A metallothionein containing a zinc finger within a four-metal cluster protects a bacterium from zinc toxicity
Claudia A. BlindauerMark D. HarrisonJohn A. ParkinsonAndrea K. RobinsonJim CavetNigel J. RobinsonPeter J. Sadler
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Abstract:
Zinc is essential for many cellular processes, including DNA synthesis, transcription, and translation, but excess can be toxic. A zinc-induced gene, smtA , is required for normal zinc-tolerance in the cyanobacterium Synechococcus PCC 7942. Here we report that the protein SmtA contains a cleft lined with Cys-sulfur and His-imidazole ligands that binds four zinc ions in a Zn 4 Cys 9 His 2 cluster. The thiolate sulfurs of five Cys ligands provide bridges between the two ZnCys 4 and two ZnCys 3 His sites, giving two fused six-membered rings with distorted boat conformations. The inorganic core strongly resembles the Zn 4 Cys 11 cluster of mammalian metallothionein, despite different amino acid sequences, a different linear order of the ligands, and presence of histidine ligands. Also, SmtA contains elements of secondary structure not found in metallothioneins. One of the two Cys 4 -coordinated zinc ions in SmtA readily exchanges with exogenous metal ( 111 Cd), whereas the other is inert. The thiolate sulfur ligands bound to zinc in this site are buried within the protein. Regions of β-strand and α-helix surround the inert site to form a zinc finger resembling the zinc fingers in GATA and LIM-domain proteins. Eukaryotic zinc fingers interact specifically with other proteins or DNA and an analogous interaction can therefore be anticipated for prokaryotic zinc fingers. SmtA now provides structural proof for the existence of zinc fingers in prokaryotes, and sequences related to the zinc finger motif can be identified in several bacterial genomes.Keywords:
Metallothionein
Zinc finger nuclease
Multizinc finger peptides are likely to reach increased prominence in the search for the “ideal” designer transcription factor for in vivo applications such as gene therapy. However, for these treatments to be effective and safe, the peptides must bind with high affinity and, more importantly, with great specificity. Our previous research has shown that zinc finger arrays can be made to bind 18 bp of DNA with picomolar affinity, but also has suggested that arrays of fingers also may bind tightly to related sequences. This work addresses the question of zinc finger DNA binding specificity. We show that by changing the way in which zinc finger arrays are constructed—by linking three two-finger domains rather than two three-finger units—far greater target specificity can be achieved through increased discrimination against mutated or closely related sequences. These new peptides have the added capability of being able to span two short gaps of unbound DNA, although still binding with picomolar affinity to their target sites. We believe that this new method of constructing zinc finger arrays will offer greater efficacy in the fields of gene therapy and in the production of transgenic organisms than previously reported zinc finger arrays.
Zinc finger nuclease
RING finger domain
Sp1 transcription factor
LIM domain
Binding selectivity
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Imidazole
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Genome editing for therapeutic applications often requires cleavage within a narrow sequence window. Here, to enable such high-precision targeting with zinc-finger nucleases (ZFNs), we have developed an expanded set of architectures that collectively increase the configurational options available for design by a factor of 64. These new architectures feature the functional attachment of the FokI cleavage domain to the amino terminus of one or both zinc-finger proteins (ZFPs) in the ZFN dimer, as well as the option to skip bases between the target triplets of otherwise adjacent fingers in each zinc-finger array. Using our new architectures, we demonstrate targeting of an arbitrarily chosen 28 bp genomic locus at a density that approaches 1.0 (i.e., efficient ZFNs available for targeting almost every base step). We show that these new architectures may be used for targeting three loci of therapeutic significance with a high degree of precision, efficiency, and specificity.
Zinc finger nuclease
Genome Engineering
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