A possible three-dimensional structure of bovine α-lactalbumin based on that of hen's egg-white lysozyme

J. Hoi. Bid. (1969) 42, 66-66

A Possible Three-dimensional Structure of Bovine a-Lactalbumin based on that of Hen’s Egg-White Lysozyme W. J. BROWNE, A. C. T. NORTH, D. C. PHILLIPS Laboratory of ikfolecular Biophysics, Department of Zoology Oxford University, Oxford, England KEITH BREW-~-, THOMAS C. VANAMAN~ AND ROBERT L. HILL

Department of Biochemistry, Duke University Medical Center Durham, North Carolina, U.S.A. (Received 3 February 1969) Bovine a-la&albumin and hen egg-white lysozyme have closely similar amino acid sequences. A model of a-lactalbumin has been constructed on the basis of the main chain conformation established for lysozyme. The side chain interactions of lysozyme are listed (Table 2) and the consequences of the side chain replacements in a-la&albumin examined. Changes in internal side chains are manner, suggesting that the model is generally interrelated in a convincing largely correct, but there are some regions where it has not been possible to deduce the conformation unequivocally. Glu 35, which acts as a proton donor in lysozyme, is absent in a-laotalbumin, in which a neighbouring histidine residue may assume a similar function. The surface cleft, which is the site of substrate binding in lysozyme, is shorter in a-la&albumin. While this would be consistent with a-la&albumin having a mono- or disaccharide 8s substrate, the biochemical evidence shows that the role of a-la&albumin in the synthesis of lactose is a complex one requiring direct interaction with the A protein.

1. Introduction Brew & Campbell (1967) first suggested that the lysozymes from higher animals and certain a-la&albumins may be structurally related because of similarities in their molecular weights, ammo acid compositions and NH,- and COOH-terminal end groups. Subsequently, Brew, Vanaman & Hill (1967) have found that the ammo acid sequence of bovine u-h&albumin is strikingly similar to that of hen egg-white lysozyme. Subject to a small number of deletions, the amino acid sequences of the two proteins (Table 1) can be aligned so that at least 45, and possibly 54, residues in a-la&albumin are identical to corresponding residues in lysozyme and at least 23 more residues are structurally similar. Furthermore, the four disulphide bonds in a-lactalbumin have been found to be homologous to the corresponding bonds in lysozyme. The proportion of identical residues is greater than that between sperm whale myoglobin and either of the chains of haemoglobin (see, e.g. Perutz, 1965); these three globin chains are known to have very similar tertiary structures despite the differences t Present address: Department $ Present address: Depsrtment Calif., U.S.A.

of Biochemistry, of Biochemistry, 66

The University, Leeds, England. Stanford University Medical Center, Stanford,

66

W. J. BROWNE

ET

TABLE

Comparison

AL.

1

of amino acid sequences of hen egg-white lysozyme ad bovine a-luctalbumin and conformation of main chain of u-la&albumin model

Residue

Lysozyme

+

a-La&albumin -

1 2

LYE Val

3

Glu Gln

-115.7

154.2 107.4

107.7 105.8

Phe

Lsu

- 69.2

156.7

117.6

4

GUY

Thr

-92.0

150.8

100.5

6

A%

-50.6

-74.0

102.6

-41.0

-61.5

111.6

7

CYS Gii

LYS CYS &ii

-47.6

-40.2

122.6

8

iz

Kl

-75.4

-33.8

107.0

9

Ala

Phe

-53.8

-60.4

105.5

10

Ala

-50.7

-50.7

6

11

Ala

Arg Glu

-45.8

-53.9

112.9 114.4

12

Met

Leu

-59.2

-40

110

13

LYS

LYE -

-35

-50

110

14

JG His

ASP Leu

I

-35

-25 -10

110

-40

15 16 17

@Y LC3U

18

Asp

LYS

19

Asn

GUY

20

TP

21 22

G Gly

23

TF Gly

-85 135 -45

-50

110 110

100

110

0

110

75 85

110

100

-15

g

GUY E-l

-75

115

24

S0r

SW

-50

165

25

izl

EtYU

-70

110 110 110

-25

110

26

cly

P,

-60

-60

110

27

Aa11

Glu

-40

-49.1

104.1

28

TOP xi

-65.8

-31.6

122.4

29

Trp vii

-82.1

-35.8

108.8

30

cya

-69.3

-44.4

109.6

31

xii

- 60.9

-44.0

115.3

32

Ala

-71.6

-29.2

112.7

w

LYE Phe Glu

-66.2

-64.6

110.9

-67.2 -81.3

-23.0 - 50.9

112.2 116.2

SC3r

- 125.6

-8.2

119.1

34t w 36 37

67.8

27.4

117.4

30

110 113.4

38

G.i Phe

39

Asn

60 -110.3

40

Thr

-69.6

-17.8

120.3

41

Gin on page 69.

-84.1

-35.7

114.7

t See footnote

109.0

POSSIBLE

STRUCTURE TABLE

1 continued

Lysozyme

a-La&albumin

42

Ala

Ala

43

Thr

Ile

44

Asn

Val

-153.8

45

Arg Am Thr

Glx

-130

ASX

Residue

46 47

I Asx

67

OF a-LACTALBUMIN

T

4

*

-30.0

142.4

-142.2

150.3

107.2

135

110

145

110

111.7

-95

-60

110

-140

-60

110

-65

110

48

-Asp

49

GUY Ser

-105

50

-60

120

110

51

Thr

-131.0

157.2

107.6

.58

-115.4

129.9

108.8

53

ASP Tyr

54

Gly

55

ii--

L,,

56

L0U

Phe

-107.6

57

Gln

54.4

58

11,

Glx Ile

114.7 119.4

-72.4

133.2

109.5

69

Asn

-76.2

152.6

115.6

60

Ser

-92.9

-2.7

125.6

61

- 125.8 GUY

Asx Asx

67.0 -42.1

35.2

rlrg

Ly3

64

Trp

Ile

63 64

TOP cys

6.5

Asn

Lys

66

Asp Gly

Asn

-132.9

Asp

67

146.3

123.0

- 179.0

122.2

-37.0 14.4

120.9

-83.3

-18.6

108.9

-133.1

-37.4

126.3

TOP

-90.8

-32.4

126.3

Cs's

-151.3

143.5

Ill.6

-85.3

140.1

106.4

73.3

8.4 -7.8

113.6 125.0

A% Thr

Gln

-134.8

17.2

69

Asp

-121.8

83.0

70

I+0

Pro

71

Gly Set-

Hi,

-61.0

11.0

111.5

Ser

-45.0

121.9

131.8

-123.8

145.8

74

G Asn

G

-114.9

76.3

75

LBU

Asn 110

68

72 73

76 71

CYS Asn

CYS A,

78

G-

Es-

79

FL

80

81

CYS ser

82

Ala

83

L0U

LYS Phe

-38.8

-42.8

121.2 106.5 127.4

82.2 107.0

-66.5

-15.9

116.2

-91.3

-12.5

107.8

43.0

48.2

113.9

-145.5

149.0

100.3

ii&

-83.2

124.5

CYS

-27.8

-50.8

105.3 116.4

Asp

-48.8

-34.5

117.5

-58.3

-31.7

108.7

-69.0

-12.6

117.8

ET AL.

W. J. BROWNE

08

TABLE 1 continued Residue

Lpozyme

a-La&albumin

4

#

7

84

Leu

-92.3

-16.6

119.1

85

ser

-61.4

-161.7

109.2

86

Ser

-62.7

ASP Ile

- 120.0 -82.6

87 88

6.5

115.1

112.9 13.1

104.9 121.6

89

Tbr

-51.9

-65.7

107.8

90

YiiL

-44.7

-62.4

113.8

91

SIX

-67.9

-60.3

111.2

92

v81

-63.7

-50.7

106.9

93

Am

- 64.3

-42.3

113.1

CYS ;il;;

-79.1

-40.2

109.7

-64.6

LYS

-67.7

-30.2 -24.9

112.0 117.2

94 95 96

-89.3

97

50

98

Ile

-45

-50

99

zl

-50

-60

100

Ser

101

Asp Gly

110 110 110

-45

-20

-110 -60 -80

-52 -40

106

-55

-35

106

-40

-30

107

-50

-20

108

-80

109

-45

-65

110 110 110 110 110 110 110 110 110

110

-61.2

-48.0

126.3

111

-61.9

-45.7

112

-63.0

-61.2

104.8 114.2

-64.8

102 103 104

ASP GUY

115 116 117

LYE GUY

118

Th

119

ASP Vctl

120 121

GlU

122

Yiii

123

Trr,

124

Ile ‘%3 GUY

125 126

120

-38.1

117.6

Leu

- 117.8

-3.7

127.1

CYS ser

- 120+

-46.1

113.6

113 114

110

Glu LYE Leu ASP GlU f Trp Gl I

POSSIBLE

STRUCTURE

69

OF a-LACTALBUMIN

TABLE 1 cun%ued Residue

Lysozyme

127

cys

127a 128

-T Am

129

Leu -

a-Lactibumin

4

G

7

cys Zl LYE IAU -

Columns 2 and 3 are reproduced from EGll et al. (1969). Columns 4, 5 end 6 indicate the conformation at the Ca atom of eaoh residue. 4 and I/ are the torsion angles about the N-Ccc and Ca-C bon& respectively. r is the sngle NCaC’. We have used the convention that the angles 4 and $ are ascribed zero values for eclipsed conformations of the main chain atoms N, Cu, C ainoe this convention appears likely to supersede that proposed by Ed&l et al. (1966). (4 and $ values in the new convention are related to the earlier values by the addition of 1800.) In addition to this change, the angles for lysozyme previously published (Blake et aE., 1967) have been changed slightly by refinement. Where the oonfornmtion in the laotalbumin model is the same as lysozyme, the angles quoted are those for lysozyme; where the conformations differ, the angles, shown in italics, were measured from the model and Bpe only accurate to 2 10’. Confornmtiomal angles are not given beyond residue 115, where the arrangement is not olearly established. t For maximum

homology

it has been suggested that the sequences in this region should be

aligned: 32

Ala

Thr

33 34

LYS Phe

I Phe

35

clu’

Thr

Ser -

Ser -

36a 36

I

E

in composition (Per&, Muirhead, Cox & Goaman, 1968). The even greater similarity between the primary structures of bovine a-lactalbumin and hen egg-white lysozyme, not least in the similarity of the disulphide bridge arrangement, has led us to consider

that the molecular conformations of the two enzymes may be very similar, and we have attempted to build a model of the c&&albumin molecule based upon that supposition. A wire skeletal model of lysozyme, to a scale of 2 cm = 1 A, was constructed as described earlier (Blake, Mair, North, Phillips & &ma, 1967) and was then modified to accommodate the a-lactalbumin sequence by changing the side chains that differ in the two molecules and by rearranging the main chain to suit the proposed deletions in them. In general, homologous side ohains were kept in the same orientation in the two models and, when different side chains were pbced in the interior of the molecule, care was taken to orient the new side chain in such a manner that it occupied nearly the same position as the corresponding side chain in lysozyme. Our conclusions are that the differences between the two molecules are not incompatible with their having closely similar conformations. For example, the hydrophobic interns1 side chains are normally replaced by other hydrophobic side chains (as is the rule with the various globin chains). Moreover, a change in one residue is often accompanied by a compensating change in a neighbouring residue in the model (not

IO

W. J. BROWNE

ET

AL.

necessarily a neighbouring residue in the sequence). For example, the following changes occur in a group of residues that pack together to form a hydrophobic internal regiont: Phe 3 -+ Leu, Leu 8 -+ Val, Met 12 + Leu, Ala 32 -+ Thr, Ile 55 --f Leu, Leu 56 -+ Phe, Ile 88 --f Leu. The residue deletions can be accommodated without causing more than local re-arrangement of the main chain conformation; again, the situation is analogous to that obtaining in the globin chains where, for instance, the loss of six residues from the haemoglobin u-chain, compared to the p-chain and myoglobin, results in a reduction in length of the CD loop, but the B and E helices have similar relative positions in all three chains. All of these changes can be accommodated so readily that the proposed model for a-lactalbumin (illustrated in Plates I and II) seems very likely to be substantially correct, though we must emphasize that the conformations of the two molecules may be different even in those regions where the primary sequences are very similar (see Perutz et al., 1968). More optimistically, we note that the relationship between these structures suggests that proteins may be structurally related even though they are functionally distinct and that the very many protein molecules may belong to a comparatively small number of families. It may be possible to derive the structures of all the members of a family relatively easily once one or two have been analysed in detail. Certainly this approach seems more promising at the moment than does the prediction of unique protein structures from chemical information alone.

2. Conformation

of the Main Chain

There are five sections of the main chain where the deletions necessary to ensure maximum homology require differences in the main chain conformation. The changes may be described most easily in terms of the a-carbon atoms and planar peptide groups, which can be thought of as rigid components linked by single bonds about which rotation is possible. (a) Residues 14 and 15 If u-carbon atoms 14 and 15 and the peptide groups following each are removed, peptide group 13 may be connected to u-carbon 16 without disturbing the positions of u-carbon atoms 13 or 17 or any of the atoms beyond them (Fig. 1). The conformation so achieved is not completely satisfactory in that the dihedral angles of u-carbon atoms 13, 16 and 17 are not all within permissible ranges (Ramachandran, Ramakrishnan t Sasisekharan, 1963). Further adjustments may easily be made to the model which cause very slight displacements of atoms beyond u-carbon 17 but permit all the dihedral angles to be brought within acceptable ranges. The replacement of both Asn 19 and Arg 21 in lysozyme by Gly in u-lactalbumin may be connected with these adjustments in conformation since the dihedral angles are less restricted where no aide chain is present. Plate I shows the resultant conformation in this region. The ribbon-like conformation exhibited by residues 17 to 22, in which alternate peptide groups lie along and across the ribbon, has been noted in other parts of lysozyme and in other enzyme molecules (see Venkatachalam, 1968). t To avoid a dual system of numbering, numbers.

residues will be denoted

by their lysozyme

sequence

PIATES I and II. Stereo photographs of the model of r-lactalbumi~~ seen from two opposite directions. Residues are numbered alld me referred to in the text. Letters indicate the six saccharide-binding sites in lysozyme. The light-colouretl balls indicate the sulphur atoms of disulphide bridges and dark-coloured balls the amino- and carboxyl-terminal groups. The mail1 (*haill is painted whit,e and arrows indicate its direction from unitlo- to citrboxyl-termirlus.

POSSIBLE

STRUCTURE

OF a-LACTALBUMIN

71

FIG. 1. The main-chain conformation of residues 11 to 17 in lysozyme (solid lines) end the corresponding region in a-la&albumin (broken lines) in which two residues are deleted. Dotted lines indicate 8 hydrogen bond.

(b) Residues 32 to 36 There is no net difference in the number of residues in this stretch but maximum homology between the sequences is achieved by omitting Lys 33 and inserting an extra residue (35a) in a-lactalbumin between Glu 35 and Ser 36. In building the model we were therefore faced with a dilemma: Should the main chain conformations, having the same total number of residues, be similar in the two molecules? Or should the main chain conformations be changed so that the side chain of the homologous residue Phe 34 occupies a similar position? We chose the tirst alternative in building the model (Plate II) but consider that there is a fair probability that the conformation is incorrect in this region. The replacements Ala 31 -+ Thr, Ala 32 -+ Thr and insertion of yet a third Thr between 35 and 36 all involve the introduction of hydrophilic groups in internal sites. Threonine is known occasionally to occur internally (e.g. residue C4 in almost all globin chains and residue B9 which has been found to be Ala, Ile, Ser or Thr in different globins) since its hydrogen-bonding potential may be satisfied by other internal groups, but it is perhaps less likely that several adjacent threonine groups would be internal. Furthermore, the rules formulated by Prothero (1966) to predict whether or not a specified sequence of residues will tend to be a-helical, which successfully indicate the approximate lengths of the lysozyme helices, suggest that residues 30 onwards will not be helical in a-lactalbumin. It appears quite possible therefore that the conformation of this stretch of the chain in a-lactalbumin will be somewhat different from that in lysozyme, but we are not able to

12

W. J. BROWNE

ET

AL.

predict a unique alternative. If we presume that the side chain of residue 38, which is Phe in lysozyme but Tyr in a-lactalbumin, remains in the same position, only a short length of main chain is in doubt; unfortunately this includes Glu 35, which is involved in the activity of lysozyme. (c) Residue 47

This residue occurs in lysozyme at the “hairpin bend” that joins the two lengths of extended chain which together form two strands of antiparallel pleated sheet. We have found that it is possible to remove residue 47, which is deleted in a-lactalbumin, and to join the peptide group of 46 to Ca 48 without appreciable disturbance of the atoms before Ca 46 or after Ca 50, so leaving the hydrogen-bonding pattern between the strands unchanged. There are several alternative ways of forming the rather sharper hairpin bend. The most satisfactory stereochemically appears to be that shown in Figure 2 where Ca 48 and Cu 49 each have dihedral angles characteristic of

FIQ. 2. The m&-chain conformation of residues 45 to 51 in lysoeyme (solid hues) and the corresponding region in a-lactalbumin (broken lines) in which one residue is deleted. Dotted lines indicate hydrogen bonds.

the right-handed u-helix. An alternative conformation for the loop in which these two atoms have dihedral angles characteristic of the left-handed a-helix is stereochemically less satisfactory. (d) Residue 100 The conformation in this region of lysozyme is rather irregular and one can quite easily remove residue 100 and close the gap. The model shown in Plate II leaves the atoms up to Ca 99 and beyond Ca 105 undisturbed and takes account of the changes in character of the side chains 102 (Gly --f Lys), 103 (Asp + Val) and 107 (Ala -+ Tyr). The effect of these changes is to form a cluster of side chains with hydrophobic character, which also include the homologous Trp 63 and Ile 98. In the model illustrated, these side chains are packed together in a satisfactory-looking manner and

POSSIBLE

STRUCTURE

OF a-LACTALBUMIN

the main chain conformation is stereochemically acceptable. It is certainly unique way of building this region of the molecule.

73

not a

(e) Residue 115 onwards The differences between the chains are more marked in this region than elsewhere with three deletions and one additional residue in u-lactalbumin and several changes of side-chain character (e.g. Asp 119 --f Leu, Vall20 + Asp). In lysozyme, the terminal carboxyl group appears to form a salt bridge with Lys 13, and the side chain of the terminal (Leu) group is in contact with other non-polar groups, Leu 25 and the aliphatic chain of Lys 13. Since all these groups are identical in a-lactalbumin, it seems highly probable that the position of the terminal residue also is the same in the two enzymes. It is therefore necessary to insert one additional residue between Cys 127 and the terminus and to remove three residues between Cys 115 and Cys 127. Neither of these can readily be done without changing the conformation of the disulphide bridge 6-127 from left-handed to right-handed?, so moving the Cfi of Cys 127 nearer to Cys 115 and further from the terminal residue. Such a change would result in a-laotalbumin having three right-handed and one left-handed bridge (30-115) whereas hen egg lysozyme has two of each. Recent measurements of circular dichroism in the spectral region to which disulphide bridges might be expected to contribute (D. G. Dalgleish, personal communication) suggest that there may well be such a difference in the chirality of the bridges in the two enzymes. Prothero’s “rules” predict that the main chain from Trp 108 to Leu 124 is likely to have the a-helical conformation. Even after reversal of the hand of disulphide bridge 6-127, the distance between Cu of 115 and Cu of 127, approximately 175 A, is too long for a substantial part of the intervening chain, comprising nine residues, to be u-helical; if the prediction according to Prothero’s rules is indeed correct,, then a substantial rearrangement starting several residues before 115 would be neoessary, and this seems to us improbable. We think it to be more likely that the chain conformations are similar at least as far as residue 115. It is clearly possible to build a stereochemically satisfactory model for the main chain between 115 and the rearranged bridge 127-6, in which hydrophobic side chains could be internal, but no uniquely favourable conformation suggests itself.

3. The Environments of the Side Chains Table 2 lists the features that we consider to be significant for the present purpose of the environments of the side chains of lysozyme and the probable environments in the model of a-lactalbumin. The comments about a-lactalbumin must be tentative for those regions where the main chain conformation differs from lysozyme and, in particular, we have not thought it worthwhile to discuss the much-changed stretch from residue 115 onwards. As described above, we have built the main chain of residues 32 to 36 as in lysozyme, so that the substitutions should be thought of as 33 Lys -+ Phe, 34 Phe + His, 35 Glu --f Thr (as shown in Table 1, rather than 33 Lys (deleted), 34 Phe, 35 Glu + His, 35a (inserted) + Thr)$. f If, on looking along the S-S bond, the S-Cfi bond from the further S atom lies at about 90’ clockwise with respect to the S-Cp bond from the nearer S atom, the bridge ia said to be righthanded, if 90” anticlockwise, left-haded. $ An mmino acid residue is underlined in this paper if the residue is the aanw in both enzymes. If the residue is dZereqt, the corresponding amino acid in the other enzyme ia named in a bracket.

74

W. J. BROWNE TABLE

ET

AL.

2

Side chain interactions in hen egg-white lysozyme and bovine a-lactulbumin Glu

E

E

Gln

E

2

Phe

surrounded by other S Largely hydrophobic groups, Leu 8 (Val), Ala 11 (Glu), Ile 55 (Leu), Ile 88 (Leu) and the hydrocarbon chains of Lys 1 (Glu) and Glu 7. -

Leu

S Points more inwards than in lysozyme. Surrounding groups remain hydrophobic, mostly different, from lysozyme: Val8 (Leu), Leu 55 (Ile), Leu 88 (Ile), CH, group of Thr 40, hydrocarbon chains of Glu 7, Glu 11 (Ala). -

3

4

Gly

E

Thr

E

4

5

Arg

E

Lys

E Main chain conformation in 123 to 125 region.

6

cys -

s

-cys

s

7

Glu

E

Glu -

E

8

Leu

I

Surrounded by Phe 3 (Leu), Ala 11 (Glu), Met 12 (Leu), Phe 38 (Tyr), Ile 55 (Leu), Ala 32 0.w.

Val

I

Many changes in surrounding groups (see column 4) but remaining hydrophobic.

9

Ala

I

Surrounded by Cys 6-Cys 127, Leu 25, Val 29-e l%(Leu) Ez 129. -

Phe

I

Spaoe to accommodate the ring largely results from the changed main-chain conformation of residues 115 to 127.

10

Ala

E

-4%

E

No impedance

11

Ala

S

Glu

S Carboxyl group can point outwards, without affecting contacts between C/3 and neighbouring hydrophobic groups (see 3, 8).

12

Met

I

Surrounded by Leu 8 (Val), Leu 17, Trp 28, Val 29, Ile 88 (Leu). -

Leu

I

13

Lys -

E

Forms salt bridge COOH.

-Lys

E

14

Arg

E

15

His

E

Forms H-bond

16

Gly

E

Conformation at Cu not favourable for non-Gly residue.

Asp

E

17

Leu -

I

Surrounded by Met 12 (Leu), Tyr 20, Trp 28, Val 92 (Ile) hy&ocarGchain of Lys 96.

Leu -

I

More remote from Leu 12 (Met).

17

1

Lys

E

2

Val

3

Forms salt bridge with Glu 7. Hydrocarbon chain runs pzlel with plane of Phe 3 (Leu).

Guanidinium group forms Hbonds to main chain CO 123 and 125.

to terminal

to Thr 89.

--

No interaction

Dihedral missible residue.

with Glu 7. See 3.

angle of Ca lies in perrange for non-glycine differs

to longer chain.

Additional neighbours are Thr 32 (Ala), Phe 56 (Leu) and Ile 92 (Val).

1

5

9

10 11

12

13 Deleted.

14

Deleted.

15

Different main chain oonformation resulting from deletions permits non-glycine residue. Salt bridge to Lys 96.

16

POSSIBLE

STRUCTURE TABLE

2 continued

18

Asp E

Lys

E

19

Asn E

Gly

E

-Tyr

S

20 Tvr s Surrounded VI%199 (Leu).

75

OF a-LACTALBUMIN

by Leu 17, Lys 96, -

18 See comments conform&ion.

on main

chain

19 20

See comments conformation.

on

main

chain

21

21

Arg

E

Gly

E

22

Gly

E

Gly -

E

23

Tyr

by Arg 2 1 (Gly), S Surrounded Val 99 (Leu), Met 105 (Ile) and Trp 111 (His).

V&l

24

Ser

S Forms H-bond NH 26.

with main chain

Ser

S Cannot form same bond because of change of 26 from Gly to Pro. Alternative bond possible to main chain NH 27.

24

25

Lou -

s

Surrounded by Ala 9 (Phe), Lys 13, Val 29, Ile 124 (Leu). Leu 129. -

Lou

s

124

25

26

Gly

S

Pro

S Conformation of Cfi in lysozyme correct for proline. Position is 2nd residue in helix as often favoured by proline.

26

27

Asn

S H-bond formed to 06 from ring NH of Trp 11 (His).

Glu

S Equivalent

27

28

Trp -

I

Surrounded by Leu 17, Tyr 20, Val 99 (Leu), %& 56(Phe), Trp 108, Met 105 (Ile). -

Trp -

I

29

Vel

I

Surrounded by Met 12 (Leu), Ala9 (Phe), Leu25, Ile 124 (Lou). -

Val

I

29

30

cya -

I

cys -

I

30

31

Ala

I

Thr

I

a-Helical in lysozyme-OH group can form H-bond back to main chain CO 27.

31

32

Ala

I

Thr

I

a-Helical in lysozyme-OH can form H-bond back to main chain CO 28. CHs-group of side chains can be accommodated as result of change of 55 to Leu from Ile.

32

33

Lys

s

Phe

S Aromatic ring accommodated.

34

Phe S Arg 114 (Leu) lies aoross outer face of ring.

His

S

22 Additional

No longer (110).

neighbour

very

near

is Trp 28. -.

Leu

bond feasible.

Additional neighbours are Val 95 (Ale) and V&l 23 (Tyr); no longer very near Ile 105 (Met).

can

readily

23

28

be

33

Can be accommodated. but in different orientation from Phe ring in lysozyme, necessitated by change of 33 to Phe from Lys and allowed by change of 114 to Leu from Arg.

34

70

W. J. RROWNE TABLE

35

Glu

S

36

Ser

I

37

ET

AL.

2 continued Thr

S a-Helical in lysozyme-OH group can form H-bond back to main chain CO 31.

35

Ser -.

I

36

Asn E

Gly

E

37

38

Phe

Tyr

S Change of 33 to Phe from Lys permits external access to hydroxyl group, but environment would be considerably changed by alteration in main chain conform&ion of residues 32 to 35.

38

39

Aan S Forma H-bond chain NH 41.

Asx

S

39

40

Thr

I

Forms H-bond to mein chain Thr CO 86 and from terminal (NH,) + . -

I

40

41

Gln

E

Glx

E

41

42

Ala -

E

Ala

E

42

43

Thr

E

Ile

E

44

Am

E

Val

E

44

45

Arg

E

Glx

E

45

46

Asn

50. S H-bond formed to 0 from Ser -

Asx

S Can make similar Asp or ksn.

47

Thr

E

48

Asp E

49

Gly

Forms internal chain CO 55.

H-bond

to main

S Surrounded by Leu 8 (Val), Lye 33 (Phe) 8nd main chain atoms of residues 4 and 6.

E

60 ser s

Cfl and Tyr 63.

Cy

to 0 from mctin

in

contaot

with

Can make similar

43

contacts.

bond whether

46

Deleted.

47

ARX E

Side chain hrts different orientetion aa 8 result of above deletion.

48

Conformation of Cu not permissible for non-(fly residue.

Glx

E

DifYelent conform&ion of resulting from rtbove deletion.

49

Forms H-bond to 06 of Asn 46 (Asx) end from NS of Asn 69 (A=+

Ser -

S

50

Ca

61

-Thr

S Forms H-bond from Tyr 63 and hydrophobic contaonetween CH, group and ring.

Thr -

S

61

52

Asp -

S Forms H-bond 59 (Asx).

Asp -

S

62

63

Tvr

S Forma H-bond to Thr from N of Asp 66 (&T.

Tyr -

S

63

54

-Gly

I

Impossible conformation at Ca for non-Gly residue; no room for C/3 atom.

(fly -

I

54

65

110

I

Surrounded by Thr 40, Leu 56 (Phe), Leu 8 (VarIle 88 (Leu), Ala 32 (Thr).

Leu

I

65

from N6 of Asn 51 and

POSSIBLE

STRUCTURE

77

OF a-LACTALBUMIN

2 wntinued

TABLE

Additional neighbour is 118 92 (V81); Phe appeara t0 fill space in “core” better than hydrophobic Leu in lyeozyme.

56

Phe

I

from Ne to main

Glx

S Not possible Glu.

Surrounded by Leu 56 (Phe), Trp 63, Leu 83 (Phe), -Ile 98. -

Be -

I

S Forms H-bonds from N3 to Ser 50 and Asp 52 and to OS from NH 61. -

Aax

S

Ser

I

ABX I

61

Arg

E

Lys

E

61

62

Trp

s

Be

S Makes closer contact with Trp 63 than Trp in lysozyme does.

62

63

Trp -

S Adjacent I10 98. -

to Ile 58. Leu 75 -(110) Trp -

neighbours are Ile 62 S Additional (Trp), Tyr 107 (Ala) and possibly Val 103 (Asp).

63

64

Cys

I

-cyfl

1

64

65

Am

E

Lys

E

65

Asn

S

66

56

Leu

I

Surrounded by Al8 32 (Thr), Al8 95 (V8l), 110 55 (Leu), Ser 91 (Asx), Ile 58, Trp 28, Trp 108. -

57

Gln

S M8kes H-bond chain CO 54.

58

Ile -

I

59

Asn

60

Forms H-bond NH 51.

from main chain

is Val

95

58

(m’3). 59

Can form similar H-bond; additional space required 8vail8ble partly as 8 result of repleoement by Asx of Thr 69; internal site would make Asp unlikely.

60

Asp

S Makes H-bond Thr 69 (Asx).

67

Gly

E

Asp

E

68

Arg

E

Gln

E

69

Thr

S Forma (A=+

Asp

S

70

Pro -

E

Pro -

E

71

Gly

E

His

E

72

Ser -

S Mekes H-bond CO 69.

Ser -

S

72

73

Arg

E

Ser

E

73

74

Asn E

75

Lou

76

-Cys S

S Adjacent

with

53 and

neighbour

57

66

H-bond

to Tyr -

Additional

if residue is in fact

Asp

to main

66

chain

Conformation of Cu atom in lysozyme only just outside permiesible range for non-Gly residue.

68 Some freedom of orientation with various H-bonding contacts possible.

Permi&ble conformation Gly residue.

for non-

71

74

110

s

cys

s

-

69

70

ABilE to Trp 03, Lys97. -

67

Additional

contaot

with Cy8 76. -

75 76

78

W. J. BROWNE

ET

AL.

TABLE 2 continued Asn

E

77

Ile -

S

78

Ser

E

79

css

s

80

E

Asp

B

81

Ala

E

Lys

E

82

83

Leu

I

Adjaoent to Ile 58, Cys 6PCys 80, Ser 91 (&). -

Phe

I

83

84

Leu

s

Surrounded by Thr 43 (Ile), Tyr 63, Gly 54, Ser 81 (Asp), a41 (GG.

La -

s

Mekes similar

85

Ser

E

Makes H-bond co 87.

Aax

E

Can make bond to same group.

86

Ser

E

Asx

E

86

87

Asp

E

Asx

E

81

88

Ile

s

Leu

s

88

89

Thr

E

Thr -

E

89

90

Ala

E

Asx

E

90

91

Ser

I

Appears to form H-bonds water molecules.

Asx

I

92

v&l

s

Adjacent to His 15 (deleted), Leu 17, Ile 88 (Leu). -

Ile

S Additional neighbours are Leu 12 (Met), Phe 50 (Leu). More open to surface as a result of deletion of His 15.

92

93

Asn E

Met

E

93

cys -

I

94

17

As-n E -

78

110 -

s

79

Pro

E

80

cys -

s

81

Ser

82

Adjacent

Adjacent

to Cys 76-Cys 94. -

to Tyr 53. -

to main

chain

Surrounded by Ala 11 (Glu), Phe 3 (Leu), Leu 8 (Val), Met 12 (Leu), Val 92 (Ile), Ser 91 (Asx), Thr 40, Ile 55 (Leu). -

to two

94 -cys I

interactions.

84

Change of 83 from Leu to Phe leaves this side chain less buried than in lysozyme. H-bonding possible to Asx 85.

95

Ala

I

Adjacent to Leu 56 (Phe), -Ile 98, Trp 108. -

Val

I

Additional

96

-Lys

s

Adjacent to Leu Val 92 (Ile). *-

Lys -

s

Could form salt bridge to Asp 16 WY).

97

Lys -

E

Lvs

E

98

Be -

I

Ile -

I

17, Tyr -

20,

Adjaoent to Ile 58, Trp 63, Ala 95 (Val), Trp58. -

neighbour

is Trp 28. -

85

91

95

96

97 Additional (Asp).

contact

with

Val 103

98

POSSIBLE

STRUCTURE

2 continued

TABLE

99

Val

Surrounded

I

by Tyr

20, Arg 21

Trp (Gly), Tyr 23 (val), Met 106 (Ile), Trp 108. -

79

OF a-LACTALBUMIN

Leu

28.

S Exposed to surface by change of 21 to Gly from Arg.

99

100

- --

Deleted.

-ASP E

Different position as a result above deletion. Salt bridge Lys 97 can still be formed. -

of 101 to

E

Lys

E

Different position deletion of 100.

of 102

103

Asp E

Val

S

Can form hydrophobic contact 103 with Trp 63, Tyr 107 (Ala), Ile 98, -_ Leu 9vVal).

104

Gly -

of Gz not perS Conformation missible for non-Gly residue; Cj3 atom would be too close to Tyr 23 (Val).

Gly -

S

104

105

Met

I

Ile

S

No longer in contact with Thr 31 105 (Ala) or Glu 27 (Am). Accessible to surface as a result of change of 23 to Val from Tyr.

106

Asn -

E

Aan

E

106

107

Ala

S Adjacent

Tyr

S

Position not uniquely defined, but 107 probably in contact with Val 103 (Asp) which intervenes between it and -Ile 98 as a result of the main chain conformation change.

108

Trp -

S Surrounded by Ile 98, Ala 95 (Val), Leu 56 (phe), Met 105 (Be), Trp 28, Glu 35 (Thr), Ala 10cyr).

Trp -

S

Additional contacts with Thr 31 108 (Ala); not now in contact with Ile 105 (Met).

109

Vel

E

Leu

E

109

110

Ala

S

Ala -

S

110

111

Trp

S Surrounded by Tyr 23 (Val), Ala 31 (Thr), Met 105 (Ile), Asn 106, Cys 115, Lys 116 (Ser); H-bond from Nr to 06 of Asn 27 (Glu).

His

S

Imidazole ring of His can occupy 111 same position as &membered ring of Trp; no longer in contact with Val23 (Tyr), Arm 106; could form H-bond or mEridge to Glu 27 (A=+

112

Arg

E

Lys

E

112

113

Asn E

Ala

E

113

114

Arg

S In contact

Leu

E

Contacts depend upon precise 114 arrangement of main chain residues 32 to 36.

115

cys -

I

Cys -

I

100

Ser

101

Asp E

102

Gly

6

s

H-bond

to main chain CO 96.

Salt bridge with Lys 97. -

Surrounded by Tyr 23 (Vat), Asn 27 (Glu), Trp 28, Ala 31 (Thr), Val99 (LeTTrp 108, Trp 111 (His).

In contact

to Ile 98; Trp 108. -

with Glu 35 (Thr).

with Phe 34 (His).

Adjacent to Trp Vall20 (Asp).

111

(His),

as a result

115

W. J. BROWNE

80

TABLE to Trp 111 (His).

2

ET

AL.

continued

116

Lys

s

Adjacent

117

Gly

E

Conformation at Ca not within permissible range for non-Gly residues.

118

Thr

S Forms H-bond co 115.

119

Asp E

120

Val

S Adjacent to Gly 26 (Pro), Asn 27 (Glu), Cys 30-Cys 115. --

Asp

121

Gln

E

Ghl

122

Ala

S

___

122

123

Trn

S Adjacent to Arg 5 (Lys), Cys 30, Lys 33 (Phe), Phe 34 (His).

Trp -

123

124

Ile

S Adjacent to Ala 9 (Phe), Leu 25, Va129, Leu 129. -

Leu

124

125

Arg

E

___

125

120

Gly

E

Conformation at Ca not within permissible range for non-Gly residues.

___

126

127

Cvs

I

Adjacent to Ag 5 (Lys), Ala 9 (Phe), Ile 124 and Leu 129. -

Cys -

I

In contact with Phe 9 (Ala) and 127 Leu 129 at least.

Glu

E

Inserted

Lys

E

128

Leu -

S

129

to main

&X

chain

Arg

E

129

I&l

s

Glu

LYS Leu

1278 128

As described under “Conformation of the Main Chain”, the large proportiou of deletions and changes in this stretch of chain make it difficult to proposo a unique conformation for it in cc-lactalbumin. It is therefore not possible to discuss the environments of individual side chains, though it may be noted that there are several hydrophobic groups available to interact with underlying groups of similar character.

In contact with Ala 9 (Phe), Lys 13, Leu 25, Ile 124 (Leu),

residue.

116 ‘17 118 119 120 121

1278

Columns I and 8 give the sequenoe number of the residue in the Zysozynze sequence. The main chain atoms of residues 32 to 36 in the a-la&albumin model are placed similarly to those in lysozyme, making the residue changes in the model Lys 33 (Phe), Phe 34 (His), Glu 35 (Thr). For the two tripeptides that are only known in composition and not in sequence (41 to 43; 90 to 92), the published tentative sequence of Brew et al. (1967), implying minimum DNA base changes from the lysozyme sequence, has been used. Columns 2 and 5 indicate the residue in lysozyme and la&albumin, respectively. Homologous residues are shown by underlining. Columns 3 and 6 indicate the general environment of the side chain: I (internal) signifies e completely buried side chain. S (surface) signifies a side chain, some, but not all, of whose atoms are buried or which is accessible to liquid on one side only. E (external) sigtzifies a side chain which projects into the surrounding liquid. Columns 4 and 7 list particular features of the environment of the side chains in lysozyme and a-lactalbumin, respectively. Underlining is used as before if the residue is the same in both enzymes. If the residue changes, this is listed and a second name given in brackets after the residue number. Note that for example, residue 9 (Ala in lysozyme, Phe in a-lactalbumin) is listed as Ala 9 (Phe) when it appears in column 4 and Phe 9 (Ala) when in column 7. The terms suwounded by, adjacent to, neighbours are used to indicate that the groups so described are close together with no other atoms intervening; they are not necessarily in van der Waals’s contact. Column 7 normally only lists differences resulting from the amino acid changes.

POSSIBLE

STRUCTURE

OF a-LACTALBUMIN

81

The side chain chsnges of internal residues tend to be co-or&ted and to preserve the hydrophobic character of those regions that are not accessible to water from the exterior. There are interesting contrasts between, for example, the groups of side chains around residue 28 and around residue 98, which are mostly unchanged in the two proteins, and the region around 12,55 and 56, where most side chains are changed in an inter-related fashion. The latter region forms an extensive hydrophobic core which includes the following amino acids: Phe 3 (Leu), Leu 8 (Val), Met 12 (Leu), Leu 17, Trp 28, Ile 55 (Leu), Leu 56 (Phe), Ile 88 (Leu), Val92 (Ile), Ala 95 (Val), -108. In each molecule there is the sa.me number of Trp and Phe and, since Leu and Ile have similar molar volumes, the difference in the volumes occupied by these eleven side chains can be estimated from the difference between the molar volumes of the side chains of Ala + Met (lysozyme) and Leu + Val (a-lactalbumin). This difference, between 82.6 cm3/mole and 118.9 cm3/mole, is 36.3 cm3/mole, which amounts to about 5% of the total volume of this hydrophobic region and corresponds to a net increase equivalent to the volume of two methyl groups. This can resdily be accommodated by slight rearrangement of other neighbouring side chains. The Pro residue that replaces Gly at position 26 occurs as the second residue of a helical stretch, a situation which has been found to be common in globin chains (Perutz, Kendrew & Watson, 1965). Although Pro cannot be incorporated in the middle of a helix, it may be an effective helix initiator, perhaps because the dihedral angle + about the Cu-N bond is constrained by the pyrrolidine ring to be approximately 120”, the value characteristic of the u-helix. Of the various changes involving Gly residues, several (16, 19, 21, 49, 102) occur near deletions and may therefore be associated with changes in the main chain conformation to or from situations where the dihedral angles of Ca do not permit a non-Gly residue. Several of the Gly that are homologous could only be replaced if the main chain conformation were changed; Gly 54 is an example where the dihedral angles of Cu are impossible for a non-Gly residue and there is insufficient room for a CJ3atom unless residue 83 is changed to Gly. In contrast to the interrelated changes of internal side chains, the replacements of external side chains appear generally to be unto-ordinated. There are, however, a number of changes that considerably affect the shape of the surface cleft that has been identified in lysozyme as the site of substrate binding. In particular, it has already been mentioned that the changes 102 Gly --f Lys, 103 Asp -+Val and 107 Ala -+ Tyr, together with the deletion of 100, appear to be conducive to the formation of a hydrophobic nucleus and such a grouping, according to our model, would block the end of the cleft normally occupied by sugar residues A and B of the substrate. Even if an alternative rearrangement is made of the main chain in this region, the replacement of 107 Ala -+ Tyr alone tends to block access to this part of the cleft. Although the charged side chains in u-lactalbumin are located on the surface and appear to be accessible to water as in lysozyme, they are distributed rather differently. It is curious that each of the eleven arginyl residues of lysozyme are changed; four are replaced by lysine, two by glutamic acid, three by uncharged residues, and two are deleted. In all, there are only two fewer basic residues in a-lactalbumin, but the close clusters of basic groups, Arg 45 and 68 in one region, Arg 61 and 73 in a second, and Arg 5, 125 and 128 in another, which form highly positively charged surface regions in lysozyme, are not found in h&albumin. Although the exact number of glutamyl

82

W. ‘J.

BROWNE

ET

AL.

and aspartyl residues and their amides has not yet been established, there must be more acidic groups than in lysozyme since the isoelectric point of a-lactalbumin is 5.1, compared with 10.1 for lysozyme. In addition to the salt bridges between Lys 13 and the terminal carboxyl group, and between Lys 97 and Asp 101, which we presume to be maintained, a number of additional pair&f residueswith opposite charges less than 5 or 6 A apart are possible in lactalbumin, as follows: Lys 18 and Lys 96 -+ Asp 16, Arg 10 -+ Glu 11, Lys 82 -+ Asp 81, His 111 -+ Glu 27, Lys 118 alu 117 or Asp 120. The lysozyme interactions Lys 1 (Glu) + Glu 7 and His 15 (deleted) --f Asp 87 are no longer possible. ?ktil the crystal structure analysis has been carried out, the predicted conformation of a-lactalbumin may perhaps best be tested against studies of the physical and chemical properties of the protein in solution. Robbins et al. (1967) have shown that the four tyrosyl residues of a-lactalbumin ionize normally on H+ titration. Reaction with acetylimidazole also reveals four normal tyrosyl groups (Gorbunoff, 1967). In contrast, the reaction of cyanuric fluoride with a-la&albumin suggests that one of the four tyrosyl groups may be unavailable to this reagent and, thus, that it is located in a somewhat more inaccessible region than the other groups. Inspection of the model shows that the four tyrosyl side chains (20, 38, 53, 107) are located near the surface but that the phenolic hydroxyl of Tyr 38 may be rather less accessible than the others. Solvent perturbation studies (Kronman, Blum & Holmes, 1966; Kronman, 1968) suggest that two of the fourt tryptophan residues are buried and two are exposed at 25°C. All four groups become accessible only when the protein is denatured by reduction of the disulphide bonds and treatment with urea. The two exposed groups become inaccessible to all but the smallest solvent molecules when the temperature is reduced to 1°C suggesting that they lie in surface crevices which change shape as the result of a conformational change of the molecule. In our model, Trp 28 is completely buried and the indole group of Trp 123 is probably accessible tosolvent as it is in lysozyme. Trp 108, which lies inxe active cleft, appears to be accessible. Trp 63, which also gin the active cleft and is accessible in lysozyme, is probably inaccessible in c+lactalbumin as a consequence of the replacement 107 Ala --f Tyr. Our model is therefore consistent with there being two buried groups and two exposed, and it is quite credible that the exposed groups become less accessible as a result of a relatively small conformational change. Comparison of the optical rotatory dispersion of a-lactalbumin and lysozyme would seem to suggest that the two molecules have similar structures. Earlier dispersion studies (Kronman et al., 1966) were not designed to compare a-lactalbumin and lysozyme directly and it is difficult to relate the published data for the two proteins. More recent studies by Aune (1968) reveal that in aqueous solutions (pH 4.5 for lysozyme, pH 6.1 for a-lactalbumin, O-1 M-KCl, 25%) the optical rotatory dispersion curves of lysozyme and a-lactalbumin are indistinguishable between 206 and 233 mp. There is a large difference in the region of aromatic absorption between 250 to 320 rnp, but at wavelengths over 320 rnp or below 250 rnp the curves approach identical rotation values. Although analysis of these data is incomplete, perhaps the observed differences can be ascribed to the different numbers of aromatic chromophores in f At the time of these experiments residues.

the molecule

we-3 believed

to include

five tryptophan

POSSIBLE

STRUCTURE

OF a-LACTALBUMIN

83

each protein. Similar conclusions have been drawn recently by Kronman (1968) on the basis of optical rotatory dispersion and circular dichroism measurements. 4. The Active

Site

The activity of lysozyme is to catalyse the hydrolysis of glycosidic bonds in the alternating /3(14) linked copolymer of N-acetyl-n-glucosamine and N-acetyl-nmuramic acid which forms one of the principal constituents of bacterial cell walls. Lysozyme cleaves the C!-0 bond following a residue of N-acetylmuramic acid. Lysozyme also cleaves the corresponding bond following any residue in chitin, the /3(14) linked homopolymer of N-acetylglucosamine. Furthermore, lysozyme is capable of catalysing transglycosylation reactions involving oligomers of the cell wall and chitin saccharides. Studies by chemical, spectroscopic and X-ray crystellogmphic methods (see, e.g. the review by Johnson, Phillips & Rupley, 1969) have led to a model of the mode of binding of substrate molecules to lysozyme and of the factors responsible for catalytic activity (Blake, Johnson, Mair, North, Phillips & Sarma, 1967). Six sugar residues appear to be bound in the surface cleft of lysozyme, designated A to F (going towards the reducing end of the polysaccharide chain) (see Plate II). Hydrolysis appears to take place between residues D and E as a result of the concerted action of Asp 52, Glu 35 (Thr) and steric factors that affect the conformation of ring D. TaG3 is a list of the interactions between the lysozyme molecule and the substrate in the model and shows which of these interacting groups are different in a-lactalbumin. It is apparent thet, while a number of interacting groups are completely changed, very many remain the same. On the assumption that residues 57 and 59 are amidated in a-lactalbumin, as they are in lysozyme, the only contacts with sugar residue C that are affected are those with residue 62 (Trp -+ He), and the only changed contacts with residue D are due to the replacement of Glu 35 by Thr. Nevertheless, the effect of the changes in the upper part of the substrate cleft appears to be to block off sites A end B. This is largely a consequence of the replacement of Ala 107 by Tyr; unless the main chain conformation is changed, it appears impossible to place the phenol ring of this residue in a position that does not prevent access to the upper pitrt of the cleft. Moreover, as described above, the deletion of residue 100 and changes in several of the neighbouring amino acids make a rearrangement of this part of the main chain, with a grouping together of several hydrophobic side chains, highly plausible. Consequently, instead of running completely across one face of the enzyme and so allowing the enzyme to be attached to any part of a polysaccharide, the surface cleft in our a-la&albumin model is blind, allowing binding of only a small oligosaccharide or of the end of a longer substrate. Even if our model is incorrect in detail, the changes in the neighbourhood of residue 100 make it unlikely that sites A and B would remain attractive saccharide binding sites in a-lactalbumin. The most highly specific interaction between lysozyme and its substrate involves the acetamido side chain of residue C which makes both hydrogen-bonded and nonpolar contacts within the enzyme cleft. These contacts appear to remain available in a-lactalbumin, though a sugar ring attached to an acetamido side chain bound to them would have to have a different orientation from that of residue C of the lysozyme substrate, again because of the replacement of Ala 107 by Tyr. Alternative methods

84

W. J. BROWNE TABLE

Interactions Sugar residue

between substrate hexasacchuride

Potential hydrogen Substrate group

ET

AL.

3 (residues

bonds ~3.26 A Enzyme group

A to F) and lysoxyme molecule Other atoms of enzyme within of substrate atoms

4A

A

N

(2)

Asp 101 -

OS2

Asp 101 Asp 103 (Val)

O’, cy, 061, 062 061,032

B

0

(6)

Asp 101 -

032

Trp 63 Asp 101 Asp 103 (Val)

Crl, ccl,

CT

w, CY, 032

022

Gln 57 (Glx) n0 58 Asn 59 (Asx) Trp 62 (Ile) Trp 63 El07 (Tyr) Trp 108 -

O’, ccc N, ‘7, Ca,C/RCy2 N, Ca,CP NC, C61, Ccl NE, C61, Ccl

Glu 35 (Thr) Asp 52 ?%57 (Glx) Am 59 (Asx) Trp 108

cs, OEZ c/l, cy, 061, OS2 O’, c’, c/3 C/3, Cy, NO32 Cy, C31, Nr

C

033) N (2) 0 (7)

Trp 62i(Ile) Ala 107 (Tyr) Am59 (Asx)

Ns 0’ N

D

0 (6) 0 (‘3)

Asp 52 x57 (Glx)

062 0’

E

0 (3) 0 (4) 0 (7) N (2)

A8p 52 Glu35(Thr) Asn 44 (Val) Glu 35 (Thr)

062 Glu OEl Am N061, NO62 Asp 0’ ?iiii Val Ala

F

0 (6) 0 (7)

Phe 34 (His) Arg 114 (La)

Notation

0’ NT1

35 (Thr) 44 (Val) 52 57 (Glx) 109 (Leu) 110

Phe 34 (His) Glu 35 (Thr) Arg 114 (Leu)

O’, cg Ne, Cy, C31, C62, Ccl, cg1

O’, cs, Cj3, Cy, cg, cy, Cy, CS,

Ocl NON, OS2 Noel

NO62

cl% CYL CY2 N C’, 0’ C’, 0’ C[, N$

aa in Table 2.

of binding which still make use of the acetamido side chain interactions are, in fact, observed in lysozyme, where the a anomers of mono- and disaccharide inhibitor molecules (in which the configuration at the reducing centre C, is such as to make the hydroxyl at this position axial to the sugar ring in the chair conformation) bind differently from the /I anomers (in which this hydroxyl is equatorial) and longer oligosaccharides (Blake, Johnson, Mair, North, Phillips & Sarma, 1967). The lower part of the cleft, where residues E and I! bind in lysozyme, is changed in detail, both in topology and in nature of the surface groups; any changes in the main chain conformation near residue 35 would cause considerable further changes in the

POSSIBLE

STRUCTURE

85

OF a-LACTALBUMIN

shape of this region of the cleft. While it would appear from our model that this part of the cleft in a-la&albumin might be able to bind saccharides, the precise mode of binding would almost certainly be different from that in lysozyme. Moreover, while one of the lysozyme residues that is directly involved in hydrolysis, Asp 52, is invariant, the other, Glu 35, is changed. However, a histidine residue is prez in the corresponding or a neighbouring position in a-lactalbumin.

5. The Activity

of a-Lactalbumin

The close similarity between the amino acid sequences of hen egg-white lysozyme and bovine a-la&albumin strongly suggests that the genes for lysozyme and a-lactalbumin may have been derived from a common ancestor and that, by a process of duplication, the ancestral gene gave rise to two genes which subsequently evolved independently (Brew et al., 1967). Our model building auggests that the similarity in amino acid sequence implies a similarity in molecular conformation. Moreover, the changes in the active site region suggest that a-lactalbumin might have a function related to, but somewhat different from, that of lysozyme. The apparent structural similarities between a-lactalbumin and lysozytne have given surprisingly little insight into the nature of the activity of a-lactalbumin. Indeed, a-lactalbumin has not been found to possess a catalytic activity but appears to act as a modifier or “specifier” protein (Brew et al., 1967). Present knowledge of the action of u-lactalbumin has been reviewed by Hill, Brew, Vanaman, Trayer & Mattock (1969). To summarize briefly, the enzyme system lactose synthetase catalyses the final step in the biosynthesis of lactose in the mammary gland by the reaction: UDP-D-galactose

lactose $ UDP.

(1) Lactose synthetase can be separated by gel filtration into two protein components, the A and B proteins, neither of which alone will promote the synthesis of lactose, as they will in combination. The B protein has been identified as u-lactalbumin (Brodbeck, Denton, Tanahashi & Ebner, 1967). Brew, Vanaman & Hill (1968) demonstrated that the A protein is a galactosyl transferase which catalyses the reaction: UDP-n-galactose

$

D-@UCOSe

+ N-acetyl-n-glucosamine

--f

-+ N-acetyl-lactosamine

f UDP. (2)

A simple explanation of the lactose synthesis process would be afforded if a-lactalbumin catalysed the glycosyl transfer reaction: N-acetyl-lactosamine

+ n-glucose + lactose + N-acetyl-n-glucosamine.

(3) Such a reaction would be quite consistent with the picture that we have developed from our model; in particular, the abbreviated cleft in a-lactalbumin would be appropriate for interaction with a disaccharide substrate, and the reaction is closely related to that of lysozyme. However, this simple explanation is clearly ruled out by the experimental observations of Brew et al. (1968) that a-lactalbumin shows no activity towards N-acetyllactosamine. Moreover, their evidence suggests that a-lactalbumin acts by modifying the substrate specificity of the A protein, so that, broadly speaking, the A protein catalyses reaction (1) in the presence of a-lactalbumin and reaction (2) in its absence. We are therefore left with the dilemma that although the model-building experiments lead us to anticipate a related activity for u-lactalbumin and lysozyme, and although they are known to be involved in closely similar types of reaction, biochemical .‘experiments suggest that they act in quite disparate ways. The dilemma can

86

W. J. BROWNE

ET

AL.

only be resolved by a more complete understanding of the function and structure of u-lactalbumin. An independent determination of the structure by X-ray crystallography has now been started (Aschaffenburg, Handford & Phillips, manuscript in preparation). REFERENCES Aune, K. (1908). Ph.D. Dissertation, Duke University. Blake, C. C. F., Johnson, L. N., Mair, G. A., North, A. C. T., Phillips, D. C. $ Sarma, V. R. (1967). Proc. Roy. Sot. B,167, 378. Blake, C. C. F., Mair, G. A., North, A. C. T., Phillips, D. C. & Serma, V. R. (1967). Proc. Roy. Sot. B,167, 365. Brew, K. & Campbell, P. N. (1967). B&hem. J. 102, 258. Brew, K., Vanaman, T. C. & Hill, R. L. (1967). J. B&Z. Chem. 242, 3747. Brew, K., Vanaman, T. C. & Hill, R. L. (1968). Proc. Nat. Acad. Sci., Wash. 59, 491. Brodbeck, U., Denton, W. L., Tanahashi, N. & Ebner, K. E. (1967). J. Biol. Chem. 242, 1391. E&all, J. T., Flory, P. J., Kendrew, J. C., Liquori, A. M., Nemethy, G., Ramachandran, G. N. & Scheraga, H. A. (1966). J. Mol. Biol. 15, 339. Gorbunoff, M. J. (1967). Biochem&ry, 6, 1606. Hill, R. L., Brew, K., Vanaman, T. C., Trayer, I. P. & Mattock, P. (1969). Brookhaven Symp. in Biology, no. 21, vol. 1, p. 139. Johnson, L. N., Phillips, D. C. & Rupley, J. A. (1969). Brookhaven Symp. in Biology, no. 2 1, vol. 1, p. 120. Kronman, M. J. (1968). B&hem. Biophys. Res. Comm. in the press. Kronman, M. J., Blum, R. & Holmes, L. G. (1966). Biochemidry, 5, 1970. Perutz, M. F. (1965). J. Mol. BioZ. 13, 646. Perutz, M. F., Kendrew, J. C. & Watson, H. C. (1965). J. Mol. Bd. 13, 669. Perutz, M. F., Muirhead, H., Cox, J. M. & Goaman, L. C. G. (1968). Nature, 219, 131. Prothero, J. W. (1966). Biophys. J. 6, 367. Ramachandran, G. N., Ramakrishnan, C. & Sasisekharan, V. (1963). J. Mol. BioZ. 7, 95. Robbins, F. M., Andreotti, R. E., Holmes, L. G. & Kronman, M. J. (1967). Biochim. Btiphye. Actu, 133, 33. Venkatachalam, C. M. (1968). Biopolymers, 6, 1425.