 |
PDBsum entry 1d1m
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Viral protein
|
PDB id
|
|
|
|
1d1m
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
 |
Contents |
 |
|
|
|
|
|
|
|
|
|
|
* Residue conservation analysis
|
|
|
|
|
References listed in PDB file
|
|
|
Key reference
|
|
|
Title
|
|
The structural basis for enhanced stability and reduced DNA binding seen in engineered second-Generation cro monomers and dimers.
|
|
|
Authors
|
|
P.B.Rupert,
A.K.Mollah,
M.C.Mossing,
B.W.Matthews.
|
|
|
Ref.
|
|
J Mol Biol, 2000,
296,
1079-1090.
[DOI no: ]
|
|
|
PubMed id
|
|
|
|
|
|
Abstract
|
|
|
It was previously shown that the Cro repressor from phage lambda, which is a
dimer, can be converted into a stable monomer by a five-amino acid insertion.
Phe58 is the key residue involved in this transition, switching from
interactions which stabilize the dimer to those which stabilize the monomer.
Structural studies, however, suggested that Phe58 did not penetrate into the
core of the monomer as well as it did into the native dimer. This was strongly
supported by the finding that certain core-repacking mutations, including in
particular, Phe58-->Trp, increased the stability of the monomer. Unexpectedly,
the same substitution also increased the stability of the native dimer. At the
same time it decreased the affinity of the dimer for operator DNA. Here we
describe the crystal structures of the Cro F58W mutant, both as the monomer and
as the dimer. The F58W monomer crystallized in a form different from that of the
original monomer. In contrast to that structure, which resembled the DNA-bound
form of Cro, the F58W monomer is closer in structure to wild-type (i.e.
non-bound) Cro. The F58W dimer also crystallizes in a form different from the
native dimer but has a remarkably similar overall structure which tends to
confirm the large changes in conformation of Cro on binding DNA. Introduction of
Trp58 perturbs the position occupied by the side-chain of Arg38, a DNA-contact
residue, providing a structural explanation for the reduction in DNA-binding
affinity.The improved thermal stability is seen to be due to the enhanced
solvent transfer free energy of Trp58 relative to Phe58, supplemented in the
dimer structure, although not the monomer, by a reduction in volume of internal
cavities.
|
|
|
|
|
|
|
|
Figure 2.
Figure 2. Superposition of the a-carbon backbone of the
original engineered monomer, Cro K56-[DGEVK], in black on the
mutant monomer, Cro K56-[DGEVK]-F58W. One of the two
crystallographically independent copies of the mutant structure
(chain A) is shown in gray and the other with open bonds. This
superposition, as well as for all other such Figures, is based
on the a-carbon atoms of residues 5-40 which includes the
a-helical core region and the helix-turn-helix.
|
|
Figure 6.
Figure 6. (a) Superposition of the mutant monomer,
Cro-[DGEVK]-F58W (open bonds), on one subunit of the native Cro
dimer (filled bonds). (b) Superposition of one subunit of the
mutant monomer, Cro-[DGEVK]-F58W (open bonds), on one subunit of
the WT Cro dimer (filled bonds) in its conformation when bound
to operator DNA. (c) Detailed view of (b) in the vicinity of
residue 58 showing the superposition of the native Cro-operator
complex (filled bonds) on one subunit of Cro-F58W. The indole
ring of Trp58 displaces Arg38' from its position in the Phe58
structure and would tend to disrupt the interaction between
Arg38' and the phosphate oxygen atom labeled S12 O2P. This
labeling is as used by the Protein Data Bank. In terms of common
conventions for the Cro system (e.g. see [Albright and Matthews
1998]), S11 O2P and S12 O2P correspond, respectively, to
phosphate groups P[E] and P[D].
|
|
|
|
|
|
The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(2000,
296,
1079-1090)
copyright 2000.
|
|
|
|
|
|
|
