Open and closed conformations of a sub-80 kDa Chagas vaccine candidate defined by a cryo-EM led integrative approach

Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, remains a significant global public health concern. It affects an estimated eight million individuals worldwide, with the majority remaining undiagnosed. Despite its profound health impact in both endemic and non-endemic areas, no vaccine is available, and the existing therapies are outdated, producing severe side effects.The 80kDa prolyl oligopeptidase of Trypanosoma cruzi (TcPOP) has been recently identified as a leading candidate for Chagas vaccine development. However, its three-dimensional structure has remained elusive for almost two decades since its discovery. We report the first three-dimensional structure of TcPOP in open and closed conformation, at a resolution of 3.0 and 2.5 Angstroms respectively, determined using single-particle cryo-electron microscopy. Multiple conformations were observed and were further characterized, using plasmonic optical tweezers.To assess the immunogenic potential of TcPOP, we immunized mice and evaluated both polyclonal and monoclonal responses against the TcPOP antigen and its homologues. The results revealed unexpected cross-reactivity across prolyl POPs from other closely related parasites, but intriguingly, not towards the human homologue.Altogether, our findings provide critical structural insights necessary to understand the immunogenicity of TcPOP for future Chagas vaccine development and diagnostic applications.Additionally, our integrative approach indicated that stage-tilted acquisition can yield biologically relevant information for challenging sub-80kDa proteins and could adequately resolve the cryoEM structures. Consequently, this comprehensive strategy can significantly enhance the success rate in determining the structures of proteins that present challenges in characterization

Cryo-EM structure of human Pol κ bound to DNA and mono-ubiquitylated PCNA

Y-family DNA polymerase κ (Pol κ) can replicate damaged DNA templates to rescue stalled replication forks. Access of Pol κ to DNA damage sites is facilitated by its interaction with the processivity clamp PCNA and is regulated by PCNA mono-ubiquitylation. Here, we present cryo-EM reconstructions of human Pol κ bound to DNA, an incoming nucleotide, and wild type or mono-ubiquitylated PCNA (Ub-PCNA). In both reconstructions, the internal PIP-box adjacent to the Pol κ Polymerase-Associated Domain (PAD) docks the catalytic core to one PCNA protomer in an angled orientation, bending the DNA exiting the Pol κ active site through PCNA, while Pol κ C-terminal domain containing two Ubiquitin Binding Zinc Fingers (UBZs) is invisible, in agreement with disorder predictions. The ubiquitin moieties are partly flexible and extend radially away from PCNA, with the ubiquitin at the Pol κ-bound protomer appearing more rigid. Activity assays suggest that, when the internal PIP-box interaction is lost, Pol κ is retained on DNA by a secondary interaction between the UBZs and the ubiquitins flexibly conjugated to PCNA. Our data provide a structural basis for the recruitment of a Y-family TLS polymerase to sites of DNA damage.This research was supported by King Abdullah University of Science and Technology through core funding (to S.M.H.) and the Competitive Research Award Grant CRG8 URF/1/4036‐01‐01 (to S.M.H. and A.D.B.), and by the Wellcome Trust (to A.D.B.). R.C. acknowledges funding from the MINECO (CTQ2016-78636-P) and to AGAUR, (2017 SGR 324). The MD project has been carried out using CSUC resources. We acknowledge The Midlands Regional Cryo-EM Facility at the Leicester Institute of Structural and Chemical Biology (LISCB), major funding from MRC (MC_PC_17136). We thank Christos Savva (LISCB, University of Leicester) for his help in cryo-EM data collection and advice on data processing.Peer reviewe

Structural insights into DNA lagging strand replication and translesion synthesis

DNA replication requires the coordination of many different enzymes at the primer terminus. The eukaryotic sliding clamp, proliferating cell nuclear antigen (PCNA), encircles DNA at primer-template junctions, providing a platform for DNA polymerases and other enzymes to bind and increasing their processivity and activity on DNA. Many DNA processing enzymes contain a conserved PIP-box motif, which binds a hydrophobic pocket on PCNA. This thesis focuses on two processes: Okazaki fragment maturation and translesion synthesis (TLS). In the former, the lagging strand polymerase (Pol) δ synthesises each Okazaki fragment upstream of the last, displacing a portion of the downstream fragment. Flap endonuclease (FEN1) cleaves the resulting flap, and a ligase enzyme seals the nick. In TLS, Pol δ stalls opposite a DNA lesion. It must then be replaced by a TLS polymerase, which can bypass the lesion, before resuming synthesis downstream. Both processes involve sequential action of enzymes. As PCNA is a homotrimer with three PIP-box binding pockets, it may recruit more than one of these enzymes simultaneously for a fast and efficient changeover. This simultaneous binding, the “toolbelt” model, has been demonstrated experimentally in archaea and bacteria. Evidence of PCNA toolbelts in eukaryotes has proven more elusive, and this thesis set out to explore the notion further. Using single-particle cryo-electron microscopy, the work presented here has uncovered structural data of different polymerases forming holoenzymes with PCNA and primer-template DNA. This work shows that Pol δ binds PCNA at the side, leaving room for a second binding partner – and indeed we show structural evidence that FEN1 can bind PCNA simultaneously, forming a toolbelt. TLS Pol κ also binds towards one side of PCNA, inviting the hypothesis of a TLS toolbelt. Together, this work provides an insight into how the interactions of enzymes with PCNA facilitates enzyme switching at the primer terminus.</p

Mechanism of human Lig1 regulation by PCNA in Okazaki fragment sealing

During lagging strand synthesis, DNA Ligase 1 (Lig1) cooperates with the sliding clamp PCNA to seal the nicks between Okazaki fragments generated by Pol δ and Flap endonuclease 1 (FEN1). We present several cryo-EM structures combined with functional assays, showing that human Lig1 recruits PCNA to nicked DNA using two PCNA-interacting motifs (PIPs) located at its disordered N-terminus (PIPN-term) and DNA binding domain (PIPDBD). Once Lig1 and PCNA assemble as two-stack rings encircling DNA, PIPN-term is released from PCNA and only PIPDBD is required for ligation to facilitate the substrate handoff from FEN1. Consistently, we observed that PCNA forms a defined complex with FEN1 and nicked DNA, and it recruits Lig1 to an unoccupied monomer creating a toolbelt that drives the transfer of DNA to Lig1. Collectively, our results provide a structural model on how PCNA regulates FEN1 and Lig1 during Okazaki fragments maturation.This research was supported by King Abdullah University of Science and Technology through core funding (to S.M.H. and A.D.B.) and the Competitive Research Award Grant CRG8 URF/1/4036‐01‐01 (to S.M.H. and A.D.B.). We acknowledge The Midlands Regional Cryo-EM Facility at the Leicester Institute of Structural and Chemical Biology (LISCB), major funding from MRC (MC_PC_17136). We thank Christos Savva and T.J. Ragan (LISCB, University of Leicester) for their help in cryo-EM data collection and advice on data processing. This project has been carried out using the resources of CSUC.Peer reviewe

Author Correction: Mechanism of human Lig1 regulation by PCNA in Okazaki fragment sealing

Peer reviewe