Theoretical Studies on Peptidoglycans. 11.
Conformations of the Disaccharide-Peptide
Subunit and the Three-Dimensional Structure of
Peptidoglycan*
R. VIRUDACHALAM and V. S. R. RAO, Molecular Biophysics U n i t ,
Indian Institute of Science, Bangalore-560012, India
Synopsis
Possible conformations of the disaccharide-peptide subunit of peptidoglycan (of Staphylococcus aureus or Micrococcus luteus)have been studied by an energy-minimization procedure. The favored conformation of the disaccharide N-acetyl-glucosaminyl-@(
1-4)-Nacetylmuramic acid (NAG-NAM) is different from that of cellulose or chitin; this disagrees
with the assumption of earlier workers. The disaccharide-peptide subunit favors three types
of conformations, among which two are compact and the third is extended. All these conformations are stabilized by intramolecular hydrogen bonds. Based on these conformations
of the subunit, two different models are proposed for the three-dimensional arrangement of
peptidoglycan in the bacterial cell wall.
INTRODUCTION
Peptidoglycan is a netlike macromolecule that surrounds the plasma
membrane of bacterial cells. It imparts rigidity to the cell wall and protects
the organism from internal osmotic pressure. Interference with its synthesis or its removal results in the loss of this rigidity and leads to the formation of a fragile protoplast or spheroplast, which under normal hypotonic
conditions swells and bursts.
This macromolecule essentially contains glycan chains interconnected
with short peptides. The disaccharide N-acetylglucosaminyl-P(1-4)-Nacetylmuramic acid (NAG-NAM) is the repeating unit of the glycan strand
(Fig. l),and the peptide that is linked to NAM is a tetrapeptide. Prior to
the last step of the biosynthesis of peptidoglycan, the peptide is actually
a pentapeptide, with the general sequence L-Ala*-y-D-Glu2-L-R3-D-Ala4~ - A l a ~The
. residue a t position 3 differs from bacteria to bacteria (lysine
in Staphylococcus aureus or Micrococcus luteus and diaminopimelic acid
in Escherichia coli), and minor variations a t position 1 are also observed.
Figure 1 summarizes the observed variations in the pentapeptide moiety
of peptidoglycan from various bacteria.
* Part of this paper was presented a t the International Symposium on Biomolecular
Structure, Conformation, Function and Evolution held a t Madras, India, January 4-7,
1978.
Biopolymers, Vol. 18, 571-589 (1979)
01979 John Wiley & Sons, Inc.
0006-3525/79/0018-0571$01.00
572
VIRUDACHALAM AND RAO
R j = L-Ornithyl, L-2-L-diomino NH
butryl, L-Lysyl, moss0
I
or L L diominopimolyl
n-$-R3
----_--__-_____-I
D - Alonino
______________-__I
0
-
Alonino
-____----_-___
L
(3)
o
(41
--_________
to
YH
u3c-c-n
--________
CO
YH
H3C-C-H
D
(51
-Coon
--_--___-_
Fig. 1. Disaccharide-peptide subunit of peptidoglycan and the observed variations in
the pentapeptide moiety.
In recent years the conformation of peptidoglycan has drawn the attention of several investigators, and various models have been
In the model of Tipper' the glycan strand was assumed to be in a chitinlike
conformation and the pentapeptide in a folded conformation. The loss
of the intramolecular hydrogen bond (as compared with chitin or cellulose)
between NAG and NAM can be compensated by a hydrogen bond between
the carbonyl group of the lactyl residue of NAM and the CH2OH group of
NAG.
In the model of Keleman and Rogers2the glycan strand of peptidoglycan
was also assumed to be in a chitinlike conformation. A pseudo-P-conformation was proposed for the tetrapeptide, since it cannot assume a purely
a-helical or @-conformationdue to the occurrence of L and D residues and
the unusual y-linkage (Fig. 1)between the D - G ~ and
u the L-LYSresidues
of the peptide. To obtain a three-dimensional structure, the glycan strands
were stacked in two separate parallel planes such that the strands in the
two planes are antiparallel to each other and are in alignment. The peptides from the two sets of glycan strands facing each other are crosslinked
to form a netlike structure. Extensive hydrogen bonding was assumed
between the stacked rings in the glycan portion of the model, and also between the extended peptide chains crosslinking the two stacks. This model
THEORY OF PEPTIDOGLYCANS. I1
573
presents peptidoglycan as a fundamentally layered and ordered structure.
Recently, Oldmixon et al.3 have considered two models for the disaccharide-pentapeptide. Their models differ from the earlier models mainly
in the conformation of the peptide portion. Depending on the conformation of the pentapeptide, one model is termed as “extended” and the other
as “compact.” In the extended model the pentapeptide is fully extended
and projects away from the surface of the glycan chain. The shape of the
pentapeptide in the extended conformation resembles a T in which the
D-Lac-L-Ala-y-D-Gluportion forms the stem and the crossbridging portion
(lysine side chain and D-Ala-D-Ala)forms the crossbar. It does not possess
any stabilizing attachments with the glycan strand. In the compact model
the pentapeptide is pressed to the surface of the glycan strand, which is
stabilized by hydrogen bonds.
According to these authors,3 the three-dimensional appearance of the
peptidoglycan layer is such that the glycan strands form one plane, over
which the crossbridging peptides form another parallel plane, the axis of
the glycan strand being perpendicular to the axis of the crossbridge. Thus,
if layers of such a model are placed one over the other, there will be alternating layers of peptides and saccharides resisting tension in different directions.
Formanek et aL4have also proposed two models, A and B, for the subunit
of peptidoglycan. In both these models the pentapeptide is arranged such
that all the NH and CO groups are involved in hydrogen-bond formation.
Model A differs from B only in the conformation of the lactyl residue. The
nature of the stacking of the glycan strands is assumed to be like that of
chitin.
In all four different models described above, the glycan strand is assumed
to be in a chitinlike conformation, and they differ only in the conformation
of the peptide moiety. It is well known that the chitin or cellulose structure
is stabilized by an intramolecular hydrogen bond between the ring oxygen
atom of the ith residue and the hydroxyl group at the C3 atom of the (i
1)th residue. Since the above type of hydrogen bond cannot form between
NAG and NAM, it is unlikely that the glycan strand of peptidoglycan assumes a chitin- or cellulose-like conformation. Moreover, none of these
models are substantiated by experiment or theory. Hence, in the present
paper an attempt has been made to study the possible conformations of
the subunit of peptidoglycan (of M . luteus or S. aureus) by theoretical
method^,^ and their arrangement in three dimensions is discussed.
+
Nomenclature, Geometry, and Fractional Charges
The numbering of the atoms and the dihedral angles of the disaccharide-pentapeptide are shown in Fig. 2. The atoms used to define the dihedral angles of NAG-NAM are listed in Table I. The definitions of the
backbone and side-chain rotational angles of the peptide segment are ac-
574
VIRUDACHALAM AND RAO
Fig. 2. Numbering of atoms and dihedral angles in the disaccharide-peptide subunit of'
peptidoglycan.
cording to the IUPAC-IUB nomenclature.6 Clockwise rotation is considered as positive.
The coordinates of the atoms of the disaccharide-pentapeptide were
generated using standard structural parameters.7-9 The sugar rings were
kept in the 4C1 conformation and the peptide units in the transplanar
conformation.
The fractional charges on the atoms of the disaccharide NAG-NAM were
calculated by the molecular orbital
T h e charges for the
peptide segment were taken from reported value^.'^,'^ Figure 3 shows the
charge distribution in the disaccharide-pentapeptide.
THEORY OF PEPTIDOGLYCANS. I1
575
TABLE I
Atoms Used to Define the Dihedral Angles for NAG-NAM
Dihedral Angle
Atoms Defining Angle
THEORETICAL
Steric Maps
I t can be seen from models that the CBsubstituent of muramic acid will
hinder the orientation of the preceding NAG but not the succeeding one.
Hence the preferred conformation of NAM-NAG will be the same as that
of the sugar residues in chitin or cellulose. The detailed analysis was
therefore restricted to NAG-NAM.
The effect of the lactyl residue on the orientation of NAG, and vice versa,
was investigated by constructing various (x,,cpo) steric maps for fixed values
of (@, p); I,P) were fixed a t all the allowed values of cellobiose. The
allowed sets of (@, p)are shown in Fig. 4. While constructing these maps,
only atoms up to Co in Fig. 2 were considered.
In the second step the disaccharide with one peptide unit (up to C;' in
Fig. 2) was considered and (a,
#o) maps were constructed for various
combinations of @, I,P and xj. (Though the range -80" to -150" is allowed
for xk, -100" and -145' are energetically favored.) The maps corresponding to two sets of (@, p,xi) are shown in Fig. 5. In all these calculations the dihedral angles were varied at 10" intervals, and the maps were
constructed according to the contact criteria."
(a,
Potential Energy Calculations
The potential energy of the molecule was computed considering nonbonded, electrostatic, and torsional contributions. The form of the functions and the constants used are the ones reported by Momany e t aL9
The disaccharide-pentapeptide can assume a large number of conformations, since many rotational angles are involved in specifying its conformation. Therefore, a systematic analysis by varying these angles a t
discrete intervals is not practical with available computer facilities. T o
simplify the calculations, the molecule was divided into three fragments;
576
VIRUDACHALAM AND RAO
H 10.2971
10.2OL1 H
n
Fig. 3. Charge distribution in the disaccharide-peptide subunit of peptidoglycan (in
fractions of an electronic charge).
the disaccharide NAG-NAM with one peptide unit (up to Cy in Fig. 2 ) is
considered as the first fragment (I),the segment from C$ to C$ as the second
fragment (TI), and the remaining portion of the pentapeptide as the third
fragment (111). The probable conformations of each of them was studied
by the Fletcher-Powell-Davidon minimization procedure.16J7
The selection of the starting conformations of fragment I for minimization was made by combining various allowed values of (@, GS),xi,and (PO,
$0). T he remaining dihedral angles of NAG-NAM were fixed in the minimum energy conformation as found in the monosaccharides (unpublished
results). About 70 conformations were minimized, and most of them lead
to three different low-energy conformations (Table 11).
THEORY OF PEPTIDOGLYCANS. I1
577
TABLE I1
Some Minimum Energy Conformations for Fragment I
S1. No.
8
v
x3
m
$0
1
2
3
52
58
64
2
-5
10
-106
- 106
-145
152
155
79
37
-150
-156
Relative
Energy
(kcal mol-')
0.00
1.48
1.68
For fragment I1 a combination of five sets of (cpl, $1) (corresponding to
the five low-energy conformations of N-acetyl "-methyl L-alanyl amidel9
and seven sets of ((a, xi,xl,x:) (corresponding to the first seven low-energy
conformations of N-acetyl "-methyl L-glutaminyl amidel9 were considered as the starting conformations for energy minimization. The angle
$2 was fixed a t -90" and -150". Similarly, in fragment 111, five sets corresponding to the low-energy conformations of N-acetyl "-methyl Lalanine amide (sign changed for D residue) were considered for ( ( ~ 4 ,$4) and
(p5, +5), and combinations of them were minimized.
Subsequently, a few low-energy conformations of fragments I1 and I11
1
/
-f
-30"
-60"
-90'
t
1
-60"
1
- 30"
I
O0
@3=
1
30'
1
SO'
90'
Fig. 4. Steric map of the disaccharides cellobiose and NAG-NAM. Regions A and B are
allowed for cellobiose and A alone is allowed for NAG-NAM; 0 , observed conformation of
cellulose or chitin.
578
VIRUDACHALAM AND RAO
00
Fig. 5. Steric map of the lactyl residue in NAG-NAM for fixed values of (@, J.", xi). Contours enclosed by the solid line correspond to (@, J.", xi) = (50°, O", -looo) and the dashed
line to (60°, lo", -145'). The 27a and 27b conformations of the lactyl residue are indicated
by the solid and open circles.
were selected, and combinations of them were considered as the starting
conformations for the pentapeptide. Since the torsional angles of lysine
$3) are not included in either of the fragments, three different
residue (a,
starting values-namely, (-60°, -60°), (-150°, 150°), and (-SO0, SO0)
-were considered. The energy of the pentapeptide was minimized with
respect to backbone angles, fixing the side groups in their favored positions.18-20 In all, about 100 conformations have been minimized, and only
those conformers whose energy is within 3 kcal mol-' are given in Table
111.
T o study the probable conformations of the disaccharide-pentapeptide,
many different low-energy conformations of the pentapeptide were combined with the low-energy conformations of the disaccharide and the energy
was minimized. In all, about 50 conformations were minimized, and only
the first 20 in the increasing order of energy are given in Table IV. Minimization of each conformation required about an hour of IBM 360144
computer time.
T H E O R Y OF PEPTIDOGLYCANS. I1
579
RESULTS AND DISCUSSION
Conformation of NAG-NAM
Figure 4 indicates that the allowed conformations of NAG-NAM are
highly restricted compared to cellobiose. Further, the favored conformation of cellulose or chitin (@ N 30", $s N -30") is disallowed for
NAG-NAM, suggesting that the glycan strand of peptidoglycan cannot
assume a conformation similar to that of chitin or cellulose. This contradicts the a s ~ u m p t i o n l -that
~ such a conformation is possible. Energy
calculations (Table 11) show that (@, p)favors a value around (50°, 0").
I t is interesting to note that this conformation is close to the solid-state
conformation of acetyl cellobiose.21 Th e xi angle favors values around
-100" or -145" (Table 11), and the preferred values of (a,
$0) seem to
depend on xi (Fig. 5 and Table 11). When xi N -loo", (a,
$ 0 ) favors
values around (150°, 40") or (150°, -150"). In the former conformation
an intramolecular hydrogen bond between the N H of L-Alal and the
CH20H of NAG is possible, and in the latter conformation the CO group
of the lactyl residue can form a hydrogen bond with the CH20H of NAG,
$0) favors
similar to the one suggested by Tipper.l When xi N -145", (a,
avalue around ( B O O , -160°), and no intramolecular hydrogen bond is possible. For the two favored values of x$,the 27a and 27b type of conformations assumed by Formanek et al.4 for the lactyl residue are disallowed (Fig.
5). However, the 27b form is very close to the allowed region.
Conformation of the Pentapeptide
Table I11 shows that the energy of the pentapeptide is minimum when
the lysine side chain is fixed at x,: = -60". For xi = 60", the energy increases by a t least 3 kcal mol-'. The backbone angles of L-Alal and L-LYS"
mostly favor values around (-SO0, 80") or (-150°, 150°), and D-Ala4 favors
(SO", -80") or (150°, -150"). Th e terminal D-Ala residue assumes a
conformation around ( ( ~ 5 ,$5) N (150°, 30"). The *angle of D-Glu residue
mostly favors a value around 90". However, when $2 = -150°, cp2 also
changes to 150". Th e remaining dihedral angles are not significantly affected by the change in $2, but the energy of the molecule increases slightly.
The angles (xi,x;, xl) favor values around (180" or 60", 180°, -80").
Conformation of the Disaccharide-Pentapeptide Subunit
Table IV shows that the disaccharide-lactyl residue portion of the subunit can assume four different conformations. Three of them are similar
to the three low-energy conformations of fragment I (Table 11), whereas
the fourth one (conformers 1and 6 of Table IV) differs in (a,
$0); it prefers
a value around (150°, -40"). In this conformation, (M, $1) favors a value
around (-170", 60°), and an intramolecular hydrogen bond between the
NH of D - G ~ uand the CH2OH of NAG is possible. T h e general features
7
6
5
4
3
2
1
S1. NO.
-82
-85
-160
-150
-167
-150
-85
-85
-154
-150
-86
-80
-159
-150
'pi
P2
84
80
86
80
96
150
78
80
89
90
84
80
84
80
$1
83
80
162
150
156
150
78
80
157
150
78
80
166
150
X%
189
180
-172
180
185
180
-172
180
-174
180
-175
180
-169
180
X::
174
180
174
180
172
170
173
180
69
70
70
70
175
180
-79
-90
-79
-100
-80
-100
-89
-90
-88
-90
-87
-90
-80
-100
xi = -60'
Xl
-85
-80
-86
-80
-84
-80
-93
-80
-85
-80
-87
-80
-86
-80
m
79
80
78
80
82
80
65
80
83
80
80
80
75
80
$3
83
80
84
80
81
80
136
150
81
80
82
80
99
150
v4
-83
-80
-82
-80
-84
-80
-155
-150
-83
-80
-82
-80
-154
-150
$4
TABLE 111
Starting and Minimized Conformations* of the Pentapeptide for $2 = -90"
162
150
161
150
158
150
159
150
154
150
160
150
160
150
P5
26
30
29
30
28
30
22
30
30
30
30
30
25
30
$5
Vexb
2.24
2.24
2.11
1.48
1.22
0.64
0.00
(kcal mol-')
$?
0
$U
c
0
>
z
c
i;l
d
U
a
-83
-85
-156
-150
-151
-150
-156
-150
-87
-80
-160
-150
-162
-150
~~
170
180
174
180
176
180
71
70
83
80
84
80
148
150
89
90
83
80
159
150
154
150
160
150
~
68
60
66
60
175
170
84
80
92
90
89
150
76
80
162
150
159
150
190
180
189
180
188
180
194
180
172
180
172
160
--167
180
-85
-80
-88
-80
-99
-80
x:t = 1800
-87
-84
-90
-80
-85
-86
-100
-80
-95
-86
-100
-80
-85
-90
-90
-80
83
80
80
80
-89
-100
85
80
89
80
128
150
81
80
84
80
83
80
84
80
82
80
84
80
58
80
82
80
81
80
82
80
89
80
In each set the second row is the starting conformation and the first row is the minimized conformation.
Vexis the excess energy over the global minimum.
14
13
12
11
10
9
8
31
30
35
30
21
30
30
30
29
30
30
30
30
30
154
150
159
150
160
150
152
150
157
150
156
150
158
150
-81
-80
-77
-80
-166
-150
-85
-80
-82
-80
-84
-80
-89
-80
3.05
2.85
1.81
1.14
3.05
2.90
2.27
10
9
8
7
6
5
4
3
2
-3
-2
0
10
10
9
10
-3
0
-3
0
-3
-3
-9
9
10
-6
-2
10
10
48
46
66
60
65
65
54
51
55
55
46
60
65
60
66
66
55
50
51
50
1
$”
SI. NO. @
a
154
155
81
80
76
79
147
137
144
136
155
150
79
80
81
81
1:16
150
137
150
xh
-102
-100
-144
-145
-147
-146
-106
-101
-103
-101
-100
-100
-146
-145
-144
-144
-101
-100
-101
-100
-37
-38
-150
-160
-141
-146
30
43
33
49
-38
-140
-146
-160
-151
-150
49
40
43
40
$0
-169
-169
-88
-86
-83
-83
-102
-95
-107
-85
-169
-86
-83
-85
-88
-88
-85
-82
-95
-85
$1
51
57
73
78
88
86
69
81
69
82
57
78
86
78
73
73
82
83
81
78
$1
X:
68
72
69
70
174
176
172
172
172
174
72
70
176
17:1
67
69
174
174
172
173
@
76
78
82
84
78
82
81
78
79
78
78
84
82
78
81
82
78
84
78
78
196
188
-176
-175
169
169
177
-173
177
180
-172
-175
167
188
-174
-176
180
-171
-173
-172
xf
-76
-73
-78
-87
-80
-74
-81
-75
-81
-80
-73
-87
-74
-89
-81
-78
-80
-79
-75
-89
xi
-85
-84
-85
-87
-92
-88
-85
-90
-85
-82
-84
-87
-88
-93
-86
-85
-82
-85
-90
-93
ca:1
79
81
79
80
69
72
75
89
79
80
81
80
72
65
77
79
80
79
89
65
$3
96
84
81
82
94
93
90
84
88
88
84
82
93
136
90
81
88
83
84
136
$4
$4
-67
-84
-81
-82
-152
- 162
-72
-87
-74
-80
-84
-82
- 162
-155
-71
-81
-80
-8:1
-87
-155
TABLE IV
Starting and Minimized Conformations” of the Disaccharide-Pentapeptide
76
80
159
160
96
80
89
80
90
80
159
160
147
159
89
80
158
162
1:1u
159
e.5
$S
17
29
33
30
49
32
49
37
48
64
29
30
32
22
40
33
64
26
37
22
xi
-60
3.83
3.35
2.94
-60
-60
2.80
2.04
2.04
1.89
1.45
1.35
0.00
(kcalmol-’)
-60
-60
-60
-60
-60
-60
-60
Vexh
0
s
k
$u
i
F
r
il
R
5n
c
tl
'I
a
67
60
64
63
63
60
63
50
60
50
62
50
56
60
45
50
67
60
60
60
8
10
-1
-1
-1
0
5
0
4
0
-1
0
8
0
6
0
7
10
-5
0
-145
-145
-1 10
-100
-110
-100
-98
-100
- 104
- 100
-104
- 100
-117
- 100
-105
-100
-144
- 145
-105
- 100
150
137
150
137
150
136
150
I65
150
136
150
74
80
151
150
166
79
80
158
163
-144
-160
-41
-52
-52
-140
44
40
40
40
43
40
-84
-140
35
40
-152
-160
-137
-140
-166
-160
-162
-160
-160
-89
-87
-153
-156
-156
-82
-85
-83
-93
-86
-94
-87
-102
-85
-173
84
76
94
92
92
83
79
83
73
78
69
76
89
78
153
162
I61
162
162
162
88
84
81
84
74
84
80
83
82
84
81
84
70
78
83
86
84
86
83
86
63
68
174
173
173
174
175
170
70
70
68
68
177
173
174
174
173
174
173
174
170
172
178
172
172
-171
-179
-170
-177
-175
-174
172
171
-172
-175
-172
-177
-172
-177
-182
68
83
-76
-72
-72
-79
-79
-87
-78
-87
80
83
-77
-89
-77
-79
-78
-79
-79
-79
-90
-85
-81
-81
-81
-85
-82
-84
-84
-87
-85
-85
--108
-93
-84
-86
-75
-86
-85
-86
78
82
80
90
90
79
82
82
81
80
78
82
59
65
79
78
77
78
77
78
84
85
82
82
82
83
87
81
82
82
87
85
146
136
86
84
85
84
84
85
In each set the second row is the starting conformation and the first row is the minimized conformation.
V,, is the excess energy over the global minimum.
20
19
18
17
16
15
14
13
12
11
-77
-81
-86
-83
-83
-83
-80
-85
-82
-82
-75
-81
-161
-155
-79
-82
-79
-82
-79
-82
156
154
70
80
162
162
156
152
158
160
158
154
158
159
160
I61
159
161
159
161
34
31
44
-6
-6
26
31
30
30
30
44
31
32
22
29
29
30
29
30
29
0
U
'J)
z
0
3
.c
F
0
tl
2
7.52
'd
M
Y
'd
-60
7.12
-60
0
7.19
7.11
-60
a
4
80
4
-60
6.93
-60
6.70
-60
5.40
-60
5.95
5.38
-60
180
4.12
-60
584
VIRUDACHALAM AND RAO
of the pentapeptide conformation are nearly the same as those described
in the earlier section.
A cursory look a t Table IV shows that among the first 10 conformations,
where the energy is less than 4 kcal mol-l, the first four (sets 1-4)are typical
conformers, and the remaining six (sets 5-10) are similar to the first four.
For example, conformer 5 is nearly the same as 4,conformers 6-9 (or 10)
differ significantly from 1 4 ,respectively, only in the (ps angle. The change
in (p5 affects the orientation of the terminal D-Ala5, but the overall shape
of the subunit remains nearly the same. The projections of the first four
low-energy conformers are shown in Figs. 6-9. In all these projections the
molecule is projected onto the plane formed by the atoms Ci, Oi, and Ci.
For clarity, only the backbone atoms are shown.
Depending on the orientation of the pentapeptide with respect to the
glycan strand, the above conformers can be divided into three broad types:
(1)compact conformation 1, (2) compact conformation 2, and (3) extended
conformation.
Compact Conformation 1
In the compact conformation 1 (Fig. 6), the segment C,U CF rises above
---
the plane of the disaccharide (the molecule is viewed from NAG to NAM,
keeping the pentapeptide on the right-hand side), and the segment C$
CT moves towards NAG. At the C;l atom of lysine, the D-Ala-D-Ala segment
and the side chain of lysine move in opposite directions, such that they are
placed across the glycan strand from above. The following hydrogen bonds
are possible in this conformation:
(1) The NH of D - G ~ udonates its hydrogen to the CH2OH oxygen of
NAG.
(2) The NH of L-Ala donates its hydrogen to the carbonyl oxygen of the
N-acetyl group of NAM.
(3) The N,H, a t the ~ - G l donates
u
its hydrogen to the carbonyl oxygen
of L-Ala (27 type).
--.
c?
Fig. 6. Projection of conformer 1 of Table IV (compact conformation 1): 0 , carbon; 0,
oxygen; 8 , nitrogen.
THEORY OF PEPTIDOGLYCANS. I1
585
Fig. 7. Projection of conformer 2 of Table IV (compact conformation 2). Symbols as in
Fig. 6.
(4) The NH of D-Ala4 donates its hydrogen to the carbonyl oxygen on
the y-carbon of D-G~u( 2 7 type).
(5) The NH of D-Ala5 donates its hydrogen to the carbonyl oxygen of
L-LYS(27 type).
Another hydrogen bond between the carbonyl group of D-Ala4and the
CHzOH group of NAM is also possible when x; = 60".
Compact Conformation 2
In the compact conformation 2 (Figs. 7 and 8), the peptide segment
C;
Cy moves down the mean plane of the disaccharide, and the segment CI -C,* is oriented nearly parallel to the axis of the glycan strand. The
crossbridging portion of the peptide (side chain of lysine and D-Ala-D-Ala)
is placed below the glycan strand. The axis of the crossbridging portion
is approximately perpendicular to that of the glycan strand. Compared
to the compact conformation 1, the above conformation has about 1.4 kcal
mol-l higher energy.
Fig. 8. Projection of conformer 3 of Table IV (compact conformation 2). Symbols as in
Fig. 6.
586
VIRUDACHALAM AND RAO
The conformer shown in Fig. 8 differs from the one shown in Fig. 7 only
in the orientation of the crossbridging portion of the peptide; in the former,
the direction of the crossbridge is opposite to that of the latter. Both these
conformers are stabilized by the following hydrogen bonds:
(1)The NH of D - G ~donates
u
its hydrogen to the carbonyl oxygen of the
lactyl residue (27 type).
(2) The N,H, a t the D-G~udonates its hydrogen to the carbonyl oxygen
of L-Ala (z7 type).
(3) The NH of D-Ala4 donates its hydrogen to the carbonyl oxygen on
the y-carbon of D-Glu (27 type).
In the conformer shown in Fig. 7, a 27-type hydrogen bond between the
NH of D-Ala5 and the carbonyl oxygen of L-LYSis also possible.
Extended Conformation
Conformer 4 of Table IV has about 2 kcal mol-' higher energy and is an
example of the extended conformation (Fig. 9). In this conformation,
atoms up to Cg of the peptide assume an extended conformation and move
away from the disaccharide. At the C$ atom, the D-Ala-D-Ala segment and
the lysine side chain (crossbridging portion of the peptide) move in opposite
directions, such that the axis of the crossbridging portion is nearly perpendicular to the axis of the glycan strand. This conformation is stabilized
by the following hydrogen bonds:
(1) The NH of L-Ala donates its hydrogen to the CHzOH oxygen of
NAG.
(2) The N,H, a t D-G~udonates its hydrogen to the carbonyl oxygen of
L-Ala (27 type).
(3) The NH of D-Ala4 donates its hydrogen to the carbonyl oxygen on
(27 type).
the y-carbon of D - G ~ u
CLa
Fig. 9. Projection of conformer 4 of Table IV (extended conformation). Symbols as in
Fig. 6.
THEORY OF PEPTIDOGLYCANS. I1
587
(4)The NH of D-Ala5 donates its hydrogen to the carbonyl oxygen of
L-LYS(27 type).
In conformer 20 of Table IV the type of hydrogen bond proposed by
Tipper1 is possible, but the peptide assumes an extended conformation
instead of a folded conformation. However, it is energetically unfavorable.
The energy of a few conformations with L-D-bend in the L-Ala-D-Glu segment and the pentapeptide in a conformation similar to that proposed by
Formanek et al.4 have also been calculated. But all of them lead to high
energy.
Hence the first four conformations of Table IV or slight modifications
of them seem to be most probable for the disaccharide-pentapeptide.
Though the overall shapes of the above conformations appear to be similar
to the “compact” and “extended” models of Oldmixon et al.,3 they differ
significantly in detail and in the hydrogen-bonding scheme.
Three-Dimensional Structure of Peptidoglycan
Based on the three types of conformations described in the earlier section,
two different models are proposed for the arrangement of peptidoglycan
in cell walls.
Covalently Linked Monolayer Structure
In Fig. 10, the repeating unit is assumed to be in the compact conformation 1, and the glycan strands are arranged parallel to one another in
the same plane. The crossbridgingportion of the peptide (lysine side chain
Fig. 10. Covalently linked monolayer structure of peptidoglycan.
588
VIRUDACHALAM AND RAO
and the D-Ala-D-Ala segment) crosses the glycan strand from above and
can be crosslinked to the neighboring strands as indicated. This leads to
an infinite sheet in which the glycan strands form one layer and the crosslinked peptides form another layer. Compact conformation 2 or the extended conformation also leads to similar arrangements. When the subunit
assumes the compact conformation, the mean plane of the pyranose ring
in the above arrangement of peptidoglycan is approximately in the plane
of the glycan strands; whereas in the extended conformation, it will be
approximately perpendicular. To form a thick peptidoglycan layer in the
cell wall it may be imagined that many monolayers are stacked one over
the other, as was pointed out by Oldmixon et al.3 The multilayer arrangement may be stabilized by secondary interactions between the
layers.
Covalently Linked Multilayer Structure
Figure 11 shows another arrangement that leads to a covalently linked
multilayer structure for peptidoglycan. In this arrangement the glycan
strands run through the edges and the axis of a hexagon, and the repeating
unit (disaccharide-peptide) assumes the compact conformations 1 and 2
alternatively. The seven strands shown in Fig. 11 form three layers-a,
b, and c. The strands (1 - l’, 2 - 2’ and 4 - 4’,5 - 5’) running on the edges
of the longitudinal faces of the hexagon form layers a and c, respectively.
The strands running on the diagonally opposite edges (3 - 3‘, 6 - 6’) and
Layer la1
Layer Ibl
Layer Icl
Fig. 11. Covalently linked multilayer structure of peptidoglycan. The glycan strands in
layers a, b, and care distinguished by giving different shadings to NAG.
THEORY OF PEPTIDOGLYCANS. I1
589
along the axis (7 - 7’) of the hexagon form layer b. The strands in the
successive layers are mutually displaced by a distance d / 2 (where d is the
side of the hexagon) along the diagonal of the hexagonal face. Since the
crossbridging peptides come alternately above and below the glycan strand,
each of the glycan strands can be covalently crosslinked with four other
strands (2 above and 2 below). This is illustrated for the strand 7 - 7’ in
Fig. 11. This pattern can repeat to form a covalently linked multilayer of
any thickness. Since all the strands in different layers are covalently linked
with one another, this arrangement will be very strong and stable and able
to withstand the high internal osmotic pressure of the cell.
In the above model, the subunits are assumed to be in the compact conformations 1and 2 alternatively. However, in reality there may be some
amount of randomness in their arrangement.
This work was partially supported by a grant from the Department of Science and Technology, India.
References
1. Tipper, D. J. (1970) Int. J . Systematic Bacteriol. 20,361-377.
2. Keleman, M. V. & Rogers, H. J. (1971) Proc. Natl. Acad. Sci. U S A 68,992-996.
3. Oldmixon, E. H., Glauser, S. & Higgins, M. L. (1974) Biopolymers 13,2037-2060.
4. Formanek, H., Formanek, S. & Wawra, H. (1974) Eur. J . Biochem. 46,279-294.
5. Ramachandran, G. N. & Sasisekharan, V. (1968) Adu. Protein Chem. 23,283-437.
6. IUPAC-IUB Commission on Biochemical Nomenclature (1970) Biochemistry 9,
3471-3479.
7. Arnott, S. & Scott, W. E. (1972) J . Chem. SOC.
Perkin Trans. 2,324-335.
8. Corey, R. B. & Pauling, L. (1953) Proc. R. Soc. London, Ser. B 141,lO-20.
9. Momany, F. A., McCuire, R. F., Burgess, A. W. & Scheraga, H. A. (1975)J. Phys. Chem.
79,2361-2381.
10. Del Re, G. (1958) J . Chem. Soc., 4031-4040.
11. Del Re, G., Pullman, B. & Yonezawa, T . (1963) Biochim. Biophys. Acta 75,153-182.
12. Berthod, H. & Pullman, A. (1965) J . Chem. Phys. 62,942-946.
13. Pullman, B. & Pullman, A. (1963) Quantum Biochemistry, Interscience, New York,
pp. 104-109.
14. Poland, D. & Scheraga, H. A. (1967) Biochemistry 6,3791-3800.
15. Srinivasan, A. R. & Rao, V. S. R. (1975) Pramana 4,95-103.
16. Fletcher, R. & Powell, M. J. D. (1963) Computer J . 6,163-168.
17. Davidon, W. C. (1959) AEC Research and Development Report, ANL 5990.
18. Lewis, P. N., Momany, F. A. & Scheraga, H. A. (1973) Isr. J . Chem. 11,121-152.
19. Bhat, T . N. (1976) Ph.D. thesis, Indian Institute of Science, Bangalore, India.
20. Bhat, T. N. & Vijayan, M. (1976) Acta Crystallogr., Sect. B. 32,891-895.
21. Marchessault, R. H. & Sundararajan, P. R. (1975) Pure Appl. Chem. 42,399-415.
Received March 29,1978
Accepted June 12,1978