Ubiquitination by HECT E3 enzymes regulates myriad processes, including tumor suppression, transcription, protein trafficking, and degradation. HECT E3s use a two-step mechanism to ligate ubiquitin to target proteins. The first step is guided by interactions between the catalytic HECT domain and the E2∼ubiquitin intermediate, which promote formation of a transient, thioester-bonded HECT∼ubiquitin intermediate. Here we report that the second step of ligation is mediated by a distinct catalytic architecture established by both the HECT E3 and its covalently linked ubiquitin. The structure of a chemically trapped proxy for an E3∼ubiquitin-substrate intermediate reveals three-way interactions between ubiquitin and the bilobal HECT domain orienting the E3∼ubiquitin thioester bond for ligation, and restricting the location of the substrate-binding domain to prioritize target lysines for ubiquitination. The data allow visualization of an E2-to-E3-to-substrate ubiquitin transfer cascade, and show how HECT-specific ubiquitin interactions driving multiple reactions are repurposed by a major E3 conformational change to promote ligation.DOI: http://dx.doi.org/10.7554/eLife.00828.001

An F 2 population (n = 151) derived from Dahl salt-sensitive (S) and Lewis rats was raised on a 8% NaCl diet for 9 weeks and analyzed for blood pressure quantitative trait loci (QTL) by use of a whole genome scan. Chromosomes 5 and 10 yielded lod scores for linkage to blood pressure that were significant; chromosomes 1, 2, 3, 8, 16, 17, and 18 gave lod scores suggestive for linkage. Chromosome 7 gave a significant signal for heart weight with a lesser effect on blood pressure. Congenic strains were constructed by introgressing Lewis low-blood-pressure QTL alleles for chromosomes 1, 5, 10, and 17 into the S genetic background. Congenic strains for chromosomes 1, 5, and 10 had significantly lower blood pressure than S, proving the existence of QTL on these chromosomes, but the chromosome 17 congenic strain failed to trap a contrasting QTL allele. The QTL allele increasing blood pressure originated from S rats for all QTL except those on chromosomes 2 and 7 in which the Lewis allele increased blood pressure. Interactions between each QTL and every other locus in the genome scan yielded significant interactions between chromosomes 10 and 4, and between chromosomes 2 and 3.More than 30 years ago, Dahl et al. (1963) selectively bred rats for sensitivity (S rats) and resistance (R rats) to the hypertensive effect of a high-salt (NaCl) diet. Inbred strains of S and R rats were subsequently developed from Dahl's selectively bred lines (Rapp and Dene 1985). These strains are the prototypic animal model for studying salt-induced hypertension.S rats develop hypertension even on a low-salt diet, but this is markedly exacerbated by increased salt intake (Dahl et al. 1963;Rapp and Dene 1985). Chromosomal regions containing blood pressure quantitative trait loci (QTL) have been detected by the candidate gene approach starting in 1972 with a biochemical genetic marker for steroid 11␤-hydroxylase Dahl 1972a, 1976), and then in 1989 when restriction fragment-length polymorphisms first became available (Rapp et al. 1989). More recently, additional chromosomal regions containing blood pressure QTL were identified around candidate genes by use of Dahl rats and more modern genetic markers

Summary Atg7 is a noncanonical, homodimeric E1 enzyme that interacts with the noncanonical E2 enzyme, Atg3, to mediate conjugation of the ubiquitin-like protein (UBL) Atg8 during autophagy. Here we report that the unique N-terminal domain of Atg7 (Atg7NTD) recruits a unique “flexible region” from Atg3 (Atg3FR). The structure of an Atg7NTD-Atg3FR complex reveals hydrophobic residues from Atg3 engaging a conserved groove in Atg7, important for Atg8 conjugation. We also report the structure of the homodimeric Atg7 C-terminal domain, which is homologous to canonical E1s and bacterial antecedents. The structures, SAXS, and cross-linking data allow modeling of a full-length, dimeric (Atg7∼Atg8-Atg3)2 complex. The model and biochemical data provide a rationale for Atg7 dimerization: Atg8 is transferred in trans from the catalytic Cys of one Atg7 protomer to Atg3 bound to the N-terminal domain of the opposite Atg7 protomer within the homodimer. The studies reveal a distinctive E1∼UBL-E2 architecture for enzymes mediating autophagy.

Core functions of autophagy are mediated by ubiquitin-like protein (UBL) cascades, in which a homodimeric E1 enzyme, Atg7, directs the UBLs Atg8 and Atg12 to their respective E2 enzymes, Atg3 and Atg10. Crystallographic and mutational analyses of yeast (Atg7 – Atg3)2 and (Atg7 –Atg10)2 complexes reveal noncanonical, multisite E1 –E2 recognition in autophagy. Atg7’s unique N-terminal domain recruits distinctive elements from the Atg3 and Atg10 ‘backsides’. This, along with E1 and E2 conformational variability, allows presentation of ‘frontside’ Atg3 and Atg10 active sites to the catalytic cysteine in the C-terminal domain from the opposite Atg7 protomer in the homodimer. Despite different modes of binding, the data suggest that common principles underlie conjugation in both noncanonical and canonical UBL cascades, whereby flexibly tethered E1 domains recruit E2s through surfaces remote from their active sites to juxtapose the E1 and E2 catalytic cysteines.

Hypertension, the most frequently diagnosed clinical condition world-wide, predisposes individuals to morbidity and mortality, yet its underlying pathological etiologies are poorly understood. So far, a large number of quantitative trait loci (QTLs) have been identified in both humans and animal models, but how they function together in determining overall blood pressure (BP) in physiological settings is unknown. Here, we systematically and comprehensively performed pair-wise comparisons of individual QTLs to create a global picture of their functionality in an inbred rat model. Rather than each of numerous QTLs contributing to infinitesimal BP increments, a modularized pattern arises: two epistatic 'blocks' constitute basic functional 'units' for nearly all QTLs, designated as epistatic module 1 (EM1) and EM2. This modularization dictates the magnitude and scope of BP effects. Any EM1 member can contribute to BP additively to that of EM2, but not to those of the same module. Members of each EM display epistatic hierarchy, which seems to reflect a related functional pathway. Rat homologues of 11 human BP QTLs belong to either EM1 or EM2. Unique insights emerge into the novel genetic mechanism and hierarchy determining BP in the Dahl salt-sensitive SS/Jr (DSS) rat model that implicate a portion of human QTLs. Elucidating the pathways underlying EM1 and EM2 may reveal the genetic regulation of BP.