O-GalNAc (N-acetylgalactosaminyl) glycosylation is an abundant posttranslational modification in mammalian cells. Dysregulation of O-GalNAc glycosylation is implicated in cancer metastasis and immune evasion; however, our mechanistic understanding remains limited due to the lack of small-molecule tools. O-GalNAc biosynthesis depends heavily on the availability of UDP-GalNAc that is biosynthesised by the cytosolic enzyme UDP-galactose-4-epimerase (GalE). Knockout studies have demonstrated that loss of GalE severely impairs O-GalNAc glycosylation, positioning GalE as a promising enzymatic therapeutic target in oncology. Here, we present an efficient workflow that combines both covalent and high-throughput crystallographic non-covalent fragment screening with structure-based design to identify GalE inhibitors. Using these strategies, we discovered a ligandable pocket adjacent to a reactive tyrosine, enabling the development of a potent, "beyond cysteine" sulfonyl fluoride covalent inhibitor as well as a derived covalent alkyne probe. Structurally-enabled fragment screening methodologies yielded nanomolar non-covalent as well as covalent binders within no more than 22 elaborated compounds. Our work demonstrates synergism in next-generation delivery of chemical matter for GalE inhibition, with the broader potential for targeting non-cysteine residues in chemical biology and therapeutic applications.

Abstract Antibiotic discovery has long relied on occupancy-driven inhibition, leaving a vast number of potential bacterial targets undrugged. 1 Targeted protein degradation offers a mechanistically distinct alternative to inhibition, yet its application to antibacterial drug discovery remains largely unexplored. 2–4 Here we describe the development of first-in-class heterobifunctional bacterial proteolysis targeting chimeras (BacPROTACs) directed against an essential Mycobacterium tuberculosis protein, 4’-phosphopantetheinyl transferase (PptT). 5 Leveraging the modular architecture of BacPROTACs, we repurposed PptT inhibitors by incorporating them into degraders, yielding compounds with markedly improved antimycobacterial activity. Integrating in vitro and cellular approaches, we developed a characterisation pipeline to assess protein degradation in bacteria, applicable to future BacPROTAC programmes. Our study establishes targeted protein degradation as a strategy for antibacterial drug discovery.

AMP-activated protein kinase (AMPK) plays an important role in maintaining energy homeostasis in mammals. AMPK is a heterotrimer of an α catalytic subunit and two regulatory subunits, β and γ. In mammals, each subunit has different isoforms (α1/α2, β1/ β2, and γ1/γ2/γ3) encoded by separate genes, leading to the potential expression of 12 AMPK complexes. Here, we show that AMPK containing the long forms of γ2 (γ2a, encoding a protein of 569 amino acids, and γ2c, 525 amino acids) binds to 14-3-3. In contrast to AMPK containing the short form of γ2 (γ2b, 328 amino acids), bacterial expression of AMPK containing the long forms of γ2 requires co-expression with 14-3-3 and prior phosphorylation of Thr172 within the α subunit. AMPKγ2-14-3-3 complexes have reduced activity compared with AMPKγ1 or AMPKγ2b but retain allosteric activation by AMP and the AMPK activator, 991. We found that two predicted 14-3-3 binding sites within γ2a (T97 and S122) were phosphorylated in the bacterially expressed AMPK complex. Furthermore, we show that a peptide spanning these two phosphorylated sites binds to 14-3-3 in vitro and determined the crystal structure of this 14-3-3-peptide co-complex. These results indicate that 14-3-3 binds to the N-terminal region of γ2a/c, reducing the activity of AMPK relative to AMPKγ1 and AMPKγ2b. Our findings reveal a new mode of regulation of AMPK containing the long forms of γ2. While the biological significance of 14-3-3 binding to AMPKγ2a/c complexes remains to be determined, our studies provide the starting point to begin to address this issue.

Abstract Hexanucleotide (GGGGCC) repeat expansions in the non-coding region of C9ORF72 are a major genetic cause of ALS/FTD and reduce C9orf72-SMCR8-WDR41 complex levels, but how this contributes to autophagy-lysosome dysfunction and previously reported mitochondrial quality-control defects in C9ORF72-ALS/FTD remains unclear. Here we identify a direct interaction between SMCR8 and the FIP200 subunit of the ULK1/2 autophagy initiation complex, mediated by two FIP200-interacting region (FIR) motifs in a disordered SMCR8 loop. Phosphorylation of these motifs by ULK1/2 or TBK1 strengthens binding and promotes ULK1/2 complex association in cells. Stabilising the SMCR8-FIP200 interaction suppresses Parkin-dependent mitophagy, whereas both stabilisation and weakening impair deferiprone-induced mitophagy, while leaving bulk autophagy, lysophagy and ivermectin-induced mitophagy largely intact. These findings define a regulated C9orf72–ULK1/2 axis and provide a mechanistic framework by which repeat-expansion-associated reduction in C9orf72 complex abundance may contribute to previously observed mitochondrial quality-control defects in C9ORF72-ALS/FTD.

Cullin-RING ligases (CRLs) are the largest family of E3 ligases, with ubiquitination activity dynamically regulated by neddylation and deneddylation by the COP9 signalosome (CSN). CSN-mediated deneddylation not only deactivates CRLs but also enables substrate receptor exchange. Although CSN is a promising drug target, the structural basis underlying its catalytic mechanism remains unclear. Here, we use cryo-electron microscopy (cryo-EM) to uncover distinct functional states of CSN-CRL (SCF) complexes, capturing key intermediates of the deneddylation cycle. We visualise an autoinhibited docking state and a catalytic intermediate in which CSN5 Ins-1 loop, RBX1 RING and neddylated Cullin WHB domains are repositioned for isopeptide cleavage. We further resolve four dissociation intermediates that define the stepwise release of CSN from its product, with RBX1 RING stabilising key interactions. Additionally, our structures locate CSNAP within a CSN3-CSN8 groove. Together, our study provides a mechanistic model for CSN function and informs the rational design of CSN-targeted therapeutics.

Zinc finger antiviral protein (ZAP) binds CpG dinucleotides in viral RNA and targets them for decay. ZAP interacts with several cofactors to form the ZAP antiviral system, including KHNYN, a multidomain endoribonuclease required for ZAPmediated RNA decay. However, it is unclear how the individual domains in KHNYN contribute to its activity. Here, we demonstrate that the KHNYN amino-terminal extended-diKH (ex-diKH) domain is required for antiviral activity and present its crystal structure. The structure belongs to a rare group of KH-containing domains, characterized by a noncanonical arrangement between two type 1 KH modules, with an additional helical bundle. N4BP1 is a KHNYN paralog with an ex-diKH domain that functionally complements the KHNYN ex-diKH domain. Interestingly, the ex-diKH domain structure is present in N4BP1-like proteins in lancelets, which are basal chordates, indicating that it is evolutionarily ancient. While many KH domains demonstrate RNA binding activity, biolayer interferometry and electrophoretic mobility shift assays indicate that the KHNYN ex-diKH domain does not bind RNA. Furthermore, residues required for canonical KH domains to bind RNA are not required for KHNYN antiviral activity. By contrast, an inter-KH domain cleft in KHNYN is a potential protein-protein interaction site, and mutations that eliminate arginine salt bridges at the edge of this cleft decrease KHNYN antiviral activity. This suggests that this domain could be a binding site for an unknown KHNYN cofactor.

Plasmodium falciparum plasmepsin X (PMX) has become a target of choice for the development of new antimalarial drugs due to its essential role across the parasite life cycle. Here we describe the 1.7 Å crystallographic structure of PMX noncovalently bound to a potent macrocyclic peptidomimetic inhibitor (7k) possessing a hydroxyethylamine (HEA) scaffold. Upon 7k binding, the enzyme adopts a novel conformation, with significant involvement of the S2'S2 loop (M526-H536) and the S2 flap (F311-G314). This results in partial closure of the active site with widespread interactions in both the prime (S') and the non-prime (S) sites of PMX. The catalytic aspartate residues D266 and D467 directly interact with the HEA pharmacophore. Docking of a 7k derivative, compound 7a, highlights a region in the S3 pocket near the S3 flexible loop (H242-F248) that may be key for ligand stabilisation. The dynamic nature of PMX and its propensity to undergo distinct types of induced fit upon inhibitor binding enables generation of potent inhibitors that target this essential malarial aspartic protease.

Signaling from the T cell antigen receptor (TCR) on CD4+ T cells plays a critical role in adaptive immune responses by inducing T cell activation, proliferation, and differentiation. Here we demonstrate that WNK1, a kinase implicated in osmoregulation in the kidney, is required in T cells to support T-dependent antibody responses. We show that the canonical WNK1-OXSR1-STK39 kinase signaling pathway is required for TCR signaling in CD4+ T cells, their subsequent entry into the cell cycle, and suppression of the ATR-mediated G2/M cell cycle checkpoint. We show that the WNK1 pathway regulates ion influx leading to water influx, potentially through AQP3, and that water influx is required for TCR-induced signaling and cell cycle entry. Thus, TCR signaling via WNK1, OXSR1, STK39 and AQP3 leads to water entry that is essential for CD4+ T cell proliferation and hence T cell-dependent antibody responses.

Autophagy is a complex intracellular degradation pathway that depends on the coordinated interplay between the core autophagy machinery and diverse membrane sources to drive the de novo formation of double-membrane vesicles, known as autophagosomes. Golgi-derived Atg9-containing vesicles are essential for this process, delivering membranes to the pre-autophagosomal structure (PAS). These vesicles contain the transmembrane proteins Atg9 and Atg27 and the peripheral membrane protein Atg23; however, the nature, function, and regulation of their interactions remain poorly understood. Here, we systematically dissect the molecular interactions between Atg9, Atg23 and Atg27, and uncover their regulation in space and time. The bipartite binding mode by which Atg23 engages Atg9 provides a structural model for how Atg23 promotes vesicle budding. Furthermore, Atg1-dependent phosphorylation of Atg9 remodels its interactions with Atg23 and Atg27 at the PAS to support autophagy initiation. Together, these findings establish a molecular and regulatory framework for the earliest steps of autophagy. ### Competing Interest Statement The authors have declared no competing interest. Cancer Research UK, https://ror.org/054225q67, CC2064 UK Medical Research Council, CC2064 Wellcome Trust, https://ror.org/029chgv08, CC2064

SAMHD1 regulates cellular nucleotide homeostasis, controlling dNTP levels by catalysing their hydrolysis into 2'-deoxynucleosides and triphosphate. In differentiated CD4+ macrophage and resting T-cells SAMHD1 activity results in the inhibition of HIV-1 infection through a dNTP blockade. In cancer, SAMHD1 desensitizes cells to nucleoside-analogue chemotherapies. Here we employ time-resolved cryogenic-EM imaging and single-particle analysis to visualise assembly, allostery and catalysis by this multi-subunit enzyme. Our observations reveal how dynamic conformational changes in the SAMHD1 quaternary structure drive the catalytic cycle. We capture five states at high-resolution in a live catalytic reaction, revealing how allosteric activators support assembly of a stable SAMHD1 tetrameric core and how catalysis is driven by the opening and closing of active sites through pairwise coupling of active sites and order-disorder transitions in regulatory domains. This direct visualisation of enzyme catalysis dynamics within an allostery-stabilised platform sets a precedent for mechanistic studies into the regulation of multi-subunit enzymes. SAMHD1 is a regulator of dNTP homeostasis and an HIV restriction factor. The authors use time-resolved cryo-EM to visualise dynamic conformational changes that drive the catalytic cycle and allosteric regulation of this multi-subunit enzyme.

Abstract One of the key events in autophagy is the formation of a double-membrane phagophore, and many regulatory mechanisms underpinning this remain under investigation. WIPI2b is among the first proteins to be recruited to the phagophore and is essential for stimulating autophagy flux by recruiting the ATG12–ATG5–ATG16L1 complex, driving LC3 and GABARAP lipidation. Here, we set out to investigate how WIPI2b function is regulated by phosphorylation. We studied two phosphorylation sites on WIPI2b, S68 and S284. Phosphorylation at these sites plays distinct roles, regulating WIPI2b’s association with ATG16L1 and the phagophore, respectively. We confirm WIPI2b is a novel ULK1 substrate, validated by the detection of endogenous phosphorylation at S284. Notably, S284 is situated within an 18-amino acid stretch, which, when in contact with liposomes, forms an amphipathic helix. Phosphorylation at S284 disrupts the formation of the amphipathic helix, hindering the association of WIPI2b with membranes and autophagosome formation. Understanding these intricacies in the regulatory mechanisms governing WIPI2b’s association with its interacting partners and membranes, holds the potential to shed light on these complex processes, integral to phagophore biogenesis.

Covalent drugs can achieve high potency with long dosing intervals. However, concerns remain about side-effects associated with off-target reactivity. Combining macrocyclic peptides with covalent warheads provides a solution to minimise off-target reactivity: the peptide enables highly specific target binding, positioning a weakly reactive warhead proximal to a suitable residue in the target. Here we demonstrate the direct discovery of covalent cyclic peptides using encoded libraries containing a weakly electrophilic cysteine-reactive fluoroamidine warhead. We combine direct incorporation of the warhead into peptide libraries using the flexible in vitro translation system with a peptide selection approach that identifies only covalent target binders. Using this approach, we identify potent and selective covalent inhibitors of the peptidyl arginine deiminase, PADI4 or PAD4, that react exclusively at the active site cysteine. We envisage this approach will enable covalent peptide inhibitor discovery for a range of related enzymes and expansion to alternative warheads in the future.

Zinc finger antiviral protein (ZAP) binds CpG dinucleotides in viral RNA and targets them for decay. ZAP interacts with several cofactors to form the ZAP antiviral system, including KHNYN, a multidomain endoribonuclease required for ZAP-mediated RNA decay. However, it is unclear how the individual domains in KHNYN contribute to its activity. Here, we demonstrate that the KHNYN amino terminal extended-diKH (ex-diKH) domain is required for antiviral activity and present its crystal structure. The structure belongs to a rare group of KH-containing domains, characterized by a non-canonical arrangement between two type-1 KH modules, with an additional helical bundle. N4BP1 is a KHNYN paralog with an ex-diKH domain that functionally complements the KHNYN ex-diKH domain. Interestingly, the ex-diKH domain structure is present in N4BP1-like proteins in lancelets, which are basal chordates, indicating that it is evolutionarily ancient. While many KH domains demonstrate RNA binding activity, biolayer interferometry and electrophoretic mobility shift assays indicate that the KHNYN ex-diKH domain does not bind RNA. Furthermore, residues required for canonical KH domains to bind RNA are not required for KHNYN antiviral activity. By contrast, an inter-KH domain cleft in KHNYN is a potential protein-protein interaction site and mutations that eliminate arginine salt bridges at the edge of this cleft decrease KHNYN antiviral activity. This suggests that this domain could be a binding site for an unknown KHNYN cofactor. ### Competing Interest Statement The authors have declared no competing interest.

Signalling through TNFR1 modulates proinflammatory gene transcription and programmed cell death, and its impairment causes autoimmune diseases and cancer. NEDD4-binding protein 1 (N4BP1) is a critical suppressor of proinflammatory cytokine production that acts as a regulator of innate immune signalling and inflammation. However, our current understanding about the molecular properties that enable N4BP1 to exert its suppressive potential remain limited. Here, we show that N4BP1 is a novel linear ubiquitin reader that negatively regulates NFκB signalling by its unique dimerization-dependent ubiquitin-binding module that we named LUBIN. Dimeric N4BP1 strategically positions two non-selective ubiquitin-binding domains to ensure preferential recognition of linear ubiquitin. Under proinflammatory conditions, N4BP1 is recruited to the nascent TNFR1 signalling complex, where it regulates duration of proinflammatory signalling in LUBIN-dependent manner. N4BP1 deficiency accelerates TNFα-induced cell death by increasing complex II assembly. Under proapoptotic conditions, caspase-8 mediates proteolytic processing of N4BP1, resulting in rapid degradation of N4BP1 by the 26 S proteasome, and acceleration of apoptosis. In summary, our findings demonstrate that N4BP1 dimerization creates a novel type of ubiquitin reader that selectively recognises linear ubiquitin which enables the timely and coordinated regulation of TNFR1-mediated inflammation and cell death.

The cGMP-dependent protein kinase (PKG) is the sole cGMP sensor in malaria parasites, acting as an essential signalling hub to govern key developmental processes throughout the parasite life cycle. Despite the importance of PKG in the clinically relevant asexual blood stages, many aspects of malarial PKG regulation, including the importance of phosphorylation, remain poorly understood. Here we use genetic and biochemical approaches to show that reduced cGMP binding to cyclic nucleotide binding domain B does not affect in vitro kinase activity but prevents parasite egress. Similarly, we show that phosphorylation of a key threonine residue (T695) in the activation loop is dispensable for kinase activity in vitro but is essential for in vivo PKG function, with loss of T695 phosphorylation leading to aberrant phosphorylation events across the parasite proteome and changes to the substrate specificity of PKG. Our findings indicate that Plasmodium PKG is uniquely regulated to transduce signals crucial for malaria parasite development.

Modular SCF (SKP1-CUL1-Fbox) ubiquitin E3 ligases orchestrate multiple cellular pathways in eukaryotes. Their variable SKP1-Fbox substrate receptor (SR) modules enable regulated substrate recruitment and sub-sequent proteasomal degradation. CAND proteins are essential for the efficient and timely exchange of SRs. To gain structural understanding of the underlying molecular mechanism, we reconstituted a human CAND1-driven exchange reaction of substrate-bound SCF alongside its co-E3 ligase DCNL1 and visualized it by cryo-EM. We describe high-resolution structural intermediates, including a ternary CAND1-SCF complex, as well as conformational and compositional intermediates representing SR-or CAND1-dissociation. We describe in molecular detail how CAND1-induced conformational changes in CUL1/RBX1 provide an optimized DCNL1-binding site and reveal an unexpected dual role for DCNL1 in CAND1-SCF dynamics. Moreover, a partially dissociated CAND1-SCF conformation accommodates cullin neddylation, leading to CAND1 displacement. Our structural findings, together with functional biochemical assays, help formulate a detailed model for CAND-SCF regulation.

Poly(ADP-ribose) polymerase (PARP) inhibitors are used in the clinic to treat BRCA-deficient breast, ovarian and prostate cancers. As their efficacy is potentiated by loss of the nucleotide salvage factor DNPH1 there is considerable interest in the development of highly specific small molecule DNPH1 inhibitors. Here, we present X-ray crystal structures of dimeric DNPH1 bound to its substrate hydroxymethyl deoxyuridine monophosphate (hmdUMP). Direct interaction with the hydroxymethyl group is important for substrate positioning, while conserved residues surrounding the base facilitate target discrimination. Glycosidic bond cleavage is driven by a conserved catalytic triad and proceeds via a two-step mechanism involving formation and subsequent disruption of a covalent glycosyl-enzyme intermediate. Mutation of a previously uncharacterised yet conserved glutamate traps the intermediate in the active site, demonstrating its role in the hydrolytic step. These observations define the enzyme's catalytic site and mechanism of hydrolysis, and provide important insights for inhibitor discovery.

Homologous recombination is a fundamental process of life. It is required for the protection and restart of broken replication forks, the repair of chromosome breaks and the exchange of genetic material during meiosis. Individuals with mutations in key recombination genes, such as BRCA2 (also known as FANCD1), or the RAD51 paralogues RAD51B, RAD51C (also known as FANCO), RAD51D, XRCC2 (also known as FANCU) and XRCC3, are predisposed to breast, ovarian and prostate cancers1–10 and the cancer-prone syndrome Fanconi anaemia11–13. The BRCA2 tumour suppressor protein—the product of BRCA2—is well characterized, but the cellular functions of the RAD51 paralogues remain unclear. Genetic knockouts display growth defects, reduced RAD51 focus formation, spontaneous chromosome abnormalities, sensitivity to PARP inhibitors and replication fork defects14,15, but the precise molecular roles of RAD51 paralogues in fork stability, DNA repair and cancer avoidance remain unknown. Here we used cryo-electron microscopy, AlphaFold2 modelling and structural proteomics to determine the structure of the RAD51B–RAD51C–RAD51D–XRCC2 complex (BCDX2), revealing that RAD51C–RAD51D–XRCC2 mimics three RAD51 protomers aligned within a nucleoprotein filament, whereas RAD51B is highly dynamic. Biochemical and single-molecule analyses showed that BCDX2 stimulates the nucleation and extension of RAD51 filaments—which are essential for recombinational DNA repair—in reactions that depend on the coupled ATPase activities of RAD51B and RAD51C. Our studies demonstrate that BCDX2 orchestrates RAD51 assembly on single stranded DNA for replication fork protection and double strand break repair, in reactions that are critical for tumour avoidance. Structural and biochemical studies of the RAD51B–RAD51C–RAD51D–XRCC2 complex reveal that it uses coupled RAD51B and RAD51C ATPase activities to promote the nucleation and extension of RAD51 nucleoprotein filaments.

Chl1 is a member of the XPD family of 5’-3’ DNA helicases, which perform a variety of roles in genome maintenance and transmission. They possess a variety of unique structural features, including the presence of a highly variable, partially-ordered insertion in the helicase domain 1. Chl1 has been shown to be required for chromosome segregation in yeast due to its role in the formation of persistent chromosome cohesion during S-phase. Here we present structural and biochemical data to show that Chl1 has the same overall domain organisation as other members of the XPD family, but with some conformational alterations. We also present data suggesting the insert domain in Chl1 regulates its DNA binding.

Neuraminidase (NA) inhibitors (NAI), oseltamivir and zanamivir, are the main antiviral medications for influenza and monitoring of susceptibility to these antivirals is routinely done by determining 50 % inhibitory concentrations (IC50) with MUNANA substrate. During 2010-2019, levels of A(H3N2) viruses presenting reduced NAI inhibition (RI) were low (~0.75 %) but varied year-on-year. The highest proportions of viruses showing RI were observed during the 2013-2014, 2016-2017 and 2017-2018 Northern Hemisphere seasons. The majority of RI viruses were found to contain positively charged NA amino acid substitutions of N329K, K/S329R, S331R or S334R, being notably higher during the 2016-2017 season. Sialidase activity kinetics were determined for viruses of RI phenotype and contemporary wild-type (WT) viruses showing close genetic relatedness and displaying normal inhibition (NI). RI phenotypes resulted from reduced sialidase activity compared to relevant WT viruses. Those containing S329R or N329K or S331R showed markedly higher Km for the substrate and Ki values for NAIs, while those with S334R showed smaller effects. Substitutions at N329 and S331 disrupt a glycosylation sequon (NDS), confirmed to be utilised by mass spectrometry. However, gain of positive charge at all three positions was the major factor influencing the kinetic effects, not loss of glycosylation. Because of the altered enzyme characteristics NAs carrying these substitutions cannot be assessed reliably for susceptibility to NAIs using standard MUNANA-based assays due to reductions in the affinity of the enzyme for its substrate and the concentration of the substrate usually used.