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The identification by X-ray crystallography of the site of attachment of an affinity label to hen egg-white lysozyme
J. Mol. Biol. (1973) ‘75, 14
The Identification by X-ray Crystallography of the Site of Attachment of an Affinity Label to Hen Egg-white Lysozyme JOHN MOULT, YUVAL ESHDAT AND NATHAN SHARON
Department of Biophysics The Weixmann Inditute of Xcience Rehovoth, Israel (Received 8 September 1972) Tetragonal crystals of hen egg-white lysozyme were treated with the active sitedirected irreversible inhibitor 2’,3’-epoxypropyl /3-glycoside of N-acetyl-n-glucosamine, ,8(l-+4)-linked dimer. The crystals were examined by X-ray crystallography, and the results compared to those obtained from crystals of the reversible complex formed between hen egg-white lysozyme and the b-phenyl glyooside of GlcNAc /3(1-+4)GloNAc~. It is concluded that the GlcNAc ,8(1-+4)GlcNAc moiety of the irreversible inhibitor occupies subsites I3 and C in the active site of the enzyme, and that the inhibitor is linked covalently to the enzyme through the carboxyl side-chain of Asp 52.
1. Introduction knowledge of the three-dimensional structure of hen egg-white lysozyme (Phillips, 1967; Blake et al., 1967a), and its substrate specificity (for review, see Chipman & Sharon, 1969), a series of specific and irreversible inhibitors (affinity labels) of the enzyme were designed (Thomas et al., 1969). These inhibitors are the 2’,3’epoxypropyl ,&glycosides of N-acetyl-lo-glucosamine, and of its @(l-+4)-linked oligomers (GlcNAc),t and (GlcNAc),. Studies with the 2’,3’-epoxypropyl j?-glycoside of (GlcNAc), ((GleNA&-Ep, Pig. 1, I) h ave led to the isolation from the irreversibly inhibited enzyme (GlcNAc),-Pr-lysozyme of a single peptide to which the (GlcNAc), moiety was attached via an ester linkage (McKelvy et al., 1970). The isolated peptide corresponded in composition to residues 46 to 53 of the sequence of hen egg-white lysozyme (Canfield, 1963b), and contained two possible sites of attachment of the affinity label, aspartic acid 48 and 52. (It has been suggested that Asp 48 is asparagine (Jolles et al., 1963; Hermann et al., 1971).) Examination of the three-dimensional model of lysozyme (Phillips, 1967; Blake et al., 1967a) shows Asp 52 to be favourably situated for reaction with the epoxide moiety on the ability label, whilst Asp 48 is some 10 A away. Thus it is expected that the site of covalent attachment of the inhibitor to the enzyme is the free carboxyl group of Asp 52. We describe here the preparation and crystallographic examination of tetragonal crystals of lysozyme which have been irreversibly inhibited by (GlcNAc),Ep, to establish the site of attachment unambiguously. Inactivation was carried out From
t Abbreviations used: GlcNAc, N-aoetyl-D-glucosami; (GTcNAc)~-E~, 2’,3’-epoxypropyl @&coside of di-(N-acetyl-D-glucosamine); (GlcNAc),-Pr-lysozyme, hen egg-white lysozyme irreversibly inactivated by (GIcNAo),-Ep. 1
1
J. MOULT,
Y.
ESHDAT
AND
N.
SHARON
CH2 OH
CH20H
NHCOCH3
NHCOCH3
FIG. 1. Chemical formula of the 2’,3’-epoxypropyl fi-glycoside of (GIcNAo)~ (I) used for affluity labelling of hen egg-white lysozyrne, and of the j3-phenyl glycoside of (GloNAc), (II).
directly on the crystals, since our preliminary experiments showed that the enzyme can be irreversibly inhibited in the crystalline state, and under the conditions used for crystallization.
2. Materials and Methods Tetragonal crystals of hen egg-white lysozyme (Alderton & Fevold, 1946) were grown at pH 4.7 in 0.02 M-acetate buffer, containing 5% NaCl. Individual crystals (space group P43212, a = b = ‘79.1 A, c = 37.9 A), about 1 mm in longest dimension, were prepared for irreversible inhibition by mounting in 1.5mm diameter quartz capillaries, with 3 cm by rubber tube attached by wax to one end. The ends of the capillary and rubber tube were sealed with wax. This arrangement facilitates the repeated sealing and unsealing of the tube necessary to prevent evaporation during the reaction and the subsequent washings. Before sealing, excess of mother liquor was removed from the tubes with a syringe and filter paper, and replaced by 60 ~1 of mother liquor containing (GIcNAc)~-Ep (60 pmol/ ml). The crystals were allowed to soak for 5 days at room temperature, during which they became badly cracked and in some cases disintegrated. At the end of this period one of the crystals was removed from the tube, wiped with filter paper and dissolved in water, the of u.v. absorption, $$ = 265 concentration being adjusted to 0.1 oh (b ased on measurement (Canfleld, 1963a)). The eneymic activity was measured using .iWicrococcz~s Zysodeii%ticus cells (Shugar, 1952) and compared to that of an unmodified crystal when brought into solution. The crystal which had been treated with the inhibitor exhibited only 20% of the activity of the unmodified enzyme. A portion of the solution of the crystals inhibited by (GlcNAc)s-Ep was subjected to exhaustive dialysis in order to remove any traces of noncovalently bound inhibitor. After lyophilisation, the material was hydrolysed in 6 M-HCl at 110°C for 22 h under vacuum. Analysis of the acid hydrolysate on an amino acid analyzer revealed the presence of 1.67 residues of glucosamine per molecule of the lysozyme. This value is in good agreement with the extent of inactivation of the dissolved crystal as determined from measurement of its enzymic activity. For the X-ray work, the modified crystals were washed 4 times with approximately O-1 ml of mother liquor (which did not contain the (GlcNAc)s-Ep) to remove excess of inhibitor, allowing at least one day for each washing. The rubber tube was then removed and the capillary sealed and transferred to a Buerger precession camera. Precession photographs with p = 18” (minimum spacing 2.5 A) were taken of the hk0, Ok1 and hhl zones. These zones are oentrosymmetric for this space group (P4,2,2). Photographs were taken with 3 6lms in a pack and exposures of 20 to 30 h, using a copper X-ray tube running at 40 kV, 28 mA with a nickel filter. No crystal was exposed for more than 50 h. The crystals were isomorphous with those of native lysozyme and showed only small changes in intensities. The complete diffraction patterns were recorded on magnetic tape using an Optronics photoscanner, and the integrated intensities of the reflections, corrected for background, were obtained by processing on an IBM 3701165 computer. Intensities within a pack were scaled together, corrected for Lorentz and polarization factors and scaled to those of the native crystal (Phillips, personal communication). Difference Fourier projections were calculated using as amplitudes the differenoes between the amplitudes of native lysozyme and of (GlcNAc)s-Pr-lysozyme, weighted with the native figures of merit, and native phases. For comparison, the same difference projections were calculated using the
-----pi---
-
,
:
INHIBITOR
ATTACHMENT
SITE
IN
LYSOZYME
3
structure factors extracted from the three-dimensional data of the reversible complex formed between lysozyme and the /%phenyl glycoside of (G~cNAc)~ (Fig. 1, II). The threedimensional structure of this complex is known (Moult, 19’70).
3. Results and Discussion Figure 2 shows the difference maps for the (GlcNAc),-Pr-lysozyme and the complex of lysozyme with the P-phenyl glycoside of (GlcNAc),. Comparison of these maps shows the same sets of peaks representing the known binding site for a disaccharide (Blake et al., 19673). In both sets of maps (I and II), the hk0 projections show two peaks, A and B, related by the 4-fold crystallographic axis, representing one binding one expects to find non-crystallosite per molecule. In the O.&l and hhl projections graphically related peaks at the same y and cl co-ordinates as in the hk0 projection, with a difference of $ in their x co-ordinates. The y co-ordinates of 0.32 for A and 0.43 for B (in the hk0 projection of (GlcNAc),-Pr-lysozyme, Fig. 2, I), correspond to 0.33 and 0.44 in the Olcl projection with z co-ordinates of 0.28 and 054. The d co-ordinates of 0.76 for A and 0.38 for B, compare with 0.74 and O-38 in the hhl projection, with z co-ordinates of 0.25 and 0.47. The 0.25 z co-ordinate is, however, poorly determined as its peak lies close to the mirror plane. The difference of 0.07 fractional units in the x co-ordinate (corresponding to a difference of 2.7 A) obtained from the Ok1 and hhl projections is considerably smaller than the size of the disaccharide (GlcNAc), viewed in this projection in the three-dimensional lysozyme-saccharide model. The corresponding co-ordinates in the different projections of the complex between lysozyme and the /Lphenyl glycoside of (GlcNAc), (Fig. 2, II) are essentially identical with those given above for (GlcNAc),-Pr-lysozyme. The maps of both crystals show other peaks, some of which are of the same order of magnitude. The presence of these peaks is to be expectejd since the three-dimensional map of the complex of lysozyme with the /Lphenyl glycoside of (GlcNAc), (Moult, 1970) contains a number of small features, some of which may be identifiable as minor conformational changes (
4
J. MOULT,
Y. ESHDAT
AND
N. SHARON
This work was supported in part by grant no. GM-19143 from the National Health, United States Public Health Service.
Institutes
of
REFERENCES Alderton, G. & Fevold, J. (1946). J. Biol. Chem. 164, 1. Blake, C. C. F., Mair, G. A., North, A. C. T., Phillips, D. C. & Sarma, V. R. (1967a). Proc. Roy. Sot. B,167, 365. Blake, C. C. F., Johnson, L. N., Mair, G. A., North, A. C. T., Phillips, D. C. & Sarma, V. R. (1967b). Proc. Roy. Sot. B,167, 378. Catield, R. E. (1963a). J. Biol. Chem. 238, 2691. Carmeld, R. E. (19633). J. BioE. Chem. 238, 2698. Chipman, D. M. & Sharon, N. (1969). Science, 165, 454. Hermann, J., Jo&%, J. & Jolles, P. (1971). Ezcrop. J. Biochem. 24, 12. Jolles, J., Jauregui-Adell, J., Bernier, I. & Jol%s, P. (1963). Biochim. biophys. Acta, 78, 668. Maron, E., Eshdat, Y. & Sharon, N. (1972). Biochim. biophys. Acta, 278, 243. McKelvy, J. F., Eshdat, Y. & Sharon, N. (1970). Israel J. Chem. 8, 170~. Moult, J. (1970). D. Phil. thesis University of Oxford. Phillips, D. C. (1967). Proc. Nat. Acad. Sci., Wash. 57, 484. Shugar, D. (1962). Biochim. biophys. Acta, 8, 302. Thomas, E. W., McKelvy, J. F. & Sharon, N. (1969). Nature, 222, 485.
The Identification by X-ray Crystallography of the Site of Attachment of an Affinity Label to Hen Egg-white Lysozyme JOHN MOULT, YUVAL ESHDAT AND NATHAN SHARON
Department of Biophysics The Weixmann Inditute of Xcience Rehovoth, Israel (Received 8 September 1972) Tetragonal crystals of hen egg-white lysozyme were treated with the active sitedirected irreversible inhibitor 2’,3’-epoxypropyl /3-glycoside of N-acetyl-n-glucosamine, ,8(l-+4)-linked dimer. The crystals were examined by X-ray crystallography, and the results compared to those obtained from crystals of the reversible complex formed between hen egg-white lysozyme and the b-phenyl glyooside of GlcNAc /3(1-+4)GloNAc~. It is concluded that the GlcNAc ,8(1-+4)GlcNAc moiety of the irreversible inhibitor occupies subsites I3 and C in the active site of the enzyme, and that the inhibitor is linked covalently to the enzyme through the carboxyl side-chain of Asp 52.
1. Introduction knowledge of the three-dimensional structure of hen egg-white lysozyme (Phillips, 1967; Blake et al., 1967a), and its substrate specificity (for review, see Chipman & Sharon, 1969), a series of specific and irreversible inhibitors (affinity labels) of the enzyme were designed (Thomas et al., 1969). These inhibitors are the 2’,3’epoxypropyl ,&glycosides of N-acetyl-lo-glucosamine, and of its @(l-+4)-linked oligomers (GlcNAc),t and (GlcNAc),. Studies with the 2’,3’-epoxypropyl j?-glycoside of (GlcNAc), ((GleNA&-Ep, Pig. 1, I) h ave led to the isolation from the irreversibly inhibited enzyme (GlcNAc),-Pr-lysozyme of a single peptide to which the (GlcNAc), moiety was attached via an ester linkage (McKelvy et al., 1970). The isolated peptide corresponded in composition to residues 46 to 53 of the sequence of hen egg-white lysozyme (Canfield, 1963b), and contained two possible sites of attachment of the affinity label, aspartic acid 48 and 52. (It has been suggested that Asp 48 is asparagine (Jolles et al., 1963; Hermann et al., 1971).) Examination of the three-dimensional model of lysozyme (Phillips, 1967; Blake et al., 1967a) shows Asp 52 to be favourably situated for reaction with the epoxide moiety on the ability label, whilst Asp 48 is some 10 A away. Thus it is expected that the site of covalent attachment of the inhibitor to the enzyme is the free carboxyl group of Asp 52. We describe here the preparation and crystallographic examination of tetragonal crystals of lysozyme which have been irreversibly inhibited by (GlcNAc),Ep, to establish the site of attachment unambiguously. Inactivation was carried out From
t Abbreviations used: GlcNAc, N-aoetyl-D-glucosami; (GTcNAc)~-E~, 2’,3’-epoxypropyl @&coside of di-(N-acetyl-D-glucosamine); (GlcNAc),-Pr-lysozyme, hen egg-white lysozyme irreversibly inactivated by (GIcNAo),-Ep. 1
1
J. MOULT,
Y.
ESHDAT
AND
N.
SHARON
CH2 OH
CH20H
NHCOCH3
NHCOCH3
FIG. 1. Chemical formula of the 2’,3’-epoxypropyl fi-glycoside of (GIcNAo)~ (I) used for affluity labelling of hen egg-white lysozyrne, and of the j3-phenyl glycoside of (GloNAc), (II).
directly on the crystals, since our preliminary experiments showed that the enzyme can be irreversibly inhibited in the crystalline state, and under the conditions used for crystallization.
2. Materials and Methods Tetragonal crystals of hen egg-white lysozyme (Alderton & Fevold, 1946) were grown at pH 4.7 in 0.02 M-acetate buffer, containing 5% NaCl. Individual crystals (space group P43212, a = b = ‘79.1 A, c = 37.9 A), about 1 mm in longest dimension, were prepared for irreversible inhibition by mounting in 1.5mm diameter quartz capillaries, with 3 cm by rubber tube attached by wax to one end. The ends of the capillary and rubber tube were sealed with wax. This arrangement facilitates the repeated sealing and unsealing of the tube necessary to prevent evaporation during the reaction and the subsequent washings. Before sealing, excess of mother liquor was removed from the tubes with a syringe and filter paper, and replaced by 60 ~1 of mother liquor containing (GIcNAc)~-Ep (60 pmol/ ml). The crystals were allowed to soak for 5 days at room temperature, during which they became badly cracked and in some cases disintegrated. At the end of this period one of the crystals was removed from the tube, wiped with filter paper and dissolved in water, the of u.v. absorption, $$ = 265 concentration being adjusted to 0.1 oh (b ased on measurement (Canfleld, 1963a)). The eneymic activity was measured using .iWicrococcz~s Zysodeii%ticus cells (Shugar, 1952) and compared to that of an unmodified crystal when brought into solution. The crystal which had been treated with the inhibitor exhibited only 20% of the activity of the unmodified enzyme. A portion of the solution of the crystals inhibited by (GlcNAc)s-Ep was subjected to exhaustive dialysis in order to remove any traces of noncovalently bound inhibitor. After lyophilisation, the material was hydrolysed in 6 M-HCl at 110°C for 22 h under vacuum. Analysis of the acid hydrolysate on an amino acid analyzer revealed the presence of 1.67 residues of glucosamine per molecule of the lysozyme. This value is in good agreement with the extent of inactivation of the dissolved crystal as determined from measurement of its enzymic activity. For the X-ray work, the modified crystals were washed 4 times with approximately O-1 ml of mother liquor (which did not contain the (GlcNAc)s-Ep) to remove excess of inhibitor, allowing at least one day for each washing. The rubber tube was then removed and the capillary sealed and transferred to a Buerger precession camera. Precession photographs with p = 18” (minimum spacing 2.5 A) were taken of the hk0, Ok1 and hhl zones. These zones are oentrosymmetric for this space group (P4,2,2). Photographs were taken with 3 6lms in a pack and exposures of 20 to 30 h, using a copper X-ray tube running at 40 kV, 28 mA with a nickel filter. No crystal was exposed for more than 50 h. The crystals were isomorphous with those of native lysozyme and showed only small changes in intensities. The complete diffraction patterns were recorded on magnetic tape using an Optronics photoscanner, and the integrated intensities of the reflections, corrected for background, were obtained by processing on an IBM 3701165 computer. Intensities within a pack were scaled together, corrected for Lorentz and polarization factors and scaled to those of the native crystal (Phillips, personal communication). Difference Fourier projections were calculated using as amplitudes the differenoes between the amplitudes of native lysozyme and of (GlcNAc)s-Pr-lysozyme, weighted with the native figures of merit, and native phases. For comparison, the same difference projections were calculated using the
-----pi---
-
,
:
INHIBITOR
ATTACHMENT
SITE
IN
LYSOZYME
3
structure factors extracted from the three-dimensional data of the reversible complex formed between lysozyme and the /%phenyl glycoside of (G~cNAc)~ (Fig. 1, II). The threedimensional structure of this complex is known (Moult, 19’70).
3. Results and Discussion Figure 2 shows the difference maps for the (GlcNAc),-Pr-lysozyme and the complex of lysozyme with the P-phenyl glycoside of (GlcNAc),. Comparison of these maps shows the same sets of peaks representing the known binding site for a disaccharide (Blake et al., 19673). In both sets of maps (I and II), the hk0 projections show two peaks, A and B, related by the 4-fold crystallographic axis, representing one binding one expects to find non-crystallosite per molecule. In the O.&l and hhl projections graphically related peaks at the same y and cl co-ordinates as in the hk0 projection, with a difference of $ in their x co-ordinates. The y co-ordinates of 0.32 for A and 0.43 for B (in the hk0 projection of (GlcNAc),-Pr-lysozyme, Fig. 2, I), correspond to 0.33 and 0.44 in the Olcl projection with z co-ordinates of 0.28 and 054. The d co-ordinates of 0.76 for A and 0.38 for B, compare with 0.74 and O-38 in the hhl projection, with z co-ordinates of 0.25 and 0.47. The 0.25 z co-ordinate is, however, poorly determined as its peak lies close to the mirror plane. The difference of 0.07 fractional units in the x co-ordinate (corresponding to a difference of 2.7 A) obtained from the Ok1 and hhl projections is considerably smaller than the size of the disaccharide (GlcNAc), viewed in this projection in the three-dimensional lysozyme-saccharide model. The corresponding co-ordinates in the different projections of the complex between lysozyme and the /Lphenyl glycoside of (GlcNAc), (Fig. 2, II) are essentially identical with those given above for (GlcNAc),-Pr-lysozyme. The maps of both crystals show other peaks, some of which are of the same order of magnitude. The presence of these peaks is to be expectejd since the three-dimensional map of the complex of lysozyme with the /Lphenyl glycoside of (GlcNAc), (Moult, 1970) contains a number of small features, some of which may be identifiable as minor conformational changes (
4
J. MOULT,
Y. ESHDAT
AND
N. SHARON
This work was supported in part by grant no. GM-19143 from the National Health, United States Public Health Service.
Institutes
of
REFERENCES Alderton, G. & Fevold, J. (1946). J. Biol. Chem. 164, 1. Blake, C. C. F., Mair, G. A., North, A. C. T., Phillips, D. C. & Sarma, V. R. (1967a). Proc. Roy. Sot. B,167, 365. Blake, C. C. F., Johnson, L. N., Mair, G. A., North, A. C. T., Phillips, D. C. & Sarma, V. R. (1967b). Proc. Roy. Sot. B,167, 378. Catield, R. E. (1963a). J. Biol. Chem. 238, 2691. Carmeld, R. E. (19633). J. BioE. Chem. 238, 2698. Chipman, D. M. & Sharon, N. (1969). Science, 165, 454. Hermann, J., Jo&%, J. & Jolles, P. (1971). Ezcrop. J. Biochem. 24, 12. Jolles, J., Jauregui-Adell, J., Bernier, I. & Jol%s, P. (1963). Biochim. biophys. Acta, 78, 668. Maron, E., Eshdat, Y. & Sharon, N. (1972). Biochim. biophys. Acta, 278, 243. McKelvy, J. F., Eshdat, Y. & Sharon, N. (1970). Israel J. Chem. 8, 170~. Moult, J. (1970). D. Phil. thesis University of Oxford. Phillips, D. C. (1967). Proc. Nat. Acad. Sci., Wash. 57, 484. Shugar, D. (1962). Biochim. biophys. Acta, 8, 302. Thomas, E. W., McKelvy, J. F. & Sharon, N. (1969). Nature, 222, 485.