title: Structural studies of an engineered zinc biosensor reveal an unanticipated mode of zinc binding.
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.
authors.affiliation: Department of Biochemistry, University of Western Ontario, London, Ontari, Canada N6A 5C1.
authors.forename: Patrick G
authors.initials: PG
authors.lastname: Telmer
authors.forename: Brian H
authors.initials: BH
authors.lastname: Shilton
mesh_terms: D001665:Binding Sites; D015374:Biosensing Techniques / Q000379:methods*; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics; D018360:Crystallography, X-Ray; D058619:Maltose-Binding Proteins; D011485:Protein Binding; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D015032:Zinc / Q000032:analysis* / Q000737:chemistry
chemical_list: D002352:Carrier Proteins; D058619:Maltose-Binding Proteins; D015032:Zinc