The Design of Metal-Binding Sites in Proteins

Regan, Lynne

doi:10.1146/annurev.bb.22.060193.001353

Cited by 158 publications

(117 citation statements)

References 30 publications

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“…The presence of positively charged residues close to the metalbinding site in the H4 tail can result in a siteselectivity association of the Ni(II) complexed tail with the negatively charged DNA backbone (41). In addition the hydrophobic environment in the entire protein is expected to enhance metal-binding capabilities because of multiple nonbonding interactions available there, as reported in the literature (42)(43)(44).…”

Section: Resultsmentioning

confidence: 90%

Molecular mechanisms in nickel carcinogenesis: modeling Ni(II) binding site in histone H4.

Zoroddu

Schinocca

Kowalik-Jankowska

et al. 2002

Environ Health Perspect

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Section: Resultsmentioning

confidence: 90%

Molecular mechanisms in nickel carcinogenesis: modeling Ni(II) binding site in histone H4.

Zoroddu

Schinocca

Kowalik-Jankowska

et al. 2002

Environ Health Perspect

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“…5) (27)(28)(29). As can be seen from Table I, single alanine mutations of these five residues in K260H did not reduce the antagonistic potency of Zn 2ϩ .…”

Section: Effects Of Znmentioning

confidence: 87%

Construction of a High Affinity Zinc Binding Site in the Metabotropic Glutamate Receptor mGluR1

Jensen

Sheppard

Jensen

et al. 2001

Journal of Biological Chemistry

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The metabotropic glutamate receptors (mGluRs) belong to family C of the G-protein-coupled receptor (GPCR) superfamily. The receptors are characterized by having unusually long amino-terminal domains (ATDs), to which agonist binding has been shown to take place. Previously, we have constructed a molecular model of the ATD of mGluR1 based on a weak amino acid sequence similarity with a bacterial periplasmic binding protein. The ATD consists of two globular lobes, which are speculated to contract from an "open" to a "closed" conformation following agonist binding. In the present study, we have created a Zn 2؉ binding site in mGluR1b by mutating the residue Lys 260 to a histidine. Zinc acts as a noncompetitive antagonist of agonist-induced IP accumulation on the K260H mutant with an IC 50 value of 2 M. Alanine mutations of three potential "zinc coligands" in proximity to the introduced histidine in K260H knock out the ability of Zn 2؉ to antagonize the agonist-induced response. Zn 2؉ binding to K260H does not appear to affect the dimerization of the receptor. Instead, we propose that binding of zinc has introduced a structural constraint in the ATD lobe, preventing the formation of a "closed" conformation, and thus stabilizing a more or less inactive "open" form of the ATD. This study presents the first metal ion site constructed in a family C GPCR. Furthermore, it is the first time a metal ion site has been created in a region outside of the seven transmembrane regions of a GPCR and the loops connecting these. The findings offer valuable insight into the mechanism of ATD closure and family C receptor activation. Furthermore, the findings demonstrate that ATD regions other than those participating in agonist binding could be potential targets for new generations of ligands for this family of receptors.

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“…On the basis of previous studies regarding natural or engineered metal-binding sites, we rationalized that mutations of solvent-exposed residue pairs i and i þ 4 of a helix [50][51][52][53] or i and i þ 3 of a helixloop 37 to a pair of either histidine or cysteine residues would induce homooligomeric assemblies upon incubation with metal. Specifically, we predicted the metal site within these assemblies to take on a tetrahedral or octahedral coordination, in which the mutated histidines or cysteines among the molecules participate in the metal coordination.…”

Section: Rationale and Design Of Mutationsmentioning

confidence: 99%

An approach to crystallizing proteins by metal‐mediated synthetic symmetrization

Laganowsky

Zhao²,

Soriaga

et al. 2011

Protein Science

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Combining the concepts of synthetic symmetrization with the approach of engineering metal-binding sites, we have developed a new crystallization methodology termed metal-mediated synthetic symmetrization. In this method, pairs of histidine or cysteine mutations are introduced on the surface of target proteins, generating crystal lattice contacts or oligomeric assemblies upon coordination with metal. Metal-mediated synthetic symmetrization greatly expands the packing and oligomeric assembly possibilities of target proteins, thereby increasing the chances of growing diffraction-quality crystals. To demonstrate this method, we designed various T4 lysozyme (T4L) and maltose-binding protein (MBP) mutants and cocrystallized them with one of three metal ions: copper (Cu 21 ), nickel (Ni 21 ), or zinc (Zn 21 ). The approach resulted in 16 new crystal structures-eight for T4L and eight for MBP-displaying a variety of oligomeric assemblies and packing modes, representing in total 13 new and distinct crystal forms for these proteins. We discuss the potential utility of the method for crystallizing target proteins of unknown structure by engineering in pairs of histidine or cysteine residues. As an alternate strategy, we propose that the varied crystallization-prone forms of T4L or MBP engineered in this work could be used as crystallization chaperones, by fusing them genetically to target proteins of interest.

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The Design of Metal-Binding Sites in Proteins

Cited by 158 publications

References 30 publications

Molecular mechanisms in nickel carcinogenesis: modeling Ni(II) binding site in histone H4.

Molecular mechanisms in nickel carcinogenesis: modeling Ni(II) binding site in histone H4.

Construction of a High Affinity Zinc Binding Site in the Metabotropic Glutamate Receptor mGluR1

An approach to crystallizing proteins by metal‐mediated synthetic symmetrization

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