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439446, 1999
Introduction
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., 1981; Kragh-Hansen, 1981; Putnam, 1984; Peters, 1985).
Such studies have revealed that HSA has a high affinity to a
very wide range of materials, including metals such as Cu21
and Zn21, 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, 1985; Fiume et al., 1988). 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., 1982). Human
Oxford University Press
S.Sugio et al.
Table I. Statistics in data collection and phasing for the tetragonal crystal
Crystal
Soaking
(mM)
Conditions
(h)
Resolution
()
No. of
refls.
Rmerge
(%)a
Completeness
(%)b
Figure of
merit
Phasing
powerc
Native
HgCl2-1
HgCl2-2
Thimerosal
Mersalyl-1
Mersalyl-2
0.53
0.50
1.04
0.51
0.50
29
24
24
24
72
3.00
3.56
3.55
3.56
4.00
3.55
26761
13534
12332
12498
9198
14118
9.9
12.5
13.4
11.1
11.1
11.3
92.0
78.2
70.9
71.3
75.4
80.9
0.461
0.464
0.427
0.415
0.452
1.665
1.708
1.232
1.265
1.552
Table II. Statistics in data collection and refinement for the triclinic crystal
pHSA
rHSA
2
93.62.38
40 737
9.4/2.4
9.7/27.2
81.4/46.9
5
93.52.37
36 149
4.4/1.5
13.4/42.8
71.3/36.1
Refinement statistics
Resolution range ()
No. of total reflections
R factorc,d
Free R factore (%)c
Completeness (%)c
50.02.5
37 461
21.8/26.0
28.2/34.7
86.2/64.8
50.02.5
33 040
20.7/29.2
29.2/42.0
77.1/48.1
No. of atoms
Protein
Solvent
460032
7
460032
4
0.014
1.72
21.0
1.59
0.014
1.75
21.1
1.69
48.5
40.7
aOverall/highest
bRmerge 5 |I I|/I .
i
i
cOverall/highest resolution
bin (2.592.50 ).
dR factor 5 |F
obs Fcalc|/|Fobs|.
eCalculated from 10% randomly selected
Fig. 2. Stereographic illustration of C 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 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.
S.Sugio et al.
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 RU200H X-ray generator. Data to a resolution of 2.38 for
pHSA and 2.37 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
(Brunger, 1992b). A real-space rotation function at a resolution
range of 10.04.0 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 rigidbody refinement followed by positional and overall temperature
factor refinements at a resolution range of 10.03.0 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 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 were then
included in the refinement, in which bulk solvent correction,
a resolution-dependent weighting scheme for reflections, and
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Fig. 4. Stereographic representation of typical double disulfide bridges (Cys200Cys246 and Cys245Cys263) shown with electron density (contour level
1.5). Disulfide bridges are drawn with thick lines.
Fig. 5. Structure around Cys34 drawn in stereo with difference electron density (contour level 2.0): Gln33, Cys34, Pro35 and Thr83 face the proteinsolvent
interface. The sulfhydryl side chain of Cys34 is toward the interior of the protein, and is surrounded by residues Pro35, His39, Val77 and Tyr84.
S.Sugio et al.
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.
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 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 for equivalent
C atoms, backbone atoms and all non-hydrogen atoms,
respectively (0.36, 0.40 and 0.76 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 . No significant correlation was found
between the residual plots of r.m.s. deviations and those of
average temperature factors.
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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 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 3 80 3 30 , 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 for all three
domains. The r.m.s. deviation between domains I and II for
corresponding C atoms is 3.1 , while that for other pairs is
4.65.0 . Although the C trace suggests a resemblance
S.Sugio et al.
References
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