.I. Mol. Bid. (1991) 221, 571-581
Crystal Structure of Human a-Lactalbumin K. Ravi Acharyat,
at l-7 A Resolution
Jingshan Ren, David I. Stuart, David C. PhillipsS
Laboratory of Molecular Biophysics Rex Richards Building, South Parks Road Oxford OX1 3QU, England
and Roger E. Fenna Department of Biochemistry and Molecular Biology University of Miami School of Medicine Miami, FL 33101, U.S.A. (Received
23 November
1990; accepted 10 May
1991)
The three-dimensional X-ray structure of human a-lactalbumin, an important component of milk, has been determined at 1.7 A (@17 nm) resolution by the method of molecular replacement, using the refined structure of baboon a-lactalbumin as the model structure. The two proteins are known to have more than 90% amino acid sequence identity and crystallize in the same orthorhombic space group, P2,2,2. The crystallographic refinement of the structure using the simulated annealing method, resulted in a crystallographic R-factor of 0.209 for the 11,373 observed reflections (F 2 2o(F)) between 8 and 1.7 ir resolution. The model comprises 983 protein atoms, 90 solvent atoms and a bound calcium ion. In the final model, the root-mean-square deviations from ideality are 0.013 A for covalent bond distances and 2.9” for bond angles. Superposition of the human and baboon u-la&albumin structures yields a root-mean-square difference of 0.67 g for the 123 structurally equivalent C” atoms. The C terminus is flexible in the human cc-lactalbumin molecule. The striking structural resemblance between a-lactalbumins and C-type lysozymes emphasizes the homologous evolutionary relationship between these two classes of proteins. Keywords:
a-lactalbumin;
milk protein; crystal structure; calcium binding; evolution
having a tight Ca ‘+ binding site (app arent affinity constant as large as IO6 to 10’ Mp’: Segawa & Sugai, 1983; Hamano et al.,1986; Berliner & Johnson, 1988; Kronman, 1989) and differs from lysozyme in its biological role. X-ray crystallographic studies of baboon a-lac to 1.7 A (1 b = 0.1 nm) resolution have revealed a novel calcium binding loop and confirmed that in the native state, a-lac and C-type lysozyme have closely similar three-dimensional structures (Stuart et al.,1986; Acharya et al., 1989). The amino acid sequence of baboon a-lac has not yet been completely determined, although it is known to be closely similar to that of human a-lac (R. Greenberg, personal communication; Table 1) and the crystallographic refinement of the baboon a-lac structure was carried out using the human a-lac arpino acid sequence as a starting point. Omit maps calculated during the course of baboon u-lac refinement indicated eight putative sequence changes (Acharya et aE.,1989; and see Table 1). Meanwhile, suitable single crystals were obtained for human a-lac and we present here the crystal structure of
1. Introduction a-Lactalbumin (a-lac$) is a globular protein secreted in the lactating mammary gland and has a relative molecular mass of 14,200. It regulates lactose biosynthesis by modulating the specificity of trans-golgi galactosyltransferase (GTase) (Hill & Brew, 1975). Comparison of amino acid sequences (Brew et al.: 1970; Findlay & Brew, 1972), gene sequences (Uandekar & Qasba, 1981; Hall et aZ., 1982) and the exon-intron organization of the genes (Qasba & Safaya, 1984) firmly established that a-lac is homologous with C-type lysozymes having evolved by divergence from a common ancestor. a-lac is a metalloprotein (Hiraoka et al.,1980) t Author to whom all correspondence should be addressed at: Department of Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, England. $ Present address: Advisory Board for the Research Councils, Elizabeth House, York Road, London SE1 7PH. England. $ Abbreviations used: a-lac, a-lactalbumin; GTase, galactosyltransferase; HEWL, hen-egg-white lysozyme. 571 0022 -%836/91/180571-11
$03.00/O
0
1991 Academic
Press
Limited
57”
K. R. Acharya
Alignment
et al.
Table 1 oJ”amino acid sequences for human and baboon a-lacs and hen-egg-white
lysozymr
Human cr-lac Baboon cc-lac HEWL
6 13 LYS-GLN-PHE-THR-LYS-CYS-GLU-LEU-SER-GLN-LEU-LEU-LYS---------ASPLYS-GLN-PHE-THR-LYS-CYS-GLU-LEU-SER-GLN-~-LEU-~---------ASPLYS-Val-PHE-Gly-Arg-CYS-GLU-LEU-Ala-Ala-Ala-Met-LYS-Arg-His-Gly12
Human a-lac Baboon or-lac HEWL
-ILE-ASP-GLY-TYR-GLY-GLY-ILE-ALA-LEU-PRO-GLU-LEU-ILE-CYS-THR-~T-ILE-ASP-GLY-TYR-GLY-Arg-ILE-ALA-LEU-PRO-GLU-LEU-ILE-CYS-THR-~T-Leu-ASP-Asn-TYR-Arg-GLY-Tyr-Ser-LEU-Gly-Asn-Trp-V~l-CYS-Al~-Ala-
20
28
14
30
28 40
Human @-lac Baboon or-lac HEWL
44
-PHE-HIS-THR-SER-GLY-TYR-ASP-THR-GLN-ALA-ILE-VAL-GLU-ASN-----PHE-HIS-THR-SER-GLY-TYR-ASP-THR-GLN-ALA-ILE-VAL-GLU-ASN-----Lys-Phe-Glu-SER-Asn-Phe-Asn-THR-GLN-ALA-Thr-Asn-Arg-ASN-Thr35
Human a-lac Baboon cl-lac HEWL
39 47 50 -ASN-GLU-SER-THR-GLU-TYR-GLY-LEU-PHE-GLN-ILE-SER-ASN-LYS-LEU-TRP-ASN-GLU-SER-THR-GLU-TYR-GLY-LEU-PHE-GLN-ILE-SER-ASN-Ala-LEU-TRP-Asp-Gly-SER-THR-Asp-TYR-GLY-Ile-Leu-GLN-ILE-Asn-Ser-~-Trp-TRP52
62
61
Human cl-lac Baboon ci-lac HEWL
Human Ct-lac Baboon a-lac HEWL
73
-CYS-LYS-SER-SER-GLN-VAL-PRO-GLN-SER-ARG-ASN-ILE-CYS-ASP-ILE-SER-CYS-LYS-SER-SER-GLN-Ser-PRO-GLN-SER-~G-ASN-ILE-CYS-ASP-ILE-Thr-CYS-Asn-Asp-Gly-Arg-Thr-PRO-Gly-SER-ARG 69 77 9: -CYS-ASP-LYS-PHE-LEU-ASP-ASP-ASP-ILE-THR-ASP-ASP-ILE-~T-CYS-~A-CYS-ASP-LYS-PHE-LEU-ASP-ASP-ASP-ILE-THR-ASP-ASP-ILE-~T-CYS-ALA-CYS-Ser-Ala-Leu-LEU-Ser-Ser-ASP-ILE-THR-Ala-Ser-Val-Asn-CYS-ALA85 96
Human cr-lac Baboon a-lac HEWL
60
79
93 100
103 107 -LYS-LYS-ILE-LEU----- ASP-ILE-LYS-GLY-ILE-ASP-TYR-TRP-LEU-ALA-HIS-LYS-LYS-ILE-LEU-----ASP-ILE-LYS-GLY-ILE-ASP-TYR-TRP-Ile-ALA-HIS-LYS-LYS-ILE-Val-Ser-ASP-Gly-Asp-GLY-Met-Asn-Ala-TRP-~-ALA-Trp100 107 111 111
117
Human cr-lac Baboon a-lac HEWL
-LYS-ALA-LEU-CYS-THR-GLU-LYS-LEU-GLU-GLN-----TRP-LEU---------CYS-LYS-ALA-LEU-CYS-THR-GLU-LYS-LEU-GLU-GLN-----TRP-LEU---------CYS-Arg-Asn-Arg-CYS-Lys-Gly-Thr-Asp-Val-GLN-Ala-TRP-Ile-Arg-Gly-CYS120 125
Human m-lac Baboon a-lac HEWL
-GLU-LYS-LEU -GLU-LYS-Glu -----Arg-E
120
123
129
The residue numbrrs are marked at the top and at the bottom of’ t.he sequrnc~ for c&w and hrn-egg-white Iysozyrnc~ H E1rI.. respectivrly. --- represents a deletion in the sequenvr used to maximize squenw idrntity. Invariant) residues arc marked in capital letters. The sequence differences between human and baboon cc-law are underlinrd. Human a-lar (Hall it ~1.. 1982): baboon cc-lat. S-rag srquencr (Acharya rt nl.. 1989): H E!&~L ((‘antirId. 1963: .lollbs PI u.. I!~Ki: (‘anfield & Liu, 1965: Imoto rt ccl.. 1972; Ibrahimi pf nl.. 1979). The sequence identities after maximizing the alignment aw: human y-la:! (88) 0 (80) 0 (38) N (Fi%) 0 (84) 0 (82) ODI (84) ODl (88) N(106) N(10.5)
OHZ(1’4)
OHi(l24)
OH9( 12.5)
Int ., internal: Cal., calcium
Comment
Water
Vontacts
Int. water
OHS(131)
N (33) 0 (50) ODI (88) OD2 (88) 0 (80) 0 (38) N (52) 0 (84) 0 (82) ODl (82) ODl (88) N( 106) 001 (33)
(R = 752AZ) ht. water (B = 7.3 AZ)
OH9(134)
Int. water (:al. ligand (R = 7.9 AZ)
OHB(135)
Int. water (R = 3Fi.5AZ)
OH@ 1%)
(hmment ht. water (H = 122 192) ht. wat,cr (H = 1:S.oAZ)
ht. watt (‘al. ligalld (R = 16.1 .V) Int.. WLtPr (R = 19% A’)
576
K. II. Acharya
b Ld) Fig. 3.
et al.
Crystal Structure
of Human
a-Lactalbumin
577
at 1.7 d
Figure 3. Comparison of human (thick line) and baboon (thin line) cc-lacs around regions (a) 40 to 47; (b) C-terminal tail: (c) Met90 and (d) calcium binding loop in human u-lac (the additional water molecule in the vicinity of the calcium binding
site hydrogen-bonded
to Asp84
and Asp87
is also shown)
and (e) calcium
to 47 form part of a P-pleated sheet and are exposed to solvent but in the crystal they are also involved in contacts with a symmetry related protein molecule. In human a-lac, Glu46 has a different confor-
mation (Fig. 3(a)). As expected, from the baboon a-lac structure, the C-terminal arm of the human a-lac molecule is rather flexible and is different to the corresponding region in baboon u-lac (Fig. 3(b)). supporting the hypothesis that the C terminus in a-lac can adopt multiple conformations (Acharya et al., 1989). In general, the overall environments of the sidechains in the two structures are very similar even though some side-chains in human a-lac have different orientations from those of the corresponding side-chains in baboon a-lac (e.g. Glu46 and Asp83). These effects are due in part to different packing interactions in the two crystals and in part to the presence of multiple conformations (e.g. MetSO, Fig. 3(c)). In such cases, only the best conformer was taken into account during the refinement of the structure and the other conformer was crudely modelled with solvent molecules. The need for more satisfactory treatments has been indicated by Kuriyan et aZ. (1986) and Gros et al. (1990).
The calcium
(b) Calcium
Lys79 Asp82 Asp84 Asp87 Asp88
Group Carbonyl Csrboxylate Carbonyl Carboxylate Carboxylate Water Water Calcium ion
0 ODl 0 ODl ODl 0 0 Cal
site in baboon
a-lat.
binding
a-lac is a calcium binding protein and possesses a novel calcium binding fold (Stuart et al., 1986; Acharya et al., 1989). The atomic structures around the calcium ion, in baboon a-lac and human a-lac superimpose well (Fig. 3(d) and (e)). The co-ordination around the calcium (Table 4) is a slightly disorted pentagonal hipyramid. The Ca-0 distances vary from 2.24 to 2.42 8, corresponding to a tight calcium binding loop in the structure. The positioning of the Asp83 side-chain is different) in human a-lac and baboon a-lac, but this does not seem to affect the geometry of the calcium binding site (Fig. 3(d) and (e)). There is an “extra” water molecule in the vicinity of the calcium binding site in human a-lac, hydrogen bonded to Asp84 (2.76 8) and Asp87 (2.91 8) (Fig. 3(d)). The structure of human a-lac confirms the previous observation in the baboon a-lac structure that the calcium binding fold only superficially resembles the “EF-hand” structure and presumably has no evolutionary relationship with other EF-hand structures (Stuart et al., 1986; Acharya et al., 1989). The biological role of calcium in a-lac is not yet
Table 4 ligands in human
and baboon a-lacs
Distance to Ca (A) Residues
binding
B-value (A’)
Human
Baboon
Human
Baboon
2.3 2.4 2.2 2.4 2.4 2.3 2.5
2.2 2.4 23 23 2.3 2.4 2.6
%4 11.0 58 10.4 7.3 191 7.9 62
139 11-7 126 7.9 l&8 16.1 21.0 11.5
-
578
K. R. Acharya et al.
7t i-
1
I
I
I
I
25
50
15
100
1
--~125
Residue numbrr (a)
62.50-
z I t 0
37*50
i @ 25.00-
I2.50-
I 0
I 25
I 50
I 100
I 75 Residue
I 125
number (0)
Figure 4. R-value plots + . human a-lac; *? baboon
for (a) the main-chain a-lat.
atoms
clear. though some evidence suggests that Ca2+ is necessary for a-lar to modify or modulate lactose synthesis (Musci & Berliner, 1985). Accordingly, cr-lac might act as a calcium modulated protein (Thompson rt al.. 1988). Recently Rao clr Brew (1989) proposed that t,ha a-lac calcium binding site has the in ~,izrofunction of imposing calcium regulation on the folding of the nascent cc-lac molecule and thereby on lactose synthesis. Kuwajima (1989) and Kuwajima rt al. (1989) propose that the calcium binding loop in a-lac may be a unique critical substructure in its folding. It is the best’ defined part. of the st,ruct,ure in the X-ray structures (as judged
and (b) the side-chain
atoms
in human
and baboon
r-law.
by the B-fac$ors; Table -l) irl both human and baboon a-lacs. Studies on the molben plobule state of guinea pig sr-lac by nuclear magnetic resonan(*e methods (Saum d trl., 19X9) concluded that thy calcium binding loop is a highly structured part of the molecule even in this partly unfolded form. a-lac can bind a variety of metal ions such as Ca2 + , Zn 2+. Ch2+, Clo2+, A13+ and Mn2+ and t,here have been a number of suggestions (Hiraoka rt nl., 1980; Murakami rt al., 1982: Rratcher & Kronman. 1984; Desmet & van (:auwelaert, 1988) that t)hrre is a Zn2+ binding site in cc-lac that is distinc+ from the calcium binding site. the occupation of’ whic.h
Crystal Structure
of Human
a-Lactalbumin
579
at 1.7 d
Table 5 Differences
in inter-molecular packing contacts ( < 34 A ) due to sequence diflerences between human and baboon a-lacs
Residue Human a-lac
Baboon a-lac
(lomment
Position
Leu
Asn
11
LYs
Tyr
13
GIY Lys
Arg Ala
20 58
Val Ser Leu Leu
Ser Thr Ile GIU
66 76 105 123
0 (11) is involved in water mediated intermolecular contacts. In baboon a-lac Am11 was more exposed to the solvent No contacts. In baboon a-lac the Tyr (OH) atom is hydrogen-bonded to Ala109 (0) No contacts NZ (58) is involved in symmetry related intermolecular contacts with the neighbouring Glu(46) side-chain atoms and is a very exposed residue. Glu(46) has a different side-chain orientation in baboon a-lac No contacts OG (76) is involved in water mediated contacts No contacts Disordered in both structures. Different conformations due to the non-rigidity of the C terminus. No inter-molecular contacts
causes the molecule to adopt an “ape-like” conformation. However, the present study provides no evidence of a second metal binding site. The cleft region, which divides the molecule into two wings in a-lac is very similar in the two structures. (c) Mobility
Figure 4(a) and (b) shows the distribution of atomic B-values (which describe the sum of the mobility and static disorder for each atom) in human and baboon a-lacs. From the plots it is clear that baboon a-lac has somewhat higher B-values for most of the atoms in comparison with human a-lac (mean B-value for all main-chain atoms of 25.5 A2 compared to 19.5 A’). A further difference is in the crystallographic refinement strategies used (baboon a-lac: PROLSQ (Konnert & Hendrickson, 1980), human a-lac: X-PLOR (Briinger, 1988, 1989; Kriinger et al., 1989)). However, since the target residual minimized in each refinement is identical that is unlikely to be a serious effect. This is likely to be due, in part,, to the radiation damage suffered during the intensity data collection for baboon a-lac crystals on the linear diffractometer and perhaps, to some extent, to undetected systematic error in the data (e.g. absorption errors). Given the well known difficulties in defining absolute B-values (Stuart & Phillips, 1986), we attach more significance to the fact that the relative mobilities are very similar for the two structures; the correlation coefficient between main-chain atom B-factors (cs,t) is 084. We have also investigated the relationship discrepancy between mobility and positional between the two molecules. The correlation coeffi-
Cj(Bj,l-(B,))(Bj.*-(B,)) + cBB
=
rgj(Bj,,
-
(B1))2Zj(Bj,
2-
@*))2]1’2
cient (&,,,S) for these two quantities has a very high value, 982; thus the regions of the molecules that differ are those of high mobility in each structure and there is no indication of any rearrangement of rigid segments of the molecule between the two structures. As mentioned before, the C terminus is mobile in human a-lac and has high B-values and, in general, it is true that the a-lac molecule has greater mobility than hen-egg-white lysozyme (HEWL) (Acharya et al., 1989). The conformational flexibility of the C terminus in cr-lac might indicate that it has a role in stabilizing the interaction of a-lac with GTase upon formation of the lactose synthetase complex, which is of important functional significance, and does not occur with C-type lysozymes. Further experiments are required to resolve this point. (d) Sequence
changes and crystal packing
The packing of the molecules in the crystals of baboon and human a-lac is similar. However, there are some water mediated interactions in the human a-lac structure that appear to be facilitated partly by the apparent amino acid differences between the two species (Table 5). Also, the differences in the side-chain orientations (mainly Glu46 and Asp83) provide additional intermolecular contacts for the stabilization of the crystal packing in human cc-lac (Table 5).
t
CB,= ~j[(Bj.l-(Bl))+(Bj,2-(B2))]X(~j-(U))
Fj(CBj,
1
-CBl))+
(Bj,2-(B2>))2
X
Zj(Dj-(D>)2]“2’
where Dj = Xbaboonj- Xhumanj;Xj is the position of atom j (after superimposition of human a-lac on to baboon a-lac); B values are isotropic temperature factors and the sum Ej, represents over all main-chain atoms.
K.
580
R. Acharya et al.
We thank Dr Richard Lyster for the generous gift of human a-lactalbumin. We thank Drs David Barford and Vasanta Subramanian for helpful discussions. We also thank Drs Helen Handoll and Diana Grace for refining the st’ructure of hen-egg-white lysozyme. The work was supported by the MRC and SERC.
Findlay. J. B. (‘. bi Brew, Ii. (1972). The complete amino Eur. J. acid sequence of human cc-la&albumin. Biochem. 27, 65-86. Fitzgerald, P. M. D. (1988), MERLOT. an integrated package of computer programs for the determination of crystal structures by molecular replacement. J. Appl. (‘rystallogr. 21. 274--278.
References
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Crystal Structure of Human
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ol-La&albumin
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3852~-3856. Qasba. P. K. & Safaya, S. K. (1984). Similarity ofnucleotide sequences of rat r-lactalbumin and chicken lysozyme genes. ,Va,ture (London), 308, 377-380. Rao. K. R. P: Brew. K. (1989). Calcium regulates folding
Edited
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581
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Bioche,m. Biophys. Res. Commun. 163. 1390-1396. Segawa, T. & Sugai. S. (1983). Tnteractions of divalent metal ions with bovine. human and goat a-lactalbumins. J. Biochem. (Tokyo), 93. 1381- 1328. Stuart, D. I. & Phillips, D. C. (1985). On the derivation of dynamic information from diffraction data. In Methods in Enzymology (Wykoff. H. W.. Hirs. C. H. W. L Timasheff, S. N., eds). vol. 115, pp. 117714l. Academic Press, Florida. Stuart, D. I., Acharya, K. R., Walker, 2u‘. 1’. C’., Smith. S. C., Lewis, M. & Phillips, D. C. (1986). I-Lactalbumin possesses a novel calcium binding loop. N&ure
(London), Sussman.
Musci. G. & Berliner, L. ,J. (1985). Probing different conformational states of bovine cc-lactalbumin: fluorescence st*udies with 4,4r-Bis[l-(phenyl-amino)8-napthalene sulfonate]. Biochemistry. 24,
at I.7 /f
J.
324. 84-87. L.
(1985).
Constrained-restrained
least
squares (CORELS) refinement of proteins and nucleic H. W.. acids. In Methods in Enzymology (Wyckoff. Hirs. C. H. W. & Timasheff, S. K’.. eds). vol. 115. pp. U-303, Academic Press, Florida. Thompson, M. P.. Groves: M. I~.. Brewer. I>. P., Farrell. H. M.. tJenness, R. & Kotts, C. E. (1988). The calcium dependent electrophoretic shift of a-lactalbumin, the modifier protein of galactosyl transferase. Biochem.
Biophys. Res. (I‘ommun. 157. 94&948.
by R. Huber
Crystal Structure of Human a-Lactalbumin K. Ravi Acharyat,
at l-7 A Resolution
Jingshan Ren, David I. Stuart, David C. PhillipsS
Laboratory of Molecular Biophysics Rex Richards Building, South Parks Road Oxford OX1 3QU, England
and Roger E. Fenna Department of Biochemistry and Molecular Biology University of Miami School of Medicine Miami, FL 33101, U.S.A. (Received
23 November
1990; accepted 10 May
1991)
The three-dimensional X-ray structure of human a-lactalbumin, an important component of milk, has been determined at 1.7 A (@17 nm) resolution by the method of molecular replacement, using the refined structure of baboon a-lactalbumin as the model structure. The two proteins are known to have more than 90% amino acid sequence identity and crystallize in the same orthorhombic space group, P2,2,2. The crystallographic refinement of the structure using the simulated annealing method, resulted in a crystallographic R-factor of 0.209 for the 11,373 observed reflections (F 2 2o(F)) between 8 and 1.7 ir resolution. The model comprises 983 protein atoms, 90 solvent atoms and a bound calcium ion. In the final model, the root-mean-square deviations from ideality are 0.013 A for covalent bond distances and 2.9” for bond angles. Superposition of the human and baboon u-la&albumin structures yields a root-mean-square difference of 0.67 g for the 123 structurally equivalent C” atoms. The C terminus is flexible in the human cc-lactalbumin molecule. The striking structural resemblance between a-lactalbumins and C-type lysozymes emphasizes the homologous evolutionary relationship between these two classes of proteins. Keywords:
a-lactalbumin;
milk protein; crystal structure; calcium binding; evolution
having a tight Ca ‘+ binding site (app arent affinity constant as large as IO6 to 10’ Mp’: Segawa & Sugai, 1983; Hamano et al.,1986; Berliner & Johnson, 1988; Kronman, 1989) and differs from lysozyme in its biological role. X-ray crystallographic studies of baboon a-lac to 1.7 A (1 b = 0.1 nm) resolution have revealed a novel calcium binding loop and confirmed that in the native state, a-lac and C-type lysozyme have closely similar three-dimensional structures (Stuart et al.,1986; Acharya et al., 1989). The amino acid sequence of baboon a-lac has not yet been completely determined, although it is known to be closely similar to that of human a-lac (R. Greenberg, personal communication; Table 1) and the crystallographic refinement of the baboon a-lac structure was carried out using the human a-lac arpino acid sequence as a starting point. Omit maps calculated during the course of baboon u-lac refinement indicated eight putative sequence changes (Acharya et aE.,1989; and see Table 1). Meanwhile, suitable single crystals were obtained for human a-lac and we present here the crystal structure of
1. Introduction a-Lactalbumin (a-lac$) is a globular protein secreted in the lactating mammary gland and has a relative molecular mass of 14,200. It regulates lactose biosynthesis by modulating the specificity of trans-golgi galactosyltransferase (GTase) (Hill & Brew, 1975). Comparison of amino acid sequences (Brew et al.: 1970; Findlay & Brew, 1972), gene sequences (Uandekar & Qasba, 1981; Hall et aZ., 1982) and the exon-intron organization of the genes (Qasba & Safaya, 1984) firmly established that a-lac is homologous with C-type lysozymes having evolved by divergence from a common ancestor. a-lac is a metalloprotein (Hiraoka et al.,1980) t Author to whom all correspondence should be addressed at: Department of Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, England. $ Present address: Advisory Board for the Research Councils, Elizabeth House, York Road, London SE1 7PH. England. $ Abbreviations used: a-lac, a-lactalbumin; GTase, galactosyltransferase; HEWL, hen-egg-white lysozyme. 571 0022 -%836/91/180571-11
$03.00/O
0
1991 Academic
Press
Limited
57”
K. R. Acharya
Alignment
et al.
Table 1 oJ”amino acid sequences for human and baboon a-lacs and hen-egg-white
lysozymr
Human cr-lac Baboon cc-lac HEWL
6 13 LYS-GLN-PHE-THR-LYS-CYS-GLU-LEU-SER-GLN-LEU-LEU-LYS---------ASPLYS-GLN-PHE-THR-LYS-CYS-GLU-LEU-SER-GLN-~-LEU-~---------ASPLYS-Val-PHE-Gly-Arg-CYS-GLU-LEU-Ala-Ala-Ala-Met-LYS-Arg-His-Gly12
Human a-lac Baboon or-lac HEWL
-ILE-ASP-GLY-TYR-GLY-GLY-ILE-ALA-LEU-PRO-GLU-LEU-ILE-CYS-THR-~T-ILE-ASP-GLY-TYR-GLY-Arg-ILE-ALA-LEU-PRO-GLU-LEU-ILE-CYS-THR-~T-Leu-ASP-Asn-TYR-Arg-GLY-Tyr-Ser-LEU-Gly-Asn-Trp-V~l-CYS-Al~-Ala-
20
28
14
30
28 40
Human @-lac Baboon or-lac HEWL
44
-PHE-HIS-THR-SER-GLY-TYR-ASP-THR-GLN-ALA-ILE-VAL-GLU-ASN-----PHE-HIS-THR-SER-GLY-TYR-ASP-THR-GLN-ALA-ILE-VAL-GLU-ASN-----Lys-Phe-Glu-SER-Asn-Phe-Asn-THR-GLN-ALA-Thr-Asn-Arg-ASN-Thr35
Human a-lac Baboon cl-lac HEWL
39 47 50 -ASN-GLU-SER-THR-GLU-TYR-GLY-LEU-PHE-GLN-ILE-SER-ASN-LYS-LEU-TRP-ASN-GLU-SER-THR-GLU-TYR-GLY-LEU-PHE-GLN-ILE-SER-ASN-Ala-LEU-TRP-Asp-Gly-SER-THR-Asp-TYR-GLY-Ile-Leu-GLN-ILE-Asn-Ser-~-Trp-TRP52
62
61
Human cl-lac Baboon ci-lac HEWL
Human Ct-lac Baboon a-lac HEWL
73
-CYS-LYS-SER-SER-GLN-VAL-PRO-GLN-SER-ARG-ASN-ILE-CYS-ASP-ILE-SER-CYS-LYS-SER-SER-GLN-Ser-PRO-GLN-SER-~G-ASN-ILE-CYS-ASP-ILE-Thr-CYS-Asn-Asp-Gly-Arg-Thr-PRO-Gly-SER-ARG 69 77 9: -CYS-ASP-LYS-PHE-LEU-ASP-ASP-ASP-ILE-THR-ASP-ASP-ILE-~T-CYS-~A-CYS-ASP-LYS-PHE-LEU-ASP-ASP-ASP-ILE-THR-ASP-ASP-ILE-~T-CYS-ALA-CYS-Ser-Ala-Leu-LEU-Ser-Ser-ASP-ILE-THR-Ala-Ser-Val-Asn-CYS-ALA85 96
Human cr-lac Baboon a-lac HEWL
60
79
93 100
103 107 -LYS-LYS-ILE-LEU----- ASP-ILE-LYS-GLY-ILE-ASP-TYR-TRP-LEU-ALA-HIS-LYS-LYS-ILE-LEU-----ASP-ILE-LYS-GLY-ILE-ASP-TYR-TRP-Ile-ALA-HIS-LYS-LYS-ILE-Val-Ser-ASP-Gly-Asp-GLY-Met-Asn-Ala-TRP-~-ALA-Trp100 107 111 111
117
Human cr-lac Baboon a-lac HEWL
-LYS-ALA-LEU-CYS-THR-GLU-LYS-LEU-GLU-GLN-----TRP-LEU---------CYS-LYS-ALA-LEU-CYS-THR-GLU-LYS-LEU-GLU-GLN-----TRP-LEU---------CYS-Arg-Asn-Arg-CYS-Lys-Gly-Thr-Asp-Val-GLN-Ala-TRP-Ile-Arg-Gly-CYS120 125
Human m-lac Baboon a-lac HEWL
-GLU-LYS-LEU -GLU-LYS-Glu -----Arg-E
120
123
129
The residue numbrrs are marked at the top and at the bottom of’ t.he sequrnc~ for c&w and hrn-egg-white Iysozyrnc~ H E1rI.. respectivrly. --- represents a deletion in the sequenvr used to maximize squenw idrntity. Invariant) residues arc marked in capital letters. The sequence differences between human and baboon cc-law are underlinrd. Human a-lar (Hall it ~1.. 1982): baboon cc-lat. S-rag srquencr (Acharya rt nl.. 1989): H E!&~L ((‘antirId. 1963: .lollbs PI u.. I!~Ki: (‘anfield & Liu, 1965: Imoto rt ccl.. 1972; Ibrahimi pf nl.. 1979). The sequence identities after maximizing the alignment aw: human y-la:! (88) 0 (80) 0 (38) N (Fi%) 0 (84) 0 (82) ODI (84) ODl (88) N(106) N(10.5)
OHZ(1’4)
OHi(l24)
OH9( 12.5)
Int ., internal: Cal., calcium
Comment
Water
Vontacts
Int. water
OHS(131)
N (33) 0 (50) ODI (88) OD2 (88) 0 (80) 0 (38) N (52) 0 (84) 0 (82) ODl (82) ODl (88) N( 106) 001 (33)
(R = 752AZ) ht. water (B = 7.3 AZ)
OH9(134)
Int. water (:al. ligand (R = 7.9 AZ)
OHB(135)
Int. water (R = 3Fi.5AZ)
OH@ 1%)
(hmment ht. water (H = 122 192) ht. wat,cr (H = 1:S.oAZ)
ht. watt (‘al. ligalld (R = 16.1 .V) Int.. WLtPr (R = 19% A’)
576
K. II. Acharya
b Ld) Fig. 3.
et al.
Crystal Structure
of Human
a-Lactalbumin
577
at 1.7 d
Figure 3. Comparison of human (thick line) and baboon (thin line) cc-lacs around regions (a) 40 to 47; (b) C-terminal tail: (c) Met90 and (d) calcium binding loop in human u-lac (the additional water molecule in the vicinity of the calcium binding
site hydrogen-bonded
to Asp84
and Asp87
is also shown)
and (e) calcium
to 47 form part of a P-pleated sheet and are exposed to solvent but in the crystal they are also involved in contacts with a symmetry related protein molecule. In human a-lac, Glu46 has a different confor-
mation (Fig. 3(a)). As expected, from the baboon a-lac structure, the C-terminal arm of the human a-lac molecule is rather flexible and is different to the corresponding region in baboon u-lac (Fig. 3(b)). supporting the hypothesis that the C terminus in a-lac can adopt multiple conformations (Acharya et al., 1989). In general, the overall environments of the sidechains in the two structures are very similar even though some side-chains in human a-lac have different orientations from those of the corresponding side-chains in baboon a-lac (e.g. Glu46 and Asp83). These effects are due in part to different packing interactions in the two crystals and in part to the presence of multiple conformations (e.g. MetSO, Fig. 3(c)). In such cases, only the best conformer was taken into account during the refinement of the structure and the other conformer was crudely modelled with solvent molecules. The need for more satisfactory treatments has been indicated by Kuriyan et aZ. (1986) and Gros et al. (1990).
The calcium
(b) Calcium
Lys79 Asp82 Asp84 Asp87 Asp88
Group Carbonyl Csrboxylate Carbonyl Carboxylate Carboxylate Water Water Calcium ion
0 ODl 0 ODl ODl 0 0 Cal
site in baboon
a-lat.
binding
a-lac is a calcium binding protein and possesses a novel calcium binding fold (Stuart et al., 1986; Acharya et al., 1989). The atomic structures around the calcium ion, in baboon a-lac and human a-lac superimpose well (Fig. 3(d) and (e)). The co-ordination around the calcium (Table 4) is a slightly disorted pentagonal hipyramid. The Ca-0 distances vary from 2.24 to 2.42 8, corresponding to a tight calcium binding loop in the structure. The positioning of the Asp83 side-chain is different) in human a-lac and baboon a-lac, but this does not seem to affect the geometry of the calcium binding site (Fig. 3(d) and (e)). There is an “extra” water molecule in the vicinity of the calcium binding site in human a-lac, hydrogen bonded to Asp84 (2.76 8) and Asp87 (2.91 8) (Fig. 3(d)). The structure of human a-lac confirms the previous observation in the baboon a-lac structure that the calcium binding fold only superficially resembles the “EF-hand” structure and presumably has no evolutionary relationship with other EF-hand structures (Stuart et al., 1986; Acharya et al., 1989). The biological role of calcium in a-lac is not yet
Table 4 ligands in human
and baboon a-lacs
Distance to Ca (A) Residues
binding
B-value (A’)
Human
Baboon
Human
Baboon
2.3 2.4 2.2 2.4 2.4 2.3 2.5
2.2 2.4 23 23 2.3 2.4 2.6
%4 11.0 58 10.4 7.3 191 7.9 62
139 11-7 126 7.9 l&8 16.1 21.0 11.5
-
578
K. R. Acharya et al.
7t i-
1
I
I
I
I
25
50
15
100
1
--~125
Residue numbrr (a)
62.50-
z I t 0
37*50
i @ 25.00-
I2.50-
I 0
I 25
I 50
I 100
I 75 Residue
I 125
number (0)
Figure 4. R-value plots + . human a-lac; *? baboon
for (a) the main-chain a-lat.
atoms
clear. though some evidence suggests that Ca2+ is necessary for a-lar to modify or modulate lactose synthesis (Musci & Berliner, 1985). Accordingly, cr-lac might act as a calcium modulated protein (Thompson rt al.. 1988). Recently Rao clr Brew (1989) proposed that t,ha a-lac calcium binding site has the in ~,izrofunction of imposing calcium regulation on the folding of the nascent cc-lac molecule and thereby on lactose synthesis. Kuwajima (1989) and Kuwajima rt al. (1989) propose that the calcium binding loop in a-lac may be a unique critical substructure in its folding. It is the best’ defined part. of the st,ruct,ure in the X-ray structures (as judged
and (b) the side-chain
atoms
in human
and baboon
r-law.
by the B-fac$ors; Table -l) irl both human and baboon a-lacs. Studies on the molben plobule state of guinea pig sr-lac by nuclear magnetic resonan(*e methods (Saum d trl., 19X9) concluded that thy calcium binding loop is a highly structured part of the molecule even in this partly unfolded form. a-lac can bind a variety of metal ions such as Ca2 + , Zn 2+. Ch2+, Clo2+, A13+ and Mn2+ and t,here have been a number of suggestions (Hiraoka rt nl., 1980; Murakami rt al., 1982: Rratcher & Kronman. 1984; Desmet & van (:auwelaert, 1988) that t)hrre is a Zn2+ binding site in cc-lac that is distinc+ from the calcium binding site. the occupation of’ whic.h
Crystal Structure
of Human
a-Lactalbumin
579
at 1.7 d
Table 5 Differences
in inter-molecular packing contacts ( < 34 A ) due to sequence diflerences between human and baboon a-lacs
Residue Human a-lac
Baboon a-lac
(lomment
Position
Leu
Asn
11
LYs
Tyr
13
GIY Lys
Arg Ala
20 58
Val Ser Leu Leu
Ser Thr Ile GIU
66 76 105 123
0 (11) is involved in water mediated intermolecular contacts. In baboon a-lac Am11 was more exposed to the solvent No contacts. In baboon a-lac the Tyr (OH) atom is hydrogen-bonded to Ala109 (0) No contacts NZ (58) is involved in symmetry related intermolecular contacts with the neighbouring Glu(46) side-chain atoms and is a very exposed residue. Glu(46) has a different side-chain orientation in baboon a-lac No contacts OG (76) is involved in water mediated contacts No contacts Disordered in both structures. Different conformations due to the non-rigidity of the C terminus. No inter-molecular contacts
causes the molecule to adopt an “ape-like” conformation. However, the present study provides no evidence of a second metal binding site. The cleft region, which divides the molecule into two wings in a-lac is very similar in the two structures. (c) Mobility
Figure 4(a) and (b) shows the distribution of atomic B-values (which describe the sum of the mobility and static disorder for each atom) in human and baboon a-lacs. From the plots it is clear that baboon a-lac has somewhat higher B-values for most of the atoms in comparison with human a-lac (mean B-value for all main-chain atoms of 25.5 A2 compared to 19.5 A’). A further difference is in the crystallographic refinement strategies used (baboon a-lac: PROLSQ (Konnert & Hendrickson, 1980), human a-lac: X-PLOR (Briinger, 1988, 1989; Kriinger et al., 1989)). However, since the target residual minimized in each refinement is identical that is unlikely to be a serious effect. This is likely to be due, in part,, to the radiation damage suffered during the intensity data collection for baboon a-lac crystals on the linear diffractometer and perhaps, to some extent, to undetected systematic error in the data (e.g. absorption errors). Given the well known difficulties in defining absolute B-values (Stuart & Phillips, 1986), we attach more significance to the fact that the relative mobilities are very similar for the two structures; the correlation coefficient between main-chain atom B-factors (cs,t) is 084. We have also investigated the relationship discrepancy between mobility and positional between the two molecules. The correlation coeffi-
Cj(Bj,l-(B,))(Bj.*-(B,)) + cBB
=
rgj(Bj,,
-
(B1))2Zj(Bj,
2-
@*))2]1’2
cient (&,,,S) for these two quantities has a very high value, 982; thus the regions of the molecules that differ are those of high mobility in each structure and there is no indication of any rearrangement of rigid segments of the molecule between the two structures. As mentioned before, the C terminus is mobile in human a-lac and has high B-values and, in general, it is true that the a-lac molecule has greater mobility than hen-egg-white lysozyme (HEWL) (Acharya et al., 1989). The conformational flexibility of the C terminus in cr-lac might indicate that it has a role in stabilizing the interaction of a-lac with GTase upon formation of the lactose synthetase complex, which is of important functional significance, and does not occur with C-type lysozymes. Further experiments are required to resolve this point. (d) Sequence
changes and crystal packing
The packing of the molecules in the crystals of baboon and human a-lac is similar. However, there are some water mediated interactions in the human a-lac structure that appear to be facilitated partly by the apparent amino acid differences between the two species (Table 5). Also, the differences in the side-chain orientations (mainly Glu46 and Asp83) provide additional intermolecular contacts for the stabilization of the crystal packing in human cc-lac (Table 5).
t
CB,= ~j[(Bj.l-(Bl))+(Bj,2-(B2))]X(~j-(U))
Fj(CBj,
1
-CBl))+
(Bj,2-(B2>))2
X
Zj(Dj-(D>)2]“2’
where Dj = Xbaboonj- Xhumanj;Xj is the position of atom j (after superimposition of human a-lac on to baboon a-lac); B values are isotropic temperature factors and the sum Ej, represents over all main-chain atoms.
K.
580
R. Acharya et al.
We thank Dr Richard Lyster for the generous gift of human a-lactalbumin. We thank Drs David Barford and Vasanta Subramanian for helpful discussions. We also thank Drs Helen Handoll and Diana Grace for refining the st’ructure of hen-egg-white lysozyme. The work was supported by the MRC and SERC.
Findlay. J. B. (‘. bi Brew, Ii. (1972). The complete amino Eur. J. acid sequence of human cc-la&albumin. Biochem. 27, 65-86. Fitzgerald, P. M. D. (1988), MERLOT. an integrated package of computer programs for the determination of crystal structures by molecular replacement. J. Appl. (‘rystallogr. 21. 274--278.
References
&OS. P.. van Gunsteren. W. F. & Hoi, MT. (:. .I. (1990). Inclusion of thermal motion in crystallographit structures by restrained molecular dynamics. Sciprlrp. 249. 1149-l 152. Hall. L.. (‘raig. R. Ii.. Edbrooke, AM.R. & (.!ampbrll. 1’. S. (1982). Comparison of the nucleotide sequence of cloned human and guinea pig pre-a-la&albumin cDN&4 with that, of chick pre-lysozyme cDNA suggests evolution from a common ancesteral gene. Nr~cl. Acids Res. 10, 3503-3515. Hamano, M.. Nitta, Ii.. Kuwajima. K. & Sugai. S. (1986). Binding strength of calcium ion to bovine r-lactal100, 1617-1622. bumin. .I. Biochem. (Tokyo).
Acharya, K. R., Stuart, D. I.. Walker. 5. P. C., Lewis, M. & Phillips, D. C. (1989). Refined structure of baboon a-lactalbumin at 1.7 A resolution. J. Mol. Biol. 208, 99-127. Baum. J.. Dobson, C’. M.. Evans, I’. A. & Hanley. (.!. (1989). Characterization of a partly folded protein by NMR methods: Studies on the molten globule state of Biochemistry, 28, 7-13. guinea pig u-lactalbumin. Berliner, L. J. & Johnson. J. D. (1988). Metal ion binding to cc-lactalbumin. In Calcium Binding ProteinsBiological Functions (Thompson, M. P., ed.), vol. 2. pp. 79-l 16, CRC Press, Boca Raton. U.S.A. Bernstein, F. C., Koetzle, T. F., Williams, G. J. B., Meyer, E. F., Jr, Brice, M. D., Rodgers, J. R., Kennard, 0.. Shimanouchi, T. & Tasumi, M. (1977). The protein data bank: a computer-based archival file for macromolecular structures. J. Mol. Biol. 112, 535-542. Bratcher, S. C. 8: Kronman, M. .J. (1984). Inter and intramolecular interactions of cc-lactalbumin XIV. Metal ion binding to N and A conformers of bovine cr-lactalbumin. J. Biol. Chem. 259, 1087-1095. Brew, K., Castellino, F. ,J., Vanaman, T. C. & Hill, R. L. (1970). The complete amino acid sequence of bovine J. Biol. Chem. 245. 4570-4582. a-lactalbumin. Briinger, A. T. (1988). Crystallographic refinement by simulated annealing. Application to 2.8 a resolution J. Mol. Biol. structure of aspartate aminotransferase. 203, 803-816. Briinger, A. T. (1989). A memory-efficient fast Fourier transformation algorithm for crystallographic refinement on supercomputers. Acta Crystal&r. sect. il. 45, 42-50. Briinger. A. T.. Kuriyan, J. & Karplus. M. (1987). Crystallographic R-factor refinement by molecular dynamics. Science, 235, 458-460. Briinger, A. T., Karplus, M. & Petsko. C. A. (1989). Crystallographic refinement by simulated annealing: application to crambin. Acta Crystallogr. sect. A, 45. 50-61. Canfield, R. E. (1963). The amino acid sequence of egg white lysozyme. J. Biol. Chem. 238, 2698-2707. Canfield, R. E. & Liu. A. K. (1965). The disulfide bonds of egg white lysozyme (muramidase). J. Biol. Chem. 240, 1997-2002. Crowther, R. A. (1972). The fast rotation function. In The Molecular Replacement Method (Rossmann, M. G.. ed.). pp. 174-178, Gordon & Breach, New York. Crowther, R. A. & Blow, D. M. (1967). A method for positioning a known molecule in an unknown crystal structure. Acta Crystallogr. 23, 544-548. Dandekar, A. M. t Qasba, P. K. (1981). Rat a-lactalbumin has a 17-residue-long COOH-terminal hydrophobic extension as judged by sequence analysis of the cDNA clones. Proc. Nat. Acad. Sci., U.S.A. 78, 4853-4857. Desmet. J. & van Canwelaert, F. (1988). Calorimetric experiments of Mn2+ binding to a-lactalbumin. Biochem. Biophys. Acta. 957, 411-419. Fenna, R. E. (1982). Crystallisation of human a-lactalbumin. J. Mol. Biol. 161, 211-215.
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Crystal Structure of Human
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