PDBsum entry 1zkb

Sugar binding, metal binding protein

PDB id: 1zkb

Contents

Protein chain

370 a.a. *

Waters ×194

* Residue conservation analysis

PDB id:

1zkb

Name:

Sugar binding, metal binding protein

Title:

Zinc-free engineered maltose binding protein

Structure:

Maltose-binding periplasmic protein. Chain: a. Fragment: residues 27-396. Synonym: maltodextrin-binding protein, mmbp. Engineered: yes. Mutation: yes

Source:

Escherichia coli. Organism_taxid: 562. Gene: male. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

2.20Å R-factor: 0.244 R-free: 0.285

Authors:

P.G.Telmer,B.H.Shilton

Key ref:

P.G.Telmer and B.H.Shilton (2005). Structural studies of an engineered zinc biosensor reveal an unanticipated mode of zinc binding. J Mol Biol, 354, 829-840. PubMed id: 16288781 DOI: 10.1016/j.jmb.2005.10.016

Date:

02-May-05 Release date: 20-Dec-05

Protein chain

P0AEX9  (MALE_ECOLI) -  Maltose/maltodextrin-binding periplasmic protein from Escherichia coli (strain K12)

Seq: 396 a.a.

Struc: 370 a.a.*

* PDB and UniProt seqs differ at 5 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.?

DOI no: 10.1016/j.jmb.2005.10.016

J Mol Biol 354:829-840 (2005)

PubMed id: 16288781

Structural studies of an engineered zinc biosensor reveal an unanticipated mode of zinc binding.

P.G.Telmer, B.H.Shilton.

ABSTRACT

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.

Selected figure(s)

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.

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

The above figures are reprinted by permission from Elsevier: J Mol Biol (2005, 354, 829-840) copyright 2005.

Figures were selected by the author.