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Article history: Human serum albumin (HSA), the major protein component in blood plasma and in extravascular spaces,
Received 17 March 2010 is known to participate in the binding and transport of a variety of endogenous and exogenous organic
Accepted 23 March 2010 compounds with anionic or electronegative features. We here report on the 3.3 Å resolution crystal struc-
Available online 27 March 2010
ture of HSA complexed with the cationic, and widely used, anesthetic lidocaine. We find that lidocaine
and HSA co-crystallise as a dimer in the unusual space group I41. The dimer consists of one HSA molecule
Keywords: without ligand and one HSA molecule with a single, bound lidocaine. HSA is a heart-shaped protein com-
Human serum albumin
posed of three homologous helical domains (I–III), which can be subdivided into two subdomains (A and
Lidocaine binding
Sulphate ion binding
B), and lidocaine binds to a unique site formed by residues from subdomain IB facing the central, inter-
Crystal structure domain crevice. In the crystal, binding seems to introduce only local conformational changes in the pro-
Conformational changes tein. According to intrinsic fluorescence experiments with aqueous HSA binding results in widespread
Intrinsic fluorescence conformational changes involving Trp214 in subdomain IIA. Results obtained with equilibrium dialysis
and isothermal titration calorimetry show that lidocaine binding is of a low affinity and occurs at one dis-
crete binding site in accordance with the X-ray data. Another crystal form of ligand-free HSA obtained in
the presence of ammonium sulphate was determined at 2.3 Å resolution revealing a sulphate ion accept-
ing cavity at the surface of subdomain IIIA. The present results contribute to a further characterisation of
the exceptional binding properties of HSA.
Ó 2010 Elsevier Inc. All rights reserved.
1047-8477/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.jsb.2010.03.014
354 K.L. Hein et al. / Journal of Structural Biology 171 (2010) 353–360
mucoid) (Schönfeld et al., 2008) however, some of them bind to 2.3. Data collection and processing
HSA (Kragh-Hansen, 1981). No structural information about such
binding sites is available. Therefore, in the present work we have Initial testing of the crystals was performed in-house with a
performed a crystallographic analysis of HSA complexed with lido- copper rotating-anode X-ray generator, whereas further testing
caine, a Na+ channel blocker widely used as a local anesthetic and using synchrotron radiation was performed at European Molecular
antiarrhythmic drug. The structural work was supplemented with Biology Laboratory outstation at Deutsches Elektronen Synchro-
intrinsic fluorescence data, equilibrium dialysis and isothermal tron (EMBL/DESY, Hamburg) or at Berliner Elektronenspeicher-
titration calorimetry. The study revealed a new crystal form of a ring-Gesellschaft für Synchrotronstrahlung (BESSY). X-ray
HSA-ligand that has not been observed before and the existence diffraction data for the HSA–lidocaine complex and HSA were col-
of a unique binding site located between domains I and III. Further- lected under cryo-conditions (110 K) using synchrotron radiation
more we compare our results to a 2.3 Å resolution crystal structure at the Swiss Light Source (SLS, Zurich) and BESSY facilities, respec-
of HSA that we have obtained with bound sulphate. tively. The data were integrated and scaled with XDS (Kabsch,
1993). The 3.3 Å data set obtained on the HSA–lidocaine complex
was processed in space group I41, and the 2.3 Å data set obtained
for HSA was processed in space group P1; data collection details
2. Materials and methods and unit cell parameters are given in Table 1. The Wilson plot
and a diffraction image from the 3.3 Å data set are shown in Sup-
2.1. Materials plementary materials.
HSA (A-7223, 95% pure), expressed in Pichia pastoris, was ob- 2.4. Structure determination
tained from Sigma–Aldrich (St. Louis, MO, USA). For removing
hydrophobic and hydrophilic ligands and additives, the protein The structures were solved by molecular replacement using
was treated with charcoal at pH 3 as described by Chen (1967), dia- PHASER (McCoy et al., 2005) and employing an array of HSA struc-
lysed extensively against deionised water, freeze-dried and then tures to identify a suitable search model. Albumin complexed with
stored at 20 °C until use. HSA (A-1887, >96% pure and essentially diflunisal (PDB 2BXE) and consisting of residues 5–78 and 88–582
fatty acid free) was also supplied by Sigma–Aldrich. provided a successful molecular replacement solution for the HSA–
Lidocaine was from Sigma–Aldrich, and [carbonyl-14C]lidocaine lidocaine complex as probed by Z-scores, electron density maps of
hydrochloride (99% pure according to the manufacturer) was from unmodelled regions and subsequent refinement statistics. Rigid
American Radiolabeled Chemicals Inc. (St. Louis, MO, USA). Other body refinement of HSA–lidocaine domains revealed significant
chemicals were also of the best grades commercially available.
Table 1
Data collection details and unit cell parameters.
2.2. Preparation and crystallisation of HSA with and without lidocaine HSA–lidocaine HSA–SO4
X-ray source Swiss Light Source–X06SA BESSY–PX2
To isolate monomeric HSA for crystallisation with lidocaine, the Wavelength (Å) 1.008 0.915
protein preparation was subjected to further purification on a Space group Tetragonal I41 Triclinic P1
Superdex S75 gel filtration column (GE Healthcare) using 50 mM Cell parameters
a (Å) 168.5 55.6
potassium phosphate with 150 mM sodium chloride, pH 7.5, as a
b (Å) 168.5 55.8
running buffer (Curry et al., 1998). Peak fractions were collected c (Å) 97.0 121.3
and analysed by SDS–PAGE. Fractions with HSA monomer were a (°) 90 80.5
pooled and concentrated in 2 mM potassium phosphate, pH 7.5, b (°) 90 90.7
to a concentration greater than 100 mg/ml. c (°) 90 63.5
Resolution range (Å) 120–3.3 (3.5–3.3) 120–2.3 (2.4–2.3)
Monomeric HSA was mixed with a 20-fold molar excess of lido- Completeness (%) 99.9 (100) 91.1 (85.6)
caine and incubated for 1–2 h. The free drug concentration was Rmrgd F (%) 16.9 (144.7) 10.4 (50.5)
then fixed by repeated cycles of concentration and dilution in Rmeas (%) 10 (171.8) 5.8 (33.6)
20 mM potassium phosphate buffer, pH 7.5, containing 0.1 mM I/r(I) 13.04 (1.2) 13.46 (3.4)
Unique reflections 20,507 51,888
lidocaine using a 10,000 MWCO centrifugal concentrator (Viva-
Redundancy 6.3 1.8
spin); the final protein concentration was 100 mg/ml or higher.
Model refinement
For crystallisation without lidocaine, defatted, freeze-dried HSA
Resolution range (Å) 45–3.3 (3.45–3.3) 28–2.3 (2.34–2.3)
was used at a concentration of 100 mg/ml. Model amino acids 1164 1150
The crystallisation experiments were carried out by vapour dif- Rwork (%)a 22.0 (32.9) 20.9 (23.5)
fusion at 19 °C using the sitting or hanging drop method. In most Rfree (%)b 26.8 (36.0) 27.0 (31.0)
cases, a 2 ll protein sample was mixed with an equal volume of Overall B-factor (Å) 142.4 44.05
Average ligand B-factor (Å) 113.2 28.7
reservoir solution in 24-well plates (Hampton Research). Crystals RMSDbonds (Å)c 0.003 0.009
of HSA–lidocaine generally appeared after 3–4 days with crystalli- RMSDangles (Å)c 0.715 1.045
sation conditions consisting of 20–21% (w/v) polyethylene glycol Ramachandran (%)d 83.9 95.1
6000, 9–10% ethylene glycol, 50/100 mM ammonium acetate, 2%
The values for the highest resolution shell are given in parentheses.
(v/v) 2-methyl-2,4-pentanediol, and 50 mM Tris–HCl, pH 7.5. Crys- Rmeas = redundancy independent R-factor (intensities). Rmrgd F = quality of
tals of HSA without lidocaine were prepared in crystallisation solu- amplitudes (F) in the scaled data set.
tions consisting of 22–25% polyethylene glycol 3350, 100–200 mM For definitions, see Diederichs and Karplus (1997).
a P P
Rwork (%) = 100 hkl|Fobs Fcalc|/ hkl Fobs, where Fobs and Fcalc are the
ammonium sulphate and 50 mM Tris–HCl, pH 7.5. For use in X-ray
observed and calculated structure factors, respectively.
diffraction data collection, crystals were cryoprotected by adding b
Rfree(%) is calculated using a randomly selected 5% sample of reflection data
reservoir buffer containing glycerol to the drop, yielding a final omitted from the refinement.
c
glycerol concentration of approximately 20%. Afterwards, the crys- Root mean square deviation from ideal bond lengths and angles.
d
tals were frozen in liquid nitrogen. Residues in most favoured regions.
K.L. Hein et al. / Journal of Structural Biology 171 (2010) 353–360 355
changes with relative displacement of domain I and III. A model and shows favourable Ramachandran plot statistics (most favoured
consisting of HSA residues 5–582 was refined by simulated anneal- regions: 83.9%). A successful molecular replacement solution for
ing using torsion angle dynamics and group B-factors using CNS HSA with sulphate was obtained using PDB 1BM0 as model. Initial
(Brünger et al., 1998). Inspection of electron density maps revealed refinement was carried out with PHENIX and poorly resolved re-
a ligand binding site in one of the two protein molecules of the gions, mainly domain IIIB, were manually rebuilt using COOT
asymmetric unit; comparable in size and overall shape to lidocaine (Emsley and Cowtan, 2004), with help from Arp/wArp (Perrakis
(Fig. 2d). A single lidocaine molecule was then included in the et al., 2001) and BUCCANEER (Cowtan, 2006). The final model
structure followed by further refinement cycles using PHENIX shows Rwork and Rfree values of 20.9% and 27.0%, respectively (Ta-
(Adams et al., 2002) and applying TLS refinement. The model yields ble 1) with 95.1% of residues in the most favourable regions of
Rwork and Rfree values of 22.0% and 26.8%, respectively (Table 1), the Ramachandran plot.
Fig. 1. Chemical structure of lidocaine on its protonated form. The pKa of the drug is Ligand binding was quantified by use of Dianorm equilibrium
7.80–7.86 (Truant and Takman, 1959), and its molecular weight is 235 Da. dialysers (Dianorm Geräte, München, Germany). The dialysis cells,
Fig. 2. Structure of HSA–lidocaine complex. (a) The asymmetric unit showing two HSA molecules of which one has bound a lidocaine molecule (green). (b) Overview of HSA
with lidocaine bound to subdomain IB in the cleft between domains I and III. The subdivision of HSA into domains (I–III) and subdomains (A and B) is shown. (c) Surface view
of the lidocaine binding site. The domains are colour-coded as follows: I, deep blue; II, marine blue; III, light blue. (d) Yellow; positive Fo Fc sa-omit difference electron
density map, with the lidocaine molecule omitted from the phasing model, contoured at 2.5r and drawn in a radius of 4 Å around lidocaine. Red; positive Fo Fc difference
electron density map before including lidocaine, contoured at 2.5r (e) 2Fo Fc electron density map contoured at 1r. (f) HSA with lidocaine (blue) superposed with HSA
(orange, PDB 1AO6) using the residues from position 100 to 200. All illustrations in this and the following structural figures were prepared using PyMol (Delano, 2002).
356 K.L. Hein et al. / Journal of Structural Biology 171 (2010) 353–360
with half-cell volumes of 250 ll carved into cylindrical disks, were lidocaine may explain why an asymmetric 2:1 HSA:lidocaine com-
assembled pair-wise, together with flat pieces of separating cellu- plex has occurred. An examination of the literature (Carter and Ho,
lose tubing. For this we used Visking size 9, / = 4.0 cm, with a 1994; Sugio et al., 1999; Ghuman et al., 2005; Lejon et al., 2008;
molecular mass cut off of 12,000–14,000 from Medicell Interna- Zunszain et al., 2008; Zhu et al., 2008) revealed that so far no other
tional Ltd., London, UK. HSA crystal has been reported to belong to this type of space group.
The experiments were carried out at 25 °C with media buffered The structure was determined by molecular replacement and re-
with 50 mM Tris–HCl, pH 7.4. Solutions of different concentrations fined at 3.3 Å resolution. The refined model has an Rfree value of
of labelled plus unlabelled ligand (25–250 lM) were prepared with 26.8% and valid stereochemistry (Table 1).
a constant albumin concentration (150 lM). Sample aliquots of The fact that lidocaine binds to only one of the two HSA mole-
175 ll containing protein and ligand were pipetted into the left- cules in the asymmetric dimer is illustrated in Fig. 2a. The binding
side of the cells, and sample of 175 ll of buffer alone were pipetted site is situated in subdomain IB in the upper part of the interdo-
into the right-side of the cells. After closure of the cells, the equi- main cleft (Fig. 2b and c). The difference electron density (Fo Fc
librium dialysers were placed in a temperature-controlled water simulated annealing omit map) shows clear density for a molecule
bath, and the cells were rotated for ca. 20 h about their horizontal the size of lidocaine at that position (Fig. 2d). Fig. 2e gives the
axes. Then the half-cells were emptied, and 100 ll portions from molecular details for binding of lidocaine (and as further indicated
the left and right compartments were used for counting of radioac- by final 2Fo Fc map). As seen from the illustration, the aromatic
tivity. Control experiments revealed that equilibrium was estab- part of lidocaine (Fig. 1) is involved in cation–p interactions with
lished within the period of time used. The recovery of Arg114, and the carbonyl group interacts with Lys190. Further sta-
radioactivity was 95.6% (±0.4%) (n = 10). The loss must be supposed bilization is provided by electrostatic interaction between the pos-
to be caused by adsorption to the Teflon of the cell compartments itively charged, tertiary amine nitrogen and the carboxylate group
and/or to the cellophane membrane. of Asp187. Apparently, the interactions between the non-aromatic
The radioactivity of lidocaine solutions which had not been dia- part of lidocaine and Asp187 and Lys190 do not cause any signifi-
lysed was used to represent the known concentration of total li- cant displacement of the two residues (Fig. 2f). By contrast, the
gand. After dialysis, the radioactivity of the samples from left- aromatic part of the ligand attracts Arg114; the residue has been
side and right-side compartments represent the concentration of moved about 4 Å. Furthermore, Arg186 is pushed aside making
unbound plus albumin-bound ligand and unbound ligand, respec- room for the incoming ligand.
tively. The association constant (K) for lidocaine binding was calcu- HSA without lidocaine was crystallized in space group P1 in the
lated by using the following equation: presence of sulphate ions, and its crystal structure was determined
by molecular replacement and refined at 2.3 Å resolution yielding a
m ¼ ðK ½LÞ=ð1 þ K ½LÞ ð1Þ
final Rfree of 27.1% (Table 1). Although HSA is known to bind inor-
In this equation, t is the average number of ligand molecules bound ganic anions such as Cl, F, SCN and SO2 4 apparently no informa-
per protein molecule, and [L] is the concentration of unbound tion exists on the exact location and construction of these sites
ligand. (Peters, 1996; Kragh-Hansen, 1981). As seen in Fig. 3a, we were
able to detect a sulphate ion accepting cavity at the surface of sub-
domain IIIA. The ion interacts with Ser480 and the backbone nitro-
2.7. Isothermal titration calorimetry
gens of Leu481 and Val482 (Fig. 3b). Additional stabilization
consists of van der Waals interactions with the Lys351 side chain
Lidocaine binding was examined using a Valerian Plotnikov (VP)
and coordination to water molecules in the cavity. Finally,
ITC MicroCalorimeter from MicroCal (Northampton, USA). De-
Lys560 from a symmetry related HSA molecule also takes part in
gassed solutions of 0.1 mM albumin in 50 mM Tris–HCl buffer,
binding, and it is likely that this interaction seals the binding site
pH 7.4 was transferred to the ITC cell, and the syringe was filled
and at the same time improves the crystal stability. The positions
with 10 mM lidocaine in the same buffer. The ITC titration was
of the great majority of residues are not affected by binding, but
done at 25 °C with an initial injection of 1 ll followed by 59 injec-
Ser480 and part of the peptide chain in which it is placed have
tions of 5 ll, all with a 10-min equilibration period between the
been slightly displaced (Fig. 3c).
injections. The heat of dilution from lidocaine alone was measured
Fig. 4a compares the structures of the two HSA molecules in the
in a similar way by having only buffer in the ITC cell. The heat of
asymmetric dimer. It is seen that domain I of HSA without ligand
dilution signal was subtracted from the binding data using ITC
(yellow) is tilted towards the middle of the molecule when com-
Data Analysis in MicroCal Origin 7.0.
pared to HSA binding lidocaine (green). Furthermore, domain III
of the ligand-free form is slightly more outer placed. The tilting
2.8. Protein Data Bank accession numbers of domain I and/or local conformational changes in subdomain
IB apparently hinder lidocaine binding. Fig. 4b–d compare our
Coordinates and structure factors have been deposited in the structures with a HSA structure found in the literature. In a review
Protein Data Bank with accession numbers 3JQZ (HSA–lidocaine) (Curry, 2004), Curry compared the ligand-free structure of HSA
and 3JRY (HSA–sulphate). published by Sugio et al. (1999) with those published by He and
Carter (1992) and his own group (Bhattacharya et al., 2000b). He
3. Results found only some small variations between the three structures,
particularly in the disposition of domain III. In the present context
3.1. Structure of the HSA–lidocaine complex and of HSA with sulphate we have used the 2.8 Å structure published by Sugio et al. (1999)
as the reference structure. Fig. 4b shows that the position of do-
Lidocaine is a tertiary amine compound, and its protonated main III of HSA–lidocaine (green) is shifted outwards as compared
form is shown in Fig. 1. The drug and HSA were dissolved in phos- with that of the same domain in the structure of Sugio et al. (red).
phate buffer, pH 7.5, at a 20:1 M ratio of ligand to protein and co- By contrast, the structure of the two other domains is very similar.
crystallised as a dimer in the unusual space group I41 (Fig. 2a). The Fig. 4c shows a more pronounced outward disposition of domain
dimer consists of one HSA molecule without ligand and one HSA III and a tilting of domain I when comparing our ligand-free form
molecule with a single, bound lidocaine (an asymmetric dimer). (yellow) with the structure of Sugio et al. (red). Finally, our 2.3 Å
A combination of crystal packing and low-affinity binding of structure with sulphate (blue) resembles the ligand-free form
K.L. Hein et al. / Journal of Structural Biology 171 (2010) 353–360 357
Fig. 3. Structure of HSA–sulphate ion complex. (a) Overview of HSA with sulphate bound at the surface of subdomain IIIA. (b) A 2Fo Fc electron density map contoured at 1r
showing the binding site for sulphate (yellow–red) with interacting residues. Lys560(sym) shown in grey is from a symmetry related HSA molecule. (c) HSA with bound
sulphate (yellow–red) superposed with HSA (orange, PDB 1AO6) using domain II (residues 208–366). The red spheres in panels (b) and (c) are water molecules.
Fig. 4. Superposition of HSA structures using the Ca-atoms of domain II (residues 208–366). (a) HSA with lidocaine (green) and HSA without lidocaine (yellow, 3.3 Å
structure) from the asymmetric dimer. (b) HSA with lidocaine (green) and HSA as determined by Sugio et al. (1999) (red, PDB 1AO6). (c) HSA without lidocaine (yellow, 3.3 Å
structure) and HSA (red, PDB 1AO6). (d) HSA with sulphate (blue, 2.3 Å structure) superposed with HSA (red, PDB 1AO6).
published by Sugio et al. (red) (Fig. 4d). The only clear difference is The three HSA structures studied in this work have also been
a pronounced outward displacement of domain III in our structure, compared with HSA saturated with myristate (fatty acid:HSA mole
which could be due to sulphate ion binding (Fig. 3c) and/or caused ratio of 12:1) (Bhattacharya et al., 2000a) and with a HSA–hemin
by contact with the other albumin molecule in the crystal dimer; complex partially saturated with myristate (fatty acid:hemin:HSA
see, e.g., Lys560 in Fig. 3b. We also found that the conformations of mole ratio of 4:1:1) (Zunszain et al., 2003). Binding of high molar
HSA with bound sulphate ion or with bound lidocaine are alike; ratios of fatty acid results in rotations of domains I and III relative
the only difference is a small variation in the relative position of to domain II (Bhattacharya et al., 2000a). Partial saturation of HSA
domain III (not shown, but see Fig. 4). In summary, a tilting of do- with myristate results in a similar rotation of domain III but in a
main I was observed for the ligand-free HSA molecule comple- less pronounced rotation of domain I (Zunszain et al., 2003). In
menting the asymmetric dimer of the lidocaine complex both fatty acid–HSA structures, domain III is placed further away
obtained at 3.3 Å resolution, and both HSA-forms obtained from the central crevice than in our structures. Although the posi-
without lidocaine, at 3.3 and 2.3 Å, were broader than the form tion of domain I, especially in the partially fatty acid loaded HSA,
found by Sugio et al. (1999) due to an outward displacement of resembles more our structures, it is placed closer to the central cre-
domain III. vice; this is especially so when comparing to HSA with lidocaine or
358 K.L. Hein et al. / Journal of Structural Biology 171 (2010) 353–360
sulphate ion. It should be noted that, in contrast to fatty acid bind- experiments resulted in a similar association constant,
ing, no rotations of domain I and III have taken place in our struc- K = 3.2(±0.3) 102 M1 (n = 5).
tures, rather it is a question of tilting of domain I and/or a pushing The inset to Fig. 5 shows that the changes in fluorescence inten-
aside of domain III. sity, resulting from addition of increasing amounts of lidocaine, can
be described fairly well by the hyperbolic curve. The curve repre-
3.2. Effect of lidocaine binding on intrinsic tryptophan fluorescence sents lidocaine binding and is constructed by using the association
constant determined by equilibrium dialysis. Thus, the results of
HSA has a single tryptophan residue (Trp214) but the crystallo- the two different methods agree on low-affinity binding of lido-
graphic studies showed that the residue is not directly involved in caine to one site on HSA.
lidocaine binding; the closest distance between the two structures Only sparse information can be found in the literature about
is about 24 Å (not shown). However, the structural features at the lidocaine binding to HSA; and only obtained by equilibrium dialysis
residue could be modified as a result of lidocaine binding. This pos- as here. Krauss et al. (1986) used [14C] ethyl–lidocaine and reported
sibility was examined for by performing intrinsic fluorescence binding to one site with a K-value, which depended somewhat on
studies with solubilised HSA. Excitation of the protein at 290 nm the type of albumin preparation used, i.e. 0.6–2.3 102 M1. Bailey
resulted in an emission spectrum with a kmax of 342 nm (Fig. 5). and Briggs (2004) measured lidocaine by a homogeneous enzyme
Addition of increasing concentrations of lidocaine caused a pro- immunoassay, and from their data it is possible to calculate a K-va-
gressive reduction of the fluorescence intensity, accompanied by lue of 5.1 102 M1, assuming binding to one site. In contrast to the
a decrease of kmax to 338 nm. These findings suggest the existence present results and to the studies just cited, Sawinski and Rapp
of lidocaine-induced conformational changes in HSA leading to a (1963) several years ago reported binding to two sites with the
decreased polarity of the environment of Trp214. same and much higher K-value, namely 1.3 105 M1. These
authors quantified unbound lidocaine in an indirect way. First,
3.3. Binding experiments the ligand was precipitated with ammonium reineckate, afterwards
it was dissolved in acetone and determined spectrophotometri-
Equilibrium dialysis revealed that binding of lidocaine was of a cally. Whether this approach, other experimental differences, or
low affinity, and that it could be described in terms of binding to the fact that the study was performed at 4 °C is the reason for the
one site. The association constant (±SEM) for binding to HSA was proposal of two binding sites with a high K-value is not clear.
calculated to be 3.1(±0.2) 102 M1 (n = 5). As a control, we also We also tried to study lidocaine binding by isothermal titration
examined binding to native HSA isolated from human serum (un- calorimetry, but reliable binding constants could not be deter-
like the recombinant source otherwise used in this study). These mined due to insufficient release of heat in our experimental
Fig. 5. Changes in HSA intrinsic fluorescence caused by lidocaine binding. The protein was excitated at 290 nm, and the effect of different ligand concentrations on the
emission spectrum is shown. The data in the inset are lidocaine-induced decreases in HSA fluorescence at kmax. The results are given as means ± SEM (n = 3). The broken curve
representing lidocaine binding is constructed by using Eq. (1) and K = 3.1 102 M1.
K.L. Hein et al. / Journal of Structural Biology 171 (2010) 353–360 359
set-up (data not shown). This indicates a very low affinity, in accor-
dance with the above estimates.
4. Discussion
The relatively large conformational changes observed for HSA Brünger, A.T., Adams, P.D., Clore, G.M., DeLano, W.L., Gros, P., Grosse-Kunstleve,
R.W., Jiang, J.S., Kuszewski, J., Nilges, M., Pannu, N.S., Read, R.J., Rice, L.M.,
(Fig. 4) are most probably caused by differences in crystal packing
Simonson, T., Warren, G.L., 1998. Crystallography & NMR system: a new
of the protein. By contrast, the changes accompanying binding of software suite for macromolecular structure determination. Acta Crystallogr. D
lidocaine (Fig. 2f) and the sulphate ion (Fig. 3c) are minor and more Biol. Crystallogr. 54, 905–921.
local. However, lidocaine binding also results in small conforma- Carter, D.C., Ho, J.X., 1994. Structure of serum albumin. Adv. Protein Chem. 45, 153–
203.
tional changes in the part of subdomain IIA which includes Chen, R.F., 1967. Removal of fatty acids from serum albumin by charcoal treatment.
Trp214. Intrinsic fluorescence measurements with solubilised J. Biol. Chem. 242, 173–181.
HSA support the view that these changes indeed are caused by Cowtan, K., 2006. The Buccaneer software for automated model building. 1. Tracing
protein chains. Acta Crystallogr. D Biol. Crystallogr. 62, 1002–1011.
lidocaine binding (Fig. 5) and not by crystal packing. Apparently, Curry, S., 2004. Plasma albumin as a fatty acid carrier. Adv. Mol. Cell. Biol. 33, 29–46.
X-ray crystallographic analyses of HSA complexed with negatively Curry, S., Mandelkow, H., Brick, P., Franks, N., 1998. Crystal structure of human
charged drugs (Ghuman et al., 2005), bilirubin (Zunszain et al., serum albumin complexed with fatty acid reveals an asymmetric distribution of
binding sites. Nat. Struct. Biol. 5, 827–835.
2008) or hemin (Zunszain et al., 2003) only revealed local confor- Delano, W.L., 2002. The PyMOL Molecular Graphics System. DeLano Scientific, San
mational changes at the respective binding sites. The only ligands Carlos, CA, USA.
found so far which cause global changes in HSA are aliphatic fatty Diederichs, K., Karplus, P.A., 1997. Improved R-factors for diffraction data analysis in
macromolecular crystallography. Nat. Struct. Biol. 4, 269–275.
acids (Curry et al., 1998). These changes are primarily caused by Emsley, P., Cowtan, K., 2004. Coot: model-building tools for molecular graphics.
fatty acid binding to site FA2 (Fig. 6a) and show as rotations of do- Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132.
mains I and III relative to domain II. Ferrer, M.L., Duchowicz, R., Carrasco, B., de la Torre, J.G., Acuna, A.U., 2001. The
conformation of serum albumin in solution: a combined phosphorescence
The characteristics of lidocaine binding to HSA differ from those
depolarization–hydrodynamic modelling study. Biophys. J. 80, 2422–2430.
found for lidocaine binding to the a-subunit of Na+ channels. Kur- Ghuman, J., Zunszain, P.A., Petitpas, I., Bhattacharya, A.A., Otagiri, M., Curry, S., 2005.
oda et al. found, by using 1H NMR spectroscopy and mutagenesis, Structural basis of the drug-binding specificity of human serum albumin. J. Mol.
p–p stacking of the aromatic rings of lidocaine and a phenylalanine Biol. 353, 38–52.
He, X.M., Carter, D.C., 1992. Atomic structure and chemistry of human serum
(Phe1489) and electrostatic attraction of the tertiary amine nitro- albumin. Nature 358, 209–215.
gen to a glutamic acid (Glu1492) in the channel protein isolated Kabsch, W., 1993. Automatic processing of rotation diffraction data from crystals of
from rat brain (Kuroda et al., 2000). Using electrophysiology and initially unknown symmetry and cell constants. J. Appl. Crystallogr. 26, 795–800.
Kragh-Hansen, U., 1981. Molecular aspects of ligand binding to serum albumin.
mutagenesis, Ahern et al. found that cation–p interaction between Pharmacol. Rev. 33, 17–53.
lidocaine and a phenylalanine (Phe1579) is essential for drug bind- Kragh-Hansen, U., Hellec, F., de Foresta, B., le Maire, M., Møller, J.V., 2001.
ing to a cardiac Na+ channel expressed in Xenopus oocytes (Ahern Detergents as probes of hydrophobic binding cavities in serum albumin and
other water-soluble proteins. Biophys. J. 80, 2898–2911.
et al., 2008). Thus, in HSA the aromatic portion of lidocaine inter- Krauss, E., Polnaszek, C.F., Scheeler, D.A., Halsall, H.B., Eckfeldt, J.H., Holtzman, J.L.,
acts with an arginine instead of a hydrophobic phenylalanine, 1986. Interaction between human serum albumin and a1-acid glycoprotein in
and the electrostatic interaction is to an aspartate and not to a glu- the binding of lidocaine to purified protein fractions and sera. J. Pharmacol. Exp.
Ther. 239, 754–759.
tamate. Furthermore, in HSA the binding is supplemented by a po- Kuroda, Y., Miyamoto, K., Tanaka, K., Maeda, Y., Ishikawa, J., Hinata, R., Otaka, A.,
lar interaction of the carbonyl group of lidocaine to a lysine residue Fujii, N., Nakagawa, T., 2000. Interactions between local anesthetics and Na+
(Fig. 2). channel inactivation gate peptides in phosphatidylserine suspensions as
studied by1H-NMR spectroscopy. Chem. Pharm. Bull. 48, 1293–1298.
The insight into the HSA–lidocaine interaction reported in this
Lejon, S., Cramer, J.F., Nordberg, P., 2008. Structural basis for the binding of
study provides a preview into the structural basis of binding of cat- naproxen to human serum albumin in the presence of fatty acids and the GA
ionic ligands and contributes to a greater understanding of the un- module. Acta Crystallogr. F64, 64–69.
ique binding properties of the protein. It will also facilitate efforts McCoy, A.J., Grosse-Kunstleve, R.W., Storoni, L.C., Read, R.J., 2005. Likelihood-
enhanced fast translation functions. Acta Crystallogr. D Biol. Crystallogr. 61,
to modify new therapeutics to control their interaction with HSA 458–464.
and thereby optimise their distribution within the human body. Olivieri, J.R., Craievich, A.F., 1995. The subdomain structure of human serum
albumin in solution under different pH conditions studied by small angle X-ray
scattering. Eur. Biophys. J. 24, 77–84.
Acknowledgments Perrakis, A., Harkiolaki, M., Wilson, K.S., Lamzin, V.S., 2001. ARP/wARP and
molecular replacement. Acta Crystallogr. D Biol. Crystallogr. 57, 1445–1450.
Peters Jr., T., 1996. All About Albumin: Biochemistry, Genetics, and Medical
This work was supported by the A.P. Møller Foundation for the Applications. Academic Press, San Diego, CA, USA.
Advancement of Medical Science and by a Hallas–Møller stipend of Petitpas, I., Bhattacharya, A.A., Twine, S., East, M., Curry, S., 2001. Crystal structure
the Novo-Nordisk Foundation. analysis of warfarin binding to human serum albumin. Anatomy of drug site I. J.
Biol. Chem. 276, 22804–22809.
Petitpas, I., Petersen, C.E., Ha, C.-E., Bhattacharya, A.A., Zunszain, P.A., Ghuman, J.,
Appendix A. Supplementary data Bhagavan, N.V., Curry, S., 2003. Structural basis of albumin–thyroxine
interactions and familial dysalbuminemic hyperthyroxinemia. Proc. Natl.
Acad. Sci. USA 100, 6440–6445.
Supplementary data associated with this article can be found, in Peyre, V., Lair, V., André, V., le Maire, G., Kragh-Hansen, U., le Maire, M., Møller, J.V.,
the online version, at doi:10.1016/j.jsb.2010.03.014. 2005. Detergent binding as a sensor of hydrophobicity and polar interactions in
the binding cavities of proteins. Langmuir 21, 8865–8875.
Sawinski, V.J., Rapp, G.W., 1963. Interaction of human serum proteins with local
References anesthetic agents. J. Dent. Res. 42, 1429–1438.
Schönfeld, D.L., Ravelli, R.B., Mueller, U., Skerra, A., 2008. The 1.8-A crystal structure
of alpha(1)-acid glycoprotein (orosomucoid) solved by UV RIP reveals the broad
Adams, P.D., Grosse-Kunstleve, R.W., Hung, L.W., Ioerger, T.R., McCoy, A.J., Moriarty,
drug-binding activity of this human plasma lipocalin. J. Mol. Biol. 384, 393–405.
N.W., Read, R.J., Sacchettini, J.C., Sauter, N.K., Terwilliger, T.C., 2002. PHENIX:
Sugio, S., Kashima, A., Mochizuki, S., Noda, M., Kobayashi, K., 1999. Crystal structure
building new software for automated crystallographic structure determination.
of human serum albumin at 2.5 Å resolution. Protein Eng. 12, 439–446.
Acta Crystallogr. D Biol. Crystallogr. 58, 1948–1954.
Truant, A.P., Takman, B., 1959. Differential physical–chemical and
Ahern, C.A., Eastwood, A.L., Dougherty, D.A., Horn, R., 2008. Electrostatic
neuropharmacologic properties of local anesthetic agents. Anesth. Analg. 38,
contributions of aromatic residues in the local anesthetic receptor of voltage-
478–484.
gated sodium channels. Circ. Res. 102, 86–94.
Zhu, L., Yang, F., Chen, L., Meehan, E.J., Huang, M., 2008. A new drug binding subsite
Bailey, D.N., Briggs, J.R., 2004. The binding of selected therapeutic drugs to human
on human serum albumin and drug–drug interaction studied by X-ray
serum a-1 acid glycoprotein and to human serum albumin in vitro. Ther. Drug
crystallography. J. Struct. Biol. 162, 40–49.
Monit. 26, 40–43.
Zunszain, P.A., Ghuman, J., Komatsu, T., Tsuchida, E., Curry, S., 2003. Crystal
Bhattacharya, A.A., Grüne, T., Curry, S., 2000a. Crystallographic analysis reveals
structural analysis of human serum albumin complexed with hemin and fatty
common modes of binding of medium and long-chain fatty acids to human
acid. BMC Struct. Biol. 3, 6.
serum albumin. J. Mol. Biol. 303, 721–732.
Zunszain, P.A., Ghuman, J., McDonagh, A.F., Curry, S., 2008. Crystallographic analysis
Bhattacharya, A.A., Curry, S., Franks, N.P., 2000b. Binding of the general anesthetics
of human serum albumin complexed with 4Z,15E-bilirubin-IXa. J. Mol. Biol.
propofol and halothane to human serum albumin. High resolution crystal
381, 394–406.
structures. J. Biol. Chem. 275, 38731–38738.