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Abstract: DNA methyltransferase 3A (DNMT3A) plays a critical role in establishing and maintaining DNA methylation patterns in vertebrates. Here we structurally and biochemically explore the interaction of DNMT3A1 with diverse modified nucleosomes indicative of different chromatin environments. A cryo-EM structure of the full-length DNMT3A1-DNMT3L complex with a H2AK119ub nucleosome reveals that the DNMT3A1 ubiquitin-dependent recruitment (UDR) motif interacts specifically with H2AK119ub and makes extensive contacts with the core nucleosome histone surface. This interaction facilitates robust DNMT3A1 binding to nucleosomes, and previously unexplained DNMT3A disease-associated mutations disrupt this interface. Furthermore, the UDR-nucleosome interaction synergises with other DNMT3A chromatin reading elements in the absence of histone ubiquitylation. H2AK119ub does not stimulate DNMT3A DNA methylation activity, as observed for the previously described H3K36me2 mark, which may explain low levels of DNA methylation on H2AK119ub marked facultative heterochromatin. This study highlights the importance of multivalent binding of DNMT3A to histone modifications and the nucleosome surface and increases our understanding of how DNMT3A1 chromatin recruitment occurs.

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Abstract: Respiratory complex I is pivotal for cellular energy conversion, harnessing energy from NADH:ubiquinone oxidoreduction to drive protons across energy-transducing membranes for ATP synthesis. Despite detailed structural information on complex I, its mechanism of catalysis remains elusive due to lack of accompanying functional data for comprehensive structure-function analyses. Here, we present the 2.3-Å resolution structure of complex I from the α-proteobacterium Paracoccus denitrificans, a close relative of the mitochondrial progenitor, in phospholipid-bilayer nanodiscs. Three eukaryotic-type supernumerary subunits (NDUFS4, NDUFS6 and NDUFA12) plus a novel L-isoaspartyl-O-methyltransferase are bound to the core complex. Importantly, the enzyme is in a single, homogeneous resting state that matches the closed, turnover-ready (active) state of mammalian complex I. Our structure reveals the elements that stabilise the closed state and completes P. denitrificans complex I as a unified platform for combining structure, function and genetics in mechanistic studies.

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Abstract: Despite treatment and other interventions, an effective prophylactic HIV vaccine is still an essential goal in the control of HIV. Inducing robust and long-lasting antibody responses is one of the main targets of an HIV vaccine. The delivery of HIV envelope glycoproteins (Env) using nanoparticle (NP) platforms has been shown to elicit better immunogenicity than soluble HIV Env. In this paper, we describe the development of a nanoparticle-based vaccine decorated with HIV Env using the SpyCatcher/SpyTag system. The Env utilised in this study, CAP255, was derived from a transmitted founder virus isolated from a patient who developed broadly neutralising antibodies. Negative stain and cryo-electron microscopy analyses confirmed the assembly and stability of the mi3 into uniform icosahedral NPs surrounded by regularly spaced CAP255 gp140 Env trimers. A three-dimensional reconstruction of CAP255 gp140 SpyTag–SpyCatcher mi3 clearly showed Env trimers projecting from the centre of each of the pentagonal dodecahedral faces of the NP. To our knowledge, this is the first study to report the formation of SpyCatcher pentamers on the dodecahedral faces of mi3 NPs. To investigate the immunogenicity, rabbits were primed with two doses of DNA vaccines expressing the CAP255 gp150 and a mosaic subtype C Gag and boosted with three doses of the NP-developed autologous Tier 2 CAP255 neutralising antibodies (Nabs) and low levels of heterologous CAP256SU NAbs.

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Abstract: The bacterial tight adherence pilus system (TadPS) assembles surface pili essential for adhesion and colonisation in many human pathogens. Pilus dynamics are powered by the ATPase CpaF (TadA), which drives extension and retraction cycles in Caulobacter crescentus through an unknown mechanism. Here we use cryogenic electron microscopy and cell-based light microscopy to characterise CpaF mechanism. We show that CpaF assembles into a hexamer with C2 symmetry in different nucleotide states. Nucleotide cycling occurs through an intra-subunit clamp-like mechanism that promotes sequential conformational changes between subunits. Moreover, a comparison of the active sites with different nucleotides bound suggests a mechanism for bidirectional motion. Conserved CpaF residues, predicted to interact with platform proteins CpaG (TadB) and CpaH (TadC), are mutated in vivo to establish their role in pilus processing. Our findings provide a model for how CpaF drives TadPS pilus dynamics and have broad implications for how other ancient type 4 filament family members power pilus assembly.

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Abstract: Transposases drive chromosomal rearrangements and the dissemination of drug-resistance genes and toxins1,2,3. Although some transposases act alone, many rely on dedicated AAA+ ATPase subunits that regulate site selectivity and catalytic function through poorly understood mechanisms. Using IS21 as a model transposase system, we show how an ATPase regulator uses nucleotide-controlled assembly and DNA deformation to enable structure-based site selectivity, transposase recruitment, and activation and integration. Solution and cryogenic electron microscopy studies show that the IstB ATPase self-assembles into an autoinhibited pentamer of dimers that tightly curves target DNA into a half-coil. Two of these decamers dimerize, which stabilizes the target nucleic acid into a kinked S-shaped configuration that engages the IstA transposase at the interface between the two IstB oligomers to form an approximately 1 MDa transpososome complex. Specific interactions stimulate regulator ATPase activity and trigger a large conformational change on the transposase that positions the catalytic site to perform DNA strand transfer. These studies help explain how AAA+ ATPase regulators—which are used by classical transposition systems such as Tn7, Mu and CRISPR-associated elements—can remodel their substrate DNA and cognate transposases to promote function.