Vol.
174,
January
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
31, 1991
COMMUNICATIONS Pages
898-902
A model for histone HSDNA interaction: Simultaneous minor and major groove binding Alain Segers, Vrije
Brussel, lnstituut voor Moleculaire Biologie, 1640 St. Genesius-Rode, Belgium
Universiteit
*Plant
Received
Genetic
Lode Wyns and lgnace Lasters*
Systems, Laboratories Brussels-ULB-UCMB-CP160, Heger, Batiment P2, 1050 Brussels, Belgium
November
29,
Paardenstraat Avenue
65, P.
1990
Using the tertiary structure of the globular domain of H5 (GH5) and based on an alternative sequence homology between GH5 and DNA-binding proteins containing the helix-turn-helix motif, a model for H5-DNA interaction is proposed. From molecular graphics it follows that helix II recognizes the major groove of the DNA, as does the second helix of the helix-turn-helix motif, while helix III makes minor groove contacts, in agreement with the hypothesis of Turnell et al. (FEBS letters 232, 263-268). In the resulting model GH5 makes contact with a full turn of DNA. 0 1'391 Academic Press, Inc.
The histones Hl and H5 bind to the nucleosomes, are involved in the formation of higher order structures of chromatin (1) and play an active role in the control of DNA replication and cell proliferation (2). However, the absence of a useful assay limits many investigations. Nevertheless, the amount of data being produced on the structure and function of the histones Hl and H5 is steadily growing (3). A breakthrough took place with the structure determination of the globular domain of H5 (GH5), using NMR-techniques (4). This will be complemented with a crystallographic analysis (5). Knowledge of the tertiary structure of GH5 induced the formulation of hypothetical binding models for GH5 to DNA (6, 7), some of them based on a possible structural homology between GH5 and the E. coli catabolite gene activator protein (CAP) (4, 8). As GH5 lacks a clear helix-turn-helix motif, the speculations of Clore et al. are mainly based on the location of the polar and charged residues in the structure. In doing so the anti-parallel helices II and III are predicted to be involved in DNA-contacts, while helix I, due to its hydrophobic nature, is presumed to make protein contacts. However, despite some structural similarities between GH5 and CAP, the sequence alignment derived from the model displays only poor homology. Even the homology between helix III of GH5 and helix F of CAP, although proposed to be structurally equivalent (4), is low. In contradiction with the results of Clore et al., the model of Mannermaa & Oikarinen
Vol.
174, No. 2, 1991
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
(8) assumes the presence of a helix-turn-helix motif in GH5, and suggests an involvement of the N-terminal part of helix III in the recognition of the major groove of the DNA. Finally, Turnell et al. (6) state that this helix III of GH5 would make minor groove contacts with the DNA. Although based on a significant sequence homology between histone Hl and pentraxins, their proposal is in conflict with both above explained models. Based on a new sequence alignment we present an alternative
structural
homology between GH5 and CAP. Helix II of GH.5 would be the functional equivalent of the C-terminal helix in the helix-turn-helix motif, present in many sequence-specific DNA-binding proteins (10). Positioning GH5 on a CAP-DNA model according to this homology is in excellent agreement with DNA-binding. In addition, the present alignment, unlike the others, is compatible with the proposal of Turnell et al., resulting in a DNA-binding mode characterized by simultaneous minor and major groove binding.
Experimental All graphical work was done on an Evans and Sutherland graphics station using the Brugel package (11). We used the coordinates (12) of the CAP-DNA model (13) obtained from the Protein Databank (14) at Brookhaven National Laboratory.
Results
and Discussion
Although GH5 does not contain the whole helix-turn-helix motif, we believe the region of GH5 comprising residues 22 to 35 to be similar to the region of the Fhelix of CAP (figure 1). This new alignment requires a shift by 18 residues with respect to the earlier proposed model of Clore et al.. In fact in our model helix II takes in the place occupied by helix III in the fit of Clore et al. (figure 2). Comparing the number of identities (4 in the present model, none in (4)), and neutral substitutions (4 versus 3), it is obvious that the new alignment is superior. In addition the present alignment in the region of helix II is in agreement with the hydrophobicity
pattern expected for a major groove binding helix (15). Also,
following the fingerprint pattern of White (16), the Gly 177 in CAP is expected to be conserved. Only our model is in agreement with this requirement. More
I
II
III
GH5
1
SASHPTYSEMIAAAIRAEKSRGGSSRQSIQKYIKSHYKVGHNADLQIKLSIRRLLAA * * * **
CAP
155
DAMTHPDGMQIKITRQEIGQIVGCSRETVGRILKMLEDQDLISAHGKTIWYGTVLG D
E
F
Fiaure 1. The proposed alignment of GH5 with CAP, based on a sequence homology with the DNA-binding region of CAP. Identities are indicated by an asterisk, alpha-helices are underlined boxes. The GH5 sequence is on top and starts at residue 1, CAP starts at 155.
Vol.
174, No. 2, 1991
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Fiaure 2. Comparison of two alignments of GH5 with CAP. The upper two sequences are aligned according to the new model. The GH5 sequence starts at residue 22 and represents the region of helix II, the CAP sequence starts at 176, representing the distal part of the DNA binding motif with helix F. Helix F is the major groove binding helix. The third sequence is that of GH5 starting at 40 and aligned according to (4). It represents the region of helix Ill. Bold typed residues are in helix structure, identities and neutral substitutions are indicated by.
support is obtained by subjecting the new alignment to the test developed by Dodd and Egan (17), which locates and statistically evaluates a potential DNAbinding region homologous to lambda cro-like proteins. Once again the new alignment agrees with an expected major groove DNA-binding helix (figure 3). We conclude that helix II of GH5 is a major groove binding helix comparable to helix F of CAP. A set of 10 C-alpha coordinates was constructed for GH5 containing Arg 26-Ser 35. The set for CAP contained Arg 180-Met 189. The resulting RMS distance between the C-alpha atoms of GH5 and CAP being 3.0 A for our fit is better than the one obtained with the fit of Clore et al., which gives 3.5 A. Because of the irregularities in the structure of helix II it is difficult to position helix I1 of GH5 on helix F of CAP using C-alpha coordinates. We preferred to use general features of the structure and decided to superimpose both helix axes. This rules out possible artifacts resulting from abberant helical parameters, since the helix axis parameter is the least affected by irregularities in the structure. Based on our assumption we positioned the GH5 structure on the model of a CAP-DNA complex. What are the consequences for the helices I and III ? CAP V G c S R E
+a1 +230 +94 +206 +I27 +104
T +225 v +121 G -34 R +159 I +102 L +68 K
+115 -___-_ +159a
‘A’ G
-106
G s S R Q
+230 -74 +206 +127 +62
S I
-2 +194
Q
+21
K
+115
Y
+140 +33 +115 ---___
I K
+1061
‘8 G H N
-106 +la -38 -20 -72 -126
A D L Q +91 I +194 K -67 L -126 s
I R
-74 +33 +127 ---_-_ -166
Fiaure 3. Comparison of the two alignments presented the new alignment, ‘B’ is the alignment found in (4). The described in (16). The higher the score the more likely the master set of proteins, all sharing the helix-turn-helix
900
in figure 2. ‘A’ indicates scores are calculated as it is to be homologous to motif.
Vol.
174, No. 2, 1991
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Fiaure 4. Stereo representation of the proposed model of DNA-binding of GH5 with a full turn of DNA, according to the new alignment. The insert displays the essential features. The axes of the alpha-helices are represented by thick lines and are labeled accordingly with roman numbers. The minor groove-binding arginines are also shown. The DNA backbone is at the left of the figure. The axis of helix I (N-terminus in the upper right corner) lies almost in the plane of the page. Helix II comes out of the page while helix Ill, being roughly antiparallel with helix II, runs into the page. The C-terminal part of helix III approaches the minor groove of the DNA very closely.
BRUGEL, we see, while fitting the axis of helix II of GH5 on the axis of helix F of CAP, that by rotating around this common
Using
the interactive
axis one can displace
graphics
package
helix I far from the DNA making
it unlikely
to be involved
in
DNA-contacts. As already pointed out in (4), due to its hydrophobic nature it is a candidate for making protein contacts. At the same time it is possible to position helix III so that its carboxyl-end
points to the minor groove of the DNA. Thus,
bringing Arg 52 and Arg 53 close to the DNA-duplex. These residues are thought to interact directly with the DNA minor groove, since they are geometrically well located (6). A preliminary study in which titration with GH5 displaces Hoechst 33258 bound to pUC18 supercoiled DNA, supports the idea that GH5 makes minor groove contacts (unpublished results). The resulting fit shows a GH5molecule with its two anti-parallel helices rougly perpendicular to the axis of the DNA duplex. Helix II lies in the major groove while helix III runs closely to the minor groove. From this follows that GH5 would make contacts with a full turn of DNA. The lysine 64 of GH5, which is strongly protected from chemical modification upon nucleosome-binding (18) is not involved in our proposed mode of binding, neither is it in any other model, since it lies outside the helices. However, because it is relatively far away from the DNA-binding site it is ideally located
to make
contacts
with
neighbouring
DNA-strands.
This
is in accordance
with the idea that GH5 has more than one binding site (7,19) (figure In the region
of helix II sequence
homology
is almost
completely
absent
4). between
the globular domains of histone Hl and H5 (7,20). This could explain the functional differencies between both types of linker histone. They could then bind non-specifically with their helix III to a narrow minor groove, while modulating the strength and sequence of binding (21,22) with the amino acid composition of helix II, and their putative adenine nucleotide binding site (23). 901
Vol.
174,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Acknowledqments We thank Dr. A. Gronenborn GH5. We thank the N.F.W.O.
for kindly providing us with the NMR-models A.S. is a fellow of the I.W.O.N.L.
References 1. 2. 43: 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
15.
Z: 18. 19. 2: 22. 23.
McGhee, J.B. and Felsenfeld, G. (1980) Annu.Rev.Biochem. 49, 1115-l 156. Sun, J-M., Wiaderkiewicz, R. and Ruiz-Carrillo, A. (1989) Science 245, 68-71. Cole, R.D. (1987) Int.J.Peptide Res. 30, 433-449. Clore. G.M.. Gronenborn. A.M.. Nilaes. M.. Sukumaran; D.K., Zarbodk J. (i98?) EM86 J. 6, 1833-l 842. Graziano, V., Gerchman, SE., Wonacott, A.J., Sweet, R.M., Wells, J.R., White, S.W. and Ramakrishnan, V. (1990) J.Mol.Biol. 212, 253-257. Turnell, W.G., Satchwell, SC. and Travers, A.A. (1988) FEBS letters 232, 263-268. Crane-Robinson, C. and Ptitsyn, O.B. (1989) Protein Engineering 2, 577582. Mannermaa, R.M. and Oikarinen, J. (1990) Biochem.Biophys.Res.Commun. 168, 254-260. McKay, D.B. and Steitz, T.A. (1981) Nature 290, 744-749. Brennan, R.G., and Matthews, B.W. (1989) J.Biol.Chem. 264, 1903-l 906. Delhaise, P., Bardiaux, M. and Wodak, S.J. (1984) J.Mol.Graph. 2, 103-106. Entry 2GAP, version of 07-MAY-86. Weber, LT. and Steitz, T.A. (1984) Proc.Natl.Acad.Sci. USA 81, 3973-3977. Berenstein, F.C., Koetzle, T.F., Williams, G.J.B., Meyer, E.F., Jr., Brice, M.D., Rodgers, J.R., Kennard, O., Shimanouchi, T., and Tasumi, M. (1977) J.Mol.Biol. 112, 535-542. Takeda, Y., Ohlendorf, D.H., Anderson, W.F., and Matthews, B.W. (1985) in: Biological macromolecules and assemblies (Jurnak, F.A. and McPherson, A. eds) Nucleic acids and interactive proteins vol. 2, pp. 223-263 John Wiley and sons. White, SW. (1987) Protein Engineering 5, 373-376. Dodd, I.B., and Egan, J.B. (1987) J.Mol.Biol. 194, 557-564. Thomas, J.O., and Wilson, C.M. (1986) EMBO J. 5, 3531-3537. Segers, A., Muyldermans, S. and Wyns, L. (1991) J.Biol.Chem. in press. Wells, D.E. (1985) NucLAcids Res. 14, rl 19-rl31. Sevall, J.S. (1988) Biochemistry 27, 5038-5044. Ristiniemi, J., and Oikarinen, J. (1989) J.Biol.Chem. 4, 2164-2174, Ristiniemi, J., and Oikarinen, J. (1988) Bioch. Bioph. Res. Comm. 153, 783-791.
902
of
174,
January
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
31, 1991
COMMUNICATIONS Pages
898-902
A model for histone HSDNA interaction: Simultaneous minor and major groove binding Alain Segers, Vrije
Brussel, lnstituut voor Moleculaire Biologie, 1640 St. Genesius-Rode, Belgium
Universiteit
*Plant
Received
Genetic
Lode Wyns and lgnace Lasters*
Systems, Laboratories Brussels-ULB-UCMB-CP160, Heger, Batiment P2, 1050 Brussels, Belgium
November
29,
Paardenstraat Avenue
65, P.
1990
Using the tertiary structure of the globular domain of H5 (GH5) and based on an alternative sequence homology between GH5 and DNA-binding proteins containing the helix-turn-helix motif, a model for H5-DNA interaction is proposed. From molecular graphics it follows that helix II recognizes the major groove of the DNA, as does the second helix of the helix-turn-helix motif, while helix III makes minor groove contacts, in agreement with the hypothesis of Turnell et al. (FEBS letters 232, 263-268). In the resulting model GH5 makes contact with a full turn of DNA. 0 1'391 Academic Press, Inc.
The histones Hl and H5 bind to the nucleosomes, are involved in the formation of higher order structures of chromatin (1) and play an active role in the control of DNA replication and cell proliferation (2). However, the absence of a useful assay limits many investigations. Nevertheless, the amount of data being produced on the structure and function of the histones Hl and H5 is steadily growing (3). A breakthrough took place with the structure determination of the globular domain of H5 (GH5), using NMR-techniques (4). This will be complemented with a crystallographic analysis (5). Knowledge of the tertiary structure of GH5 induced the formulation of hypothetical binding models for GH5 to DNA (6, 7), some of them based on a possible structural homology between GH5 and the E. coli catabolite gene activator protein (CAP) (4, 8). As GH5 lacks a clear helix-turn-helix motif, the speculations of Clore et al. are mainly based on the location of the polar and charged residues in the structure. In doing so the anti-parallel helices II and III are predicted to be involved in DNA-contacts, while helix I, due to its hydrophobic nature, is presumed to make protein contacts. However, despite some structural similarities between GH5 and CAP, the sequence alignment derived from the model displays only poor homology. Even the homology between helix III of GH5 and helix F of CAP, although proposed to be structurally equivalent (4), is low. In contradiction with the results of Clore et al., the model of Mannermaa & Oikarinen
Vol.
174, No. 2, 1991
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
(8) assumes the presence of a helix-turn-helix motif in GH5, and suggests an involvement of the N-terminal part of helix III in the recognition of the major groove of the DNA. Finally, Turnell et al. (6) state that this helix III of GH5 would make minor groove contacts with the DNA. Although based on a significant sequence homology between histone Hl and pentraxins, their proposal is in conflict with both above explained models. Based on a new sequence alignment we present an alternative
structural
homology between GH5 and CAP. Helix II of GH.5 would be the functional equivalent of the C-terminal helix in the helix-turn-helix motif, present in many sequence-specific DNA-binding proteins (10). Positioning GH5 on a CAP-DNA model according to this homology is in excellent agreement with DNA-binding. In addition, the present alignment, unlike the others, is compatible with the proposal of Turnell et al., resulting in a DNA-binding mode characterized by simultaneous minor and major groove binding.
Experimental All graphical work was done on an Evans and Sutherland graphics station using the Brugel package (11). We used the coordinates (12) of the CAP-DNA model (13) obtained from the Protein Databank (14) at Brookhaven National Laboratory.
Results
and Discussion
Although GH5 does not contain the whole helix-turn-helix motif, we believe the region of GH5 comprising residues 22 to 35 to be similar to the region of the Fhelix of CAP (figure 1). This new alignment requires a shift by 18 residues with respect to the earlier proposed model of Clore et al.. In fact in our model helix II takes in the place occupied by helix III in the fit of Clore et al. (figure 2). Comparing the number of identities (4 in the present model, none in (4)), and neutral substitutions (4 versus 3), it is obvious that the new alignment is superior. In addition the present alignment in the region of helix II is in agreement with the hydrophobicity
pattern expected for a major groove binding helix (15). Also,
following the fingerprint pattern of White (16), the Gly 177 in CAP is expected to be conserved. Only our model is in agreement with this requirement. More
I
II
III
GH5
1
SASHPTYSEMIAAAIRAEKSRGGSSRQSIQKYIKSHYKVGHNADLQIKLSIRRLLAA * * * **
CAP
155
DAMTHPDGMQIKITRQEIGQIVGCSRETVGRILKMLEDQDLISAHGKTIWYGTVLG D
E
F
Fiaure 1. The proposed alignment of GH5 with CAP, based on a sequence homology with the DNA-binding region of CAP. Identities are indicated by an asterisk, alpha-helices are underlined boxes. The GH5 sequence is on top and starts at residue 1, CAP starts at 155.
Vol.
174, No. 2, 1991
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Fiaure 2. Comparison of two alignments of GH5 with CAP. The upper two sequences are aligned according to the new model. The GH5 sequence starts at residue 22 and represents the region of helix II, the CAP sequence starts at 176, representing the distal part of the DNA binding motif with helix F. Helix F is the major groove binding helix. The third sequence is that of GH5 starting at 40 and aligned according to (4). It represents the region of helix Ill. Bold typed residues are in helix structure, identities and neutral substitutions are indicated by.
support is obtained by subjecting the new alignment to the test developed by Dodd and Egan (17), which locates and statistically evaluates a potential DNAbinding region homologous to lambda cro-like proteins. Once again the new alignment agrees with an expected major groove DNA-binding helix (figure 3). We conclude that helix II of GH5 is a major groove binding helix comparable to helix F of CAP. A set of 10 C-alpha coordinates was constructed for GH5 containing Arg 26-Ser 35. The set for CAP contained Arg 180-Met 189. The resulting RMS distance between the C-alpha atoms of GH5 and CAP being 3.0 A for our fit is better than the one obtained with the fit of Clore et al., which gives 3.5 A. Because of the irregularities in the structure of helix II it is difficult to position helix I1 of GH5 on helix F of CAP using C-alpha coordinates. We preferred to use general features of the structure and decided to superimpose both helix axes. This rules out possible artifacts resulting from abberant helical parameters, since the helix axis parameter is the least affected by irregularities in the structure. Based on our assumption we positioned the GH5 structure on the model of a CAP-DNA complex. What are the consequences for the helices I and III ? CAP V G c S R E
+a1 +230 +94 +206 +I27 +104
T +225 v +121 G -34 R +159 I +102 L +68 K
+115 -___-_ +159a
‘A’ G
-106
G s S R Q
+230 -74 +206 +127 +62
S I
-2 +194
Q
+21
K
+115
Y
+140 +33 +115 ---___
I K
+1061
‘8 G H N
-106 +la -38 -20 -72 -126
A D L Q +91 I +194 K -67 L -126 s
I R
-74 +33 +127 ---_-_ -166
Fiaure 3. Comparison of the two alignments presented the new alignment, ‘B’ is the alignment found in (4). The described in (16). The higher the score the more likely the master set of proteins, all sharing the helix-turn-helix
900
in figure 2. ‘A’ indicates scores are calculated as it is to be homologous to motif.
Vol.
174, No. 2, 1991
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Fiaure 4. Stereo representation of the proposed model of DNA-binding of GH5 with a full turn of DNA, according to the new alignment. The insert displays the essential features. The axes of the alpha-helices are represented by thick lines and are labeled accordingly with roman numbers. The minor groove-binding arginines are also shown. The DNA backbone is at the left of the figure. The axis of helix I (N-terminus in the upper right corner) lies almost in the plane of the page. Helix II comes out of the page while helix Ill, being roughly antiparallel with helix II, runs into the page. The C-terminal part of helix III approaches the minor groove of the DNA very closely.
BRUGEL, we see, while fitting the axis of helix II of GH5 on the axis of helix F of CAP, that by rotating around this common
Using
the interactive
axis one can displace
graphics
package
helix I far from the DNA making
it unlikely
to be involved
in
DNA-contacts. As already pointed out in (4), due to its hydrophobic nature it is a candidate for making protein contacts. At the same time it is possible to position helix III so that its carboxyl-end
points to the minor groove of the DNA. Thus,
bringing Arg 52 and Arg 53 close to the DNA-duplex. These residues are thought to interact directly with the DNA minor groove, since they are geometrically well located (6). A preliminary study in which titration with GH5 displaces Hoechst 33258 bound to pUC18 supercoiled DNA, supports the idea that GH5 makes minor groove contacts (unpublished results). The resulting fit shows a GH5molecule with its two anti-parallel helices rougly perpendicular to the axis of the DNA duplex. Helix II lies in the major groove while helix III runs closely to the minor groove. From this follows that GH5 would make contacts with a full turn of DNA. The lysine 64 of GH5, which is strongly protected from chemical modification upon nucleosome-binding (18) is not involved in our proposed mode of binding, neither is it in any other model, since it lies outside the helices. However, because it is relatively far away from the DNA-binding site it is ideally located
to make
contacts
with
neighbouring
DNA-strands.
This
is in accordance
with the idea that GH5 has more than one binding site (7,19) (figure In the region
of helix II sequence
homology
is almost
completely
absent
4). between
the globular domains of histone Hl and H5 (7,20). This could explain the functional differencies between both types of linker histone. They could then bind non-specifically with their helix III to a narrow minor groove, while modulating the strength and sequence of binding (21,22) with the amino acid composition of helix II, and their putative adenine nucleotide binding site (23). 901
Vol.
174,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Acknowledqments We thank Dr. A. Gronenborn GH5. We thank the N.F.W.O.
for kindly providing us with the NMR-models A.S. is a fellow of the I.W.O.N.L.
References 1. 2. 43: 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
15.
Z: 18. 19. 2: 22. 23.
McGhee, J.B. and Felsenfeld, G. (1980) Annu.Rev.Biochem. 49, 1115-l 156. Sun, J-M., Wiaderkiewicz, R. and Ruiz-Carrillo, A. (1989) Science 245, 68-71. Cole, R.D. (1987) Int.J.Peptide Res. 30, 433-449. Clore. G.M.. Gronenborn. A.M.. Nilaes. M.. Sukumaran; D.K., Zarbodk J. (i98?) EM86 J. 6, 1833-l 842. Graziano, V., Gerchman, SE., Wonacott, A.J., Sweet, R.M., Wells, J.R., White, S.W. and Ramakrishnan, V. (1990) J.Mol.Biol. 212, 253-257. Turnell, W.G., Satchwell, SC. and Travers, A.A. (1988) FEBS letters 232, 263-268. Crane-Robinson, C. and Ptitsyn, O.B. (1989) Protein Engineering 2, 577582. Mannermaa, R.M. and Oikarinen, J. (1990) Biochem.Biophys.Res.Commun. 168, 254-260. McKay, D.B. and Steitz, T.A. (1981) Nature 290, 744-749. Brennan, R.G., and Matthews, B.W. (1989) J.Biol.Chem. 264, 1903-l 906. Delhaise, P., Bardiaux, M. and Wodak, S.J. (1984) J.Mol.Graph. 2, 103-106. Entry 2GAP, version of 07-MAY-86. Weber, LT. and Steitz, T.A. (1984) Proc.Natl.Acad.Sci. USA 81, 3973-3977. Berenstein, F.C., Koetzle, T.F., Williams, G.J.B., Meyer, E.F., Jr., Brice, M.D., Rodgers, J.R., Kennard, O., Shimanouchi, T., and Tasumi, M. (1977) J.Mol.Biol. 112, 535-542. Takeda, Y., Ohlendorf, D.H., Anderson, W.F., and Matthews, B.W. (1985) in: Biological macromolecules and assemblies (Jurnak, F.A. and McPherson, A. eds) Nucleic acids and interactive proteins vol. 2, pp. 223-263 John Wiley and sons. White, SW. (1987) Protein Engineering 5, 373-376. Dodd, I.B., and Egan, J.B. (1987) J.Mol.Biol. 194, 557-564. Thomas, J.O., and Wilson, C.M. (1986) EMBO J. 5, 3531-3537. Segers, A., Muyldermans, S. and Wyns, L. (1991) J.Biol.Chem. in press. Wells, D.E. (1985) NucLAcids Res. 14, rl 19-rl31. Sevall, J.S. (1988) Biochemistry 27, 5038-5044. Ristiniemi, J., and Oikarinen, J. (1989) J.Biol.Chem. 4, 2164-2174, Ristiniemi, J., and Oikarinen, J. (1988) Bioch. Bioph. Res. Comm. 153, 783-791.
902
of
