Osaka Laboratories, Yoshitomi Pharmaceutical Industries Ltd,2-25-1, Shodai-Ohtani, Hirakata, Osaka 573-1153, Japan
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Abstract |
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Keywords: crystal structure/human serum albumin/recombinant protein
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Introduction |
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Materials and methods |
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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 150255 mg/ml protein, 50 mM potassium phosphate (pH 5.05.5), 3038% (v/v) polyethylene glycol 400 and 5 mM sodium azide at 293 K over 23 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., 1989; Carter and He, 1990
), 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 ( = 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., 1992
). Data collection statistics are summarized in Table I
. Isomorphous difference Patterson maps, calculated at 4.0 Å resolution using the XtalView program package (McRee, 1993
), 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, 1994
), 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 I
. The initial phases were then improved and extended to 3.0 Å through iterative cycles of solvent flattening (Wang, 1985
) implemented in the PHASES program (Furey, 1994
). 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, 1992b
) 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., 1986
). 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, 1990
). The procedure was iterated using the CCP4 program suite (Collaborative Computational Project Number 4, 1994
) and SQUASH (Zhang and Main, 1990
), 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 10.03.0 Å resolution. No further attempts were made to refine this model with the data from the tetragonal crystal form.
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pHSA was purchased from Miles Inc. (catalog no. 82-301), and rHSA was prepared using the procedure described previously (Sumi et al., 1993; Ohi et al., 1994
; 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 0.31.2 M at a pH of 4.55.0. 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 120240 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 7.08.0), 2026% (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 288293 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;
, 91.1/91.0°; ß, 103.5/103.4°;
, 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, 1974
; Rao et al., 1976
; Carter et al., 1989
; Carter and He, 1990
; He and Carter, 1992
; Ho et al., 1993
; Carter and Ho, 1994
).
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 II. Structural determination of pHSA in the triclinic crystal was performed using molecular replacement and crystallographic refinement facilities implemented in X-PLOR (Brünger, 1992b
). A real-space rotation function at a resolution range of 10.04.0 Å followed by a PC-refinement with the structure of the tetragonal crystal as a search model gave two unique solutions (7.8
and 7.6
) 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 10.03.0 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 50.02.5 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, 1992a
) 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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Results and discussion |
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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., 1977), 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 5582 (4600 non-hydrogen atoms), and seven water molecules, whereas two copies of HSA (residues 5582) and four water molecules are involved in the current coordinates of rHSA. An electron density corresponding to residues 14 and residues 583585 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 II 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, 1965
; Laskowski et al., 1993
). Average coordinate error estimated from a Luzzati analysis (Luzzati, 1952
) 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 1520 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 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
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
-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 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 1
. It consists of three domains, I (residues 1195), II (196383) and III (384585), which are not only topologically identical, as predicted from amino acid sequence comparison, but also very similar in three-dimensional structure (Figure 2
). The overall r.m.s. deviation of the corresponding C
atoms is 3.78 Å for all three domains. The r.m.s. deviation between domains I and II for corresponding C
atoms is 3.1 Å, while that for other pairs is 4.65.0 Å. Although the C
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 2
). Each domain can be further divided into subdomains a and b, which are further described in the next section.
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It has been said from physicochemical studies that HSA is a flexible protein that easily changes its molecular shape (Carter and Ho, 1994). A couple of interesting features are revealed from a comparison of our defatted HSA with that in complex with myristates (Curry et al., 1998
). 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 -helices, respectively (Figure 2
). Subdomain a is composed in each case of a cluster of four
-helices (a-h1 to a-h4) flanked by two short
-helices (a-h5 and a-h6), while subdomain b in each case has another cluster of four
-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 2
. 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, 1992
).
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 4). 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, 1994
).
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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 2A). Its sulfhydryl group is oxidized by cysteine or glutathione in 3040% of HSA molecules in the bloodstream to form an intermolecular disulfide linkage (Peters, 1985
). 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 5
). Although Cys34 is located at the surface of the protein, its S
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
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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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 C). 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., 1975
, 1976
). The bilirubin binding site overlaps with this locus (Brown and Shockley, 1982
). 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 6A
). Site II, the putative binding site for tryptophane, thyroxine, octanoate and other drugs (Sudlow et al., 1975
), 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 6B
). Unlike subdomains IIa and IIIa, subdomain Ia has no pocket near its hydrophobic core (Figure 2A
). 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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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 1). 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.
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Conclusion |
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Acknowledgments |
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Notes |
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2 To whom all correspondence should be addressed
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References |
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Received November 22, 1998; revised January 27, 1999; accepted February 24, 1999.