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Different segmental flexibility of human serum transferrin and lactoferrin
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 275, No. 1, November 15, pp. 181-12341989
Different Segmental Flexibility of Human Serum Transferrin R. VfGH,* *Institute
L. CSER,t F. KILAR,*
AND
and Lactoferrin
I. SIMON*,l
of Enzymology, Biological Research Center, Hungarian Academy of Sciences, H-1502, P.O. Box 7, Budapest, Hungary, and tCentra1 Research Institute for Physics, Hungarian Academy of Sciences, H-1525, P.O. Bw 49, Budapest, Hungary Received May 2,1989, and in revised form July 6,1989
X-ray diffraction studies show that the diferric (holo) forms of human serum transferrin and lactoferrin have almost the same conformation in crystal. In solution, however, the two proteins exhibit different characteristics. The differences are even more pronounced in the apo forms. Small-angle X-ray and neutron scattering data show that lactoferrin is less compact, in apo and holo forms, than the corresponding forms of transferrin in solution. The comparison of primary structures of the two proteins suggests that one of the interdomain hinge regions is significantly longer in lactoferrin than its counterpart in transferrin. The difference in flexibility due to the long hinge region in lactoferrin may be responsible for many of the differences in the physicochemical charaCteriPtiCS Of the tW0 proteins. o 1989 Academic Press, k.
Human serum transferrin and lactoferrin are iron-binding glycoproteins (M, 80,000) (1). Although their physiological roles are completely different, viz, transferrin mediates iron exchange between tissues while lactoferrin kills bacteria by removing iron, their structures are very similar. There is an almost 60% homology in their amino acid sequences (2) and recent X-ray diffraction studies on their holo, or differic, forms revealed practically the same structure (3, 4). Both proteins contain an N-terminal lobe and a C-terminal lobe as a result of a gene duplication. There are two domain boundaries in each lobe. In differic forms not only their functions but many of their physicochemical parameters in solution are quite different, in contrast to their similarities in crystal form. In apo forms the differences are even more pronounced (5,6). We have studied the small-angle X-ray and neutron scattering of apo- and holotransferrin and apo- and hololactoferrin
1 To whom correspondence
and made an attempt to explain the observed differences between the parameters characteristic of the solution conformation of transferrin and lactoferrin by comparing their sequence data. MATERIALS
AND
METHODS
Lactoferrin was a generous gift of Dr. K. Nemeth, Institute of Haematology and Blood Transfusion, while transferrin, from pooled human sera, was obtained from Boehring-Werke AG (Marburg) and was used without further purification. The chelating agent nitrilotriacetic acid (NTA)’ was a Fluka (Buchs) product, while all other reagents were Reanal (Budapest) products of reagent grade. Differic transferrin and lactoferrin were prepared by addition of stoichiometric amounts of Fe(NTA)$to the apo proteins. The homogeneity and purity of the samples were checked by urea gel electrophoresis according to the method of Makey and Seal (7). All samples used in the scattering experiments were in 10 mM Tris-HCl buffer, pH 7.4. Small-angle X-ray scattering experiments were carried out on a Dron-1 type X-ray analytical instru-
a Abbreviation
should be addressed. 181
used: NTA, nitrilotriacetic
acid.
0003-9861/89 $3.00 Copyright All rights
0 1989 by Academic Press, Inc. of reproduction in any form reserved.
182
ViGH ET AL.
I
0.1
I 1
I
-
2 (2~~~10‘~rad21
FIG. 1. Guinier plots of small-angle X-ray scattering pattern of apolactoferrin (Lf), hololactoferrin (Lf-Fez), apotransferrin (Tf), and holotransferrin (Tf-Fez). I, intensity of scattered X-ray; 28,scattering angle. Protein concentration is 25 mg/ml. Inset, concentration dependence of the apparent radii of gyration.
ment (USSR) combined with a Shimadzu (Japan) small-angle camera. Cu K, X-ray (X = 0.154 nm) was used, the Cu K, line was removed by a Ni filter and the continuous part of the spectrum was eliminated by a differential discriminator. The collimation correction, based on Lake’s iteration procedure, was performed as reported earlier (8). Small-angle neutron scattering experiments were carried out on a time-of-flight small angle scattering device at the IBR-2 pulsed reactor of the Joint Institute for Nuclear Research in Dubna, USSR (9). Radii of gyration were determined from the slope of the Guinier plots (lo), i.e., log I vs (28) (2) in the range of 5-13 mrad scattering angles in the X-ray
TABLE I RADII OF GYRATION OF APO- AND HOLOTRANSFERRIN AND APO- AND HOLOLACTOFERRIN
Rgbm)
Transferrin Lactoferrin
X-ray Neutron X-ray Neutron
APO
Ho10
3.30 * 0.03 3.32 + 0.05 3.64 f 0.03 3.73 20.05
3.15 + 0.03 3.22 -+0.05 3.40 rt 0.03 3.55 k 0.05
scattering experiments and In I vs &a in the range of 0.2-0.8 nm-’ scattering vector in the neutron scattering experiments. The apparent R8 values were determined in the range of lo-30 mg/ml protein concentration and extrapolated to zero concentration. The size of the interdomain “hinge” region was estimated from the amino acid sequences around the variable parts in the primary structure reported as domain boundaries in the X-ray diffraction studies (3,4x RESULTS AND DISCUSSION
The radii of gyration of apo- and holotransferrin and apo- and hololactoferrin are determined from the Guinier plots of Figs. 1 and 2 and listed in Table I. In our preceding paper the effect of iron binding on the conformation of transferrin was studied by means of small-angle X-ray scattering (11). The observed decrease of the radius of gyration upon stepwise iron saturation of the transferrin was explained as a result of the alteration of the relative positions of the two lobes. In the present study we observed about the same decrease of Rg of transferrin upon iron saturation by means of neutron scattering.
HUMAN
SERUM
TRANSFERRIN
005
0.1
AND
183
LACTOFERRIN
Ln I t
0
Q*[nm-*I
FIG. 2. Guinier plots of small-angle neutron scattering pattern of apolactoferrin (Lf), hololactoferrin (Lf-Fez), apotransferrin (Tf), and holotransferrin (Tf-Fez). I, intensity of scattered neutrons; Q, scattering vector. Protein concentration is 25 mg/ml. Inset, concentration dependence of the apparent radii of gyration.
The change in Rg is even more pronounced for lactoferrin. The Rg values of lactoferrin are significantly greater than the respective values of transferrin. Even for the diferric forms, where X-ray diffraction shows very similar structures, the differences are 8-10%. The scattering data indicate that in solution hololactoferrin is less compact than holotransferrin, the apo forms are less compact than the corresponding holo forms, and the difference between the conformations in apo forms is greater than that in holo forms. A macromolecule with five interdomain hinge regions is quite flexible and may occur in an equilibrium of many conformers. Ligand binding, as shown in Ref. (ll), is one of the factors which can shift this equilibrium. Intermolecular interactions during crystallization may be another. Our small-angle scattering data indicate that at least one of the two proteins (lactoferrin), appears to be in a more compact structure in the crystal state than its average structure in solution. To find the origin of different compactness in solution we focused our attention
on the interdomain hinge regions of these proteins. There are various procedures to localize domains in the 3D structure of proteins (12), but none of them gives the exact start and termination of the domain. Therefore, it is still not easy to identify precisely the size of the interdomain hinge regions from the X-ray data alone. However, in general, consonant regions of homologous proteins belong to the structural domains, while the variable segments around the positions reported as domain boundaries correspond to the interdomain hinge region.
TABLE SUGGESTED
Transferrin Lactoferrin Transferrin Lactoferrin Transferrin Lactoferrin
II
SHORT AND LONG INTERDOMAIN HINGE REGIONS
86-94 84-92
245-248 248-251
328-343 332-345 419-426 582-585 433-446 605-608
Note. Long regions are underlined.
N lobe Between lobes C lobe
134
VfGH
By comparing the sequence data of Ref. (2) to the X-ray data of Refs. (3) and (4), we suggest the interdomain regions listed in Table II. The data indicate that the C-terminal lobe of lactoferrin may contain a long hinge region (14 residues) whereas the corresponding hinge region in transferrin is short (8 residues). The difference in flexibility due to the long hinge region in the C-terminal lobe of lactoferrin may be responsible for many of the differences in the physicochemical characteristics of the two proteins. ACKNOWLEDGMENT This work was supported by Grant OTKA 318 from the Hungarian Academy of Sciences. REFERENCES 1. AISEN, P., AND LISTOWSKY, I. (1980) Annu. Rev.
Biochem 49,357-393. 2. METZ-BOUTIGLJE, M. H., JOLL~S J., MAZURIER, J., SCHOENTGEN, F., LEGRAND, D., SPIK, G., MONTREUIL, J., AND JOLL&, P. (1984) Eur. J. Biothem 145,659-676.
ET AL. 3. ANDERSON, B. F., BAKER, H. M., DODSON, E. J., NORRIS, G. E., RUMBALL, S. V., WATERS, J. M., AND BAKER, E. N. (1987) Prop. NatL Acad Sci.
USA 84,1’769-1773. 4. BAILEY, S., EVANS, R. W., GARRATT, R. C., GORINSKY, B., HASNAIN, S., HORSBURGH, C., JHOTI, H., LINDLEY, P. F., MYDIN, A., SARRA, R., AND WATSON, J. L. (1988) Biochemistry 27, 58045812. 5. AISEN, P., AND LIEBMAN, A. (1972) Biochiwz. Bab
phys. Acta 257,314-322. 6. HARRINGTON, J. P., STUART, J., AND JONES, A. (1987) Int. J B&hem. lO, lOOl-1008. 7. MAKEY, D. G., AND SEAL, U. S. (1976) Biochim. Biophys. Acta 453,250-256. 8. KILAR, F., SIMON, I., LAKATOS, S., VONDERVISZT, F., MEDGYESI, G. A., AND ZAVODSZKY, P. (1985) Eur. J. Biochem 147,17-25. 9. VAGOV, V. A., KUNCHENKO, A. B., OSTANEVICH, Yu. M., AND SALAMATTIN, I. M. (1983) Joint Institute for Nuclear Research Report P14-831 898, Dubna, USSR. [In Russian] 10. GUINIER, A., FOURNET, G. (1955) Small Angle Scattering of X-Rays, Wiley, New York. 11. KIL~R, F., AND SIMON, I. (1985) Biophys. .I 48,799802. 12. JANIN, J., AND WODAK, S. J. (1983) Prog. Biophys.
Mol. BioL 42.21-78.
Different Segmental Flexibility of Human Serum Transferrin R. VfGH,* *Institute
L. CSER,t F. KILAR,*
AND
and Lactoferrin
I. SIMON*,l
of Enzymology, Biological Research Center, Hungarian Academy of Sciences, H-1502, P.O. Box 7, Budapest, Hungary, and tCentra1 Research Institute for Physics, Hungarian Academy of Sciences, H-1525, P.O. Bw 49, Budapest, Hungary Received May 2,1989, and in revised form July 6,1989
X-ray diffraction studies show that the diferric (holo) forms of human serum transferrin and lactoferrin have almost the same conformation in crystal. In solution, however, the two proteins exhibit different characteristics. The differences are even more pronounced in the apo forms. Small-angle X-ray and neutron scattering data show that lactoferrin is less compact, in apo and holo forms, than the corresponding forms of transferrin in solution. The comparison of primary structures of the two proteins suggests that one of the interdomain hinge regions is significantly longer in lactoferrin than its counterpart in transferrin. The difference in flexibility due to the long hinge region in lactoferrin may be responsible for many of the differences in the physicochemical charaCteriPtiCS Of the tW0 proteins. o 1989 Academic Press, k.
Human serum transferrin and lactoferrin are iron-binding glycoproteins (M, 80,000) (1). Although their physiological roles are completely different, viz, transferrin mediates iron exchange between tissues while lactoferrin kills bacteria by removing iron, their structures are very similar. There is an almost 60% homology in their amino acid sequences (2) and recent X-ray diffraction studies on their holo, or differic, forms revealed practically the same structure (3, 4). Both proteins contain an N-terminal lobe and a C-terminal lobe as a result of a gene duplication. There are two domain boundaries in each lobe. In differic forms not only their functions but many of their physicochemical parameters in solution are quite different, in contrast to their similarities in crystal form. In apo forms the differences are even more pronounced (5,6). We have studied the small-angle X-ray and neutron scattering of apo- and holotransferrin and apo- and hololactoferrin
1 To whom correspondence
and made an attempt to explain the observed differences between the parameters characteristic of the solution conformation of transferrin and lactoferrin by comparing their sequence data. MATERIALS
AND
METHODS
Lactoferrin was a generous gift of Dr. K. Nemeth, Institute of Haematology and Blood Transfusion, while transferrin, from pooled human sera, was obtained from Boehring-Werke AG (Marburg) and was used without further purification. The chelating agent nitrilotriacetic acid (NTA)’ was a Fluka (Buchs) product, while all other reagents were Reanal (Budapest) products of reagent grade. Differic transferrin and lactoferrin were prepared by addition of stoichiometric amounts of Fe(NTA)$to the apo proteins. The homogeneity and purity of the samples were checked by urea gel electrophoresis according to the method of Makey and Seal (7). All samples used in the scattering experiments were in 10 mM Tris-HCl buffer, pH 7.4. Small-angle X-ray scattering experiments were carried out on a Dron-1 type X-ray analytical instru-
a Abbreviation
should be addressed. 181
used: NTA, nitrilotriacetic
acid.
0003-9861/89 $3.00 Copyright All rights
0 1989 by Academic Press, Inc. of reproduction in any form reserved.
182
ViGH ET AL.
I
0.1
I 1
I
-
2 (2~~~10‘~rad21
FIG. 1. Guinier plots of small-angle X-ray scattering pattern of apolactoferrin (Lf), hololactoferrin (Lf-Fez), apotransferrin (Tf), and holotransferrin (Tf-Fez). I, intensity of scattered X-ray; 28,scattering angle. Protein concentration is 25 mg/ml. Inset, concentration dependence of the apparent radii of gyration.
ment (USSR) combined with a Shimadzu (Japan) small-angle camera. Cu K, X-ray (X = 0.154 nm) was used, the Cu K, line was removed by a Ni filter and the continuous part of the spectrum was eliminated by a differential discriminator. The collimation correction, based on Lake’s iteration procedure, was performed as reported earlier (8). Small-angle neutron scattering experiments were carried out on a time-of-flight small angle scattering device at the IBR-2 pulsed reactor of the Joint Institute for Nuclear Research in Dubna, USSR (9). Radii of gyration were determined from the slope of the Guinier plots (lo), i.e., log I vs (28) (2) in the range of 5-13 mrad scattering angles in the X-ray
TABLE I RADII OF GYRATION OF APO- AND HOLOTRANSFERRIN AND APO- AND HOLOLACTOFERRIN
Rgbm)
Transferrin Lactoferrin
X-ray Neutron X-ray Neutron
APO
Ho10
3.30 * 0.03 3.32 + 0.05 3.64 f 0.03 3.73 20.05
3.15 + 0.03 3.22 -+0.05 3.40 rt 0.03 3.55 k 0.05
scattering experiments and In I vs &a in the range of 0.2-0.8 nm-’ scattering vector in the neutron scattering experiments. The apparent R8 values were determined in the range of lo-30 mg/ml protein concentration and extrapolated to zero concentration. The size of the interdomain “hinge” region was estimated from the amino acid sequences around the variable parts in the primary structure reported as domain boundaries in the X-ray diffraction studies (3,4x RESULTS AND DISCUSSION
The radii of gyration of apo- and holotransferrin and apo- and hololactoferrin are determined from the Guinier plots of Figs. 1 and 2 and listed in Table I. In our preceding paper the effect of iron binding on the conformation of transferrin was studied by means of small-angle X-ray scattering (11). The observed decrease of the radius of gyration upon stepwise iron saturation of the transferrin was explained as a result of the alteration of the relative positions of the two lobes. In the present study we observed about the same decrease of Rg of transferrin upon iron saturation by means of neutron scattering.
HUMAN
SERUM
TRANSFERRIN
005
0.1
AND
183
LACTOFERRIN
Ln I t
0
Q*[nm-*I
FIG. 2. Guinier plots of small-angle neutron scattering pattern of apolactoferrin (Lf), hololactoferrin (Lf-Fez), apotransferrin (Tf), and holotransferrin (Tf-Fez). I, intensity of scattered neutrons; Q, scattering vector. Protein concentration is 25 mg/ml. Inset, concentration dependence of the apparent radii of gyration.
The change in Rg is even more pronounced for lactoferrin. The Rg values of lactoferrin are significantly greater than the respective values of transferrin. Even for the diferric forms, where X-ray diffraction shows very similar structures, the differences are 8-10%. The scattering data indicate that in solution hololactoferrin is less compact than holotransferrin, the apo forms are less compact than the corresponding holo forms, and the difference between the conformations in apo forms is greater than that in holo forms. A macromolecule with five interdomain hinge regions is quite flexible and may occur in an equilibrium of many conformers. Ligand binding, as shown in Ref. (ll), is one of the factors which can shift this equilibrium. Intermolecular interactions during crystallization may be another. Our small-angle scattering data indicate that at least one of the two proteins (lactoferrin), appears to be in a more compact structure in the crystal state than its average structure in solution. To find the origin of different compactness in solution we focused our attention
on the interdomain hinge regions of these proteins. There are various procedures to localize domains in the 3D structure of proteins (12), but none of them gives the exact start and termination of the domain. Therefore, it is still not easy to identify precisely the size of the interdomain hinge regions from the X-ray data alone. However, in general, consonant regions of homologous proteins belong to the structural domains, while the variable segments around the positions reported as domain boundaries correspond to the interdomain hinge region.
TABLE SUGGESTED
Transferrin Lactoferrin Transferrin Lactoferrin Transferrin Lactoferrin
II
SHORT AND LONG INTERDOMAIN HINGE REGIONS
86-94 84-92
245-248 248-251
328-343 332-345 419-426 582-585 433-446 605-608
Note. Long regions are underlined.
N lobe Between lobes C lobe
134
VfGH
By comparing the sequence data of Ref. (2) to the X-ray data of Refs. (3) and (4), we suggest the interdomain regions listed in Table II. The data indicate that the C-terminal lobe of lactoferrin may contain a long hinge region (14 residues) whereas the corresponding hinge region in transferrin is short (8 residues). The difference in flexibility due to the long hinge region in the C-terminal lobe of lactoferrin may be responsible for many of the differences in the physicochemical characteristics of the two proteins. ACKNOWLEDGMENT This work was supported by Grant OTKA 318 from the Hungarian Academy of Sciences. REFERENCES 1. AISEN, P., AND LISTOWSKY, I. (1980) Annu. Rev.
Biochem 49,357-393. 2. METZ-BOUTIGLJE, M. H., JOLL~S J., MAZURIER, J., SCHOENTGEN, F., LEGRAND, D., SPIK, G., MONTREUIL, J., AND JOLL&, P. (1984) Eur. J. Biothem 145,659-676.
ET AL. 3. ANDERSON, B. F., BAKER, H. M., DODSON, E. J., NORRIS, G. E., RUMBALL, S. V., WATERS, J. M., AND BAKER, E. N. (1987) Prop. NatL Acad Sci.
USA 84,1’769-1773. 4. BAILEY, S., EVANS, R. W., GARRATT, R. C., GORINSKY, B., HASNAIN, S., HORSBURGH, C., JHOTI, H., LINDLEY, P. F., MYDIN, A., SARRA, R., AND WATSON, J. L. (1988) Biochemistry 27, 58045812. 5. AISEN, P., AND LIEBMAN, A. (1972) Biochiwz. Bab
phys. Acta 257,314-322. 6. HARRINGTON, J. P., STUART, J., AND JONES, A. (1987) Int. J B&hem. lO, lOOl-1008. 7. MAKEY, D. G., AND SEAL, U. S. (1976) Biochim. Biophys. Acta 453,250-256. 8. KILAR, F., SIMON, I., LAKATOS, S., VONDERVISZT, F., MEDGYESI, G. A., AND ZAVODSZKY, P. (1985) Eur. J. Biochem 147,17-25. 9. VAGOV, V. A., KUNCHENKO, A. B., OSTANEVICH, Yu. M., AND SALAMATTIN, I. M. (1983) Joint Institute for Nuclear Research Report P14-831 898, Dubna, USSR. [In Russian] 10. GUINIER, A., FOURNET, G. (1955) Small Angle Scattering of X-Rays, Wiley, New York. 11. KIL~R, F., AND SIMON, I. (1985) Biophys. .I 48,799802. 12. JANIN, J., AND WODAK, S. J. (1983) Prog. Biophys.
Mol. BioL 42.21-78.