Muse Oke

STRUCTURE OF THE DNA REPAIR HELICASE HEL308 REVEALS DNA BINDING AND AUTOINHIBITORY DOMAINS

Hel308 is a superfamily 2 helicase conserved in eukaryotes and archaea. It is thought to function in the early stages of recombination following replication fork arrest and has a specificity for removal of the lagging strand in model replication forks. A homologous helicase constitutes the N-terminal domain of human DNA polymerase Q. The Drosophila homologue mus301 is implicated in double strand break repair and meiotic recombination. We have solved the high resolution crystal structure of Hel308 from the crenarchaeon Sulfolobus solfataricus, revealing a five-domain structure with a central pore lined with essential DNA binding residues. The fifth domain is shown to act as an autoinhibitory domain or molecular brake, clamping the single-stranded DNA extruded through the central pore of the helicase structure to limit the helicase activity of the enzyme. This provides an elegant mechanism to tune the processivity of the enzyme to its functional role. Hel308 can displace streptavidin from a biotinylated DNA molecule, and this activity is only partially inhibited when the DNA is pre-bound with abundant DNA-binding proteins RPA or Alba1, whereas pre-binding with the recombinase RadA has no effect on activity. These data suggest that one function of the enzyme may be in the removal of bound proteins at stalled replication forks and recombination intermediates.

Extensive DNA mimicry by the ArdA anti-restriction protein and its role in the spread of antibiotic resistance

The ardA gene, found in many prokaryotes including important pathogenic species, allows associated mobile genetic elements to evade the ubiquitous Type I DNA restriction systems and thereby assist the spread of resistance genes in bacterial populations. As such, ardA contributes to a major healthcare problem. We have solved the structure of the ArdA protein from the conjugative transposon Tn916 and find that it has a novel extremely elongated curved cylindrical structure with defined helical grooves. The high density of aspartate and glutamate residues on the surface follow a helical pattern and the whole protein mimics a 42-base pair stretch of B-form DNA making ArdA by far the largest DNA mimic known. Each monomer of this dimeric structure comprises three alpha–beta domains, each with a different fold. These domains have the same fold as previously determined proteins possessing entirely different functions. This DNA mimicry explains how ArdA can bind and inhibit the Type I restriction enzymes and we demonstrate that 6 different ardA from pathogenic bacteria can function in Escherichia coli hosting a range of different Type I restriction systems.

Structure of the heterotrimeric PCNA from Sulfolobus solfataricus

The structure of the heterotrimeric PCNA complex from S. sulfataricus is reported to 2.3 Å.

AcsD catalyzes enantioselective citrate desymmetrization in siderophore biosynthesis

Bacterial pathogens need to scavenge iron from their host for growth and proliferation during infection. They have evolved several strategies to do this, one being the biosynthesis and excretion of small, high-affinity iron chelators known as siderophores. The biosynthesis of siderophores is an important area of study, not only for potential therapeutic intervention, but also to illuminate new enzyme chemistries. Two general pathways for siderophore biosynthesis exist: the well-characterized nonribosomal peptide synthetase (NRPS)-dependent pathway and the NRPS-independent (NIS) pathway, which relies on a different family of sparsely-investigated synthetases. Here, we report structural and biochemical studies of AcsD from Pectobacterium (formerly Erwinia) chrysanthemi, a NIS synthetase involved in achromobactin biosynthesis. The structures of ATP and citrate complexes provide a mechanistic rationale for stereospecific formation of an enzyme-bound (3R)-citryl-adenylate, which reacts with L-serine to form a likely achromobactin precursor. AcsD is a novel acyl adenylate-forming enzyme with a new fold and chemical catalysis strategy.

Crystal structure and silica condensing activities of silicatein alpha-cathepsin L chimeras.

Cathepsin L mutants with the ability to condense silica from solution have been generated and a 1.5 A crystal structure of one of these chimeras allows us to rationalise the catalytic mechanism of silicic acid condensation.

A Dimeric Rep Protein Initiates Replication of a Linear Archaeal Virus Genome: Implications for the Rep Mechanism and Viral Replication

ABSTRACT The Rudiviridae are a family of rod-shaped archaeal viruses with covalently closed, linear double-stranded DNA (dsDNA) genomes. Their replication mechanisms remain obscure, although parallels have been drawn to the Poxviridae and other large cytoplasmic eukaryotic viruses. Here we report that a protein encoded in the 34-kbp genome of the rudivirus SIRV1 is a member of the replication initiator (Rep) superfamily of proteins, which initiate rolling-circle replication (RCR) of diverse viruses and plasmids. We show that SIRV Rep nicks the viral hairpin terminus, forming a covalent adduct between an active-site tyrosine and the 5′ end of the DNA, releasing a 3′ DNA end as a primer for DNA synthesis. The enzyme can also catalyze the joining reaction that is necessary to reseal the DNA hairpin and terminate replication. The dimeric structure points to a simple mechanism through which two closely positioned active sites, each with a single tyrosine residue, work in tandem to catalyze DNA nicking and joining. We propose a novel mechanism for rudivirus DNA replication, incorporating the first known example of a Rep protein that is not linked to RCR. The implications for Rep protein function and viral replication are discussed.

The Respiratory Arsenite Oxidase: Structure and the Role of Residues Surrounding the Rieske Cluster

The arsenite oxidase (Aio) from the facultative autotrophic Alphaproteobacterium Rhizobium sp. NT-26 is a bioenergetic enzyme involved in the oxidation of arsenite to arsenate. The enzyme from the distantly related heterotroph, Alcaligenes faecalis, which is thought to oxidise arsenite for detoxification, consists of a large α subunit (AioA) with bis-molybdopterin guanine dinucleotide at its active site and a 3Fe-4S cluster, and a small β subunit (AioB) which contains a Rieske 2Fe-2S cluster. The successful heterologous expression of the NT-26 Aio in Escherichia coli has resulted in the solution of its crystal structure. The NT-26 Aio, a heterotetramer, shares high overall similarity to the heterodimeric arsenite oxidase from A. faecalis but there are striking differences in the structure surrounding the Rieske 2Fe-2S cluster which we demonstrate explains the difference in the observed redox potentials (+225 mV vs. +130/160 mV, respectively). A combination of site-directed mutagenesis and electron paramagnetic resonance was used to explore the differences observed in the structure and redox properties of the Rieske cluster. In the NT-26 AioB the substitution of a serine (S126 in NT-26) for a threonine as in the A. faecalis AioB explains a −20 mV decrease in redox potential. The disulphide bridge in the A. faecalis AioB which is conserved in other betaproteobacterial AioB subunits and the Rieske subunit of the cytochrome bc 1 complex is absent in the NT-26 AioB subunit. The introduction of a disulphide bridge had no effect on Aio activity or protein stability but resulted in a decrease in the redox potential of the cluster. These results are in conflict with previous data on the betaproteobacterial AioB subunit and the Rieske of the bc 1 complex where removal of the disulphide bridge had no effect on the redox potential of the former but a decrease in cluster stability was observed in the latter.

Structural and functional characterisation of a conserved archaeal RadA paralog with antirecombinase activity.

DNA recombinases (RecA in bacteria, Rad51 in eukarya and RadA in archaea) catalyse strand exchange between homologous DNA molecules, the central reaction of homologous recombination, and are among the most conserved DNA repair proteins known. RecA is the sole protein responsible for this reaction in bacteria, whereas there are several Rad51 paralogs that cooperate to catalyse strand exchange in eukaryotes. All archaea have at least one (and as many as four) RadA paralog, but their function remains unclear. Herein, we show that the three RadA paralogs encoded by the Sulfolobus solfataricus genome are expressed under normal growth conditions and are not UV inducible. We demonstrate that one of these proteins, Sso2452, which is representative of the large archaeal RadC subfamily of archaeal RadA paralogs, functions as an ATPase that binds tightly to single-stranded DNA. However, Sso2452 is not an active recombinase in vitro and inhibits D-loop formation by RadA. We present the high-resolution crystal structure of Sso2452, which reveals key structural differences from the canonical RecA family recombinases that may explain its functional properties. The possible roles of the archaeal RadA paralogs in vivo are discussed.

Unusual Chromophore and Cross‐Links in Ranasmurfin: A Blue Protein from the Foam Nests of a Tropical Frog

Ranasmurfin is an unusual blue protein isolated from the nests of a Malaysian tree frog, Polypedates leucomystax,[1] showing the rich chemical diversity displayed by biomolecular foams. Many species of tropical frogs use foams to protect delicate eggs and developing embryos against environmental challenges. These nests act as miniature ecosystems containing a spectrum of novel proteins and other macromolecules with functions related to foam stabilization and adhesion, resistance to microbial degradation, predation, or dehydration, providing a biocompatible environment for embryonic development.Thisworkformspartofourwiderstudyofthe intriguing physical and chemical properties of biofoams as unusual examples of biological soft matter.[2]

Crystallization of Ranasmurfin, a blue-coloured protein from Polypedates leucomystax

A novel blue protein from frog nests has been crystallized.