 |
PDBsum entry 1lax
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Sugar binding protein
|
PDB id
|
|
|
|
1lax
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
 |
Contents |
 |
|
|
|
|
|
|
|
|
|
|
|
|
* Residue conservation analysis
|
|
|
|
|
References listed in PDB file
|
|
|
Key reference
|
|
|
Title
|
|
Crystal structure of a defective folding protein.
|
|
|
Authors
|
|
F.A.Saul,
M.Mourez,
B.Vulliez-Le normand,
N.Sassoon,
G.A.Bentley,
J.M.Betton.
|
|
|
Ref.
|
|
Protein Sci, 2003,
12,
577-585.
[DOI no: ]
|
|
|
PubMed id
|
|
|
|
|
|
Abstract
|
|
|
Maltose-binding protein (MBP or MalE) of Escherichia coli is the periplasmic
receptor of the maltose transport system. MalE31, a defective folding mutant of
MalE carrying sequence changes Gly 32-->Asp and Ile 33-->Pro, is either degraded
or forms inclusion bodies following its export to the periplasmic compartment.
We have shown previously that overexpression of FkpA, a heat-shock periplasmic
peptidyl-prolyl isomerase with chaperone activity, suppresses MalE31 misfolding.
Here, we have exploited this property to characterize the maltose transport
activity of MalE31 in whole cells. MalE31 displays defective transport behavior,
even though it retains maltose-binding activity comparable with that of the
wild-type protein. Because the mutated residues are in a region on the surface
of MalE not identified previously as important for maltose transport, we have
solved the crystal structure of MalE31 in the maltose-bound state in order to
characterize the effects of these changes. The structure was determined by
molecular replacement methods and refined to 1.85 A resolution. The conformation
of MalE31 closely resembles that of wild-type MalE, with very small
displacements of the mutated residues located in the loop connecting the first
alpha-helix to the first beta-strand. The structural and functional
characterization provides experimental evidence that MalE31 can attain a
wild-type folded conformation, and suggest that the mutated sites are probably
involved in the interactions with the membrane components of the maltose
transport system.
|
|
|
|
|
|
|
|
Figure 1.
Figure 1. FkpA prevents MalE31 aggregation. Cells
expressing chromosomally encoded MalE-wt or MalE31, transformed
by pTrc99 or pTfkp, were grown in rich medium at 37°C for 3 h,
and then fractionated from spheroplasts. Periplasmic (soluble)
and membrane (insoluble) fractions, corresponding to 5 x 10^9
cells, were analyzed on SDS-polyacrylamide (12.5%) gel stained
by Coomassie blue. (Lane 1), Strain PD28 (  malE)
carrying pTrc99. (Lane 2) Strain MC4100 (malE) carrying pTrc99.
(Lane 3) Strain JMB5 (malE31) carrying pTrc99. (Lane 4) JMB5
cells carrying pTfkp.
|
|
Figure 5.
Figure 5. Schematic view of the MalE31 structure. The
mutated residues at positions 32 and 33 (top left) occur in an
 ß turn at an
exposed position in the amino-terminal domain. Positions 13, 14,
38, and 63 located in the N-domain correspond to mutations
affecting maltose transport (see text). The bound maltose
substrate (middle) is located in a deep cleft between the amino-
and carboxy-terminal domains.
|
|
|
|
|
|
The above figures are
reprinted
by permission from the Protein Society:
Protein Sci
(2003,
12,
577-585)
copyright 2003.
|
|
|
Secondary reference #1
|
|
|
Title
|
|
Degradation versus aggregation of misfolded maltose-Binding protein in the periplasm of escherichia coli.
|
|
|
Authors
|
|
J.M.Betton,
N.Sassoon,
M.Hofnung,
M.Laurent.
|
|
|
Ref.
|
|
J Biol Chem, 1998,
273,
8897-8902.
[DOI no: ]
|
|
|
PubMed id
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Figure 1.
Fig. 1. Fractionation of soluble and insoluble MalE
variants. Cells grown in rich medium containing 0.2% maltose at
30 °C for 3 h were fractionated from spheroplasts.
Periplasmic (soluble) and membrane fractions (insoluble),
corresponding to 10^8 bacteria, were separated by
SDS-polyacrylamide gel electrophoresis (12.5% acrylamide). The
gels were stained with Coomassie Blue or silver-stained. A,
strains harboring pHCME31 or p  709
plasmid. B, strains harboring pHCME-I33P or p 709 plasmid.
|
|
Figure 5.
Fig. 5. Effect of overproduced MalE31 on DegP. Fractions
from cellular fractionation experiment shown in Fig. 1A were
separated by SDS-polyacrylamide gel electrophoresis, and
immunoblot analysis of the gel was performed by using a rabbit
antibody directed against DegP. A, whole cell extracts. B,
periplasmic and membrane fractions. Lanes 1, pHCME31 in pop6590.
Lanes 2, pHCME31 in NS2. Lanes 3, pHCME31 in pop6499. Lanes 4, p
709 in
pop6499.
|
|
|
|
|
|
The above figures are
reproduced from the cited reference
with permission from the ASBMB
|
|
|
Secondary reference #2
|
|
|
Title
|
|
Folding of a mutant maltose-Binding protein of escherichia coli which forms inclusion bodies.
|
|
|
Authors
|
|
J.M.Betton,
M.Hofnung.
|
|
|
Ref.
|
|
J Biol Chem, 1996,
271,
8046-8052.
|
|
|
PubMed id
|
|
|
|
|
|
|
|
|
|
|
|
|
