Tyrosyl DNA phosphodiesterase I (Tdp1) is a member of the phospholipase D superfamily and hydrolyzes 3′phospho-DNA adducts via two conserved catalytic histidines, one acting as the lead nucleophile and the second as a general acid/base. Substitution of the second histidine specifically to arginine contributes to the neurodegenerative disease SCAN1. We investigated the catalytic role of this histidine in the yeast protein (His432) using a combination of X-ray crystallography, biochemistry, yeast genetics and theoretical chemistry. The structures of wild type Tdp1 and His432Arg both show a phosphorylated form of the nucleophilic histidine that is not observed in the structure of His432Asn. The phosphohistidine is stabilized in the His432Arg structure by the guanidinium group that also restricts access of a nucleophilic water molecule to the Tdp1-DNA intermediate. Biochemical analyses confirm that His432Arg forms an observable and unique Tdp1-DNA adduct during catalysis. Substitution of His432 by Lys does not affect catalytic activity or yeast phenotype, but substitution with Asn, Gln, Leu, Ala, Ser and Thr all result in severely compromised enzymes and Top1-camptothecin dependent lethality. Surprisingly, His432Asn did not show a stable covalent Tdp1-DNA intermediate which suggests another catalytic defect. Theoretical calculations revealed that the defect resides in the nucleophilic histidine and that the pKa of this histidine is crucially dependent upon the second histidine and the incoming phosphate of the substrate. This represents a unique example of substrate-activated catalysis that applies to the entire phospholipase D superfamily.
Phosphatidic acid is the central intermediate in membrane phospholipid synthesis and is generated by two acyltransferases in a pathway conserved in all life forms. The second step in this pathway is catalyzed by 1-acyl-sn-glycero-3-phosphate acyltransferase, called PlsC in bacteria. The crystal structure of PlsC from Thermotoga maritima reveals an unusual hydrophobic/aromatic N-terminal two-helix motif linked to an acyltransferase αβ domain that contains the catalytic HX4D motif. PlsC dictates the acyl chain composition of the 2-position of phospholipids, and the acyl chain selectivity ‘ruler’ is an appropriately placed and closed hydrophobic tunnel. This was confirmed by site-directed mutagenesis and membrane composition analysis of Escherichia coli cells expressing the mutated proteins. MD simulations reveal that the two-helix motif represents a novel substructure that firmly anchors the protein to one leaflet of the membrane. This binding mode allows the PlsC active site to acylate lysophospholipids within the membrane bilayer using soluble acyl donors.
Staphylococcal species are a leading cause of bacterial drug-resistant infections and associated mortality. One strategy to combat bacterial drug resistance is to revisit compromised targets, and to circumvent resistance mechanisms using structure-assisted drug discovery. The folate pathway is an ideal candidate for this approach. Antifolates target an essential metabolic pathway, and the necessary detailed structural information is now available for most enzymes in this pathway. Dihydropteroate synthase (DHPS) is the target of the sulfonamide class of drugs, and its well characterized mechanism facilitates detailed analyses of how drug resistance has evolved. Here, we surveyed clinical genetic sequencing data in S. aureus to distinguish natural amino acid variations in DHPS from those that are associated with sulfonamide resistance. Five mutations were identified, F17L, S18L, T51M, E208K, and KE257_dup. Their contribution to resistance and their cost to the catalytic properties of DHPS were evaluated using a combination of biochemical, biophysical and microbiological susceptibility studies. These studies show that F17L, S18L, and T51M directly lead to sulfonamide resistance while unexpectedly increasing susceptibility to trimethoprim, which targets the downstream enzyme dihydrofolate reductase. The secondary mutations E208K and KE257_dup restore trimethoprim susceptibility closer to wild-type levels while further increasing sulfonamide resistance. Structural studies reveal that these mutations appear to selectively disfavor the binding of the sulfonamides by sterically blocking an outer ring moiety that is not present in the substrate. This emphasizes that new inhibitors must be designed that strictly stay within the substrate volume in the context of the transition state.
Summary Bacteriophage T4 provides an important model system for studying the mechanism of homologous recombination. We have determined the crystal structure of the T4 UvsX recombinase, and the overall architecture and fold closely resembles that of RecA, including a highly conserved ATP binding site. Based on this new structure, we reanalyzed EM reconstructions of UvsX-DNA filaments and docked the UvsX crystal structure into two different filament forms, a compressed filament generated in the presence of ADP and an elongated filament generated in the presence of ATP and aluminum fluoride. In these reconstructions, the ATP binding site sits at the protomer interface, as in the RecA filament crystal structure. However, the environment of the ATP binding site is altered in the two filament reconstructions, suggesting that nucleotide cannot be as easily accommodated at the protomer interface of the compressed filament. Finally, we show that the phage helicase UvsW completes the UvsX-promoted strand-exchange reaction, allowing the generation of simple nicked circular product rather than complex networks of partially exchanged substrates.
The UvsY recombination mediator protein is critical for efficient homologous recombination in bacteriophage T4 and is the functional analog of the eukaryotic Rad52 protein. During T4 homologous recombination, the UvsX recombinase has to compete with the prebound gp32 single-stranded binding protein for DNA-binding sites and UvsY stimulates this filament nucleation event. We report here the crystal structure of UvsY in four similar open-barrel heptameric assemblies and provide structural and biophysical insights into its function. The UvsY heptamer was confirmed in solution by centrifugation and light scattering, and thermodynamic analyses revealed that the UvsY-ssDNA interaction occurs within the assembly via two distinct binding modes. Using surface plasmon resonance, we also examined the binding of UvsY to both ssDNA and the ssDNAgp32 complex. These analyses confirmed that ssDNA can bind UvsY and gp32 independently and also as a ternary complex. They also showed that residues located on the rim of the heptamer are required for optimal binding to ssDNA, thus identifying the putative ssDNAbinding surface. We propose a model in which UvsY promotes a helical ssDNA conformation that disfavors the binding of gp32 and initiates the assembly of the ssDNA-UvsX filament.homologous recombination | structural modification | DNA binding | DNA architecture | crystallography H omologous recombination (HR) involves the exchange of strands between homologous DNA molecules and has fundamental roles in double-stranded DNA (dsDNA) break repair, the rescue of stalled replication forks, and recombination-dependent replication (1). Defects in HR are associated with genetic instability, chromosomal abnormalities, and cancer (1). HR is performed by an ATP-dependent recombinase, RecA in prokaryotes and Rad51 in eukaryotes, that creates a filament with approximately sixfold helical symmetry. The filament first binds single-stranded DNA (ssDNA) and then samples incoming dsDNA to search for homology and promote the exchange reaction (2, 3). Key insights into the mechanism have been provided by structural (4) and dynamics (5, 6) studies of the HR filament. ssDNA-binding proteins, RPA in eukaryotes and SSB in prokaryotes, protect the ssDNA from nucleases and remove secondary structures during HR, but they also block the binding of the recombinase (3). This obstacle is overcome by the recombination mediator proteins (RMPs) (7), Rad52 in eukaryotes and RecOR in prokaryotes, that stimulate the exchange of the ssDNA-binding proteins for the recombinase.Phage T4 is able to process DNA in isolation from the host Escherichia coli by encoding all of the necessary DNA metabolic proteins. The T4 UvsXYW system encodes the core HR machinery (8) comprising UvsX (the recombinase), UvsW (the SF2 remodeling helicase), and UvsY (the RMP). Together with the T4 ssDNA-binding protein gp32 (9, 10), these three proteins can efficiently perform HR in an in vitro reconstituted system (11). UvsY is a 15.8-kDa protein with properties that are consistent with its rol...
Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is a highly conserved eukaryotic DNA repair enzyme that hydrolyses 3′phospho-DNA adducts, such as the 3′phospho-tyrosyl intermediate stabilized by camptothecins (CPT) poisoning of DNA topoisomerase I (Top1). Tdp1 catalytic mechanism contains two conserved His residues: The nucleophilic attack of the N-terminal histidine on the Top1-DNA complex releases Top1 and forms a 3′ phosph-histidyl intermediate. The second histidine activates a water molecule to hydrolyze the Tdp1-DNA complex. In humans, a mutation of the second His to Arg (H493R) is associated with the recessive neurodegenerative disease SCAN1. This mutant is defective in processing CPT-induced 3′ phospho-tyrosyl linkages and accumulates Tdp1-DNA complexes in vitro. The analogous yeast SCAN1 mutant (H432R) also enhances cell sensitivity to CPT. However, replacing His432 with Asn (H432N) or Gln (H432Q) induced an even more severe catalytic defect and a Top1-dependent lethality in the absence of CPT. As with SCAN1, these phenotypes were recessive and suppressed by wild-type Tdp1. Our findings suggest that Asn or Gln at this position precludes protonation of the Top1 active site tyrosine phenoxyanion, allowing for the regeneration of the original 3′phospho-tyrosyl linkage. Our recent crystal structures of the H432R and H432N mutants support such a ping-pong reaction mechanism. Surprisingly, however, substituting Ala for the first His (H182A), which should abolish the catalytic activity and therefore the catalytic defects of the Tdp1H432N mutant, did not abolish the Top1-dependent lethality of this Tdp1 mutant. Moreover, expression of the single H182A mutant also induced Top1-dependent toxicity. These data indicate the mutant enzymes are active, leading us to posit that in the absence of His182 but presents of the smaller Ala, the adjacent conserved His181, may rotate into the active site and act as a nucleophile to resolve the Top1-DNA intermediate. Consistent with this model, mutating His181 to Ala suppressed the Top1-dependent lethality of the H182A and H432N mutants. Rotation of His181 is allowed by the small and flexible Ala in the position of His182, thus, a more conservative substitution, such as Phe, should repress His181 rotation and its related toxicity. Indeed, the single His182 to Phe (H182F) mutant showed cell viability. These results indicate that His181 only rotate into the catalytic pocket when position 182 is substituted with a small and flexible residue, such as Ala. Related studies of the corresponding human Top1 and Tdp1 mutants induced similar lethal phenotypes in yeast, supporting a conservation of catalytic mechanism amongst Tdp1 orthologs. Our findings also indicate that the non-conserved N-terminal residues regulate Tdp1-protein interactions, which impact cellular levels of potentially lethal Top1- or Tdp1-DNA covalent complexes, such as those induced by CPT. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3645.
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