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PDBsum entry 1zkb
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Sugar binding, metal binding protein
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PDB id
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1zkb
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References listed in PDB file
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Key reference
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Title
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Structural studies of an engineered zinc biosensor reveal an unanticipated mode of zinc binding.
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Authors
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P.G.Telmer,
B.H.Shilton.
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Ref.
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J Mol Biol, 2005,
354,
829-840.
[DOI no: ]
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PubMed id
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Abstract
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Protein engineering was used previously to convert maltose-binding protein (MBP)
into a zinc biosensor. Zn(2+) binding by the engineered MBP was thought to
require a large conformational change from "open" to
"closed", similar to that observed when maltose is bound by the
wild-type protein. We show that although this re-designed MBP molecule binds
Zn(2+) with high affinity as previously reported, it does not adopt a closed
conformation in solution as assessed by small-angle X-ray scattering.
High-resolution crystallographic studies of the engineered Zn(2+)-binding MBP
molecule demonstrate that Zn(2+) is coordinated by residues on the N-terminal
lobe only, and therefore Zn(2+) binding does not require the protein to adopt a
fully closed conformation. Additional crystallographic studies indicate that
this unexpected Zn(2+) binding site can also coordinate Cu(2+) and Ni(2+) with
only subtle changes in the overall conformation of the protein. This work
illustrates that the energetic barrier to domain closure, which normally
functions to maintain MBP in an open concentration in the absence of ligand, is
not easily overcome by protein design. A comparison to the mechanism of
maltose-induced domain rearrangement is discussed.
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Figure 2.
Figure 2. EZ-MBP-HA remains open in solution when bound to
Zn2+. The experimental SAXS curves for Zn2+-free and Zn2+-bound
EZ-MBP-HA were compared to the crystal structures of open and
closed forms of wild-type MBP. All scattering curves are
represented as a plot of the momentum transfer, Q (a function of
the scattering angle), versus the natural logarithm of the
scattering intensity. In each case, differences between the
scattering intensities in the two experiments are plotted as a
percentage of the total signal. (a) Experimental SAXS curves for
wild-type MBP in the closed ligand-bound form (red) and the open
ligand-free form (black) were overlaid, illustrating the
expected SAXS changes when the protein undergoes conformational
change from open to closed. (b) A superposition of SAXS curves
from Zn2+-free (black) and Zn2+-bound EZ-MBP-HA (red) shows that
there is little, if any, conformational change in response to
Zn2+ binding. To find whether EZ-MBP-HA exists in an open or
closed conformation in solution, experimental SAXS data from
Zn2+-bound MBP (black) were compared with theoretical scattering
from (c) closed MBP and (d) open MBP, both shown in red.
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Figure 4.
Figure 4. The Zn2+ binding site of EZ-MBP-HA. (a) The
structure of the Zn2+-binding site of EZ-MBP-HA. The refined
structure of the Zn2+-bound protein was used to calculate a
2F[O] -F[C] electron density map (gold, contoured at 1s). To
positively identify bound Zn ions, data were collected at the
absorption peak for Zn2+(1.2824 Å) and used to calculate
an anomalous difference map (red, contoured at 3 s). (b) The
structure of EZ-MBP-HA in the absence of Zn2+. The refined
structure of Zn2+-free EZ-MBP-HA was used to calculate a
2F[O]-F[C] electron density map (contoured at 1s). (c) A
superposition of the Zn2+-free (yellow) and Zn2+-bound (grey)
structures of EZ-MBP-HA, showing the movement of H63 and H66 to
coordinate Zn2+ atoms. Figures were prepared using Spock,26
Raster3D,27 and SwissPDBViewer.28
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(2005,
354,
829-840)
copyright 2005.
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