Characterizing ATP processing by the AAA+ protein p97 at the atomic level.

The human enzyme p97 regulates various cellular pathways by unfolding hundreds of protein substrates in an ATP-dependent manner, making it an essential component of protein homeostasis and an impactful pharmacological target. The hexameric complex undergoes substantial conformational changes throughout its catalytic cycle. Here we elucidate the molecular motions that occur at the active site in the temporal window immediately before and after ATP hydrolysis by merging cryo-EM, NMR spectroscopy and molecular dynamics simulations. p97 populates a metastable reaction intermediate, the ADP·Pi state, which is poised between hydrolysis and product release. Detailed snapshots reveal that the active site is finely tuned to trap and eventually discharge the cleaved phosphate. Signalling pathways originating at the active site coordinate the action of the hexamer subunits and couple hydrolysis with allosteric conformational changes. Our multidisciplinary approach enables a glimpse into the sophisticated spatial and temporal orchestration of ATP handling by a prototype AAA+ protein.

Nucleosome dyad determines the H1 C-terminus collapse on distinct DNA arms.

Nucleosomes are symmetric structures. However, binding of linker histones generates an inherently asymmetric H1-nucleosome complex, and whether this asymmetry is transmitted to the overall nucleosome structure, and therefore also to chromatin, is unclear. Efforts to investigate potential asymmetry due to H1s have been hampered by the DNA sequence, which naturally differs in each gyre. To overcome this issue, we designed and analyzed by cryo-EM a nucleosome reconstituted with a palindromic (601L) 197-bp DNA. As in the non-palindromic 601 sequence, H1 restricts linker DNA flexibility but reveals partial asymmetrical unwrapping. However, in contrast to the non-palindromic nucleosome, in the palindromic nucleosome H1 CTD collapses to the proximal linker. Molecular dynamics simulations show that this could be dictated by a slightly tilted orientation of the globular domain (GD) of H1, which could be linked to the DNA sequence of the nucleosome dyad.

Allosteric control of Ubp6 and the proteasome via a bidirectional switch.

The proteasome recognizes ubiquitinated proteins and can also edit ubiquitin marks, allowing substrates to be rejected based on ubiquitin chain topology. In yeast, editing is mediated by deubiquitinating enzyme Ubp6. The proteasome activates Ubp6, whereas Ubp6 inhibits the proteasome through deubiquitination and a noncatalytic effect. Here, we report cryo-EM structures of the proteasome bound to Ubp6, based on which we identify mutants in Ubp6 and proteasome subunit Rpt1 that abrogate Ubp6 activation. The Ubp6 mutations define a conserved region that we term the ILR element. The ILR is found within the BL1 loop, which obstructs the catalytic groove in free Ubp6. Rpt1-ILR interaction opens the groove by rearranging not only BL1 but also a previously undescribed network of three interconnected active-site-blocking loops. Ubp6 activation and noncatalytic proteasome inhibition are linked in that they are eliminated by the same mutations. Ubp6 and ubiquitin together drive proteasomes into a unique conformation associated with proteasome inhibition. Thus, a multicomponent allosteric switch exerts simultaneous control over both Ubp6 and the proteasome.

Structural basis for safe and efficient energy conversion in a respiratory supercomplex.

Proton-translocating respiratory complexes assemble into supercomplexes that are proposed to increase the efficiency of energy conversion and limit the production of harmful reactive oxygen species during aerobic cellular respiration. Cytochrome bc complexes and cytochrome aa3 oxidases are major drivers of the proton motive force that fuels ATP generation via respiration, but how wasteful electron- and proton transfer is controlled to enhance safety and efficiency in the context of supercomplexes is not known. Here, we address this question with the 2.8 Å resolution cryo-EM structure of the cytochrome bcc-aa3 (III2-IV2) supercomplex from the actinobacterium Corynebacterium glutamicum. Menaquinone, substrate mimics, lycopene, an unexpected Qc site, dioxygen, proton transfer routes, and conformational states of key protonable residues are resolved. Our results show how safe and efficient energy conversion is achieved in a respiratory supercomplex through controlled electron and proton transfer. The structure may guide the rational design of drugs against actinobacteria that cause diphtheria and tuberculosis.

Structural basis of antagonism of human APOBEC3F by HIV-1 Vif.

HIV-1 virion infectivity factor (Vif) promotes degradation of the antiviral APOBEC3 (A3) proteins through the host ubiquitin-proteasome pathway to enable viral immune evasion. Disrupting Vif-A3 interactions to reinstate the A3-catalyzed suppression of human immunodeficiency virus type 1 (HIV-1) replication is a potential approach for antiviral therapeutics. However, the molecular mechanisms by which Vif recognizes A3 proteins remain elusive. Here we report a cryo-EM structure of the Vif-targeted C-terminal domain of human A3F in complex with HIV-1 Vif and the cellular cofactor core-binding factor beta (CBFß) at 3.9-Å resolution. The structure shows that Vif and CBFß form a platform to recruit A3F, revealing a direct A3F-recruiting role of CBFß beyond Vif stabilization, and captures multiple independent A3F-Vif interfaces. Together with our biochemical and cellular studies, our structural findings establish the molecular determinants that are critical for Vif-mediated neutralization of A3F and provide a comprehensive framework of how HIV-1 Vif hijacks the host protein degradation machinery to counteract viral restriction by A3F.

Transposon molecular domestication and the evolution of the RAG recombinase.

Domestication of a transposon (a DNA sequence that can change its position in a genome) to give rise to the RAG1-RAG2 recombinase (RAG) and V(D)J recombination, which produces the diverse repertoire of antibodies and T cell receptors, was a pivotal event in the evolution of the adaptive immune system of jawed vertebrates. The evolutionary adaptations that transformed the ancestral RAG transposase into a RAG recombinase with appropriately regulated DNA cleavage and transposition activities are not understood. Here, beginning with cryo-electron microscopy structures of the amphioxus ProtoRAG transposase (an evolutionary relative of RAG), we identify amino acid residues and domains the acquisition or loss of which underpins the propensity of RAG for coupled cleavage, its preference for asymmetric DNA substrates and its inability to perform transposition in cells. In particular, we identify two adaptations specific to jawed-vertebrates-arginine 848 in RAG1 and an acidic region in RAG2-that together suppress RAG-mediated transposition more than 1,000-fold. Our findings reveal a two-tiered mechanism for the suppression of RAG-mediated transposition, illuminate the evolution of V(D)J recombination and provide insight into the principles that govern the molecular domestication of transposons.

FipoQ/FBXO33, a Cullin-1-based ubiquitin ligase complex component modulates ubiquitination and solubility of polyglutamine disease protein.

Polyglutamine (polyQ) diseases describe a group of progressive neurodegenerative disorders caused by the CAG triplet repeat expansion in the coding region of the disease genes. To date, nine such diseases, including spinocerebellar ataxia type 3 (SCA3), have been reported. The formation of SDS-insoluble protein aggregates in neurons causes cellular dysfunctions, such as impairment of the ubiquitin-proteasome system, and contributes to polyQ pathologies. Recently, the E3 ubiquitin ligases, which govern substrate specificity of the ubiquitin-proteasome system, have been implicated in polyQ pathogenesis. The Cullin (Cul) proteins are major components of Cullin-RING ubiquitin ligases (CRLs) complexes that are evolutionarily conserved in the Drosophila genome. In this study, we examined the effect of individual Culs on SCA3 pathogenesis and found that the knockdown of Cul1 expression enhances SCA3-induced neurodegeneration and reduces the solubility of expanded SCA3-polyQ proteins. The F-box proteins are substrate receptors of Cul1-based CRL. We further performed a genetic modifier screen of the 19 Drosophila F-box genes and identified F-box involved in polyQ pathogenesis (FipoQ) as a genetic modifier of SCA3 degeneration that modulates the ubiquitination and solubility of expanded SCA3-polyQ proteins. In the human SK-N-MC cell model, we identified that F-box only protein 33 (FBXO33) exerts similar functions as FipoQ in modulating the ubiquitination and solubility of expanded SCA3-polyQ proteins. Taken together, our study demonstrates that Cul1-based CRL and its associated F-box protein, FipoQ/FBXO33, modify SCA3 protein toxicity. These findings will lead to a better understanding of the disease mechanism of SCA3 and provide insights for developing treatments against SCA3. Cover Image for this issue: doi: 10.1111/jnc.14510.

A Near-Atomic Structure of the Dark Apoptosome Provides Insight into Assembly and Activation.

In Drosophila, the Apaf-1-related killer (Dark) forms an apoptosome that activates procaspases. To investigate function, we have determined a near-atomic structure of Dark double rings using cryo-electron microscopy. We then built a nearly complete model of the apoptosome that includes 7- and 8-blade ß-propellers. We find that the preference for dATP during Dark assembly may be governed by Ser325, which is in close proximity to the 2' carbon of the deoxyribose ring. Interestingly, ß-propellers in V-shaped domains of the Dark apoptosome are more widely separated, relative to these features in the Apaf-1 apoptosome. This wider spacing may be responsible for the lack of cytochrome c binding to ß-propellers in the Dark apoptosome. Our structure also highlights the roles of two loss-of-function mutations that may block Dark assembly. Finally, the improved model provides a framework to understand apical procaspase activation in the intrinsic cell death pathway.

A near atomic structure of the active human apoptosome.

In response to cell death signals, an active apoptosome is assembled from Apaf-1 and procaspase-9 (pc-9). Here we report a near atomic structure of the active human apoptosome determined by cryo-electron microscopy. The resulting model gives insights into cytochrome c binding, nucleotide exchange and conformational changes that drive assembly. During activation an acentric disk is formed on the central hub of the apoptosome. This disk contains four Apaf-1/pc-9 CARD pairs arranged in a shallow spiral with the fourth pc-9 CARD at lower occupancy. On average, Apaf-1 CARDs recruit 3 to 5 pc-9 molecules to the apoptosome and one catalytic domain may be parked on the hub, when an odd number of zymogens are bound. This suggests a stoichiometry of one or at most, two pc-9 dimers per active apoptosome. Thus, our structure provides a molecular framework to understand the role of the apoptosome in programmed cell death and disease.

Structural mechanism of ligand activation in human calcium-sensing receptor.

Human calcium-sensing receptor (CaSR) is a G-protein-coupled receptor (GPCR) that maintains extracellular Ca(2+) homeostasis through the regulation of parathyroid hormone secretion. It functions as a disulfide-tethered homodimer composed of three main domains, the Venus Flytrap module, cysteine-rich domain, and seven-helix transmembrane region. Here, we present the crystal structures of the entire extracellular domain of CaSR in the resting and active conformations. We provide direct evidence that L-amino acids are agonists of the receptor. In the active structure, L-Trp occupies the orthosteric agonist-binding site at the interdomain cleft and is primarily responsible for inducing extracellular domain closure to initiate receptor activation. Our structures reveal multiple binding sites for Ca(2+) and PO4(3-) ions. Both ions are crucial for structural integrity of the receptor. While Ca(2+) ions stabilize the active state, PO4(3-) ions reinforce the inactive conformation. The activation mechanism of CaSR involves the formation of a novel dimer interface between subunits.

Expanded polyglutamine domain possesses nuclear export activity which modulates subcellular localization and toxicity of polyQ disease protein via exportin-1.

Polyglutamine (polyQ) diseases are a group of late-onset, progressive neurodegenerative disorders caused by CAG trinucleotide repeat expansion in the coding region of disease genes. The cell nucleus is an important site of pathology in polyQ diseases, and transcriptional dysregulation is one of the pathologic hallmarks observed. In this study, we showed that exportin-1 (Xpo1) regulates the nucleocytoplasmic distribution of expanded polyQ protein. We found that expanded polyQ protein, but not its unexpanded form, possesses nuclear export activity and interacts with Xpo1. Genetic manipulation of Xpo1 expression levels in transgenic Drosophila models of polyQ disease confirmed the specific nuclear export role of Xpo1 on expanded polyQ protein. Upon Xpo1 knockdown, the expanded polyQ protein was retained in the nucleus. The nuclear disease protein enhanced polyQ toxicity by binding to heat shock protein (hsp) gene promoter and abolished hsp gene induction. Further, we uncovered a developmental decline of Xpo1 protein levels in vivo that contributes to the accumulation of expanded polyQ protein in the nucleus of symptomatic polyQ transgenic mice. Taken together, we first showed that Xpo1 is a nuclear export receptor for expanded polyQ domain, and our findings establish a direct link between protein nuclear export and the progressive nature of polyQ neurodegeneration.

Crystallization and preliminary crystallographic analysis of the central domain of Drosophila Dribble, a protein that is essential for ribosome biogenesis.

Dribble (DBE) is a Drosophila protein that is essential for ribosome biogenesis. Bioinformatics analysis revealed a folded central domain of DBE which is flanked by structural disorder in the N- and C-terminal regions. The protein fragment spanning amino-acid residues 16-197 (DBE(16-197)) was produced for structural determination. In this report, the crystallization and preliminary X-ray diffraction data analysis of the DBE(16-197) protein domain are described. Crystals of DBE(16-197) were grown by the sitting-drop vapour-diffusion method at 289 K using ammonium phosphate as a precipitant. The crystals belonged to space group P2(1)2(1)2(1). Data were collected that extended to beyond 2 A resolution.