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Summary The broad-spectrum antibiotic d-cycloserine (DCS) is a key component of regimens used to treat multi- and extensively drug-resistant tuberculosis. DCS, a structural analogue of d-alanine, binds to and inactivates two essential enzymes involved in peptidoglycan biosynthesis, alanine racemase (Alr) and d-Ala:d-Ala ligase. Inactivation of Alr is thought to proceed via a mechanism-based irreversible route, forming an adduct with the pyridoxal 5’-phosphate cofactor, leading to bacterial death. Inconsistent with this hypothesis, Mycobacterium tuberculosis Alr activity can be detected after exposure to clinically relevant DCS concentrations. To address this paradox, we investigated the chemical mechanism of Alr inhibition by DCS. Inhibition of M. tuberculosis Alr and other Alrs is reversible, mechanistically revealed by a previously unidentified DCS-adduct hydrolysis. Dissociation and subsequent rearrangement to a stable substituted oxime explains Alr reactivation in the cellular milieu. This knowledge provides a novel route for discovery of improved Alr inhibitors against M. tuberculosis and other bacteria.

Summary RET receptor tyrosine kinase plays vital developmental and neuroprotective roles in metazoans. GDNF family ligands (GFLs) when bound to cognate GFRα co-receptors recognize and activate RET stimulating its cytoplasmic kinase function. The principles for RET ligand-co-receptor recognition are incompletely understood. Here, we report a crystal structure of the cadherin-like module (CLD1-4) from zebrafish RET revealing interdomain flexibility between CLD2 and CLD3. Comparison with a cryo-electron microscopy structure of a ligand-engaged zebrafish RETECD-GDNF-GFRα1a complex indicates conformational changes within a clade-specific CLD3 loop adjacent to the co-receptor. Our observations indicate that RET is a molecular clamp with a flexible calcium-dependent arm that adapts to different GFRα co-receptors, while its rigid arm recognizes a GFL dimer to align both membrane-proximal cysteine-rich domains. We also visualize linear arrays of RETECD-GDNF-GFRα1a suggesting that a conserved contact stabilizes higher-order species. Our study reveals that ligand-co-receptor recognition by RET involves both receptor plasticity and strict spacing of receptor dimers by GFL ligands., Graphical abstract, Highlights • X-ray structure of zebrafish RETCLD1-4 module reveals conformational flexibility • Conformational differences between RETCLD1-4 and a liganded RETECD cryo-EM structure • Spatial separation of RETECD CRD dimer C termini imposed by each ligand dimer • Differences in GDNF and GDF15 co-receptor engagement of RET and multimerization evidence, Adams et al. use X-ray crystallography and cryo-electron microscopy to probe conformational changes in RET due to binding of GDNF ligand-GFRα1 co-receptor. The study shows a two-site clamping mechanism with flexible adaptations near the RET calcium sites but near identical spacings of the RET cysteine-rich domains established by GDNF/GDF15 ligand dimers.

The Arp2/3 complex regulates many cellular processes by stimulating formation of branched actin filament networks. Because three of its seven subunits exist as two different isoforms, mammals produce a family of Arp2/3 complexes with different properties that may be suited to different physiological contexts. To shed light on how isoform diversification affects Arp2/3 function, we determined a 4.2 Å resolution cryo-EM structure of the most active human Arp2/3 complex containing ARPC1B and ARPC5L, and compared it with the structure of the least active ARPC1A-ARPC5-containing complex. The architecture of each isoform-specific Arp2/3 complex is the same. Strikingly, however, the N-terminal half of ARPC5L is partially disordered compared to ARPC5, suggesting that this region of ARPC5/ARPC5L is an important determinant of complex activity. Confirming this idea, the nucleation activity of Arp2/3 complexes containing hybrid ARPC5/ARPC5L subunits is higher when the ARPC5L N-terminus is present, thereby providing insight into activity differences between the different Arp2/3 complexes., Summary: The Arp2/3 complex stimulates formation of branched actin filament networks and exhibits isoform diversity in mammals. We show how different Arp2/3 subunit isoforms contribute to differences in complex function.

This study characterizes the interaction between the carboxy-terminal (ERLI) motif of the essential polarity protein Crb and the Pals1/Stardust PDZ-domain protein. Structures of human Pals1 PDZ with and without a Crb peptide are described, explaining the highly conserved nature of the ERLI motif and revealing a sterically blocked peptide-binding groove in the absence of ligand., Many components of epithelial polarity protein complexes possess PDZ domains that are required for protein interaction and recruitment to the apical plasma membrane. Apical localization of the Crumbs (Crb) transmembrane protein requires a PDZ-mediated interaction with Pals1 (protein-associated with Lin7, Stardust, MPP5), a member of the p55 family of membrane-associated guanylate kinases (MAGUKs). This study describes the molecular interaction between the Crb carboxy-terminal motif (ERLI), which is required for Drosophila cell polarity, and the Pals1 PDZ domain using crystallography and fluorescence polarization. Only the last four Crb residues contribute to Pals1 PDZ-domain binding affinity, with specificity contributed by conserved charged interactions. Comparison of the Crb-bound Pals1 PDZ structure with an apo Pals1 structure reveals a key Phe side chain that gates access to the PDZ peptide-binding groove. Removal of this side chain enhances the binding affinity by more than fivefold, suggesting that access of Crb to Pals1 may be regulated by intradomain contacts or by protein–protein interaction.

Sister-chromatid cohesion is established by Eco1-mediated acetylation on two conserved tandem lysines in the cohesin Smc3 subunit. However, the molecular basis of Eco1 substrate recognition and acetylation in cohesion is not fully understood. Here, we discover and rationalize the substrate specificity of Eco1 using mass spectrometry coupled with in-vitro acetylation assays and crystallography. Our structures of the X. laevis Eco2 (xEco2) bound to its primary and secondary Smc3 substrates demonstrate the plasticity of the substrate-binding site, which confers substrate specificity by concerted conformational changes of the central β hairpin and the C-terminal extension.

The functions of cohesin are central to genome integrity, chromosome organization and transcription regulation through its prevention of premature sister-chromatid separation and the formation of DNA loops. The loading of cohesin onto chromatin depends on the Scc2–Scc4 complex; however, little is known about how it stimulates the cohesion-loading activity. Here we determine the large ‘hook' structure of Scc2 responsible for catalysing cohesin loading. We identify key Scc2 surfaces that are crucial for cohesin loading in vivo. With the aid of previously determined structures and homology modelling, we derive a pseudo-atomic structure of the full-length Scc2–Scc4 complex. Finally, using recombinantly purified Scc2–Scc4 and cohesin, we performed crosslinking mass spectrometry and interaction assays that suggest Scc2–Scc4 uses its modular structure to make multiple contacts with cohesin., The cohesin complex maintains genome integrity by ensuring correct sister-chromatid segregation during mitosis and meiosis. Here, Chao et al. present a pseudo-atomic model of the full-length Scc2–Scc4 cohesin loader complex and reveal key Scc2 surfaces crucial for cohesin loading.

The phosphoserine/threonine binding protein 14-3-3 stimulates the catalytic activity of protein kinase C-epsilon (PKCepsilon) by engaging two tandem phosphoserine-containing motifs located between the PKCepsilon regulatory and catalytic domains (V3 region). Interaction between 14-3-3 and this region of PKCepsilon is essential for the completion of cytokinesis. Here, we report the crystal structure of 14-3-3zeta bound to a synthetic diphosphorylated PKCepsilon V3 region revealing how a consensus 14-3-3 site and a divergent 14-3-3 site cooperate to bind to 14-3-3 and so activate PKCepsilon. Thermodynamic data show a markedly enhanced binding affinity for two-site phosphopeptides over single-site 14-3-3 binding motifs and identifies Ser 368 as a gatekeeper phosphorylation site in this physiologically relevant 14-3-3 ligand. This dual-site intra-chain recognition has implications for other 14-3-3 targets, which seem to have only a single 14-3-3 motif, as other lower affinity and cryptic 14-3-3 gatekeeper sites might exist.

Mutations in the park2 gene, encoding the RING-inBetweenRING-RING E3 ubiquitin ligase parkin, cause 50% of autosomal recessive juvenile Parkinsonism cases. More than 70 known pathogenic mutations occur throughout parkin, many of which cluster in the inhibitory amino-terminal ubiquitin-like domain, and the carboxy-terminal RING2 domain that is indispensable for ubiquitin transfer. A structural rationale showing how autosomal recessive juvenile Parkinsonism mutations alter parkin function is still lacking. Here we show that the structure of parkin RING2 is distinct from canonical RING E3 ligases and lacks key elements required for E2-conjugating enzyme recruitment. Several pathogenic mutations in RING2 alter the environment of a single surface-exposed catalytic cysteine to inhibit ubiquitination. Native parkin adopts a globular inhibited conformation in solution facilitated by the association of the ubiquitin-like domain with the RING-inBetweenRING-RING C-terminus. Autosomal recessive juvenile Parkinsonism mutations disrupt this conformation. Finally, parkin autoubiquitinates only in cis, providing a molecular explanation for the recessive nature of autosomal recessive juvenile Parkinsonism., Mutations in the E3 ubiquitin ligase parkin are associated with juvenile Parkinson’s disease. Here the authors report the solution structure of the Parkin RING2 domain, revealing how disease-associated mutations affect its function and providing a molecular explanation for the recessive nature of the disease.

Summary Atypical protein kinase C (aPKC) is a key apical-basal polarity determinant and Par complex component. It is recruited by Par3/Baz (Bazooka in Drosophila) into epithelial apical domains through high-affinity interaction. Paradoxically, aPKC also phosphorylates Par3/Baz, provoking its relocalization to adherens junctions (AJs). We show that Par3 conserved region 3 (CR3) forms a tight inhibitory complex with a primed aPKC kinase domain, blocking substrate access. A CR3 motif flanking its PKC consensus site disrupts the aPKC kinase N lobe, separating P-loop/αB/αC contacts. A second CR3 motif provides a high-affinity anchor. Mutation of either motif switches CR3 to an efficient in vitro substrate by exposing its phospho-acceptor site. In vivo, mutation of either CR3 motif alters Par3/Baz localization from apical to AJs. Our results reveal how Par3/Baz CR3 can antagonize aPKC in stable apical Par complexes and suggests that modulation of CR3 inhibitory arms or opposing aPKC pockets would perturb the interaction, promoting Par3/Baz phosphorylation., Graphical Abstract, Highlights • Sequences flanking the Par3 CR3 consensus PKC site cooperate to inhibit aPKC • A Par3 CR3 inhibitory arm disrupts aPKC P-loop/αB/αC contacts and αC-helix position • Mutating either CR3 arm switches Par3 into an efficient aPKC substrate in vitro • Equivalent Bazooka substitutions alter its apical localization to AJs in vivo, Par3 is required for aPKC membrane recruitment, yet it polarizes to adherens junctions upon phosphorylation. Soriano et al. show that Par3 antagonizes active aPKC kinase by separating crucial N-lobe contacts. Disrupting high-affinity Par3 contacts switches it to an efficient aPKC substrate and polarizes Par3/Bazooka from apical domains to adherens junctions.

SummaryThe RET receptor tyrosine kinase is essential to vertebrate development and implicated in multiple human diseases. RET binds a cell surface bipartite ligand comprising a GDNF family ligand and a GFRα coreceptor, resulting in RET transmembrane signaling. We present a hybrid structural model, derived from electron microscopy (EM) and low-angle X-ray scattering (SAXS) data, of the RET extracellular domain (RETECD), GDNF, and GFRα1 ternary complex, defining the basis for ligand recognition. RETECD envelopes the dimeric ligand complex through a composite binding site comprising four discrete contact sites. The GFRα1-mediated contacts are crucial, particularly close to the invariant RET calcium-binding site, whereas few direct contacts are made by GDNF, explaining how distinct ligand/coreceptor pairs are accommodated. The RETECD cysteine-rich domain (CRD) contacts both ligand components and makes homotypic membrane-proximal interactions occluding three different antibody epitopes. Coupling of these CRD-mediated interactions suggests models for ligand-induced RET activation and ligand-independent oncogenic deregulation.