Crystal Structure of the R-Protein of the Multisubunit ATP-Dependent Restriction Endonuclease NgoAVII

The restriction endonuclease (REase) NgoAVII iscomposed of two proteins, R.NgoAVII and N.NgoAVII,and shares features of both Type II restriction en-zymes and Type I/III ATP-dependent restriction en-zymes (see accompanying paper Zaremba et al.,2014). Here we present crystal structures of theR.NgoAVII apo-protein and the R.NgoAVII C-terminaldomain bound to a specific DNA. R.NgoAVII is com-posed of two domains: an N-terminal nucleolytic PLDdomain; and a C-terminal B3-like DNA-binding do-main identified previously in BfiI and EcoRII REases,and in plant transcription factors. Structural compar-ison of the B3-like domains of R.NgoAVII, EcoRII, BfiIand the plant transcription factors revealed a con-served DNA-binding surface comprised of N- andC-arms that together grip the DNA. The C-arms ofR.NgoAVII, EcoRII, BfiI and plant B3 domains are sim-ilar in size, but the R.NgoAVII N-arm which makes themajority of the contacts to the target site is muchlonger. The overall structures of R.NgoAVII and BfiIare similar; however, whilst BfiI has stand-alone cat-alytic activity, R.NgoAVII requires an auxiliary cog-nate N.NgoAVII protein and ATP hydrolysis in or-der to cleave DNA at the target site. The structureswe present will help formulate future experiments toexplore the molecular mechanisms of intersubunitcrosstalk that control DNA cleavage by R.NgoAVIIand related endonucleases

Structural mechanisms of the degenerate sequence recognition by Bse634I restriction endonuclease

Restriction endonuclease Bse634I recognizes and cleaves the degenerate DNA sequence 5'-R/CCGGY-3' (R stands for A or G; Y for T or C, '/' indicates a cleavage position). Here, we report the crystal structures of the Bse634I R226A mutant complexed with cognate oligoduplexes containing ACCGGT and GCCGGC sites, respectively. In the crystal, all potential H-bond donor and acceptor atoms on the base edges of the conserved CCGG core are engaged in the interactions with Bse634I amino acid residues located on the α6 helix. In contrast, direct contacts between the protein and outer base pairs are limited to van der Waals contact between the purine nucleobase and Pro203 residue in the major groove and a single H-bond between the O2 atom of the outer pyrimidine and the side chain of the Asn73 residue in the minor groove. Structural data coupled with biochemical experiments suggest that both van der Waals interactions and indirect readout contribute to the discrimination of the degenerate base pair by Bse634I. Structure comparison between related enzymes Bse634I (R/CCGGY), NgoMIV (G/CCGGC) and SgrAI (CR/CCGGYG) reveals how different specificities are achieved within a conserved structural core

Structure of the metal-independent restriction enzyme BfiI reveals fusion of a specific DNA-binding domain with a nonspecific nuclease

Among all restriction endonucleases known to date, BfiI is unique in cleaving DNA in the absence of metal ions. BfiI represents a different evolutionary lineage of restriction enzymes, as shown by its crystal structure at 1.9-Å resolution. The protein consists of two structural domains. The N-terminal catalytic domain is similar to Nuc, an EDTA-resistant nuclease from the phospholipase D superfamily. The C-terminal DNA-binding domain of BfiI exhibits a β-barrel-like structure very similar to the effector DNA-binding domain of the Mg2+-dependent restriction enzyme EcoRII and to the B3-like DNA-binding domain of plant transcription factors. BfiI presumably evolved through domain fusion of a DNA-recognition element to a nonspecific nuclease akin to Nuc and elaborated a mechanism to limit DNA cleavage to a single double-strand break near the specific recognition sequence. The crystal structure suggests that the interdomain linker may act as an autoinhibitor controlling BfiI catalytic activity in the absence of a specific DNA sequence. A PSI-BLAST search identified a BfiI homologue in a Mesorhizobium sp. BNC1 bacteria strain, a plant symbiont isolated from an EDTA-rich environment

Design of [(2-pyrimidinylthio)acetyl]benzenesulfonamides as inhibitors of human carbonic anhydrases.

A series of [(2-pyrimidinylthio)acetyl]benzenesulfonamides were designed and synthesized. Their binding affinities as inhibitors of several recombinant human carbonic anhydrase (CA) isozymes were determined by isothermal titration calorimetry (ITC) and thermal shift assay (TSA). A group of compounds containing a chlorine atom in the benzenesulfonamide ring were found to exhibit higher selectivity but lower binding affinity toward tested CAs. The crystal structures of selected compounds in complex with CA II were determined to atomic resolution. Docking studies were performed to compare the binding modes of experimentally determined crystallographic structures with computational prediction of the pyrimidine derivative binding to CA II. Several compounds bound to select CAs with single-digit nanomolar affinities and could be used as leads for inhibitor development toward a select CA isozyme

Structure of the metal-independent restriction enzyme BfiI reveals fusion of a specific DNA-binding domain with a nonspecific nuclease

Among all restriction endonucleases known to date, BfiI is unique in cleaving DNA in the absence of metal ions. BfiI represents a different evolutionary lineage of restriction enzymes, as shown by its crystal structure at 1.9-Å resolution. The protein consists of two structural domains. The N-terminal catalytic domain is similar to Nuc, an EDTA-resistant nuclease from the phospholipase D superfamily. The C-terminal DNA-binding domain of BfiI exhibits a β-barrel-like structure very similar to the effector DNA-binding domain of the Mg2+-dependent restriction enzyme EcoRII and to the B3-like DNA-binding domain of plant transcription factors. BfiI presumably evolved through domain fusion of a DNA-recognition element to a nonspecific nuclease akin to Nuc and elaborated a mechanism to limit DNA cleavage to a single double-strand break near the specific recognition sequence. The crystal structure suggests that the interdomain linker may act as an autoinhibitor controlling BfiI catalytic activity in the absence of a specific DNA sequence. A PSI-BLAST search identified a BfiI homologue in a Mesorhizobium sp. BNC1 bacteria strain, a plant symbiont isolated from an EDTA-rich environment