Chen Shen, Mihir Vohra, Pengfei Zhang, Xianrong Mao, Matthew D. Figley, Jian Zhu, Yo Sasaki, Hao Wu, Aaron DiAntonio, and Jeffrey Milbrandt. 1/26/2021. “Multiple domain interfaces mediate SARM1 autoinhibition.” Proceedings of the National Academy of Sciences, 118, 4. Publisher's VersionAbstract
Axon degeneration is an active program of subcellular self-destruction that drives pathology in the injured and diseased nervous system. SARM1 is an inducible NAD+ hydrolase and the central executioner of axon loss. In healthy axons, the SARM1 NADase is autoinhibited. With injury or disease, this autoinhibition is relieved and SARM1 depletes NAD+, inducing a metabolic crisis and subsequent axon loss. Here we combine peptide screening, cryo-electron microscopy, and site-directed mutagenesis with analysis of axonal metabolomics and axon degeneration to define five domain interactions within and across SARM1 protomers that are required to maintain an inactive SARM1 octamer. These structural insights may enable the development of SARM1 inhibitors that stabilize this autoinhibited conformation and thereby block axon degeneration.Axon degeneration is an active program of self-destruction mediated by the protein SARM1. In healthy neurons, SARM1 is autoinhibited and, upon injury autoinhibition is relieved, activating the SARM1 enzyme to deplete NAD+ and induce axon degeneration. SARM1 forms a homomultimeric octamer with each monomer composed of an N-terminal autoinhibitory ARM domain, tandem SAM domains that mediate multimerization, and a C-terminal TIR domain encoding the NADase enzyme. Here we discovered multiple intramolecular and intermolecular domain interfaces required for SARM1 autoinhibition using peptide mapping and cryo-electron microscopy (cryo-EM). We identified a candidate autoinhibitory region by screening a panel of peptides derived from the SARM1 ARM domain, identifying a peptide mediating high-affinity inhibition of the SARM1 NADase. Mutation of residues in full-length SARM1 within the region encompassed by the peptide led to loss of autoinhibition, rendering SARM1 constitutively active and inducing spontaneous NAD+ and axon loss. The cryo-EM structure of SARM1 revealed 1) a compact autoinhibited SARM1 octamer in which the TIR domains are isolated and prevented from oligomerization and enzymatic activation and 2) multiple candidate autoinhibitory interfaces among the domains. Mutational analysis demonstrated that five distinct interfaces are required for autoinhibition, including intramolecular and intermolecular ARM-SAM interfaces, an intermolecular ARM-ARM interface, and two ARM-TIR interfaces formed between a single TIR and two distinct ARM domains. These autoinhibitory regions are not redundant, as point mutants in each led to constitutively active SARM1. These studies define the structural basis for SARM1 autoinhibition and may enable the development of SARM1 inhibitors that stabilize the autoinhibited state.All study data are included in the article and/or supporting information. The atomic coordinates and cryo-EM map have been deposited in the Protein Data Bank (PDB), (PDB ID code 7KNQ) (46), and the EM Data Resource, (ID code EMD-22954) (47).
Travis Walton, Hao Wu, and Alan Brown. 1/20/2021. “Structure of a microtubule-bound axonemal dynein.” Nature Communications, 12, 1, Pp. 477. Publisher's VersionAbstract
Axonemal dyneins are tethered to doublet microtubules inside cilia to drive ciliary beating, a process critical for cellular motility and extracellular fluid flow. Axonemal dyneins are evolutionarily and biochemically distinct from cytoplasmic dyneins that transport cargo, and the mechanisms regulating their localization and function are poorly understood. Here, we report a single-particle cryo-EM reconstruction of a three-headed axonemal dynein natively bound to doublet microtubules isolated from cilia. The slanted conformation of the axonemal dynein causes interaction of its motor domains with the neighboring dynein complex. Our structure shows how a heterotrimeric docking complex specifically localizes the linear array of axonemal dyneins to the doublet microtubule by directly interacting with the heavy chains. Our structural analysis establishes the arrangement of conserved heavy, intermediate and light chain subunits, and provides a framework to understand the roles of individual subunits and the interactions between dyneins during ciliary waveform generation.
Katherine J. Susa, Shaun Rawson, Andrew C. Kruse, and Stephen C. Blacklow. 1/15/2021. “Cryo-EM structure of the B cell co-receptor CD19 bound to the tetraspanin CD81.” Science, 371, 6526, Pp. 300–305. Publisher's VersionAbstract
A core component of the immune system are B cells, which are activated by infection and then mature to provide long-lived immunity. Activation is initiated when a cell surface B cell receptor, in association with its coreceptor, recognizes an antigen. Susa et al. report a structure of the B cell receptor CD81 in complex with its co receptor, CD19. CD81 alone binds to cholesterol, but the conformational changes associated with binding to CD19 occlude the cholesterol-binding pocket. Regulating cholesterol binding could play a role in the activation mechanism. The structure also provides a basis for the design of immunotherapies.Science, this issue p. 300Signaling through the CD19-CD81 co-receptor complex, in combination with the B cell receptor, is a critical determinant of B cell development and activation. It is unknown how CD81 engages CD19 to enable co-receptor function. Here, we report a 3.8-angstrom structure of the CD19-CD81 complex bound to a therapeutic antigen-binding fragment, determined by cryo–electron microscopy (cryo-EM). The structure includes both the extracellular domains and the transmembrane helices of the complex, revealing a contact interface between the ectodomains that drives complex formation. Upon binding to CD19, CD81 opens its ectodomain to expose a hydrophobic CD19-binding surface and reorganizes its transmembrane helices to occlude a cholesterol binding pocket present in the apoprotein. Our data reveal the structural basis for CD19-CD81 complex assembly, providing a foundation for rational design of therapies for B cell dysfunction.
Tobias Herrmann, Raúl Torres, Eric N. Salgado, Cristina Berciu, Daniel Stoddard, Daniela Nicastro, Simon Jenni, and Stephen C. Harrison. 1/13/2021. “Functional refolding of the penetration protein on a non-enveloped virus.” Nature, 590, 7847, Pp. 666-670. Publisher's VersionAbstract
A non-enveloped virus requires a membrane lesion to deliver its genome into a target cell1. For rotaviruses, membrane perforation is a principal function of the viral outer-layer protein, VP42,3. Here we describe the use of electron cryomicroscopy to determine how VP4 performs this function and show that when activated by cleavage to VP8* and VP5*, VP4 can rearrange on the virion surface from an `upright' to a `reversed' conformation. The reversed structure projects a previously buried `foot' domain outwards into the membrane of the host cell to which the virion has attached. Electron cryotomograms of virus particles entering cells are consistent with this picture. Using a disulfide mutant of VP4, we have also stabilized a probable intermediate in the transition between the two conformations. Our results define molecular mechanisms for the first steps of the penetration of rotaviruses into the membranes of target cells and suggest similarities with mechanisms postulated for other viruses.
Tianshu Xiao, Jianming Lu, Jun Zhang, Rebecca I. Johnson, Lindsay G. A. McKay, Nadia Storm, Christy L. Lavine, Hanqin Peng, Yongfei Cai, Sophia Rits-Volloch, Shen Lu, Brian D. Quinlan, Michael Farzan, Michael S. Seaman, Anthony Griffiths, and Bing Chen. 1/11/2021. “A trimeric human angiotensin-converting enzyme 2 as an anti-SARS-CoV-2 agent.” Nature Structural & Molecular Biology. Publisher's VersionAbstract
Effective intervention strategies are urgently needed to control the COVID-19 pandemic. Human angiotensin-converting enzyme 2 (ACE2) is a membrane-bound carboxypeptidase that forms a dimer and serves as the cellular receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). ACE2 is also a key negative regulator of the renin–angiotensin system that modulates vascular functions. We report here the properties of a trimeric ACE2 ectodomain variant, engineered using a structure-based approach. The trimeric ACE2 variant has a binding affinity of \textasciitilde60þinspace}pM for the spike protein of SARS‑CoV‑2 (compared with 77þinspace}nM for monomeric ACE2 and 12–22þinspace}nM for dimeric ACE2 constructs), and its peptidase activity and the ability to block activation of angiotensin II receptor type 1 in the renin–angiotensin system are preserved. Moreover, the engineered ACE2 potently inhibits SARS‑CoV‑2 infection in cell culture. These results suggest that engineered, trimeric ACE2 may be a promising anti-SARS-CoV-2 agent for treating COVID-19.
L. Robert Hollingsworth, Liron David, Yang Li, Andrew R. Griswold, Jianbin Ruan, Humayun Sharif, Pietro Fontana, Elizabeth L. Orth-He, Tian-Min Fu, Daniel A. Bachovchin, and Hao Wu. 1/8/2021. “Mechanism of filament formation in UPA-promoted CARD8 and NLRP1 inflammasomes.” Nature Communications, 12, 1, Pp. 189. Publisher's VersionAbstract
NLRP1 and CARD8 are related cytosolic sensors that upon activation form supramolecular signalling complexes known as canonical inflammasomes, resulting in caspase−1 activation, cytokine maturation and/or pyroptotic cell death. NLRP1 and CARD8 use their C-terminal (CT) fragments containing a caspase recruitment domain (CARD) and the UPA (conserved in UNC5, PIDD, and ankyrins) subdomain for self-oligomerization, which in turn form the platform to recruit the inflammasome adaptor ASC (apoptosis-associated speck-like protein containing a CARD) or caspase-1, respectively. Here, we report cryo-EM structures of NLRP1-CT and CARD8-CT assemblies, in which the respective CARDs form central helical filaments that are promoted by oligomerized, but flexibly linked, UPAs surrounding the filaments. Through biochemical and cellular approaches, we demonstrate that the UPA itself reduces the threshold needed for NLRP1-CT and CARD8-CT filament formation and signalling. Structural analyses provide insights on the mode of ASC recruitment by NLRP1-CT and the contrasting direct recruitment of caspase-1 by CARD8-CT. We also discover that subunits in the central NLRP1CARD filament dimerize with additional exterior CARDs, which roughly doubles its thickness and is unique among all known CARD filaments. Finally, we engineer and determine the structure of an ASCCARD–caspase-1CARD octamer, which suggests that ASC uses opposing surfaces for NLRP1, versus caspase-1, recruitment. Together these structures capture the architecture and specificity of the active NLRP1 and CARD8 inflammasomes in addition to key heteromeric CARD-CARD interactions governing inflammasome signalling.
Kazuki Kato, Sadeem Ahmad, Zixiang Zhu, Janet M. Young, Xin Mu, Se Hoon Park, Harmit S. Malik, and Sun Hur. 12/16/2020. “Structural analysis of RIG-I-like receptors reveals ancient rules of engagement between diverse RNA helicases and TRIM ubiquitin ligases.” Molecular Cell. Publisher's VersionAbstract
Summary RNA helicases and E3 ubiquitin ligases mediate many critical functions in cells, but their actions have largely been studied in distinct biological contexts. Here, we uncover evolutionarily conserved rules of engagement between RNA helicases and tripartite motif (TRIM) E3 ligases that lead to their functional coordination in vertebrate innate immunity. Using cryoelectron microscopy and biochemistry, we show that RIG-I-like receptors (RLRs), viral RNA receptors with helicase domains, interact with their cognate TRIM/TRIM-like E3 ligases through similar epitopes in the helicase domains. Their interactions are avidity driven, restricting the actions of TRIM/TRIM-like proteins and consequent immune activation to RLR multimers. Mass spectrometry and phylogeny-guided biochemical analyses further reveal that similar rules of engagement may apply to diverse RNA helicases and TRIM/TRIM-like proteins. Our analyses suggest not only conserved substrates for TRIM proteins but also, unexpectedly, deep evolutionary connections between TRIM proteins and RNA helicases, linking ubiquitin and RNA biology throughout animal evolution.
Miao Gui, Meisheng Ma, Erica Sze-Tu, Xiangli Wang, Fujiet Koh, Ellen D. Zhong, Bonnie Berger, Joseph H. Davis, Susan K. Dutcher, Rui Zhang, and Alan Brown. 12/14/2020. “Structures of radial spokes and associated complexes important for ciliary motility.” Nature Structural & Molecular Biology, 28, 1, Pp. 29-37. Publisher's VersionAbstract
In motile cilia, a mechanoregulatory network is responsible for converting the action of thousands of dynein motors bound to doublet microtubules into a single propulsive waveform. Here, we use two complementary cryo-EM strategies to determine structures of the major mechanoregulators that bind ciliary doublet microtubules in Chlamydomonas reinhardtii. We determine structures of isolated radial spoke RS1 and the microtubule-bound RS1, RS2 and the nexin−dynein regulatory complex (N-DRC). From these structures, we identify and build atomic models for 30 proteins, including 23 radial-spoke subunits. We reveal how mechanoregulatory complexes dock to doublet microtubules with regular 96-nm periodicity and communicate with one another. Additionally, we observe a direct and dynamically coupled association between RS2 and the dynein motor inner dynein arm subform c (IDAc), providing a molecular basis for the control of motor activity by mechanical signals. These structures advance our understanding of the role of mechanoregulation in defining the ciliary waveform.
Longfei Wang, Di Wu, Carol V. Robinson, Hao Wu, and Tian-Min Fu. 10/15/2020. “Structures of a Complete Human V-ATPase Reveal Mechanisms of Its Assembly.” Molecular Cell. Publisher's Version
Pranav N. M. Shah, David J. Filman, Krishanthi S. Karunatilaka, Emma L. Hesketh, Elisabetta Groppelli, Mike Strauss, and James M. Hogle. 10/2/2020. “Cryo-EM structures reveal two distinct conformational states in a picornavirus cell entry intermediate.” PLOS Pathogens, 16, 9, Pp. 1-26. Publisher's VersionAbstract
Author summary Nonenveloped viruses need to provide mechanisms that allow their genomes to be delivered across membranes. This process remains poorly understood. For enteroviruses such as poliovirus, genome delivery involves a program of conformational changes that include expansion of the particle and externalization of two normally internal peptides, VP4 and the VP1 N-terminus, which then insert into the cell membrane, triggering endocytosis and the creation of pores that facilitate the transfer of the viral RNA genome across the endosomal membrane. This manuscript describes five high-resolution cryo-EM structures of altered poliovirus particles that represent a number of intermediates along this pathway. The structures reveal several surprising findings, including the discovery of a new intermediate that is expanded, but has not yet externalized the membrane interactive peptides; the clear identification of a unique exit site for the VP1 N-terminus; the demonstration that the externalized VP1 N-terminus partitions between two different sites in a temperature-dependent fashion; direct visualization of an amphipathic helix at the N-terminus of VP1 in an ideal position for interaction with cellular membranes; and the observation that a significant portion of VP4 remains inside the particle and accounts for a density feature that had previously been ascribed to part of the viral RNA. These findings represent significant additions to our understanding of the cell entry process of an important class of human pathogens.
Jeong Joo Kim, Anant Gharpure, Jinfeng Teng, Yuxuan Zhuang, Rebecca J. Howard, Shaotong Zhu, Colleen M. Noviello, Richard M. Walsh, Erik Lindahl, and Ryan E. Hibbs. 9/2/2020. “Shared structural mechanisms of general anaesthetics and benzodiazepines.” Nature. Publisher's VersionAbstract
Most general anaesthetics and classical benzodiazepine drugs act through positive modulation of $\gamma$-aminobutyric acid type A (GABAA) receptors to dampen neuronal activity in the brain1–5. However, direct structural information on the mechanisms of general anaesthetics at their physiological receptor sites is lacking. Here we present cryo-electron microscopy structures of GABAA receptors bound to intravenous anaesthetics, benzodiazepines and inhibitory modulators. These structures were solved in a lipidic environment and are complemented by electrophysiology and molecular dynamics simulations. Structures of GABAA receptors in complex with the anaesthetics phenobarbital, etomidate and propofol reveal both distinct and common transmembrane binding sites, which are shared in part by the benzodiazepine drug diazepam. Structures in which GABAA receptors are bound by benzodiazepine-site ligands identify an additional membrane binding site for diazepam and suggest an allosteric mechanism for anaesthetic reversal by flumazenil. This study provides a foundation for understanding how pharmacologically diverse and clinically essential drugs act through overlapping and distinct mechanisms to potentiate inhibitory signalling in the brain.
Yongfei Cai, Jun Zhang, Tianshu Xiao, Hanqin Peng, Sarah M. Sterling, Richard M. Walsh, Shaun Rawson, Sophia Rits-Volloch, and Bing Chen. 7/21/2020. “Distinct conformational states of SARS-CoV-2 spike protein.” Science. Publisher's VersionAbstract
Intervention strategies are urgently needed to control the SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) pandemic. The trimeric viral spike (S) protein catalyzes fusion between viral and target cell membranes to initiate infection. Here we report two cryo-EM structures, derived from a preparation of the full-length S protein, representing its prefusion (2.9Å resolution) and postfusion (3.0Å resolution) conformations, respectively. The spontaneous transition to the postfusion state is independent of target cells. The prefusion trimer has three receptor-binding domains clamped down by a segment adjacent to the fusion peptide. The postfusion structure is strategically decorated by N-linked glycans, suggesting possible protective roles against host immune responses and harsh external conditions. These findings advance our understanding of SARS-CoV-2 entry and may guide development of vaccines and therapeutics.
Stephen M. Hinshaw and Stephen C. Harrison. 7/16/2020. “The Structural Basis for Kinetochore Stabilization by Cnn1/CENP-T.” Current Biology. Publisher's VersionAbstract
Summary Chromosome segregation depends on a regulated connection between spindle microtubules and centromeric DNA. The kinetochore mediates this connection and ensures it persists during anaphase, when sister chromatids must transit into daughter cells uninterrupted. The Ctf19 complex (Ctf19c) forms the centromeric base of the kinetochore in budding yeast. Biochemical experiments show that Ctf19c members associate hierarchically when purified from cell extract [1], an observation that is mostly explained by the structure of the complex [2]. The Ctf3 complex (Ctf3c), which is not required for the assembly of most other Ctf19c factors, disobeys the biochemical assembly hierarchy when observed in dividing cells that lack more basal components [3]. Thus, the biochemical experiments do not completely recapitulate the logic of centromeric Ctf19c assembly. We now present a high-resolution structure of the Ctf3c bound to the Cnn1-Wip1 heterodimer. Associated live-cell imaging experiments provide a mechanism for Ctf3c and Cnn1-Wip1 recruitment to the kinetochore. The mechanism suggests feedback regulation of Ctf19c assembly and unanticipated similarities in kinetochore organization between yeast and vertebrates.
David Tomasek, Shaun Rawson, James Lee, Joseph S. Wzorek, Stephen C. Harrison, Zongli Li, and Daniel Kahne. 6/11/2020. “Structure of a nascent membrane protein as it folds on the BAM complex.” Nature. Publisher's VersionAbstract
Mitochondria, chloroplasts and Gram-negative bacteria are encased in a double layer of membranes. The outer membrane contains proteins with a $\beta$-barrel structure1,2. $\beta$-Barrels are sheets of $\beta$-strands wrapped into a cylinder, in which the first strand is hydrogen-bonded to the final strand. Conserved multi-subunit molecular machines fold and insert these proteins into the outer membrane3–5. One subunit of the machines is itself a $\beta$-barrel protein that has a central role in folding other $\beta$-barrels. In Gram-negative bacteria, the $\beta$-barrel assembly machine (BAM) consists of the $\beta$-barrel protein BamA, and four lipoproteins5–8. To understand how the BAM complex accelerates folding without using exogenous energy (for example, ATP)9, we trapped folding intermediates on this machine. Here we report the structure of the BAM complex of Escherichia coli folding BamA itself. The BamA catalyst forms an asymmetric hybrid $\beta$-barrel with the BamA substrate. The N-terminal edge of the BamA catalyst has an antiparallel hydrogen-bonded interface with the C-terminal edge of the BamA substrate, consistent with previous crosslinking studies10–12; the other edges of the BamA catalyst and substrate are close to each other, but curl inward and do not pair. Six hydrogen bonds in a membrane environment make the interface between the two proteins very stable. This stability allows folding, but creates a high kinetic barrier to substrate release after folding has finished. Features at each end of the substrate overcome this barrier and promote release by stepwise exchange of hydrogen bonds. This mechanism of substrate-assisted product release explains how the BAM complex can stably associate with the substrate during folding and then turn over rapidly when folding is complete.
Xuewu Sui, Kun Wang, Nina L. Gluchowski, Shane D. Elliott, Maofu Liao, Tobias C. Walther, and Robert V. Farese. 5/13/2020. “Structure and catalytic mechanism of a human triacylglycerol-synthesis enzyme.” Nature. Publisher's VersionAbstract
Triacylglycerols store metabolic energy in organisms and have industrial uses as foods and fuels. Excessive accumulation of triacylglycerols in humans causes obesity and is associated with metabolic diseases1. Triacylglycerol synthesis is catalysed by acyl-CoA diacylglycerol acyltransferase (DGAT) enzymes2–4, the structures and catalytic mechanisms of which remain unknown. Here we determined the structure of dimeric human DGAT1, a member of the membrane-bound O-acyltransferase (MBOAT) family, by cryo-electron microscopy at approximately 3.0 \AA resolution. DGAT1 forms a homodimer through N-terminal segments and a hydrophobic interface, with putative active sites within the membrane region. A structure obtained with oleoyl-CoA substrate resolved at approximately 3.2 \AA shows that the CoA moiety binds DGAT1 on the cytosolic side and the acyl group lies deep within a hydrophobic channel, positioning the acyl-CoA thioester bond near an invariant catalytic histidine residue. The reaction centre is located inside a large cavity, which opens laterally to the membrane bilayer, providing lipid access to the active site. A lipid-like density–-possibly representing an acyl-acceptor molecule–-is located within the reaction centre, orthogonal to acyl-CoA. Insights provided by the DGAT1 structures, together with mutagenesis and functional studies, provide the basis for a model of the catalysis of triacylglycerol synthesis by DGAT.
Benjamin J Orlando and Maofu Liao. 2020. “ABCG2 transports anticancer drugs via a closed-to-open switch.” Nat Commun, 11, 1, Pp. 2264.Abstract
ABCG2 is an ABC transporter that extrudes a variety of compounds from cells, and presents an obstacle in treating chemotherapy-resistant cancers. Despite recent structural insights, no anticancer drug bound to ABCG2 has been resolved, and the mechanisms of multidrug transport remain obscure. Such a gap of knowledge limits the development of novel compounds that block or evade this critical molecular pump. Here we present single-particle cryo-EM studies of ABCG2 in the apo state, and bound to the three structurally distinct chemotherapeutics. Without the binding of conformation-selective antibody fragments or inhibitors, the resting ABCG2 adopts a closed conformation. Our cryo-EM, biochemical, and functional analyses reveal the binding mode of three chemotherapeutic compounds, demonstrate how these molecules open the closed conformation of the transporter, and establish that imatinib is particularly effective in stabilizing the inward facing conformation of ABCG2. Together these studies reveal the previously unrecognized conformational cycle of ABCG2.
Xudong Wu, Marc Siggel, Sergey Ovchinnikov, Wei Mi, Vladimir Svetlov, Evgeny Nudler, Maofu Liao, Gerhard Hummer, and Tom A. Rapoport. 2020. “Structural basis of ER-associated protein degradation mediated by the Hrd1 ubiquitin ligase complex.” Science, 368, 6489. Publisher's VersionAbstract
Misfolded endoplasmic reticulum (ER) proteins are retrotranslocated into the cytosol, polyubiquitinated, and degraded by the proteasome in a process known as ER-associated protein degradation (ERAD). ERAD of misfolded luminal ER proteins (ERAD-L) is mediated by the Hrd1 complex, composed of the ubiquitin ligase Hrd1 and four additional proteins (Hrd3, Der1, Usa1, and Yos9). Wu et al. report a cryo–electron microscopy structure of the active Hrd1 complex from yeast and, based on this structure, developed a model for how substrates are recognized and retrotranslocated. They propose that Hrd3 and Yos9 jointly create a luminal binding site for misfolded glycoproteins. Hrd1 and Der1 form “half-channels” juxtaposed in a thinned section of the ER membrane, which allows a polypeptide loop of an ERAD-L substrate to move through it.Science, this issue p. eaaz2449INTRODUCTIONProtein homeostasis in the endoplasmic reticulum (ER) is maintained by a quality control system. When a newly synthesized ER protein misfolds, it is ultimately retrotranslocated into the cytosol, polyubiquitinated, and degraded by the proteasome, a pathway referred to as ER-associated protein degradation (ERAD). ERAD alleviates cytotoxic stress imposed by protein misfolding and is implicated in numerous diseases. ERAD is found in all eukaryotic cells but is best studied for the ERAD-L pathway in Saccharomyces cerevisiae, which disposes of misfolded glycoproteins in the ER lumen. The glycan attached to these proteins is first trimmed by glycosidases to generate a terminal α1,6-mannose residue. This residue, together with an unfolded polypeptide segment, targets the substrate to the Hrd1 complex, which is composed of the multispanning ubiquitin ligase Hrd1 and four additional proteins (Hrd3, Der1, Usa1, and Yos9). The Hrd1 complex mediates the retrotranslocation of the polypeptide into the cytosol, where it is polyubiquitinated, extracted from the membrane by the Cdc48 adenosine triphosphatase complex, and, finally, degraded by the proteasome.RATIONALEThe mechanism of ERAD-L remains poorly understood. Arguably the most mysterious aspect is how misfolded proteins cross the ER membrane, which normally presents a barrier to macromolecules. How ERAD-L substrates are recognized and distinguished from properly folding intermediates is also unclear. Answers to these questions require structural information on the Hrd1 complex.RESULTSHere, we report a structure of the active Hrd1 complex from S. cerevisiae, as determined by cryo–electron microscopy (cryo-EM) analysis of two subcomplexes. Our structures, biochemical data, and experiments in vivo indicate that the Hrd1 complex functions as a monomer in ERAD-L. Hrd3 and Yos9 jointly create a luminal binding site that recognizes misfolded glycoproteins. The α1,6-mannose residue binds to the mannose 6-phosphate receptor homology (MRH) domain of Yos9, and the polypeptide segment downstream of the glycan attachment site is likely accommodated in a groove of the luminal domain of Hrd3. Hrd1 and the rhomboid-like Der1 protein are linked by Usa1 on the cytosolic side of the membrane. Both Der1 and Hrd1 have lateral gates that face one another within the membrane and possess luminal and cytosolic cavities, respectively. Both proteins distort the membrane region between the lateral gates, making it much thinner than a normal phospholipid bilayer, an observation supported by molecular dynamics simulations. The structures and photocrosslinking experiments indicate that the retrotranslocation of an ERAD-L substrate is initiated by loop insertion of the polypeptide into the membrane, with one strand of the loop interacting with Der1 and the other with Hrd1.CONCLUSIONOur results lead to a model for the mechanism of retrotranslocation through the Hrd1 complex. The pathway across the membrane is formed by two “half-channels” corresponding to the luminal and cytosolic cavities of Der1 and Hrd1, respectively. These half-channels are juxtaposed in a thinned membrane region. The substrate inserts into the retrotranslocon as a hairpin that is hydrophilic on both sides. These features contrast with the Sec61 channel, which accepts substrates with a hydrophobic signal or transmembrane segment forming one side of the loop. This segment exits the lateral gate into the lipid environment and is not translocated, while the other side of the loop moves through the membrane in an entirely hydrophilic environment. The structural features of the retrotranslocon can facilitate movement of a fully hydrophilic substrate through a thinned and thus distorted membrane, a paradigm that may be replicated in other protein translocation systems.Initiation of ERAD-L revealed by cryo-EM and photocrosslinking.(A) Side view of a space-filling model of the Hrd1 complex, based on structures of the Hrd1 Usa1-Der1-Hrd3 and Hrd3-Yos9 subcomplexes. (B) Hypothetical position of a glycosylated ERAD-L substrate in the Hrd1 complex (dashed blue line). Substrate-interacting amino acid residues in Hrd1 and Der1 (red and orange, respectively) were determined by photocrosslinking. N, N terminus; C, C terminus; DHFR, dihydrofolate reductase; TM, transmembrane helix. (C) Model for the first three stages of retrotranslocation.Misfolded luminal endoplasmic reticulum (ER) proteins undergo ER-associated degradation (ERAD-L): They are retrotranslocated into the cytosol, polyubiquitinated, and degraded by the proteasome. ERAD-L is mediated by the Hrd1 complex (composed of Hrd1, Hrd3, Der1, Usa1, and Yos9), but the mechanism of retrotranslocation remains mysterious. Here, we report a structure of the active Hrd1 complex, as determined by cryo–electron microscopy analysis of two subcomplexes. Hrd3 and Yos9 jointly create a luminal binding site that recognizes glycosylated substrates. Hrd1 and the rhomboid-like Der1 protein form two “half-channels” with cytosolic and luminal cavities, respectively, and lateral gates facing one another in a thinned membrane region. These structures, along with crosslinking and molecular dynamics simulation results, suggest how a polypeptide loop of an ERAD-L substrate moves through the ER membrane.
Sandeep K Singh, Miao Gui, Fujiet Koh, Matthew CJ Yip, and Alan Brown. 2020. “Structure and activation mechanism of the BBSome membrane protein trafficking complex.” Edited by Andrew P Carter. eLife, 9, Pp. e53322. Publisher's VersionAbstract
Bardet-Biedl syndrome (BBS) is a currently incurable ciliopathy caused by the failure to correctly establish or maintain cilia-dependent signaling pathways. Eight proteins associated with BBS assemble into the BBSome, a key regulator of the ciliary membrane proteome. We report the electron cryomicroscopy (cryo-EM) structures of the native bovine BBSome in inactive and active states at 3.1 ­and 3.5 Å resolution, respectively. In the active state, the BBSome is bound to an Arf-family GTPase (ARL6/BBS3) that recruits the BBSome to ciliary membranes. ARL6 recognizes a composite binding site formed by BBS1 and BBS7 that is occluded in the inactive state. Activation requires an unexpected swiveling of the b-propeller domain of BBS1, the subunit most frequently implicated in substrate recognition, which widens a central cavity of the BBSome. Structural mapping of disease-causing mutations suggests that pathogenesis results from folding defects and the disruption of autoinhibition and activation.
Brianna Lowey, Aaron T. Whiteley, Alexander F. A. Keszei, Benjamin R. Morehouse, Ian T. Mathews, Sadie P. Antine, Victor J. Cabrera, Dmitry Kashin, Percy Niemann, Mohit Jain, Frank Schwede, John J. Mekalanos, Sichen Shao, Amy S.Y. Lee, and Philip J. Kranzusch. 2020. “CBASS Immunity Uses CARF-Related Effectors to Sense 3′–5′- and 2′–5′-Linked Cyclic Oligonucleotide Signals and Protect Bacteria from Phage Infection.” Cell, 182, 1, Pp. 38-49.e17. Publisher's VersionAbstract
Summary cGAS/DncV-like nucleotidyltransferase (CD-NTase) enzymes are immune sensors that synthesize nucleotide second messengers and initiate antiviral responses in bacterial and animal cells. Here, we discover Enterobacter cloacae CD-NTase-associated protein 4 (Cap4) as a founding member of a diverse family of >2,000 bacterial receptors that respond to CD-NTase signals. Structures of Cap4 reveal a promiscuous DNA endonuclease domain activated through ligand-induced oligomerization. Oligonucleotide recognition occurs through an appended SAVED domain that is an unexpected fusion of two CRISPR-associated Rossman fold (CARF) subunits co-opted from type III CRISPR immunity. Like a lock and key, SAVED effectors exquisitely discriminate 2′–5′- and 3′–5′-linked bacterial cyclic oligonucleotide signals and enable specific recognition of at least 180 potential nucleotide second messenger species. Our results reveal SAVED CARF family proteins as major nucleotide second messenger receptors in CBASS and CRISPR immune defense and extend the importance of linkage specificity beyond mammalian cGAS-STING signaling.
Roman Sloutsky, Noelle Dziedzic, Matthew J. Dunn, Rachel M. Bates, Ana P. Torres-Ocampo, Sivakumar Boopathy, Brendan Page, John G. Weeks, Luke H. Chao, and Margaret M. Stratton. 2020. “Heterogeneity in human hippocampal CaMKII transcripts reveals allosteric hub-dependent regulation.” Science Signaling, 13, 641. Publisher's VersionAbstract
Members of the calcium/calmodulin-dependent protein kinase II (CaMKII) family of oligomeric serine and threonine kinases mediate numerous processes, including long-term memory formation. Each CaMKII subunit contains a kinase domain, a linker region, and a hub domain, which mediates oligomerization. Sloutsky et al. sequenced at least 70 CaMKII-encoding transcripts expressed in the human hippocampus, a primary memory center in the brain. Structural and functional analyses revealed interactions between the kinase and hub domains that affected the sensitivity of CaMKII variants to activation by calcium/calmodulin. Together, these data suggest an additional role for the hub domain in regulating CaMKII activation, which may provide a therapeutic target.Calcium/calmodulin-dependent protein kinase II (CaMKII) plays a central role in Ca2+ signaling throughout the body. In the hippocampus, CaMKII is required for learning and memory. Vertebrate genomes encode four CaMKII homologs: CaMKIIα, CaMKIIβ, CaMKIIγ, and CaMKIIδ. All CaMKIIs consist of a kinase domain, a regulatory segment, a variable linker region, and a hub domain, which is responsible for oligomerization. The four proteins differ primarily in linker length and composition because of extensive alternative splicing. Here, we report the heterogeneity of CaMKII transcripts in three complex samples of human hippocampus using deep sequencing. We showed that hippocampal cells contain a diverse collection of over 70 CaMKII transcripts from all four CaMKII-encoding genes. We characterized the Ca2+/CaM sensitivity of hippocampal CaMKII variants spanning a broad range of linker lengths and compositions. The effect of the variable linker on Ca2+/CaM sensitivity depended on the kinase and hub domains. Moreover, we revealed a previously uncharacterized role for the hub domain as an allosteric regulator of kinase activity, which may provide a pharmacological target for modulating CaMKII activity. Using small-angle x-ray scattering and single-particle cryo–electron microscopy (cryo-EM), we present evidence for extensive interactions between the kinase and the hub domains, even in the presence of a 30-residue linker. Together, these data suggest that Ca2+/CaM sensitivity in CaMKII is homolog dependent and includes substantial contributions from the hub domain. Our sequencing approach, combined with biochemistry, provides insights into understanding the complex pool of endogenous CaMKII splice variants.