Google Scholar).3 The domains are color-coded as follows: red, domain I; green, domain II; blue, domain III. The A and B sub-domains within each domain are depicted in darkand light shades, respectively. The fatty acids are represented by space-filling models colored by atom type (gray, carbon; red, oxygen). All figures were prepared using Bobscript and Raster3D (20Merritt E.A. Bacon D.J. Methods Enzymol. 1997; 227: 505-524Crossref Scopus (3873) Google Scholar, 40Esnouf R.M. J. Mol. Graphics. 1997; 15: 133-138Google Scholar, 41Kraulis P.J. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar).View Large Image Figure ViewerDownload (PPT) Most experiments were carried out using fat-free recombinant HSA,1 prepared by charcoal treatment (16Sogami M. Foster J.F. Biochemistry. 1968; 7: 2172-2182Crossref PubMed Scopus (161) Google Scholar) at low pH. This was supplied at a concentration of 250 mg ml−1 in 145 mm NaCl by Dr. John Woodrow of Delta Biotechnology Limited (Nottingham, UK). The halothane-myristate complex was formed using protein that, in addition, originally contained 40 mm octanoate (C8:0) and 15 mg liter−1 Tween 80. In both cases the protein was further purified on a Superdex S75 gel filtration column (Amersham Pharmacia Biotech) with a phosphate running buffer (50 mmpotassium phosphate, 150 mm sodium chloride, pH 7.5) to remove dimers and polymers of HSA, exactly as described previously (14Curry S. Mandelkow H. Brick P. Franks N. Nat. Struct. Biol. 1998; 5: 827-835Crossref PubMed Scopus (1171) Google Scholar). After combining the appropriate fractions, the running buffer was exchanged with a storage buffer (50 mm potassium phosphate, pH 7.0), and the protein was concentrated using an Amicon 30-kDa molecular mass cut-off centrifugal concentrator (Millipore, Watford, Hertfordshire, UK) to greater than 80 mg ml−1and stored at 4 °C. All chemicals were obtained from Sigma unless otherwise stated. Crystals of native HSA were grown by vapor diffusion at 4 °C using the sitting drop configuration. Crystals were first grown with a reservoir of 28–30% (w/v) polyethylene glycol 3350, 50 mm potassium phosphate, pH 7.0. After 2–3 months, large stacked plates were observed in some drops, but these crystals were rarely single and diffracted poorly. However, using these crystals as seeds and equilibrating with a lower concentration of polyethylene glycol 25–26% (w/v), crystals were obtained with dimensions of approximately 0.2 × 0.3 × 0.2 mm in 4–6 weeks. These crystals diffracted to high resolution (2.1 Å). For the propofol complex, an identical crystallization procedure was followed except that a saturating concentration of propofol (approximately 4 mm in 25–26% polyethylene glycol) was maintained throughout. The propofol was a gift from Zeneca Pharmaceuticals, Alderley Park, Macclesfield, UK. Co-crystallization with propofol generally resulted in larger crystals than those obtained in the absence of propofol. Native propofol-free crystals could be readily obtained by back-soaking in solutions that contained progressively less propofol while at the same time progressively increasing the polyethylene glycol concentration up to 32% (w/v). Complexes with halothane were prepared by exposing native crystals to chosen partial pressures of halothane in 1-mm sealed glass capillaries at room temperature. The partial pressure was set by using mixtures of halothane and hexadecane at defined mole ratios. To the extent that halothane and hexadecane mix ideally, the vapor pressure of halothane above such a mixture can, according to Raoult's Law, be taken to be proportional to its mole fraction. The maximum partial pressure of halothane that could be used with native crystals before a significant deterioration in the diffraction patterns was observed was 15% of the saturated vapor pressure, which would correspond to a partial pressure of 5% atm, or 2.6 mm in free aqueous solution. To prepare the halothane-myristate complex, crystals with myristate were first prepared (14Curry S. Mandelkow H. Brick P. Franks N. Nat. Struct. Biol. 1998; 5: 827-835Crossref PubMed Scopus (1171) Google Scholar) before exposure to halothane, as described above. In the presence of myristate we found that a much higher concentration of halothane could be used (60% of the saturated vapor pressure, which would correspond to a partial pressure of 20% atm, or 10.5 mm in free aqueous solution) before lattice disorder in the crystals reduced the resolution of the diffraction patterns. Data were collected to high resolution at the synchrotrons in Daresbury (SRS, UK) and in Hamburg (DESY, Germany). At Daresbury (beamline 9.6), short exposure times (2–3 s) were used to minimize radiation damage, which was evident with longer exposures. In Hamburg (beamline X-11), the exposure times were 20–30 s. All data was processed using MOSFLM. 2A. Leslie, personal communication. Details of the data collection are given in Table I.Table IData collection details and unit cell parametersNative HSAHSA-propofolHSA-halothaneHSA-myristate-halothaneX-ray sourceDaresbury 9.6Daresbury 9.6Hamburg X11Daresbury 9.6Wavelength (Å)0.8700.8700.9090.870Space GroupTriclinic P1Triclinic P1Triclinic P1Monoclinic C2 a(Å)54.855.454.6188.9 b(Å)55.655.655.039.1 c(Å)120.3120.5120.096.7 α81.281.181.490.0 β91.190.690.8105.4 γ64.365.565.590.0Resolution range (Å)36.3–2.629.9–2.215.0–2.446.0–2.4Independent reflections37,95662,87048,00126,988Multiplicity 1-aValues for the outermost resolution shell are given in parentheses.2.0 (2.0)1.9 (1.6)1.9 (1.8)3.5 (3.4)Completeness (%) 1-aValues for the outermost resolution shell are given in parentheses.97.5 (97.3)96.1 (93.4)95.7 (87.7)99.1 (98.6)Rmerge(%)a,b4.5 (25.1)4.6 (29.6)4.9 (26.7)4.9 (27.8)I/ς1 1-aValues for the outermost resolution shell are given in parentheses.4.0 (1.3)7.6 (2.2)8.1 (2.2)8.6 (2.6)b Rmerge (%) = 100 × Σh Σj ‖ Ihj −Ih ‖ /Σh Σj Ihj, where Ih is the weighted mean intensity of the symmetry related reflectionsIhj.1-a Values for the outermost resolution shell are given in parentheses. Open table in a new tab b Rmerge (%) = 100 × Σh Σj ‖ Ihj −Ih ‖ /Σh Σj Ihj, where Ih is the weighted mean intensity of the symmetry related reflectionsIhj. The structure of native HSA was determined using molecular replacement with the program AMoRe (17Navaza J. Acta Crystallogr. Sect. A. 1994; 50: 157-163Crossref Scopus (5027) Google Scholar). The coordinates of the search model were those of "molecule A" in the 2.5-Å structure of HSA (Brookhaven code 1AO6) recently determined by Sugio et al. (15Sugio S. Kashima A. Mochizuki S. Noda M. Kobayashi K. Protein Eng. 1999; 12: 439-446Crossref PubMed Scopus (1481) Google Scholar). Rigid-body refinement was carried out using the program X-PLOR (18Brünger A.T. Kuriyan J. Karplus M. Science. 1987; 235: 458-460Crossref PubMed Scopus (2126) Google Scholar) followed by restrained least squares crystallographic refinement. For the structure containing both halothane and myristate, the HSA coordinates of the previously determined HSA-myristate structure (14Curry S. Mandelkow H. Brick P. Franks N. Nat. Struct. Biol. 1998; 5: 827-835Crossref PubMed Scopus (1171) Google Scholar) were used before rigid-body refinement. The coordinates for propofol were taken from the Cambridge Structural data base (19Fletcher D.A. McMeeking R.F. Parkin D.J. J. Chem. Inf. Comput. Sci. 1996; 36: 746-749Crossref Scopus (1150) Google Scholar), and those for halothane were calculated assuming standard stereochemistry. At the resolution of our data, the two enantiomers of halothane would have been indistinguishable and we arbitrarily chose to model theR enantiomer. After the addition of water molecules as well as fatty acid and anesthetic molecules where appropriate, all of the refined models had good stereochemistry (Table II), with no main-chain dihedral angles lying in disallowed regions of the Ramachandran plot (not shown). Coordinates and structure factors have been deposited in the Protein Data Bank; identification codes are given in Table II.Table IIModel refinementNative HSAHSA-propofolHSA-halothaneHSA-myristate-halothanePDB ID1e781e7a1e7b1e7cModeled amino acids5-5825-5825-5803-584Number of water molecules601205727Resolution range (Å)36.3–2.629.9–2.215.0–2.446.0–2.4Rmodel(%) 2-aRmodel (%) = 100 × Σhkl ‖ Fobs −Fcalc ‖ /Σhkl Fobs whereFobs and Fcalc are the observed and calculated structure factors, respectively.24.724.827.023.0Rfree(%) 2-bRfree (%) is theRmodel (%) calculated using a randomly selected 5% sample of reflection data omitted from refinement.27.727.230.328.1Root mean square deviation from ideal bond lengths (Å)0.0060.0070.0060.007Root mean square deviation from ideal bond angles (°)1.11.21.21.2Average B-factor (Å2)75.459.976.151.32-a Rmodel (%) = 100 × Σhkl ‖ Fobs −Fcalc ‖ /Σhkl Fobs whereFobs and Fcalc are the observed and calculated structure factors, respectively.2-b Rfree (%) is theRmodel (%) calculated using a randomly selected 5% sample of reflection data omitted from refinement. Open table in a new tab In the absence of fatty acids, HSA crystallized in a P1 space group with unit cell dimensions (Table I) that have not been observed before despite the fact that our crystallization conditions were similar to those used by others (13He X.M. Carter D.C. Nature. 1992; 358: 209-215Crossref PubMed Scopus (3452) Google Scholar, 15Sugio S. Kashima A. Mochizuki S. Noda M. Kobayashi K. Protein Eng. 1999; 12: 439-446Crossref PubMed Scopus (1481) Google Scholar). The native HSA structure that we have determined is essentially identical to those previously published, with only minor differences in the flexible subdomain IIIB (Fig. 1 A), due no doubt to differences in crystal packing. For comparison, Fig. 1 Bshows the HSA structure in the presence of myristate (14Curry S. Mandelkow H. Brick P. Franks N. Nat. Struct. Biol. 1998; 5: 827-835Crossref PubMed Scopus (1171) Google Scholar) 3Bhattacharya, A. A., Grüne, T., and Curry, S. (2000) J. Mol. Biol. 303, 721–732. and the locations of eight fatty acid binding sites. For the crystals containing propofol, the quality of the difference electron density allowed the positions and orientations of two propofol molecules to be unambiguously determined. One molecule (PR1) binds in subdomain IIIA, and the other (PR2) binds in subdomain IIIB (Fig. 2). The propofol molecule in IIIA (Fig. 2 B and Table III) binds in an apolar pocket with the phenolic hydroxyl group, making a hydrogen bond (3.1 Å) with the main-chain carbonyl oxygen of Leu-430 and with the aromatic ring of the anesthetic sandwiched between the side chains of Leu-453 and Asn-391. One of the two isopropyl groups makes numerous apolar contacts at one end of the pocket, whereas the other is exposed at the aqueous entrance, although it too makes close contacts with several side chains (Asn-391, Leu-407, Arg-410, and Tyr-411). The mouth of the binding pocket opens onto a network of five hydrogen-bonded water molecules that are further stabilized by interactions with Ser-489, Arg-410, and Tyr-411. The electron density for this solvent-exposed isopropyl group is much better defined (indicating a higher degree of order) than that of the isopropyl group, which is deeper in the pocket. The only conformational adjustment that takes place on propofol binding to this pocket is a 120° rotation of the side chain of Val 433, which moves to accommodate the inner isopropyl group. Comparisons with structures that contain fatty acids suggest that this propofol molecule would compete for ligand binding at fatty acid binding site FA3 and also disrupt the binding of fatty acid at site FA4 (via interactions with Arg-410, which coordinates the fatty acid carboxyl group) (14Curry S. Mandelkow H. Brick P. Franks N. Nat. Struct. Biol. 1998; 5: 827-835Crossref PubMed Scopus (1171) Google Scholar, 23Curry S. Brick P. Franks N.P. Biochim. Biophys. Acta. 1999; 1441: 131-140Crossref PubMed Scopus (450) Google Scholar)3.Table IIIPropofol binding sitesAnestheticBinding locationInteractions with hydroxylResidues lining cavity wallsPropofol 1IIIA (FA3)Leu-430 carbonyl OLeu-387, Ile-388, Asn-391, Cys-392, Phe-403, Leu-407, Arg-410, Tyr-411, Val-433, Gly-434, Cys-438, Ala-449, Leu-453Propofol 2IIIB (FA5)S579Phe-502, Phe-507, Phe-509, Ala-528, Glu-531, Leu-532, His-535, Val-547, Phe-551, Val-576, Gln-580 Open table in a new tab The second propofol molecule (Fig. 2 C, Table III) binds in a cavity located in sub-domain IIIB that is mainly lined by aromatic residues (Phe-502, Phe-507, Phe-509, and Phe-551). The anesthetic is sandwiched between the side chains of Phe-502 and Leu-532, which make close contacts with the propofol aromatic ring. The aliphatic portion of Glu-531 and the side chain of His-535, situated approximately 4 Å from the base of the propofol molecule, close off this end of the pocket. The hydroxyl group of Ser-579 makes a hydrogen bond (2.9 Å) with the propofol hydroxyl. The entrance to the binding pocket is quite polar, with several well-ordered water molecules and a number of polar residues in close proximity. As with the first propofol site, there are only a few minor local conformational changes on binding, the most marked of these being a 90° rotation about the Cα-Cβ bond of Phe-507, which moves the side chain away from the center of the binding pocket (there are also minor movements in the aromatic rings of Phe-502 and Phe-509). Superposition of the fatty acid structures3indicates that the binding of this propofol molecule could be prevented by ligands that bind to fatty acid binding site FA5. It is probable that the first of the two propofol binding sites (PR1 in sub-domain IIIA) has the highest affinity because, during one experiment where the crystals were partially back-soaked and the propofol concentration was reduced, the electron density for the second propofol molecule PR2 disappeared, whereas that for the first molecule was easily interpretable (data not shown). When crystals of HSA were exposed to halothane vapor, we found that a maximum concentration of around 15% of the saturated vapor pressure could be used before there was a noticeable deterioration in the resolution of the diffraction pattern. With myristate-containing crystals, a significantly higher concentration could be used (60% of the saturated vapor pressure) before this occurred. At the lower concentration and in the absence of fatty acid, the difference electron density showed three "high affinity" halothane binding sites (molecules HAL1, HAL2, and HAL3; Fig. 3 A, Table IV). (Although the position of the electron-dense bromine atom was always clear, there was some ambiguity about the relative positions of the chlorine atom and the CF3 group. In most cases the shape of the density was used to guide positioning of the slightly bulkier CF3 group, but because the data are limited to 2.4 Å resolution and the model B-factors are relatively high, the orientations modeled cannot be regarded as definitive.) Two of these halothane molecules (HAL1 and HAL2) bind within a solvent-exposed trough at the interface between subdomains IIA and IIB, which can also bind a fatty acid molecule (FA6). At the higher halothane concentration, a third molecule (HAL4) also binds in the trough (see Figs. 3 B and4 A), adjacent to HAL1 and HAL2. The strongest density was observed for the central halothane molecule HAL1, which binds in an amphiphilic environment formed on the one side by the polar groups of Arg-209 and Glu-534, which interact via a salt bridge (that also involves Asp-324), and on the other side by the aliphatic portion of Lys-212 and the side chains of Ala-213 and Leu-327. The second molecule (HAL2) is in a predominantly apolar environment (Ala-213, Leu-347, Ala-350 and the aliphatic portion of K351), although a polar interaction is provided by Arg-209. The third molecule (HAL4) in the trough only binds at much higher concentrations and makes relatively few interactions with neighboring side chains. Even at the higher halothane concentration there was, within experimental error, no significant change in the local structure, despite the competitive displacement of myristate.Table IVHalothane binding sitesAnestheticBinding locationResidues lining cavity wallsHalothane 1IIA-IIBArg-209, Ala-210, Ala-213, Leu-347, Ala-350, Lys-351, Glu-354, Lys-212(FA6)Halothane 2IIA-IIBArg-209, Lys-212, Ala-213, Val-216, Asp-324, Leu-327, Leu-331(FA6)Halothane 3IIIAIle-388, Asn-391, Phe-403, Leu-407, Leu-430, Val-433, Gly-434, Cys-438, Ala-449, Leu-453(FA3 & 4)Halothane 4IIA-IIBVal-216, Phe-228, Ser-232, Val-235, Val-325(FA6)Halothane 5IIALeu-238, His-242, Arg-257, Leu-260, Ile-264, Ser-287, Ile-290, Ala-291(FA7)Halothane 6IIATrp-214, Arg-218, Leu-219, Arg-222, Phe-223, Leu-238, Ala-291(FA7)Halothane 7IA-IIAAla-26, Tyr-30, Leu-66, His-67, Phe-70, Asn-99, Asp-249, Leu-250, Leu-251(FA8)Halothane 8IA-IBAla-21, Leu-135, Lys-136, Leu-139, Leu-155, Ala-158, Lys-159, Lys-162 Open table in a new tab Figure 4Details of halothane binding sites. A, halothane binding sites at the interface between subdomains IIA and IIB. B, halothane site in subdomain IIIA.C, halothane sites in subdomain IIA. D, halothane site at the interface between subdomains IA and IIA. The difference electron density is an Fo − Fcomit map calculated at 4 ς. Some of the amino acid side chains that are close to the halothane molecules are shown as ball andstick models (a complete list is given in Table IV). Note that in D only 11 of the 14 carbon atoms of myristate are shown because, due to disorder, the terminal carbons were not observed in the electron density map.View Large Image Figure ViewerDownload (PPT) At the lower halothane concentration, in addition to the two molecules HAL1 and HAL2 at the IIA/IIB interface, a third high affinity molecule (HAL3) is present in subdomain IIIA (Fig. 3, A andB). This molecule binds in a site that overlaps with the methylene tail of the fatty acid bound in site FA3 and with the first propofol molecule (PF1). HAL3 makes numerous close, mainly apolar, interactions within the binding pocket (Table IV and Fig. 4 B). The bromine atom interacts with the sulfur of Cys-438, the main chain of Gly-434 and makes additional (hydrophobic) contacts with Phe-403 and the side chain of Asn-391 (Fig. 4 B). At the higher halothane concentration, electron density appears for molecules HAL5 and HAL6 within a binding site in subdomain IIA that can also bind fatty acid FA7. These two halothane molecules (see Fig. 3 B and 4 C) lie adjacent to one another in a predominantly apolar environment, although both molecules also interact with polar groups. The main chain carbonyl oxygen of Arg-257 contacts halothane HAL5, whereas its charged guanidinium side chain interacts with the bromine atom of the anesthetic. Similarly, the bromine atom of HAL6 is close to the guanidinium of Arg-222. HAL6 is also within 5 Å of Trp-214, which has been implicated in halothane binding to HSA (7Eckenhoff R.G. J. Biol. Chem. 1996; 271: 15521-15526Crossref PubMed Scopus (64) Google Scholar). With the HSA structure in the presence of myristate and at the higher halothane concentration, we observed strong electron density for two more halothane molecules (HAL7 and HAL8). One of these (HAL7) binds at the interface between subdomains IA and IIA (Fig. 3 B and4 D) in a cavity that is formed as a consequence of the fatty acid-induced conformational change (Ref. 14Curry S. Mandelkow H. Brick P. Franks N. Nat. Struct. Biol. 1998; 5: 827-835Crossref PubMed Scopus (1171) Google Scholar and Fig. 1). This conformational change rotates domain I relative to domain II to create a largely apolar cavity that is flanked on one side by the methylene tail of the fatty acid bound to FA2. The bromine atom is coordinated by several polar interactions (Tyr-30, His-67, Asn-99, and Asp-249). Binding of HAL7 displaces the myristate from site FA8. The other halothane molecule, HAL8, present in the HSA-myristate crystals (Fig. 3 B), binds in a solvent-exposed niche that is formed by the parallel side chains of Lys-136, Lys-159, and Lys-162 (not shown). The orientations of these side chains that form the hydrophobic cavity are determined very largely by interactions with a symmetry-related HSA molecule in the crystal, suggesting that the binding site for halothane HAL8 is a crystallographic artifact. A number of general statements can be made about the nature of the propofol and halothane binding sites on HSA and the effects these anesthetics have on the protein structure. First, only a relatively small number of discrete sites are involved. In all cases these are pre-formed pockets or clefts on the protein that are, in almost all cases, capable of binding natural ligands (i.e. fatty acids). Second, the only changes we observed in local structure were two side-chain conformational changes on propofol binding (see "Results"), and there was no evidence in the pharmacologi