No abstract

PARP1 and PARP2 produce poly(ADP-ribose) in response to DNA breaks. HPF1 regulates PARP1/2 catalytic output, most notably permitting serine modification with ADP-ribose. However, PARP1 is substantially more abundant in cells than HPF1, challenging whether HPF1 can pervasively modulate PARP1. Here, we show biochemically that HPF1 efficiently regulates PARP1/2 catalytic output at sub-stoichiometric ratios matching their relative cellular abundances. HPF1 rapidly associates/dissociates from multiple PARP1 molecules, initiating serine modification before modification initiates on glutamate/aspartate, and accelerating initiation to be more comparable to elongation reactions forming poly(ADP-ribose). This “hit and run” mechanism ensures HPF1 contributions to PARP1/2 during initiation do not persist and interfere with PAR chain elongation. We provide structural insights into HPF1/PARP1 assembled on a DNA break, and assess HPF1 impact on PARP1 retention on DNA. Our data support the prevalence of serine-ADP-ribose modification in cells and the efficiency of serine-ADP-ribose modification required for an acute DNA damage response.

Iron acquisition at the outer membrane (OM) of Gram-negative bacteria is powered by the proton motive force (PMF) of the cytoplasmic membrane (CM), harnessed by the CM-embedded complex of ExbB, ExbD, and TonB. Its stoichiometry, ensemble structural features, and mechanism of action are unknown. By panning combinatorial phage libraries, periplasmic regions of dimerization between ExbD and TonB were predicted. Using overexpression of full-length His 6 -tagged exbB-exbD and S-tagged tonB, we purified detergent-solubilized complexes of ExbB-ExbD-TonB from Escherichia coli. Protein-detergent complexes of ϳ230 kDa with a hydrodynamic radius of ϳ6.0 nm were similar to previously purified ExbB 4 -ExbD 2 complexes. Significantly, they differed in electronegativity by native agarose gel electrophoresis. The stoichiometry was determined to be ExbB 4 -ExbD 1 -TonB 1 . Single-particle electron microscopy agrees with this stoichiometry. Two-dimensional averaging supported the phage display predictions, showing two forms of ExbD-TonB periplasmic heterodimerization: extensive and distal. Three-dimensional (3D) particle classification showed three representative conformations of ExbB 4 -ExbD 1 -TonB 1 . Based on our structural data, we propose a model in which ExbD shuttles a proton across the CM via an ExbB interprotein rearrangement. Proton translocation would be coupled to ExbD-mediated collapse of extended TonB in complex with ligand-loaded receptors in the OM, followed by repositioning of TonB through extensive dimerization with ExbD. Here we present the first report for purification of the ExbBExbD-TonB complex, molar ratios within the complex (4:1:1), and structural biology that provides insights into 3D organization. IMPORTANCEReceptors in the OM of Gram-negative bacteria allow entry of iron-bound siderophores that are necessary for pathogenicity. Numerous iron-acquisition strategies rely upon a ubiquitous and unique protein for energization: TonB. Complexed with ExbB and ExbD, the Ton system links the PMF to OM transport. Blocking iron uptake by targeting a vital nanomachine holds promise in therapeutics. Despite much research, the stoichiometry, structural arrangement, and molecular mechanism of the CM-embedded ExbB-ExbD-TonB complex remain unreported. Here we demonstrate in vitro evidence of ExbB 4 -ExbD 1 -TonB 1 complexes. Using 3D EM, we reconstructed the complex in three conformational states that show variable ExbD-TonB heterodimerization. Our structural observations form the basis of a model for TonB-mediated iron acquisition.

Gram-negative bacteria rely on the ExbB-ExbD-TonB system for the import of essential nutrients. Despite decades of research, the stoichiometry, subunit organization, and mechanism of action of the membrane proteins of the Ton system remain unclear. We copurified ExbB with ExbD as an ∼240 kDa protein-detergent complex, measured by light scattering and by native gels. Quantitative Coomassie staining revealed a stoichiometry of ExbB4-ExbD2. Negative stain electron microscopy and 2D analysis showed particles of ∼10 nm diameter in multiple structural states. Nanogold labeling identified the position of the ExbD periplasmic domain. Random conical tilt was used to reconstruct the particles in three structural states followed by sorting of the single particles and refinement of each state. The different states are interpreted by coordinated structural rearrangements between the cytoplasmic domain and the periplasmic domain, concordant with in vivo predictions.

The XRCC1–DNA ligase IIIα complex (XL) is critical for DNA single-strand break repair, a key target for PARP inhibitors in cancer cells deficient in homologous recombination. Here, we combined biophysical approaches to gain insights into the shape and conformational flexibility of the XL as well as XRCC1 and DNA ligase IIIα (LigIIIα) alone. Structurally-guided mutational analyses based on the crystal structure of the human BRCT–BRCT heterodimer identified the network of salt bridges that together with the N-terminal extension of the XRCC1 C-terminal BRCT domain constitute the XL molecular interface. Coupling size exclusion chromatography with small angle X-ray scattering and multiangle light scattering (SEC-SAXS–MALS), we determined that the XL is more compact than either XRCC1 or LigIIIα, both of which form transient homodimers and are highly disordered. The reduced disorder and flexibility allowed us to build models of XL particles visualized by negative stain electron microscopy that predict close spatial organization between the LigIIIα catalytic core and both BRCT domains of XRCC1. Together our results identify an atypical BRCT–BRCT interaction as the stable nucleating core of the XL that links the flexible nick sensing and catalytic domains of LigIIIα to other protein partners of the flexible XRCC1 scaffold.

SignificanceThe overproduction and purification of membrane proteins are intrinsically difficult, making their analysis challenging. We purified the TraE membrane protein from a bacterial conjugation system that is involved in plasmid transfer. Our results suggest that this protein forms hexamers with a central pore, and we also show that it binds to the TraD protein. The structure of TraE is completely different from that of the previously characterized periplasmic domain. This has intriguing implications for the role of TraE and of its interaction partner TraD in substrate translocation across the bacterial cell envelope. This work makes an important contribution to understanding of the mechanism of plasmid transfer, contributing to the design of approaches to inhibit the spread of antibiotic resistance genes.

Nutrient import across Gram-negative bacteria's outer membrane is powered by the proton-motive force, delivered by the cytoplasmic membrane protein complex ExbB-ExbD-TonB. Having purified the ExbB4-ExbD2 complex in the detergent dodecyl maltoside, we substituted amphipol A8-35 for detergent, forming a water-soluble membrane protein/amphipol complex. Properties of the ExbB4-ExbD2 complex in detergent or in amphipols were compared by gel electrophoresis, size exclusion chromatography, asymmetric flow field-flow fractionation, thermal stability assays, and electron microscopy. Bound detergent and fluorescently labeled amphipol were assayed quantitatively by 1D NMR and analytical ultracentrifugation, respectively. The structural arrangement of ExbB4-ExbD2 was examined by EM, small-angle X-ray scattering, and small-angle neutron scattering using a deuterated amphipol. The amphipol-trapped ExbB4-ExbD2 complex is slightly larger than its detergent-solubilized counterpart. We also investigated a different oligomeric form of the two proteins, ExbB6-ExbD4, and propose a structural arrangement of its transmembrane α-helical domains.

The eukaryotic DNA replication fork is a hub of enzymes that continuously act to synthesize DNA, propagate DNA methylation and other epigenetic marks, perform quality control, repair nascent DNA, and package this DNA into chromatin. Many of the enzymes involved in these spatiotemporally correlated processes perform their functions by binding to proliferating cell nuclear antigen (PCNA). A long-standing question has been how the plethora of PCNA-binding enzymes exert their activities without interfering with each other. As a first step towards deciphering this complex regulation, we studied how Chromatin Assembly Factor 1 (CAF-1) binds to PCNA. We demonstrate that CAF-1 binds to PCNA in a heretofore uncharacterized manner that depends upon a cation-pi (π) interaction. An arginine residue, conserved among CAF-1 homologs but absent from other PCNA-binding proteins, inserts into the hydrophobic pocket normally occupied by proteins that contain canonical PCNA interaction peptides (PIPs). Mutation of this arginine disrupts the ability of CAF-1 to bind PCNA and to assemble chromatin. The PIP of the CAF-1 p150 subunit resides at the extreme C-terminus of an apparent long α-helix (119 amino acids) that has been reported to bind DNA. The length of that helix and the presence of a PIP at the C-terminus are evolutionarily conserved among numerous species, ranging from yeast to humans. This arrangement of a very long DNA-binding coiled-coil that terminates in PIPs may serve to coordinate DNA and PCNA binding by CAF-1.