Crystal structure of human serum albumin at 2.5 Å resolution

S. Sugio1, A. Kashima, S. Mochizuki, M. Noda and K. Kobayashi2

Osaka Laboratories, Yoshitomi Pharmaceutical Industries Ltd,2-25-1, Shodai-Ohtani, Hirakata, Osaka 573-1153, Japan


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Conclusion
 References
 
A new triclinic crystal form of human serum albumin (HSA), derived either from pool plasma (pHSA) or from a Pichia pastoris expression system (rHSA), was obtained from polyethylene glycol 4000 solution. Three-dimensional structures of pHSA and rHSA were determined at 2.5 Å resolution from the new triclinic crystal form by molecular replacement, using atomic coordinates derived from a multiple isomorphous replacement work with a known tetragonal crystal form. The structures of pHSA and rHSA are virtually identical, with an r.m.s. deviation of 0.24 Å for all C{alpha} atoms. The two HSA molecules involved in the asymmetric unit are related by a strict local twofold symmetry such that the C{alpha} atoms of the two molecules can be superimposed with an r.m.s. deviation of 0.28 Å in pHSA. Cys34 is the only cysteine with a free sulfhydryl group which does not participate in a disulfide linkage with any external ligand. Domains II and III both have a pocket formed mostly of hydrophobic and positively charged residues and in which a very wide range of compounds may be accommodated. Three tentative binding sites for long-chain fatty acids, each with different surroundings, are located at the surface of each domain.

Keywords: crystal structure/human serum albumin/recombinant protein


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Conclusion
 References
 
Human serum albumin (HSA) is the most abundant protein found in plasma and shows a typical blood concentration of 5 g/100 ml. Its physiological and pharmacological properties have been extensively studied over several decades (Fehske et al., 1981Go; Kragh-Hansen, 1981Go; Putnam, 1984Go; Peters, 1985Go). Such studies have revealed that HSA has a high affinity to a very wide range of materials, including metals such as Cu2+ and Zn2+, fatty acids, amino acids, metabolites such as bilirubin and for many drug compounds. The most important physiological role of the protein is therefore thought to be to bring such solutes in the bloodstream to their target organs, as well as to maintain the pH and osmotic pressure of plasma. In addition to its ordinary clinical applications, such as hypovolemic shock treatment, many investigators have attempted to utilize HSA as a carrier to deliver various drugs to their specific targets (Yapel, 1985Go; Fiume et al., 1988Go). The primary sequence of HSA shows that the protein is a single polypeptide with 585 residues containing 17 pairs of disulfide bridges and one free cysteine (Dugiaczyk et al., 1982Go). Human serum albumin, as well as serum albumin from other species, has been found to consist of three homologous domains probably derived through gene multiplication (Brown, 1976Go). Despite many investigations using hydrodynamics, small-angle X-ray diffraction, electron microscopy and structural prediction, its three-dimensional molecular structure has remained largely unknown. Several crystal forms of HSA were reported in the 1970's (McClure and Craven, 1974Go; Rao et al., 1976Go), but they provided no structural information due to their poor reproducibility. The first crystal structure of HSA at low resolution was reported by Carter and co-workers in 1989 (Carter et al., 1989Go; Carter and He, 1990Go), and its refined structure at 2.8 Å resolution was published by the same group (He and Carter, 1992Go). The research group has also succeeded through extensive efforts in producing half a dozen different crystal forms of both HSA and non-human serum albumin, improving the resolution of their crystal structures and enhancing the knowledge of the diverse chemistry of serum albumin (Ho et al., 1993Go; Carter and Ho, 1994Go; Carter et al., 1994Go). Recently, a couple of structures of HSA have been published by another research group (Curry et al., 1998Go), in which five myristates are accommodated in fatty acid sites. Not only did these studies shed light on the structural features of the protein, but knowledge of the three-dimensional structure also contributed to the clarification of how the protein binds various ligands. In this article, we describe a new crystal form of defatted HSA and discuss various molecular aspects of the protein as determined at the highest resolution so far reported.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Conclusion
 References
 
Crystallization of the tetragonal crystal

pHSA was purchased from Sigma Chemical Co. (catalog no. A-3782), and was used for crystallization without further purification. Crystallization took place via a conventional hanging drop procedure. Plate-shaped yellowish crystals (1.0 x 1.0 x 0.2 mm) were grown from a solution containing 150–255 mg/ml protein, 50 mM potassium phosphate (pH 5.0–5.5), 30–38% (v/v) polyethylene glycol 400 and 5 mM sodium azide at 293 K over 2–3 weeks. Preliminary diffraction studies revealed that the crystals belonged to the tetragonal space group P4212 with cell dimensions a = 187.1 Å, c = 80.5 Å. This crystal form is isomorphous to that previously reported (Carter et al., 1989Go; Carter and He, 1990Go), containing one HSA molecule in an asymmetric unit. Due to their high solvent content (77%), diffraction of these crystals only reaches a resolution of 3.0 Å.

Structure determination of the tetragonal crystal

Intensity data from a native crystal were collected with a Rigaku R-AIXS IV area-detector on a Rigaku FR X-ray generator ({lambda} = 1.5418 Å). Intensity data from derivative crystals were collected with a Rigaku R-AXIS IIc area-detector on a Rigaku RU-200H X-ray generator. Data to 3.0 Å resolution for the native crystal and to 3.6 or 4.0 Å for derivatives were measured at 293 K. All diffraction data were processed with PROCESS (Sato et al., 1992Go). Data collection statistics are summarized in Table IGo. Isomorphous difference Patterson maps, calculated at 4.0 Å resolution using the XtalView program package (McRee, 1993Go), revealed that crystals soaked in HgCl2, thimerosal or mersalyl could be used for phasing with multiple isomorphous replacement. Heavy-atom sites for the HgCl2 derivative were located using the HASSP program (Terwilliger, 1994Go), and refined using a correlation search method implemented in XtalView. Subsequent cross difference Fourier syntheses with phases derived solely from the HgCl2 derivative clearly showed all the heavy-atom sites for other derivatives. Initial phases at 3.6 Å resolution were obtained via the multiple isomorphous replacement method with all derivatives. Phasing statistics are listed in Table IGo. The initial phases were then improved and extended to 3.0 Å through iterative cycles of solvent flattening (Wang, 1985Go) implemented in the PHASES program (Furey, 1994Go). In the first electron density map, a number of helical features were clear and easy to interpret, and a `poly-alanine' model containing a few cysteine and proline residues was built using the molecular graphics program QUANTA 4.1 (Molecular Simulation Inc.). The initial atomic model consisted of 1806 non-hydrogen atoms from 359 residues, and was subjected to stereochemically restrained least-squares refinement in X-PLOR (Brünger, 1992bGo) with the parameter set proposed by Engh and Huber (1991). Sequence assignment of the atomic model was carried out using the amino acid sequence derived from the analysis of genomic DNA (Minghetti et al., 1986Go). Further improvement of phases were achieved using a density modification procedure in which the partial model contribution was taken into account as well as solvent flattening and histogram matching (Zhang and Main, 1990Go). The procedure was iterated using the CCP4 program suite (Collaborative Computational Project Number 4, 1994Go) and SQUASH (Zhang and Main, 1990Go), and no additional features emerged on the subsequent electron density map after the fourth cycle of phase refinement. The last cycle of density modification provided an atomic model containing 3794 out of the 4652 possibly existing non-hydrogen atoms (81.6%) of the HSA molecule, giving a crystallographic R factor of 31.4% at 10.0–3.0 Å resolution. No further attempts were made to refine this model with the data from the tetragonal crystal form.


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Table I. Statistics in data collection and phasing for the tetragonal crystal
 
Crystallization of the triclinic crystal

pHSA was purchased from Miles Inc. (catalog no. 82-301), and rHSA was prepared using the procedure described previously (Sumi et al., 1993Go; Ohi et al., 1994Go; Yokoyama and Ohmura, 1995). Prior to crystallization, fatty acids were removed from the pHSA and rHSA solutions with powder-activated charcoal at pH 3.0 with 1.0 N hydrochloric acid. After removal of the charcoal, the solution was neutralized with 1.0 N sodium hydroxide. An aliquot of the solution was then loaded on a POROS 20-CM (PerSeptive Biosystems) which had been equilibrated with 0.1 M sodium acetate (pH 4.5). Elution was carried out with a linear salt gradient at NaCl concentrations of 0.3–1.2 M at a pH of 4.5–5.0. Fractions corresponding to the main peak were pooled, and the entire column work was repeated until all the protein solution had been processed by the Pharmacia FPLC system. The pooled fraction was concentrated by ultrafiltration, and dialyzed against 10 mM potassium phosphate (pH 7.0) containing 5 mM sodium azide. The purified HSA was diluted to 120–240 mg/ml with 5 mM sodium azide. Crystallization was performed using a hanging drop procedure where the reservoir solution contained 50 mM potassium phosphate (pH 7.0–8.0), 20–26% (w/v) polyethylene glycol 4000 and 5 mM sodium azide. A few prismatic crystals with an average size of 0.4 mm for each edge were grown at 288–293 K within a month. Preliminary diffraction studies revealed that crystals of both pHSA and rHSA obtained from the above procedure diffract X-rays at up to 2.4 Å resolution, belonged to the same space group, i.e. triclinic P1, and have virtually identical unit cell parameters: a, 59.7/59.4 Å; b, 97.0/96.9 Å; c, 59.7/59.2 Å; {alpha}, 91.1/91.0°; ß, 103.5/103.4°; {gamma}, 75.1/74.9° (pHSA/rHSA). Two HSA molecules are involved in the asymmetric unit with solvent content of 50/49%. The unit cell volume of these crystals, 324600/319700 Å3, is significantly different from that of any other crystal form of serum albumin so far reported (McClure and Craven, 1974Go; Rao et al., 1976Go; Carter et al., 1989Go; Carter and He, 1990Go; He and Carter, 1992Go; Ho et al., 1993Go; Carter and Ho, 1994Go).

Structural determination of the triclinic crystal

Diffraction data of the triclinic crystal form were measured at 288 or 293 K with a Rigaku R-AXIS IIc on a Rigaku RU-200H X-ray generator. Data to a resolution of 2.38 Å for pHSA and 2.37 Å for rHSA were collected from two and five crystals, respectively, and were processed in the same way as described above. Data collection statistics are summarized in Table IIGo. Structural determination of pHSA in the triclinic crystal was performed using molecular replacement and crystallographic refinement facilities implemented in X-PLOR (Brünger, 1992bGo). A real-space rotation function at a resolution range of 10.0–4.0 Å followed by a PC-refinement with the structure of the tetragonal crystal as a search model gave two unique solutions (7.8{sigma} and 7.6{sigma}) corresponding to two molecules in an asymmetric unit. A subsequent translation search showed unambiguously a solution from which the relative positions of these molecules were derived. A rigid-body refinement followed by positional and overall temperature factor refinements at a resolution range of 10.0–3.0 Å provided a reasonable molecular packing in the triclinic crystal lattice with a crystallographic R factor of 38.6%. The atomic model obtained above was then subjected to a refinement procedure where simulated annealing from 1000 to 300 K was followed by conventional positional and overall temperature factor refinements at 3.0 Å resolution. The resulting model was manually revised using three-dimensional graphics. Hundreds of side chain atoms which had not been observed in the tetragonal crystal were identified and added to the model. All reflections within a resolution range of 50.0–2.5 Å were then included in the refinement, in which bulk solvent correction, a resolution-dependent weighting scheme for reflections, and tightly restrained individual isotropic temperature factors were incorporated. Although a local twofold symmetry was observed between HSA molecules in the asymmetric unit, no restraints based on this relation were applied during the refinement process. Isolated peaks with significant height in Fo-Fc difference maps were carefully chosen as ordered water molecules if they were located near the protein molecules. A free R factor (Brünger, 1992aGo) for 10% of randomly selected reflections was monitored for every cycle of the refinement to assess progress. Crystallographic R factor and free R factor for the current atomic model were 21.8 and 28.2%, respectively.


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Table II. Statistics in data collection and refinement for the triclinic crystal
 
The triclinic structure of rHSA was refined in the same way as described above, but with the refined structure of pHSA as the starting model. Current R and free R were 20.7 and 29.2% at 50–2.5 Å resolution. The refinement statistics for both pHSA and rHSA are shown in Table IIGo. Atomic coordinates and structure factors of triclinic pHSA and rHSA have been deposited in the Brookhaven Protein Data Bank with accession codes 1AO6 and 1BM0, respectively.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Conclusion
 References
 
Structural determination and quality

The structural determination of HSA was performed using two different crystal forms which reveal complementary properties. As the atomic coordinates of HSA had not been deposited in the Brookhaven Protein Data Bank (Bernstein et al., 1977Go), isomorphous replacement methods had to be employed to solve the initial structure of the protein. Tetragonal crystals were suitable for these experiments because of their high tolerance of the changing conditions of the mother liquor during heavy-atom derivative preparations. On the other hand, all attempts to transfer triclinic crystals from the mother liquor to any other solution failed. This was probably due to the marked difference in packing density between the two crystal forms. The solvent content of tetragonal crystals is very high (77%) and HSA molecules are loosely packed in the crystal lattice, while in triclinic crystals solvent content is rather low (50%) and molecular packing is consequently tighter. After the partial molecular model had been made, the subsequent structure refinement proceeded using intensity data collected from the triclinic crystals, which diffract better (up to 2.4 Å resolution) than the tetragonal crystals (below 3.0 Å).

The current atomic coordinates of pHSA consist of two HSA molecules, each of which contains residues 5–582 (4600 non-hydrogen atoms), and seven water molecules, whereas two copies of HSA (residues 5–582) and four water molecules are involved in the current coordinates of rHSA. An electron density corresponding to residues 1–4 and residues 583–585 of the HSA molecule is not clearly observed, probably due to conformational flexibility at both termini. Geometrical analysis of the atomic coordinates shown in Table IIGo indicates that bond distances, bond angles and dihedral angles were in good agreement with the ideal values proposed by Engh and Huber (1991). The peptide linkages at Pro96 adopt the cis-conformation. A Ramachandran plot (data not shown) reveals that the backbone conformation for all residues falls into either energetically favorable or allowed regions (Ramakrishnan and Ramachandran, 1965Go; Laskowski et al., 1993Go). Average coordinate error estimated from a Luzzati analysis (Luzzati, 1952Go) was about 0.3 Å on R (0.4 Å on free R) for both pHSA and rHSA. The Fo-Fc difference map from the last refinement cycle showed no significant features attributable to mistracing or incorrect atomic coordinates. Average temperature factors for pHSA and rHSA significantly differ residue by residue, i.e. those for exposed residues tended to be 100 Å2, while those for buried ones stayed at around 15–20 Å2, the result of which was the overall temperature factors, 48.5 Å2 for pHSA and 40.7 Å2 for rHSA, were rather large.

Local symmetry in the triclinic lattice

Two HSA molecules are involved in the asymmetric unit of the triclinic crystal, although HSA shows a mono-dispersive state even in condensed solutions. The local twofold symmetry is rather strict, although no assumption was made during the refinement process regarding non-crystallographic symmetry restraints. The rotation angle to superimpose two crystallographically independent molecules in the asymmetric unit is 179.8°, and translation vector length is less than 0.15 Å for both pHSA and rHSA. These two independent molecules in the pHSA structure have virtually identical structures, with r.m.s. deviations of 0.28, 0.31 and 0.66 Å for equivalent C{alpha} atoms, backbone atoms and all non-hydrogen atoms, respectively (0.36, 0.40 and 0.76 Å for rHSA). Further analysis of the residue basis clearly showed that the r.m.s. deviations for C{alpha} atoms are fairly constant throughout the polypeptide chain. All the regions in which structural deviations are relatively distinct are found in loop conformations which connect two {alpha}-helices, and r.m.s. deviations in these regions remain below 1.2 Å. No significant correlation was found between the residual plots of r.m.s. deviations and those of average temperature factors.

Overall structure

As can be expected from the isomorphism of crystals, the refined structures of pHSA and rHSA are virtually identical, with an r.m.s. deviation of 0.24 Å for all C{alpha} atoms. Only the structure of pHSA will therefore be described and discussed. HSA is a helical protein with turns and extended loops, and resembles a heart shape, with approximate dimensions of 80 x 80 x 30 Å, as illustrated in Figure 1Go. It consists of three domains, I (residues 1–195), II (196–383) and III (384–585), which are not only topologically identical, as predicted from amino acid sequence comparison, but also very similar in three-dimensional structure (Figure 2Go). The overall r.m.s. deviation of the corresponding C{alpha} atoms is 3.78 Å for all three domains. The r.m.s. deviation between domains I and II for corresponding C{alpha} atoms is 3.1 Å, while that for other pairs is 4.6–5.0 Å. Although the C{alpha} trace suggests a resemblance between domains I and II, the actual shapes of domains II and III look more similar than those of I and II (Figure 2Go). Each domain can be further divided into subdomains a and b, which are further described in the next section.



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Fig. 1. Schematic drawing of the HSA molecule. Each subdomain is marked with a different color (yellow for subdomain Ia; green, Ib; red, IIa; magenta, IIb; blue, IIIa; and cyan, IIIb). N- and C-termini are marked as N and C, respectively. Arg117, Lys351 and Lys475, which may be binding sites for long-chain fatty acids, are colored white.

 


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Fig. 2. Stereographic illustration of C{alpha} traces of three domains (A, domain I; B, domain II; C, domain III): the traces are superimposed by a least-squares method with the corresponding C{alpha} atoms, which are determined using a multiple sequence alignment of the HOMOLOGY program (Molecular Simulation, Inc.), and are drawn in the same direction. All disulfide bands are drawn as thick lines. The side chains of Cys34 (in A), and residues forming site I (in C) and site II (in C) are also shown as thick lines.

 
Although all three domains of the HSA molecule have similar three-dimensional structures, their assembly is highly asymmetric (Figure 1Go). Domains I and II are almost perpendicular to each other to form a T-shaped assembly in which the tail of subdomain IIa is attached to the interface region between subdomains Ia and Ib by hydrophobic interactions and hydrogen bonds. In contrast, domain III protrudes from subdomain IIb at an angel of about 45° to form a Y-shaped assembly for domains II and III. Domain III interacts only with subdomain IIb. These features make the HSA molecule heart-shaped. There are few contacts between domains I and III due to a large channel formed by subdomains Ib, IIIa and IIIb. The two helices at the C-terminus of domain III show very high temperature factors (>75 Å2) due to their few interactions with other parts of the molecule.

It has been said from physicochemical studies that HSA is a flexible protein that easily changes its molecular shape (Carter and Ho, 1994Go). A couple of interesting features are revealed from a comparison of our defatted HSA with that in complex with myristates (Curry et al., 1998Go). First of all, the relative arrangement of these three domains are largely changed when fatty acids bind to the protein. Secondly, each domain retains its conformation during complex formation, except for that of two C-terminal helices in domain III which move towards the outside of the molecule. These features imply that the molecular flexibility of HSA in different conditions is mainly due to the relative motions of its domain structures.

Subdomains

Each domain can be further divided into subdomains a and b, which are composed of six and four {alpha}-helices, respectively (Figure 2Go). Subdomain a is composed in each case of a cluster of four {alpha}-helices (a-h1 to a-h4) flanked by two short {alpha}-helices (a-h5 and a-h6), while subdomain b in each case has another cluster of four {alpha}-helices (b-h1 to b-h4). A long extended loop traverses the two subdomains to link them together. The helix clusters found in subdomains a and b are also similar in three-dimensional structure, and are approximately related by a pseudo-twofold axis situated between helices a-h2 and b-h2, as shown in Figure 2Go. As the last helices (b-h4) of domains I and II are fused with the first helices (a-h1) of the next domains, the total number of helices in a HSA molecule is 28 rather than 30 (He and Carter, 1992Go).

Marked common structural features are observed between the subdomains, not only in polypeptide chain folding but also in their unique disulfide bond topology. There are 35 cysteine residues in HSA, 34 of which form 17 disulfide bridges. Sixteen of these participate in the formation of eight so-called double disulfide bridges (Figures 3 and 4GoGo). The two adjoining cysteine residues in the amino acid sequence on helices a-h3 and b-h3 lie at the center of this unique folding topology. The first bridge connects helices h3 and h1, which are spatially aligned nearly perpendicular to each other, while the second links helices h3 and h4, which are nearly parallel. Consequently, three helices (h2, h3 and h4) join to form a curved wall at one side of the subdomain. Another series of disulfide bridges is found around helices a-h4, a-h5 and a-h6, where two consecutive cysteine residues are located on each a-h5 helix. Residues having aliphatic and aromatic side chains are clustered at the inner surface of the helix wall to form the hydrophobic core of the subdomain, and are partly capped by helix h1. This topological feature is seen in every subdomain except Ia-h3. Sequence comparison of serum albumins of various species suggests that simultaneous mutation of two cysteine residues may have occurred at the end of helix Ia-h3 in ancient proteins (Carter and Ho, 1994Go).



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Fig. 3. Schematic drawing of secondary structure elements and disulfide bridges of HSA. Helices are represented by rectangles, and loops and turns by thin lines. Disulfide bridges are drawn with thick lines. The sequence nomenclature is derived from Minghetti et al. (1986).

 


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Fig. 4. Stereographic representation of typical double disulfide bridges (Cys200–Cys246 and Cys245–Cys263) shown with electron density (contour level 1.5{sigma}). Disulfide bridges are drawn with thick lines.

 
Free sulfhydryl group at Cys34

Cys34, located in a loop between helices Ia-h2 and Ia-h3, is the only cysteine residue that does not participate in any disulfide bridges (Figure 2AGo). Its sulfhydryl group is oxidized by cysteine or glutathione in 30–40% of HSA molecules in the bloodstream to form an intermolecular disulfide linkage (Peters, 1985Go). In the current structure, however, none of the features on the final difference electron density map is observed around the sulfhydryl side chain of Cys34 (Figure 5Go). Although Cys34 is located at the surface of the protein, its S{gamma} atom is toward the interior and is surrounded by side chains of Pro35, His39, Val77 and Tyr84, which prevent the sulfhydryl group from coupling with the external counterparts. Ion-spray mass spectrometry revealed that the purified HSA used for crystallization contains much fewer oxidized species at Cys34 than the commercially available substance (data not shown), and that triclinic crystals grew only from solution of the former and not of the latter. This observation suggests that the molecular species mainly involved in the triclinic crystals is one whose Cys34 is free of oxidation. When the protein is in solution, the phenolic side chain of Tyr84 may flip over to enable the external disulfide counterparts to bind to the S{gamma} atom of Cys34, or the backbone conformation may change to bring the free sulfhydryl group toward the exterior of the protein.



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Fig. 5. Structure around Cys34 drawn in stereo with difference electron density (contour level 2.0{sigma}): Gln33, Cys34, Pro35 and Thr83 face the protein–solvent interface. The sulfhydryl side chain of Cys34 is toward the interior of the protein, and is surrounded by residues Pro35, His39, Val77 and Tyr84.

 
Binding sites for drugs and other compounds

In both subdomains IIa and IIIa, a part of the hydrophobic core is further surrounded by the first few residues of the extended loop together with helices a-h5 and a-h6 to form a pocket (Figures 2B and CGo). The pocket of subdomain IIa seems to correspond to the so-called site I, which is thought to be a binding site for salicylates, sulfonamides and a number of other drugs (Sudlow et al., 1975Go, 1976Go). The bilirubin binding site overlaps with this locus (Brown and Shockley, 1982Go). The inside wall of the pocket is formed by hydrophobic side chains, whereas the entrance of the pocket is surrounded by positively charged residues, i.e., Arg257, Arg222, Lys199, His242, Arg218 and Lys195 (Figure 6AGo). Site II, the putative binding site for tryptophane, thyroxine, octanoate and other drugs (Sudlow et al., 1975Go), would correspond to the pocket of subdomain IIIa, which is almost the same size as site I. The pocket is lined by hydrophobic side chains and the double disulfide bridges of helix IIIa-h3. The side chain of Arg410 is located at the mouth of the pocket while the hydroxyl of Tyr411 faces toward the inside of the pocket (Figure 6BGo). Unlike subdomains IIa and IIIa, subdomain Ia has no pocket near its hydrophobic core (Figure 2AGo). Due to relaxation of helix Ia-h4, the latter is no longer parallel to helix Ia-h3. The side chains of the residues on helix Ia-h4 bury the putative pocket, and subdomain IIa interacts with them to seal this region.



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Fig. 6. Stereographic illustration of ligand binding site I (A) and site II (B) with charge distribution overlayed on the illustrations as dot surfaces (blue for positive and red for negative charges, respectively). Charge distribution is derived from electrostatic potential calculations using the DelPhi program (Molecular Simulation, Inc.). Helices h1, h2, h3, h4 and h6 in subdomains IIa and IIIa are colored magenta, yellow, green, orange and cyan, respectively. Helix Ib-h3 (only shown in A) is colored grey.

 
Camerman et al. (1976) proposed that the backbone nitrogens of the first three residues as well as the imidazole of His3 form a binding site of the Cu2+ ion. No significant evidence regarding metal binding can be drawn from our refined coordinates due to disorder at the N-terminus.

It is well known that HSA is able to accommodate fatty acids. Reed (1986) proposed that palmitic acid is bound to Lys473, Lys349 and Lys116 in bovine serum albumin (equivalent to Lys475, Lys351 and Arg116 in HSA, respectively). Lys475 and Lys351 are located on the molecular surface (Figure 1Go). The Lys475 on helix IIIa-h5, however, projects from the molecular surface. Two more clefts are found near Lys351 on helix IIb-h3. One is at the interface between subdomains IIa and IIb and the other between subdomains IIb and IIIa. Arg117 is located in a crevice formed by the helix cluster of subdomain Ib and the extended loop between subdomains Ia and Ib.


    Conclusion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Conclusion
 References
 
We determined the X-ray structures of HSA, derived from human pooled plasma or from a Pichia pastoris expression system, at a resolution of 2.5 Å, and demonstrated that their three-dimensional structures are identical within the margin of experimental error. HSA consists of three similar helical domains with eight pairs of double disulfide bridges. Each domain is further divided into two subdomains. Cys34, the only cysteine with a free sulfhydryl group, does not participate in a disulfide linkage with any external ligand. Deep hydrophobic pockets with positively charged entrances are located at similar positions in subdomains IIa and IIIa, and are thought to correspond to the so-called site I and site II binding sites for various compounds. Subdomain Ia does not have such a pocket. Three possible binding sites for long-chain fatty acids are located on the surface of the molecule, but their structural environments are entirely different. Our proposed structure for HSA, whose atomic coordinates are now available from PDB, will serve as a guide for researchers investigating the biochemical properties and medicinal applications of this protein.


    Acknowledgments
 
Some of the diffraction experiments were carried out using the Rigaku R-AXIS IV area-detector and FR X-ray generator of the Nara Institute for Science and Technology. We thank Dr Toshio Hakoshima for permitting the use of this equipment.


    Notes
 
1 Present address: Mitsubishi Chemical Corporation, Yokohama 227-8502, Japan Back

2 To whom all correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Conclusion
 References
 
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Received November 22, 1998; revised January 27, 1999; accepted February 24, 1999.