Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Miami, Florida, FL 33101, USA and 2 Department of Biology and Biochemistry, University of Bath, Bath, UK
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Abstract |
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Keywords: flexibility/-lactalbumin/lactose synthase/lysozyme/protein structure/thermal stability
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Introduction |
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The evolutionary divergence of LA from an ancestral LZ has been accompanied by changes in stability and folding/unfolding behavior that may result from its adaptation to a new function. -Lactalbumin is less stable than LZ and has a native structure that is strongly dependent on a tightly bound Ca2+ ion (Hiroaka et al., 1980
; Kronman, 1989
; Rao and Brew, 1989
; Ewbank and Creighton, 1993a
,b
). Certain substructures of LA assume different conformations in various crystal forms and metal complexes (Acharya et al. 1989
, 1991
; Harata and Muraki, 1992
; Ren et al., 1993
; Pike et al., 1996
).
-Lactalbumin also adopts a stable molten globule (MG) conformation at room temperature, on removal of the structural Ca2+ ion, at low pH or under other mildly destabilizing conditions (Kronman, 1989
; Kuwajima, 1989
); a third conformer, that is intermediate between the native and MG states (pre-MG state), is stable in the pH 3.74.1 range (Lala and Kaul, 1992
; Gussakovsky and Haas, 1995
). The tendency of LA to assume alternative conformations and stable partially denatured states suggests that folding and unfolding processes are less cooperative than in most proteins, possibly reflecting the lower stability of the native structure. The similarity of the LA MG state to an early intermediate in the folding pathway has stimulated many studies of its structure and properties and LA has become a model protein for numerous studies of protein folding (Kuwajima, 1989
; Alexandrescu et al., 1993
; Uchiyama et al., 1995
; Balbach et al., 1997
; Schulman et al., 1997
; Wu and Kim, 1997
; Pfeil, 1998
; Song et al., 1998
; Wu and Kim, 1998
; and references therein). The observation that an aggregated missfolded form of human LA causes apoptosis in tumor but not in normal cells (Håkansson et al., 1995; Svensson et al., 1999
) suggests that partially folded states of LA may be biologically significant.
The structures of LAs and LZs are divided into two lobes by a cleft, a larger -helical lobe composed of residues 134 and 86123 and a smaller lobe (residues 3585) containing a small antiparallel ß-sheet and a disulfide loop (Figure 1
). The Ca2+-binding site is located at the junction of the lobes (Acharya et al., 1989
) and, in LZ, the cleft is the binding site for the extended oligosaccharide substrate. In LA, Tyr103 replaces Ala or Pro in different LZ lines and extends the contacts between the lobes by interacting with Trp60 of the ß-lobe and Trp104 of the
-lobe to block one end of the cleft (Acharya et al., 1989
). The shortened cleft is compatible with the activity of LA that utilizes part of the lower cleft and an adjacent surface (aromatic cluster I) of the helical lobe (Grobler et al., 1994a
; Malinovskii et al., 1996
).
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mLA has been previously used in nuclear magnetic resonance spectroscopic studies of folding (Balbach et al., 1996) and its crystallographic structure has been reported (Pike et al., 1996
). An N-terminal methionyl extension is present in mLA that is absent from bovine LA but is necessary for intracellular expression in Escherichia coli. In a similar form of recombinant bovine LA, the additional residue has been found to reduce thermal stability (Ishikawa et al., 1998
) but detailed functional and structural studies show that it does not affect structure or activity (Grobler et al., 1994a
; Pike et al., 1996
). The present observations suggest that the lower stability of LA results, in part, from enhanced local flexibility of regions surrounding the functional site of LA including unique substitutions that are required for activity.
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Materials and methods |
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Reagents and kits for molecular biology and reagents for enzyme assays were from the same sources as in previous studies (Grobler et al. 1994a; Malinovskii et al., 1996
). Bovine milk LA was purchased from Sigma Chemical Corp. and goat milk LA was purified as described previously (MacGillivray et al., 1979
).
Mutagenesis, protein expression, folding and purification
Mutants of mLA were constructed and expressed as described previously (Grobler et al., 1994a). Proteins were expressed in E.coli strain BL21(DE3) as inclusion bodies, extracted, partially purified in the denatured state, folded and finally purified by gel filtration and ion exchange chromatography in the absence of denaturants and presence of 0.1 mM CaCl2. The procedures are as described by Malinovskii et al. (1996).
CD spectroscopy and thermal equilibrium unfolding
Near and far UV CD spectra for bLA and mLA variants and thermal unfolding profiles were measured with a JASCO 720 spectropolarimeter fitted with a Neslab RTE-110 water bath that can be programmed for constant temperature or to produce designed temperature gradients. For the acquisition of spectra, generally 20 scans were collected at a rate of 100 nm/min and averaged and smoothed. Far UV spectra (200250 nm) were determined using a path length of 0.1 cm and near UV spectra with a path length of 1 cm. CD spectra were determined and thermal unfolding studies carried out with protein samples dissolved at a concentrations of 0.30.5 mg/ml in 20 mM TrisHCl pH 7.4 containing 1 mM CaCl2. Molten globule spectra were determined in 10 mM HCl.
The stability of LA is highly sensitive to the binding of Ca2+ to the primary calcium-binding site. Both bovine LA (Kronman et al., 1981; Aramini et al., 1992
) and human LA (Chandra et al., 1998
) have a second weaker Ca-binding site which could also influence stability. To saturate the primary structural binding site (Kd
0.2 µM; see Kronman et al., 1981
; Kronman, 1989
), but not the secondary site which has a Kd of approximately 3 mM (Aramini et al., 1992
), 1 mM CaCl2 was also added to the buffer used for stability measurements. For thermal unfolding the temperature was increased at a rate of less than 50°C/h and data were collected at 0.1 or 0.2°C intervals using a response time of 4 s. For all mutants, the change in ellipticity was monitored at 270 or 275 nm, but for a few, the change in ellipticity at 222 nm was also measured in a separate experiment.
Thermal unfolding data were analyzed by calculating native and unfolded state ellipticity at different temperatures by linear regression analysis of baseline data in temperature ranges below and above the unfolding transition. These were then used to calculate fractional unfolding across the transition range. A van't Hoff analysis of data between 15 and 85% unfolding was used to determine Tm, Hm and
Sm. Data were also analyzed by fitting to equations that include the temperature dependence of
Hm (
Cp). However, as pointed out previously (Shi and Kirsch, 1995), this produces little change in the value obtained for Tm.
Hm has the largest errors in all of these fits, being susceptible to errors in the baseline ellipticity for the native and unfolded states. Changes in Tm are the most reliable measurement of stability changes and were used to calculate the stability at the Tm of mLA using the relationship:
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Where GT is the free energy of unfolding at temperature T (Kelvin), Tm is the mid-point of the unfolding transition in K,
HTm is the enthalpy of unfolding at Tm and
Cp is the heat capacity change for unfolding. A value of 5.6 kJ/mol/K was used for the heat capacity change based on the relationship of Tm and
Hm for LA variants.
Other methods
Oligonucleotide synthesis and automated DNA sequencing were performed by Dr Rudolf Werner, Department of Biochemistry and Molecular Biology, University of Miami School of Medicine.
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Results |
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The recombinant form of bovine LA, mLA, which differs from bovine LA in having valine substituted for Met90 and in the N-terminal methionyl extension, was expressed, folded and purified as previously described (Grobler et al., 1994a; Malinovskii et al., 1996
). The N-terminal residue is designated Met-1 and the second residue, Glu1 so that the sequence numbering corresponds to that of bovine and other eutherian LAs (Grobler et al., 1994b
). mLA is closely similar in crystallographic structure to baboon, buffalo, guinea-pig and human LAs (Pike et al., 1996
). The mutants investigated here had been previously expressed as part of a study of structurefunction relationships in LA, and were characterized functionally by measuring two kinetic parameters that describe distinct facets of LA activity: KiLA, the equilibrium dissociation constant of LA from a galactosyltransferaseMn2+UDPgalactose complex, and Kmglc, the extrapolated Km for glucose at a saturating concentration of LA (see Grobler et al., 1994a
; Malinovskii et al., 1996
).
Figure 1 shows the sites of mutations which include residues of the hydrophobic box, a largely buried cluster of aromatic and aliphatic side chains, aromatic cluster I, a surface cluster with a high proportion of aromatic residues, and residues located in the molecular cleft. The hydrophobic box bridges the two lobes of LA, and aromatic cluster I is on the surface of the
-helical lobe. Mutants were designed principally using homology-based substitutions for residues that are unique to LA and structurally conservative substitutions at other sites.
Structures and thermal stability of LA variants
The near and far UV CD spectra of the mLA variants indicate that most mutations do not perturb the tertiary or secondary structure (Figure 2). The mutant Tyr103Ala, previously expressed by Grobler and co-workers (1994a) was found to have a molten globule-like structure, since it lacked a near-UV CD spectrum, but had a far-UV CD spectrum that indicated the presence of a high secondary structure content. In the present study, this mutant was purified by a different procedure, described by Malinovskii et al. (1996), to produce a preparation with a near-UV CD spectrum that has reduced ellipticity relative to the wild-type protein and a modified far-UV CD spectrum (Figure 2
). The form of this mutant characterized here appears to have been lost in the earlier study during separation by HPLC anion exchange chromatography. This suggests that this substitution perturbs the tertiary structure and also reduces native-state stability. In contrast, the Tyr103Pro mutant has spectra that are closely similar to the wild-type protein (Grobler et al., 1994a
). Analysis of the far-UV CD spectrum of the Tyr103Ala mutant suggests that the secondary structure content is similar to the wild-type protein, apart from a slight reduction in helix content.
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Structural effects of mutations
The effect of select mutations were modeled in the human LA structure (Acharya et al., 1991) using O (Jones et al., 1991
), conducting energy minimization and dynamics simulations using X-PLOR (Brunger, 1992
). The Tyr103Pro mutation was also modeled in the mLA structure (molecule A from the 1HFZ pdb file) using HyperChem. In the dynamics simulations, the water was removed and heating was simulated to 3000 or 4000 K followed by cooling to 300 K. Regions of the protein distant from the mutation site were constrained.
Molecular modeling of the Tyr103Pro mutant suggests that this substitution produces a more open cleft of the type found in LZ (Figure 1, insert). Ile101 is displaced by more than 11 Å, and Asp102 moves by 10 Å to within H-bonding distance of Lys99. The accessibility of Trp104 to solvent is also increased. The Leu110His mutation is predicted to alter the orientation of the side chains of Phe31 and His32, and the structure of residues 113118 including Lys114, whose side chain is displaced by 10 Å. The modeled Lys114Asn mutation suggests a local change that allows the formation of an H-bond between the Asn side chain and the adjacent peptide group. Crystallographic studies of these and other mLA mutants are currently in progress.
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Discussion |
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Several LZ-based substitutions introduced into mLA reduce stability (see Table I) suggesting that the environments and interactions of these residues are not conserved between these proteins. Tyr103 is the source of a structural difference between LA and LZ, since it provides additional tertiary structure interactions between the two lobes and blocks access to the upper cleft in LA. The substitution of Ala, which is present at this site in most lysozymes, produces a mutant with perturbed structure and low activity and stability, indicating that the interactions of Tyr103 have a major stabilizing effect on the native structure (~10 kJ/mol). In contrast, the Pro103 mutant is fully active, has normal structure and activity, and is only slightly less stable than the wild-type protein. Modeling indicates that the Pro103 mutant has an open cleft structure that can bind the extended oligosaccharide substrate in lysozyme (as expected since a group of lysozymes have proline at this site). It therefore appears that Pro103 stabilizes mLA by its demonstrated effect on the entropy of the unfolded protein (Matthews et al., 1987
) in contrast to Tyr which increases tertiary interactions between the two lobes. A lysozyme mutant containing the equivalent of Tyr103 has not been described but this substitution is expected to be incompatible with the bacteriolytic activity, since it blocks subsites A and B of the cleft and prevents binding of the natural substrate (Brown et al., 1969
). Searches of the dBEST database with BLAST reveal the presence of two human and one mouse testis-specific ESTs that encode homologues of LZ and LA, that have a tyrosine at the site corresponding to Tyr103 (Figure 5
). One human sequence has the catalytic Glu and Asp of LZ but the second has substitutions for both of these residues; the mouse sequence appears to be the orthologue of the latter. The properties of these proteins have not been determined but their sequences suggest that they represent new lines of the LA/LZ superfamily that have novel functions. These findings support the view that sequence variation at this site reflects the interplay of functional and structural requirements during natural selection. In the LZs the dominant selective effect is associated with the need for an open cleft for activity, whereas in the LAs and other homologues that do not require an open cleft, tyrosine is an acceptable alternative to proline for enhancing native state stability.
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In contrast, mutants with increased thermal stability have substitutions for residues in the adjacent more flexible regions of structure. Substitutions for Leu110 have large effects on LA activity yet two, Arg and His, increase stability while Glu is destabilizing. Substitutions at this site have similar effects on stability in LZ and LA. In human LZ, Arg115 corresponds to Leu110 of LA. A histidine substitution increases thermal stability (Shih and Kirsch, 1995), whereas a glutamate substitution produces a loss in activity and stability. The effects of the Glu substitution appear to reflect changes in the interaction of residue 115 with Trp34, that corresponds to His23 in LA (Harata et al., 1993
). One aspect of the role of Leu110 in LA involves its effects on the environment of His32, a residue that is the most crucial for LA function, and may include transmission of conformational changes in the flexible loop to His32 (Pike et al., 1996
). A nonpolar residue is not found at the site corresponding to Leu110 of LA in any LZ and the introduction of such a residue in LA appears to have been a key step in the functional divergence of LA from LZ that resulted in a reduced molecular stability. His32 and Leu110 affect the ability of LA at saturation to promote glucose binding to ß-GT1, indicating a direct role in activity, and it appears that these residues have been selected for activity but not for optimal protein stability (see Shoichet et al., 1995
).
The different LA crystallographic structures underscore the alternative conformations that can be assumed by regions that surround the active site, specifically the flexible loop (residues 105110) and residues 112119. Previous studies have shown that when bovine LA binds to GT, the reactivity of Lys114 to acylation by acetic anhydride is increased twofold and that of Lys5 is reduced threefold, but the other 10 lysines and the -amino group are not affected (Richardson and Brew, 1980
). Lys5 is adjacent to the LA functional site and its decreased reactivity is likely to result from reduced accessibility in the proteinprotein complex, but the increased reactivity of Lys114 is more readily explained by a change in conformation that reduces the pK of the
-amino group or increases its nucleophilicity. Together, the structural and chemical data suggest that enhanced local flexibility may be important for LA activity (Pike et al., 1996
).
The substitution of Asn for Lys114 generates a protein with a 40-fold lower affinity for galactosyltransferase and a Tm increased by more than 10°C, effects that can be reasonably expected to have a common basis. Lys114 is a solvent-exposed residue that, based on its high crystallographic B factor in different LA structures, has a high level of flexibility. The low activity of the Asn114 mutant, and unchanged activities of the Glu and Gln mutants, suggests that the Asn substitution has a significant structural effect. Additional side-chain to backbone interactions are suggested by modeling, that could generate a more fixed structure in this region. Reduced flexibility can explain the large increase in enthalpic stabilization in this mutant (about 50 kJ/mol at 56.2°C) which is partly compensated by increased entropy of unfolding. The net stabilizing effect reflects an increase in structural cooperativity produced by the reduced local flexibility (Creighton, 1983). Structural and biophysical studies of this mutant are in progress to test this proposal.
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Acknowledgments |
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Notes |
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References |
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Acharya,K.R., Stuart,D.I., Walker,N.P.C., Lewis,M. and Phillips,D.C. (1989) J. Mol. Biol., 208, 99127.[ISI][Medline]
Alexandrescu,A.T., Evans,P.A., Pitkeathly,M., Baum,J. and Dobson,C.M. (1993) Biochemistry, 32, 17071718.[ISI][Medline]
Anderson,P.J., Brooks,C.L. and Berliner,L.J. (1997) Biochemistry, 36, 1164811654.[ISI][Medline]
Aramini,J.M., Drakenberg,T., Hiraoki,T., Ke,Y., Nitta,K. and Vogel,H.J. (1992) Biochemistry, 31, 67616778.[ISI][Medline]
Balbach,J., Forge,V., Lau,W.S., van Nuland,N.A., Brew,K. and Dobson,C.M. (1996) Science, 274, 11611163.
Balbach,J., Forge,V., Lau,W.S., Jones,J.A., van Nuland,N.A. and Dobson,C.M. (1997) Proc. Natl. Acad. Sci. USA, 94, 71827185.
Brew,K. and Grobler,J.A. (1992) -Lactalbumin. In Fox,P. (ed.), Advanced Dairy Chemistry, Vol. 1. pp. 191229.
Brown,W.J., North,A.C.T., Phillips,D.C., Brew,K., Vanaman,T.C. and Hill,R.L. (1969) J. Mol. Biol., 42, 6586.[ISI][Medline]
Brunger,A.T. (1992) X-PLOR: Version 3.1. A system for Crystallography and NMR. Yale University Press, New Haven.
Chandra,N., Brew,K. and Acharya,K.R. (1998) Biochemistry, 37, 47674772.[ISI][Medline]
Chaudhuri,T.K. et al. (1999) J. Mol. Biol., 285, 11791194.[ISI][Medline]
Creighton,T.E. (1983) Biopolymers, 22, 4958.[ISI][Medline]
Ewbank,J.J. and Creighton,T.E. (1993a) Biochemistry, 32, 36773693.[ISI][Medline]
Ewbank,J.J. and Creighton,T.E. (1993b) Biochemistry, 32, 36943707.[ISI][Medline]
Grobler,J.A., Wang,M., Pike,A.C.W. and Brew,K. (1994a) J. Biol. Chem., 269, 51065114.
Grobler,J., Rao,K.R., Pervaiz,S. and Brew,K. (1994b) Arch. Biochem. Biophys., 313, 360366.[ISI][Medline]
Gussakovsky,E.E. and Haas,E. (1995) Protein Sci., 4, 23192326.
HåkanssonA., Zhitotovsky,B., Orrenius,S., Sabharwal,H. and Svanborg,C. (1995) Proc. Natl. Acad. Sci. USA, 92, 80648068.[Abstract]
Harata,K. and Muraki,M. (1992) J. Biol. Chem., 267, 14191421.
Harata,K., Muraki,M., and Jigami,Y. (1993) J. Mol. Biol., 233, 524535[ISI][Medline]
Hill,R.L. and Brew,K. (1975) Adv. Enzymol. Rel. Areas Mol. Biol., 43, 411489.[ISI][Medline]
Hiroaka,Y., Segawa,Y., Kuwajima,K., Sugai,S. and Murai,N. (1980) Biochem. Biophys. Res. Commun., 93, 10981104.
Ishikawa,N., Chiba,T., Chen,L.T., Shimizu,A., Ikeguchi,M. and Sugai,S. (1998) Protein Engng, 11, 333335.[Abstract]
Jones,T.A., Zou,J.Y., Cowan,S.W. and Kjeldgaard,M. (1991) Acta Crystallogr., A47, 110119.[ISI]
Kornegay,J.R., Schilling,J.W. and Wilson,A.C. (1994) Mol. Biol. Evol., 11, 921928.[Abstract]
Kronman,M.J. (1989) CRC Crit. Rev. Biochem. Mol. Biol., 24, 565667.
Kronman,M.J., Sinha,S.K. and Brew,K. (1981) J. Biol. Chem., 256, 85828587.
Kuwajima,K. (1989) Proteins, 6, 87103.[ISI][Medline]
Lala,A.K. and Kaul,P. (1992) J. Biol.Chem., 267, 1991419918.
MacGillivray,R.T.A., Barnes,K. and Brew,K. (1979) Arch. Biochem. Biophys., 197, 404414.[ISI][Medline]
Malinovskii,V.A., Tian,J., Grobler,J.A. and Brew,K. (1996) Biochemistry, 35, 97109715.[ISI][Medline]
Matthews,B.W., Nicholson,H. and Becktel,W.J. (1987) Proc. Natl. Acad. Sci. USA, 84, 66636667.[Abstract]
McKenzie,H.A. and White,F.H.,Jr. (1991) Adv. Protein Chem., 41, 173315.[ISI][Medline]
Pfeil,W. (1998) Proteins, 30, 4348.[ISI][Medline]
Pike,A.C.W., Brew,K. and Acharya,K.R. (1996) Structure, 4, 691703.[ISI][Medline]
Rao,K.R. and Brew,K. (1989) Biochem. Biophys. Res. Commun., 163, 13901396.[ISI][Medline]
Ren,J., Stuart,D.I. and Acharya,K.R. (1993) J. Biol. Chem., 268, 1929219298.
Richardson,R.H. and Brew,K. (1980) J. Biol. Chem., 255, 33773385.
Sarker,G. and Sommer,S.S. (1990) Biotechniques, 8, 404407.[ISI][Medline]
Schulman,B.A., Kim,P.S., Dobson,C.M., and Redfield,C. (1997) Nature Struct. Biol., 8, 630634.
Shaw,D.C., Messer,M., Scrivener,A.M., Nicholas,K.R. and Griffiths,M. (1993) Biochim. Biophys. Acta., 1161, 177186.[ISI][Medline]
Schulman,B.A., Kim,P.S., Dobson,C.M. and Redfield,C. (1997) Nature Struct. Biol., 4, 630634.[ISI][Medline]
Shih,P. and Kirsch,J.F. (1995) Protein Sci., 4, 20632072.
Shoichet,B.K., Baase,W.A., Kuroki,R. and Matthews,B.W. (1995) Proc. Natl. Acad. Sci. USA, 92, 452456.[Abstract]
Song,J., Bai,P., Luo,L. and Peng,Z.Y. (1998) J. Mol. Biol., 280, 167174.[ISI][Medline]
Svensson,M., Sabharwal,H., Håkansson,A., Mossberg,A-K., Lipniunas,P., Leffler,H., Svanborg,C. and Linse,S. (1999) J. Biol. Chem., 274, 63886396.
Uchiyama,H., Perez-Prat,E.M., Watanabe,K., Kumagai,I. and Kuwajima,K. (1995) Protein Engng, 8, 11531161.[Abstract]
Vanderheeren,G. and Hanssens,I. (1994) J. Biol. Chem., 269, 70907094.
Wu,L.C. and Kim,P.S. (1997) Proc. Natl. Acad. Sci. USA, 94, 1431414319.
Wu,L.C. and Kim,P.S. (1998) J. Mol. Biol., 280, 175182.[ISI][Medline]
Xie,D., Bhakuni,V. and Freire,E, (1991) Biochemistry, 30, 1067310678.[ISI][Medline]
Received January 22, 1999; revised March 23, 1999; accepted March 25, 1999.