Biology Reference
In-Depth Information
B. MutL is an Mn
2þ
-Dependent Endonuclease
The endonuclease activity of MutL
a
is strictly dependent on manganese,
and the importance of Asp699 and Glu705 (
D
QHAX
2
E
X
4
E) for metal binding
has been demonstrated.
67
However, the spatial arrangement of these residues
in the structure
B. subtilis
MutL suggests that they are unlikely to form a single
metal ion-binding site (
Fig. 6
).
96
Mutation of Asp462 or His464 (
D
Q
H
AX
2-
EX
4
E) in
B. subtilis
MutL, or the equivalent residues in human PMS2 or
yeast PMS1, abrogates endonuclease activity and mismatch repair func-
tion.
67,94,96,99,117
Similarly, mutation of Asp523 (
D
QHAX
2
EX
4
E) in yeast
MLH3 disrupts the activities of the protein in mismatch repair and meiotic
crossing over.
118
Collectively, these results reveal the importance of these two
conserved residues and suggest that their side chains, and potentially the main
and/or side chains within the
b
1-
b
2 loop, could define the binding site of the
catalytic metal.
Interestingly, while the endonuclease activity is strictly dependent on man-
ganese,
67,96
human PMS2 and
B. subtilis
MutL bind zinc.
99
Accordingly, two Zn
2
þ
metal ions are found in the structure of
B. subtilis
MutL (
Fig. 4
).
96
The Zn
2
þ
ion occupying ''site A'' is defined by the side chain of Glu468 from the DQHAX
2-
E
X
4
E motif, as well as those of Cys604 and His606 from the
C
P
H
GRP motif
(
Fig. 7
A). A well-ordered water molecule completes the tetrahedral coordination
of this site. The Zn
2
þ
ion bound at ''site B'' is coordinated by the side chains of
His464 and Glu468 from the DQ
H
AX
2
E
X
4
E motif and loosely contacts the
side chain of Cys573 from the S
C
Kmotif(
Fig. 7
A). The usual coordination
distances for Zn
2
þ
metal ions range from 2.11 to 2.39
˚
; however, the thiol
group of Cys573 is located 2.67-2.93
˚
away, indicating that this residue
does not belong to the first coordination shell of the Zn
2
þ
ion bound to ''site
B.''
120,121
Studies in solution show that mutation of Glu468, Cys604, or His606
abrogates Zn
2
þ
binding, whereas mutation of His464 does not affect
metal binding, suggesting that ''site A,'' but not ''site B,'' is a bona fide Zn
2
þ
-
binding site.
96
Numerous studies have mined protein structure databases to analyze the
coordination numbers, geometry, and composition of metal-binding sites found in
proteins.
120-127
Based on these studies, the coordination number and residue
composition of the Zn
2
þ
-binding site of MutL (''site A'') would preclude Mn
2
þ
binding, suggesting that the catalytic metal ion binds elsewhere. However, if Zn
2
þ
is not mimicking the binding of the catalytic metal ion, what is its role?
C. The Regulatory Zn
2þ
-Binding Site
In addition to the structure of
B. subtilis
MutL bound to Zn
2
þ
, two other
independent structures of its apo-form are available.
96
Superimposition
of these structures reveals that the relative orientation between the two
