THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 275, No. 49, Issue of December 8, pp. 38731 38738, 2000
© 2000 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
Binding of the General Anesthetics Propofol and Halothane to
Human Serum Albumin
HIGH RESOLUTION CRYSTAL STRUCTURES*
Received for publication, June 22, 2000, and in revised form, August 11, 2000
Published, JBC Papers in Press, August 11, 2000, DOI 10.1074/jbc.M005460200
Ananyo A. Bhattacharya, Stephen Curry! , and Nicholas P. Franks!
From the Biophysics Section, The Blackett Laboratory, Imperial College of Science, Technology and Medicine,
London SW7 2BW, United Kingdom
Human serum albumin (HSA) is one of the most abun- on the forces that are involved in anesthetic-protein interac-
dant proteins in the circulatory system and plays a key tions and virtually no information on the molecular architec-
role in the transport of fatty acids, metabolites, and
tures of anesthetic binding sites.
drugs. For many drugs, binding to serum albumin is a
The lack of direct structural information is due at least in
critical determinant of their distribution and pharma-
part to the fact that the most likely targets for general anes-
cokinetics; however, there have as yet been no high
thetics are thought to be neuronal ion channels. These are, of
resolution crystal structures published of drug-albumin
course, integral membrane proteins and have proven to be
complexes. Here we describe high resolution crystal
exceptionally difficult to crystallize in a form that is suitable for
structures of HSA with two of the most widely used
high resolution x-ray diffraction analysis. However, there are
general anesthetics, propofol and halothane. In addi-
several soluble proteins to which anesthetics are known to
tion, we describe a crystal structure of HSA complexed
bind, and studies with these proteins have provided valuable
with both halothane and the fatty acid, myristate. We
information on the nature of anesthetic binding sites. Most of
show that the intravenous anesthetic propofol binds at
this work has been done with serum proteins and luciferase
two discrete sites on HSA in preformed pockets that
enzymes, but so far the only example of an anesthetic-sensitive
have been shown to accommodate fatty acids. Similarly
protein for which there is also high resolution structural data is
we show that the inhalational agent halothane binds (at
firefly luciferase (3).
concentrations in the pharmacologically relevant
Perhaps the most extensively studied anesthetic binding pro-
range) at three sites that are also fatty acid binding loci.
At much higher halothane concentrations, we have tein is serum albumin, and there have been numerous attempts
identified additional sites that are occupied. All of the to characterize the binding sites involved (4 8), none of them,
higher affinity anesthetic binding sites are amphiphilic
however, using direct structural techniques. This protein is not
in nature, with both polar and apolar parts, and anes-
only amenable to high resolution structural analysis but, more
thetic binding causes only minor changes in local
importantly, is known to play a key role in the pharmacological
structure.
actions of several general anesthetics.
The importance of serum albumin in anesthetic pharmacol-
ogy derives from its high concentration in the circulatory sys-
How general anesthetics exert their effects in the central
tem (approximately 0.6 mM in plasma) and from its ability to
nervous system has remained a puzzle for more than 150 years,
bind an extraordinarily diverse range of drugs (including most
but there is now a growing consensus that they act by binding
anesthetics), metabolites, and fatty acids (for reviews, see Refs.
directly to protein targets (1). The identity of these targets,
9 11). In several cases more than 50% of a clinically adminis-
however, remains uncertain, although a large body of evidence
tered general anesthetic will be bound to serum albumin, and
is accumulating on the functional effects of general anesthetics
in some cases, such as the intravenous agent propofol, approx-
on a variety of possible candidates (1, 2). Most of these data
imately 80% is bound (12). Consequently, any changes in the
come from electrophysiological measurements, coupled more
interactions between an anesthetic and serum albumin, either
recently with the techniques of molecular genetics. Although
by fatty acids or other drugs competing for binding or by ge-
these approaches are crucial in understanding the actions of
netic modifications in the protein itself, could result in signif-
general anesthetics, they give at best only indirect information
icant changes in the pharmacologically active concentration of
the anesthetic.
* This work was supported by grants from the Medical Research Although a high resolution structure of human serum albu-
Council, London and the Biotechnology and Biological Sciences Re-
min was published some years ago (13), the unavailability of
search Council, Swindon, United Kingdom. The costs of publication of
the three-dimensional coordinates did not encourage others to
this article were defrayed in part by the payment of page charges. This
extend this work. Curry et al. (14) subsequently published a
article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact. high resolution structure of the protein that identified the
The atomic coordinates and structure factors (code 1e78 (native HSA),
principal fatty acid binding sites, and this was followed by the
1e7a (HSA-propofol),: 1e7b (HSA-halothane), 1e7c (HSA-myristate-hal-
publication of an independent determination of the native
othane)) have been deposited in the Protein Data Bank, Research Col-
structure (15). The protein is heart-shaped and contains 585
laboratory for Structural Bioinformatics, Rutgers University, New Bruns-
wick, NJ (http:/ amino acids. It is organized into three homologous domains
/www.rcsb.org/).
! To whom correspondence should be addressed: Biophysics Section,
(labeled I-III), and each domain consists of two sub-domains (A
The Blackett Laboratory, Imperial College of Science, Technology, and
and B) that share common structural elements (Fig. 1). In this
Medicine, Prince Consort Rd., London SW7 2BW, UK. Tel.: 004420-
paper we have used x-ray crystallography to provide high res-
7594-7629; Fax: 004420-7589-0191; E-mail: s.curry@ic.ac.uk or n.
franks@ic.ac.uk. olution information on the nature and locations of the principal
This paper is available on line at http://www.jbc.org 38731
Downloaded from www.jbc.org by guest, on May 26, 2012
38732 Propofol and Halothane Binding Sites on Human Serum Albumin
Zeneca Pharmaceuticals, Alderley Park, Macclesfield, UK. Co-crystal-
binding sites for two of the most widely used general anesthet-
lization with propofol generally resulted in larger crystals than those
ics, the intravenous agent propofol and the inhalational agent
obtained in the absence of propofol. Native propofol-free crystals could
halothane (see Structures I and II).
be readily obtained by back-soaking in solutions that contained progres-
sively less propofol while at the same time progressively increasing the
EXPERIMENTAL PROCEDURES
polyethylene glycol concentration up to 32% (w/v).
Protein Purification Most experiments were carried out using fat-
Complexes with halothane were prepared by exposing native crystals
free recombinant HSA,1 prepared by charcoal treatment (16) at low pH.
to chosen partial pressures of halothane in 1-mm sealed glass capillar-
1
This was supplied at a concentration of 250 mg ml in 145 mM NaCl by
ies at room temperature. The partial pressure was set by using mix-
Dr. John Woodrow of Delta Biotechnology Limited (Nottingham, UK).
tures of halothane and hexadecane at defined mole ratios. To the extent
The halothane-myristate complex was formed using protein that, in
that halothane and hexadecane mix ideally, the vapor pressure of
1
addition, originally contained 40 mM octanoate (C8:0) and 15 mg liter
halothane above such a mixture can, according to Raoult s Law, be
Tween 80. In both cases the protein was further purified on a Superdex
taken to be proportional to its mole fraction. The maximum partial
S75 gel filtration column (Amersham Pharmacia Biotech) with a phos-
pressure of halothane that could be used with native crystals before a
phate running buffer (50 mM potassium phosphate, 150 mM sodium
significant deterioration in the diffraction patterns was observed was
chloride, pH 7.5) to remove dimers and polymers of HSA, exactly as
15% of the saturated vapor pressure, which would correspond to a
described previously (14). After combining the appropriate fractions,
partial pressure of 5% atm, or 2.6 mM in free aqueous solution. To
the running buffer was exchanged with a storage buffer (50 mM potas-
prepare the halothane-myristate complex, crystals with myristate were
sium phosphate, pH 7.0), and the protein was concentrated using an
first prepared (14) before exposure to halothane, as described above. In
Amicon 30-kDa molecular mass cut-off centrifugal concentrator (Milli-
the presence of myristate we found that a much higher concentration of
1
pore, Watford, Hertfordshire, UK) to greater than 80 mg ml and
halothane could be used (60% of the saturated vapor pressure, which
stored at 4 °C. All chemicals were obtained from Sigma unless other-
would correspond to a partial pressure of 20% atm, or 10.5 mM in free
wise stated.
aqueous solution) before lattice disorder in the crystals reduced the
Crystallization and Complex Formation Crystals of native HSA
resolution of the diffraction patterns.
were grown by vapor diffusion at 4 °C using the sitting drop configura-
Data Collection and Processing Data were collected to high resolu-
tion. Crystals were first grown with a reservoir of 28 30% (w/v) poly-
ethylene 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 poly-
ethylene 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 saturat-
ing concentration of propofol (approximately 4 mM in 25 26% polyeth-
ylene glycol) was maintained throughout. The propofol was a gift from
1
STRUCTURES I AND II.
The abbreviation used is: HSA, human serum albumin.
TABLE I
Data collection details and unit cell parameters
HSA-myristate-
Native HSA HSA-propofol HSA-halothane
halothane
X-ray source Daresbury 9.6 Daresbury 9.6 Hamburg X11 Daresbury 9.6
Wavelength (Å) 0.870 0.870 0.909 0.870
Space Group Triclinic P1 Triclinic P1 Triclinic P1 Monoclinic C2
a (Å) 54.8 55.4 54.6 188.9
b (Å) 55.6 55.6 55.0 39.1
c (Å) 120.3 120.5 120.0 96.7
81.2 81.1 81.4 90.0
91.1 90.6 90.8 105.4
64.3 65.5 65.5 90.0
Resolution range (Å) 36.3 2.6 29.9 2.2 15.0 2.4 46.0 2.4
Independent reflections 37,956 62,870 48,001 26,988
Multiplicitya 2.0 (2.0) 1.9 (1.6) 1.9 (1.8) 3.5 (3.4)
Completeness (%)a 97.5 (97.3) 96.1 (93.4) 95.7 (87.7) 99.1 (98.6)
Rmerge (%)a,b 4.5 (25.1) 4.6 (29.6) 4.9 (26.7) 4.9 (27.8)
I/ 1a 4.0 (1.3) 7.6 (2.2) 8.1 (2.2) 8.6 (2.6)
a
Values for the outermost resolution shell are given in parentheses.
b
Rmerge (%) 100 Ihj Ih / Ihj, where Ih is the weighted mean intensity of the symmetry related reflections Ihj.
h j h j
TABLE II
Model refinement
Native HSA HSA-propofol HSA-halothane HSA-myristate-halothane
PDB ID 1e78 1e7a 1e7b 1e7c
Modeled amino acids 5-582 5-582 5-580 3-584
Number of water molecules 60 120 57 27
Resolution range (Å) 36.3 2.6 29.9 2.2 15.0 2.4 46.0 2.4
Rmodel (%)a 24.7 24.8 27.0 23.0
Rfree (%)b 27.7 27.2 30.3 28.1
Root mean square deviation from ideal bond lengths 0.006 0.007 0.006 0.007
(Å)
Root mean square deviation from ideal bond angles (°) 1.1 1.2 1.2 1.2
Average B-factor (Å2) 75.4 59.9 76.1 51.3
a
Rmodel (%) 100 Fobs Fcalc / Fobs where Fobs and Fcalc are the observed and calculated structure factors, respectively.
hkl hkl
b
Rfree (%) is the Rmodel (%) calculated using a randomly selected 5% sample of reflection data omitted from refinement.
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Propofol and Halothane Binding Sites on Human Serum Albumin 38733
tion 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.2 Details of
the data collection are given in Table I.
Structure Determination and Model Refinement The structure of
native HSA was determined using molecular replacement with the
program AMoRe (17). 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. (15).
Rigid-body refinement was carried out using the program X-PLOR
(18) 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 (14)
were used before rigid-body refinement. The coordinates for propofol
were taken from the Cambridge Structural data base (19), 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 the R
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.
RESULTS
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 condi-
tions were similar to those used by others (13, 15). 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. 1A), due no doubt to differ-
ences in crystal packing. For comparison, Fig. 1B shows the
HSA structure in the presence of myristate (14)3 and the loca-
tions of eight fatty acid binding sites.
For the crystals containing propofol, the quality of the dif-
ference 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. 2B 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 FIG. 1. The structure of HSA and the locations of fatty acid
binding sites. The native structure of HSA (A) and the structure of
makes numerous apolar contacts at one end of the pocket,
HSA in the presence of myristate (B), showing the locations of eight
whereas the other is exposed at the aqueous entrance, although
fatty acid binding sites. Fatty acids FA4 and FA8 are shown in a darker
it too makes close contacts with several side chains (Asn-391,
shade of gray for clarity of presentation. Further details on the fatty
Leu-407, Arg-410, and Tyr-411). The mouth of the binding
acid binding sites have been published elsewhere (14, 23).3 The domains
are color-coded as follows: red, domain I; green, domain II; blue, domain
pocket opens onto a network of five hydrogen-bonded water
III. The A and B sub-domains within each domain are depicted in dark
molecules that are further stabilized by interactions with Ser-
and light shades, respectively. The fatty acids are represented by space-
489, Arg-410, and Tyr-411. The electron density for this sol-
filling models colored by atom type (gray, carbon; red, oxygen). All
vent-exposed isopropyl group is much better defined (indicat-
figures were prepared using Bobscript and Raster3D (20, 40 41).
ing a higher degree of order) than that of the isopropyl group,
which is deeper in the pocket. The only conformational adjust- The second propofol molecule (Fig. 2C, Table III) binds in a
ment that takes place on propofol binding to this pocket is a cavity located in sub-domain IIIB that is mainly lined by aro-
120° rotation of the side chain of Val 433, which moves to matic residues (Phe-502, Phe-507, Phe-509, and Phe-551). The
accommodate the inner isopropyl group. Comparisons with anesthetic is sandwiched between the side chains of Phe-502
structures that contain fatty acids suggest that this propofol and Leu-532, which make close contacts with the propofol
molecule would compete for ligand binding at fatty acid binding aromatic ring. The aliphatic portion of Glu-531 and the side
site FA3 and also disrupt the binding of fatty acid at site FA4 chain of His-535, situated approximately 4 Å from the base of
(via interactions with Arg-410, which coordinates the fatty acid the propofol molecule, close off this end of the pocket. The
carboxyl group) (14, 23)3. 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
2
A. Leslie, personal communication.
3
number of polar residues in close proximity. As with the first
Bhattacharya, A. A., Grüne, T., and Curry, S. (2000) J. Mol. Biol.
303, 721 732. propofol site, there are only a few minor local conformational
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38734 Propofol and Halothane Binding Sites on Human Serum Albumin
FIG. 2. The propofol binding sites on HSA. A, HSA with propofol showing the locations of the two propofol binding sites. Site PR1, which is
within sub-domain IIIA (B), and site PR2, which is within sub-domain IIIB (C) are shown. The dashed lines represent hydrogen bonds. The
difference electron density is an Fo Fc omit map calculated at 4 . The amino acid side chains that are close to the propofol molecules are shown
as ball and stick models (a complete list is given in Table III).
TABLE III
Propofol binding sites
Anesthetic Binding location Interactions with hydroxyl Residues lining cavity walls
Propofol 1 IIIA (FA3) Leu-430 carbonyl O Leu-387, Ile-388, Asn-391, Cys-392, Phe-403, Leu-407, Arg-410, Tyr-411, Val-433,
Gly-434, Cys-438, Ala-449, Leu-453
Propofol 2 IIIB (FA5) S579 Phe-502, Phe-507, Phe-509, Ala-528, Glu-531, Leu-532, His-535, Val-547,
Phe-551, Val-576, Gln-580
changes on binding, the most marked of these being a 90° HAL1, which binds in an amphiphilic environment formed on
rotation about the C -C bond of Phe-507, which moves the the one side by the polar groups of Arg-209 and Glu-534, which
side chain away from the center of the binding pocket (there are interact via a salt bridge (that also involves Asp-324), and on
also minor movements in the aromatic rings of Phe-502 and the other side by the aliphatic portion of Lys-212 and the side
Phe-509). Superposition of the fatty acid structures3 indicates chains of Ala-213 and Leu-327. The second molecule (HAL2) is
that the binding of this propofol molecule could be prevented by in a predominantly apolar environment (Ala-213, Leu-347, Ala-
ligands that bind to fatty acid binding site FA5. It is probable 350 and the aliphatic portion of K351), although a polar inter-
that the first of the two propofol binding sites (PR1 in sub- action is provided by Arg-209. The third molecule (HAL4) in the
domain IIIA) has the highest affinity because, during one ex- trough only binds at much higher concentrations and makes
periment where the crystals were partially back-soaked and relatively few interactions with neighboring side chains. Even
the propofol concentration was reduced, the electron density for at the higher halothane concentration there was, within exper-
the second propofol molecule PR2 disappeared, whereas that imental error, no significant change in the local structure,
for the first molecule was easily interpretable (data not shown). despite the competitive displacement of myristate.
When crystals of HSA were exposed to halothane vapor, we At the lower halothane concentration, in addition to the two
found that a maximum concentration of around 15% of the molecules HAL1 and HAL2 at the IIA/IIB interface, a third
saturated vapor pressure could be used before there was a high affinity molecule (HAL3) is present in subdomain IIIA
noticeable deterioration in the resolution of the diffraction pat- (Fig. 3, A and B). This molecule binds in a site that overlaps
tern. With myristate-containing crystals, a significantly higher with the methylene tail of the fatty acid bound in site FA3 and
concentration could be used (60% of the saturated vapor pres- with the first propofol molecule (PF1). HAL3 makes numerous
sure) before this occurred. At the lower concentration and in close, mainly apolar, interactions within the binding pocket
the absence of fatty acid, the difference electron density showed (Table IV and Fig. 4B). The bromine atom interacts with the
three high affinity halothane binding sites (molecules HAL1, sulfur of Cys-438, the main chain of Gly-434 and makes addi-
HAL2, and HAL3; Fig. 3A, Table IV). (Although the position of tional (hydrophobic) contacts with Phe-403 and the side chain
the electron-dense bromine atom was always clear, there was of Asn-391 (Fig. 4B).
some ambiguity about the relative positions of the chlorine At the higher halothane concentration, electron density ap-
atom and the CF3 group. In most cases the shape of the density pears for molecules HAL5 and HAL6 within a binding site in
was used to guide positioning of the slightly bulkier CF3 group, subdomain IIA that can also bind fatty acid FA7. These two
but because the data are limited to 2.4 Å resolution and the halothane molecules (see Fig. 3B and 4C) lie adjacent to one
model B-factors are relatively high, the orientations modeled another in a predominantly apolar environment, although both
cannot be regarded as definitive.) Two of these halothane mol- molecules also interact with polar groups. The main chain
ecules (HAL1 and HAL2) bind within a solvent-exposed trough carbonyl oxygen of Arg-257 contacts halothane HAL5, whereas
at the interface between subdomains IIA and IIB, which can its charged guanidinium side chain interacts with the bromine
also bind a fatty acid molecule (FA6). At the higher halothane atom of the anesthetic. Similarly, the bromine atom of HAL6 is
concentration, a third molecule (HAL4) also binds in the trough close to the guanidinium of Arg-222. HAL6 is also within5Åof
(see Figs. 3B and 4A), adjacent to HAL1 and HAL2. The strong- Trp-214, which has been implicated in halothane binding to
est density was observed for the central halothane molecule HSA (7).
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Propofol and Halothane Binding Sites on Human Serum Albumin 38735
With the HSA structure in the presence of myristate and at
the higher halothane concentration, we observed strong elec-
tron density for two more halothane molecules (HAL7 and
HAL8). One of these (HAL7) binds at the interface between
subdomains IA and IIA (Fig. 3B and 4D) in a cavity that is
formed as a consequence of the fatty acid-induced conforma-
tional change (Ref. 14 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. 3B), 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.
DISCUSSION
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 in-
volved. 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 pharmacologically relevant range of concentrations (see
below) of global changes in protein structure. In the case of
propofol, there were no generalized changes in structure even
at saturating concentrations of the drug, whereas the same was
true for halothane at concentrations up to 5% atm in the
absence of fatty acid and up to 20% atm in the presence of fatty
acid. Only above these concentrations did we see evidence of
crystal disorder, but this could have been a consequence of
crystal contacts being disrupted rather than due to a confor-
mational change in the protein.
It has been shown (21) that inhalational anesthetics shift the
denaturation temperature of BSA to higher temperatures (pre-
sumably as a consequence of the anesthetics binding to the
FIG. 3. The halothane binding sites on HSA. A, HSA with halo-
folded rather than the unfolded state), and it has also been thane at a low concentration, showing three halothane binding sites. B,
HSA, with halothane at high concentration, and myristate, showing
shown (22) that the fluorescent anisotropy of two tryptophan
seven halothane binding sites and five fatty acid binding sites. The
residues in BSA are increased in the presence of anesthetics.
anesthetics and fatty acids are represented by space-filling models
On the basis of these two observations it has been proposed (22)
colored by atom type (gray, carbon; red, oxygen; brown, bromine; dark
that anesthetics may exert their effects on proteins at the green, chlorine; light green, fluorine).
molecular level by attenuating the movement of the local amino
acid side chains, which is in turn postulated to stabilize certain FA5, for the molecule in IIIB. The propofol molecule in IIIB not
protein conformations and, hence, affect function. One predic- only binds weaker than the molecule in IIIA (because electron
tion would be that amino acids that line anesthetic binding density for this molecule was the first to disappear when the
sites should show reduced crystallographic temperature factors propofol concentration was reduced), but it also binds in a site
when anesthetics bind. However, although the HSA/myristate/ that almost certainly accommodates the most tightly binding
halothane structure does have an average temperature factor fatty acid (23, 24). For these reasons one can safely conclude
that is significantly lower than the structure with myristate that, at pharmacologically relevant concentrations of propofol
alone, the amino acids directly in contact with the anesthetics in the blood (which are many times lower than the concentra-
have temperature factors reduced to the same extent as those tions present in our crystals), only a single propofol binding site
amino acids that do not contribute to binding interactions. would be occupied (the site in subdomain IIIA). This site in
Propofol binds at two sites, one in subdomain IIIA and one in subdomain IIIA has previously been identified crystallographi-
subdomain IIIB. In both cases the aromatic ring lies within an cally (13) as one of the two most important drug binding sites
apolar pocket, with the phenolic hydroxyl group making a (termed site II by Sudlow et al. (25, 26)) and one that can also
hydrogen bond, in the one case (IIIA) with a main-chain car- accommodate diazepam, ibuprofen, and other aromatic drugs.
bonyl oxygen and in the other case (IIIB) with a serine hydroxyl Our data with propofol showing only two discrete binding
(Fig. 2). Both propofol molecules would compete for fatty acid sites, even at saturating concentrations, are very difficult to
binding: FA3 and possibly FA4, for the molecule in IIIA, and reconcile with some recent binding studies that have concluded
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38736 Propofol and Halothane Binding Sites on Human Serum Albumin
TABLE IV
Halothane binding sites
Anesthetic Binding location Residues lining cavity walls
Halothane 1 IIA-IIB Arg-209, Ala-210, Ala-213, Leu-347, Ala-350, Lys-351, Glu-354, Lys-212
(FA6)
Halothane 2 IIA-IIB Arg-209, Lys-212, Ala-213, Val-216, Asp-324, Leu-327, Leu-331
(FA6)
Halothane 3 IIIA Ile-388, Asn-391, Phe-403, Leu-407, Leu-430, Val-433, Gly-434, Cys-438, Ala-449, Leu-453
(FA3 & 4)
Halothane 4 IIA-IIB Val-216, Phe-228, Ser-232, Val-235, Val-325
(FA6)
Halothane 5 IIA Leu-238, His-242, Arg-257, Leu-260, Ile-264, Ser-287, Ile-290, Ala-291
(FA7)
Halothane 6 IIA Trp-214, Arg-218, Leu-219, Arg-222, Phe-223, Leu-238, Ala-291
(FA7)
Halothane 7 IA-IIA Ala-26, Tyr-30, Leu-66, His-67, Phe-70, Asn-99, Asp-249, Leu-250, Leu-251
(FA8)
Halothane 8 IA-IB Ala-21, Leu-135, Lys-136, Leu-139, Leu-155, Ala-158, Lys-159, Lys-162
that propofol binds to a large number (around 15) of saturable weakly to proteins could exert their effects by shifting the
sites (27) or that propofol causes protein unfolding that results equilibria between functionally distinct conformational states
in the absence of any saturable sites (12). It is possible that (e.g. the open and closed states of an ion channel).
these binding studies were somehow confounded by the pres- Which of the halothane sites are pharmacologically relevant?
ence of fatty acids (no particular precautions were taken to This is a difficult question to answer with certainty. The low
exclude them), and more work is clearly needed to resolve the halothane concentration we used (5% atm) was still signifi-
apparent discrepancy between these binding studies and our cantly higher than the maximum concentration likely to be
crystallographic results. used for maintenance of anesthesia, so those binding sites that
At the low halothane concentration and in the absence of were only populated at the higher concentration (HAL4, HAL5,
fatty acid, only three halothane binding sites were well occu- and HAL6) are most unlikely to be important. However, all
pied (HAL1, HAL2, and HAL3). However, we could not discern three of the halothane molecules that bind at the lower con-
any key features of these binding sites that distinguished them centration (HAL1, HAL2, and HAL3) are potentially displace-
from the lower affinity sites that were occupied at the higher able by fatty acid, and between 0.1 and 2 molecules of fatty acid
halothane concentration. All of the binding sites were predom- is thought to bind under normal physiological conditions. The
inantly apolar, although most also showed evidence of signifi- halothane molecules HAL1 and HAL2 are probably less sus-
cant polar interactions between charged or polar amino acids ceptible to displacement than HAL3 because in the myristate
and the polarizable halogen atoms, particularly the bromine. structure the halothane molecules HAL1 and HAL2 were able
The possible importance of polar interactions between proteins to displace the fatty acid FA6, whereas in contrast, the fatty
and halogenated compounds has been noted before (3, 28), and acid FA3 was able to prevent the binding of halothane HAL3.
the likelihood that general anesthetic binding sites are am- In addition, other evidence3 suggests that FA3 binds more
phiphilic in nature has been stressed by our group (29, 30) and tightly than FA6. Finally, it might be that there is sufficient
others (31 33). fatty acid in the blood to induce the conformational change that
Interestingly, as was the case with propofol, all of the halo- results in the formation of the binding site for HAL7, which
thane molecules bound within pre-formed pockets or clefts. would also make this site (in addition to those for HAL1, HAL2,
Furthermore (leaving aside halothane HAL8, whose binding and HAL3) potentially relevant pharmacologically.
site was artifactually formed by crystal contacts), all of the Because of the promiscuous nature of HSA-drug interac-
binding sites were also binding sites for fatty acids. Indeed, in tions, the possibility that the free, pharmacologically active
the crystal structure at the high halothane concentration and concentrations of co-administered drugs could be affected by
in the presence of myristate, the fatty acid has clearly been their competing for common binding sites on the protein has
displaced in sites FA6 (by HAL1, HAL2, and HAL4), FA7 (by often been considered (11). For example, the volatile anesthetic
HAL5 and HAL6), and FA8 (by HAL7). This is entirely consist- enflurane has been shown (36) to displace diazepam from HSA
ent with the work of Dubois et al. (5) and Dubois and Evers (6) in vitro, and the in vivo pharmacokinetics of thiopental are
on the related protein bovine serum albumin that showed hal- known to be significantly affected (37) by the presence of hal-
othane and other volatile anesthetics competed with fatty acids othane. Our finding that propofol binds with highest affinity to
for binding. In addition, the two halothane molecules, HAL5 a site in subdomain IIIA that can also bind a benzodiazepine
and HAL6, bind within a site that has been identified (13) as (13) suggests that there might be a significant interaction
a key drug binding locus on HSA ( site I of Sudlow et al. between these drugs (which are often co-administered). How-
(25, 26)). ever, a common binding site does not guarantee a pharmaco-
Although halothane binding to HAL7 can displace myristate logically relevant interaction. Although a high percentage of
bound to FA8, this site is not occupied by fatty acids with longer both drugs may be bound to HSA, for either drug the percent-
chains,3 which are much more prevalent in normal plasma (34). age of HSA molecules that are involved in binding could still be
Thus, under normal physiological conditions, the binding of very small (because the plasma concentration of HSA is very
HAL7 would be expected to increase rather than decrease due much greater than the total drug concentration). Indeed, a
to the presence of fatty acids whose binding is responsible for brief report (12) concluded that diazepam did not displace
the formation of the cavity within which HAL7 binds. This bound propofol; nonetheless this potential interaction has yet
observation supports an early suggestion (35) that anesthetics to be extensively studied.
might act by stabilizing certain conformational states of a Perhaps paradoxically, it is the relatively weaker binding
protein simply because binding sites appear fortuitously in that drugs such as the volatile general anesthetics that might be
state. Thus even anesthetics that bind intrinsically very more effective at competing with other drugs for binding to
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Propofol and Halothane Binding Sites on Human Serum Albumin 38737
FIG. 4. Details 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 Fc omit map calculated at 4 . Some of the amino acid side chains that are close to the halothane molecules are shown as ball
and stick 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.
HSA. This is because they are present at sufficiently high In summary, we have shown that two widely used general
concentrations to interact, at least potentially, with a large anesthetics, propofol and halothane, bind to a small number of
fraction of the HSA molecules. From our data we can conclude
discrete sites on HSA in the pharmacologically relevant range
that halothane (and perhaps other volatile anesthetics) could
of concentrations. These sites are preformed amphiphilic pock-
compete for the binding of propofol in subdomain IIIA. We are
ets or clefts on the protein, and anesthetic binding causes only
not aware of any binding studies that have investigated this
very minor changes in local structure.
possibility. Similarly, it is possible that halothane molecules
HAL5 and HAL6 might displace so-called site I drugs. How- Acknowledgments We thank Delta Biotechnology Ltd. for purified
recombinant HSA and the staff at Daresbury SRS (UK) and at DESY
ever, this seems much less likely because these halothane
Hamburg (Germany) for help with data collection. We acknowledge the
molecules clearly bind rather weakly (electron density only
use of the Engineering and Physical Sciences Research Council chem-
appears at higher halothane concentrations), and available
ical data base service at Daresbury, and we are very grateful to Peter
binding data show that a variety of volatile anesthetics are
Brick for helpful comments on the manuscript and Bill Lieb for many
relatively ineffective at displacing phenytoin and warfarin (38, stimulating discussions. A. Bhattacharya acknowledges the award of a
Ph.D. studentship from the Medical Research Council.
39), which are classed as site I drugs.
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38738 Propofol and Halothane Binding Sites on Human Serum Albumin
Acta 1430, 46 56
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