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Summary: Kv7.x (KCNQ) voltage-gated potassium channels form the cardiac and auditory IKs current and the neuronal M-current. The five Kv7 subtypes have distinct assembly preferences encoded by a C-terminal cytoplasmic assembly domain, the A-domain Tail. Here, we present the high-resolution structure of the Kv7.4 A-domain Tail together with biochemical experiments that show that the domain is a self-assembling, parallel, four-stranded coiled coil. Structural analysis and biochemical studies indicate conservation of the coiled coil in all Kv7 subtypes and that a limited set of interactions encode assembly specificity determinants. Kv7 mutations have prominent roles in arrhythmias, deafness, and epilepsy. The structure together with biochemical data indicate that A-domain Tail arrhythmia mutations cluster on the solvent-accessible surface of the subunit interface at a likely site of action for modulatory proteins. Together, the data provide a framework for understanding Kv7 assembly specificity and the molecular basis of a distinct set of Kv7 channelopathies. [Copyright &y& Elsevier]

E2 conjugating enzymes play a central role in ubiquitin and ubiquitin-like protein (ublp) transfer cascades: the E2 accepts the ublp from the E1 enzyme and then the E2 often interacts with an E3 enzyme to promote ublp transfer to the target. We report here the crystal structure of a complex between the C-terminal domain from NEDD8''s heterodimeric E1 (APPBP1-UBA3) and the catalytic core domain of NEDD8''s E2 (Ubc12). The structure and associated mutational analyses reveal molecular details of Ubc12 recruitment by NEDD8''s E1. Interestingly, the E1''s Ubc12 binding domain resembles ubiquitin and recruits Ubc12 in a manner mimicking ubiquitin''s interactions with ubiquitin binding domains. Structural comparison with E2-E3 complexes indicates that the E1 and E3 binding sites on Ubc12 may overlap and raises the possibility that crosstalk between E1 and E3 interacting with an E2 could influence the specificity and processivity of ublp transfer. [Copyright &y& Elsevier]

The UEV domain of the TSG101 protein functions in both HIV-1 budding and the vacuolar protein sorting (VPS) pathway, where it binds ubiquitylated proteins as they are sorted into vesicles that bud into late endosomal compartments called multivesicular bodies (MVBs). TSG101 UEV-ubiquitin interactions are therefore important for delivery of both substrates and hydrolytic enzymes to lysosomes, which receive proteins via fusion with MVBs. Here, we report the crystal structure of the TSG101 UEV domain in complex with ubiquitin at 2.0 A˚ resolution. TSG101 UEV contacts the Ile44 surface and an adjacent loop of ubiquitin through a highly solvated interface. Mutations that disrupt the interface inhibit MVB sorting, and the structure also explains how the TSG101 UEV can independently bind its ubiquitin and Pro-Thr/Ser-Ala-Pro peptide ligands. Remarkably, comparison with mapping data from other UEV and related E2 proteins indicates that although the different E2/UEV domains share the same structure and have conserved ubiquitin binding activity, they bind through very different interfaces. [Copyright &y& Elsevier]

E1 enzymes initiate ubiquitin-like protein (ubl) transfer cascades by catalyzing adenylation of the ubl''s C terminus. An E1''s selectivity for its cognate ubl is essential because the E1 subsequently coordinates the ubl with its correct downstream pathway. We report here the structure of the 120 kDa quaternary complex between human APPBP1-UBA3, a heterodimeric E1, its ubl NEDD8, and ATP. The E1 selectively recruits NEDD8 through a bipartite interface, involving a domain common to all ubl activating enzymes including bacterial ancestors, and also eukaryotic E1-specific sequences. By modeling ubiquitin into the NEDD8 binding site and performing mutational analysis, we identify a single conserved arginine in APPBP1-UBA3 that acts as a selectivity gate, preventing misactivation of ubiquitin by NEDD8''s E1. NEDD8 residues that interact with E1 correspond to residues in ubiquitin important for binding the proteasome and other ubiquitin-interacting proteins, suggesting that the conjugation and recognition machineries have coevolved for each specific ubl. [Copyright &y& Elsevier]

Nurr1, a nuclear receptor essential for the development, maintenance, and survival of midbrain dopaminergic neurons, is a potential therapeutic target for Parkinson's disease, a neurological disorder characterized by the degeneration of these same neurons. Efforts to identify Nurr1 agonists have been hampered by the recognition that it lacks several classic regulatory elements of nuclear receptor function, including the canonical ligand-binding pocket. Here we report that the dopamine metabolite 5,6-dihydroxyindole (DHI) binds directly to and modulates the activity of Nurr1. Using biophysical assays and X-ray crystallography, we show that DHI binds to the ligand-binding domain within a non-canonical pocket, forming a covalent adduct with Cys566. In cultured cells and zebrafish, DHI stimulates Nurr1 activity, including the transcription of target genes underlying dopamine homeostasis. These findings suggest avenues for developing synthetic Nurr1 ligands to ameliorate the symptoms and progression of Parkinson's disease. • The dopamine metabolite 5,6-dihydroxyindole (DHI) binds directly to Nurr1 • DHI forms a covalent adduct with Nurr1, reacting as the indolequinone with Cys566 • The Nurr1-metabolite structure reveals a previously unreported ligand-binding pocket • DHI stimulates the transcription of Nurr1 target genes underlying dopamine homeostasis Nurr1, a critical regulator of dopaminergic neuron health and potential therapeutic target for Parkinson's disease, lacks the canonical nuclear receptor ligand-binding pocket. Here, Bruning et al. demonstrate that the receptor binds to a dopamine metabolite and show that the metabolite drives the expression of cellular machinery underlying dopamine homeostasis. [ABSTRACT FROM AUTHOR]

Maintenance of genome stability following DNA damage requires origin-independent reinitiation of DNA replication at repaired replication forks. In E. coli, PriA, PriB, PriC, and DnaT play critical roles in recognizing repaired replication forks and reloading the replisome onto the template to reinitiate DNA replication. Here, we report the 2.0 A resolution crystal structure of E. coli PriB, revealing a dimer that consists of a single structural domain formed by two oligonucleotide/oligosaccharide binding (OB) folds. Structural similarity of PriB to single-stranded DNA binding proteins reveals insights into its mechanisms of DNA binding. The structure further establishes a putative protein interaction surface that may contribute to the role of PriB in primosome assembly by facilitating interactions with PriA and DnaT. This is the first high-resolution structure of a protein involved in oriC-independent replisome loading and provides unique insight into mechanisms of replication restart in E. coli.