TMEM106B is a receptor mediating ACE2-independent SARS-CoV-2 cell entry.

SARS-CoV-2 is associated with broad tissue tropism, a characteristic often determined by the availability of entry receptors on host cells. Here, we show that TMEM106B, a lysosomal transmembrane protein, can serve as an alternative receptor for SARS-CoV-2 entry into angiotensin-converting enzyme 2 (ACE2)-negative cells. Spike substitution E484D increased TMEM106B binding, thereby enhancing TMEM106B-mediated entry. TMEM106B-specific monoclonal antibodies blocked SARS-CoV-2 infection, demonstrating a role of TMEM106B in viral entry. Using X-ray crystallography, cryogenic electron microscopy (cryo-EM), and hydrogen-deuterium exchange mass spectrometry (HDX-MS), we show that the luminal domain (LD) of TMEM106B engages the receptor-binding motif of SARS-CoV-2 spike. Finally, we show that TMEM106B promotes spike-mediated syncytium formation, suggesting a role of TMEM106B in viral fusion. Together, our findings identify an ACE2-independent SARS-CoV-2 infection mechanism that involves cooperative interactions with the receptors heparan sulfate and TMEM106B.

Structural basis of σ54 displacement and promoter escape in bacterial transcription.

Gene transcription is a fundamental cellular process carried out by RNA polymerase (RNAP). Transcription initiation is highly regulated, and in bacteria, transcription initiation is mediated by sigma (σ) factors. σ recruits RNAP to the promoter DNA region, located upstream of the transcription start site (TSS) and facilitates open complex formation, where double-stranded DNA is opened up into a transcription bubble and template strand DNA is positioned inside RNAP for initial RNA synthesis. During initial transcription, RNAP remains bound to σ and upstream DNA, presumably with an enlarging transcription bubble. The release of RNAP from upstream DNA is required for promoter escape and processive transcription elongation. Bacteria sigma factors can be broadly separated into two classes with the majority belonging to the σ70 class, represented by the σ70 that regulates housekeeping genes. σ54 forms a class on its own and regulates stress response genes. Extensive studies on σ70 have revealed the molecular mechanisms of the σ70 dependent process while how σ54 transitions from initial transcription to elongation is currently unknown. Here, we present a series of cryo-electron microscopy structures of the RNAP-σ54 initial transcribing complexes with progressively longer RNA, which reveal structural changes that lead to promoter escape. Our data show that initially, the transcription bubble enlarges, DNA strands scrunch, reducing the interactions between σ54 and DNA strands in the transcription bubble. RNA extension and further DNA scrunching help to release RNAP from σ54 and upstream DNA, enabling the transition to elongation.

The DNA replication initiation protein DnaD recognises a specific strand of the Bacillus subtilis chromosome origin.

Genome replication is a fundamental biological activity shared by all organisms. Chromosomal replication proceeds bidirectionally from origins, requiring the loading of two helicases, one for each replisome. However, the molecular mechanisms underpinning helicase loading at bacterial chromosome origins (oriC) are unclear. Here we investigated the essential DNA replication initiation protein DnaD in the model organism Bacillus subtilis. A set of DnaD residues required for ssDNA binding was identified, and photo-crosslinking revealed that this ssDNA binding region interacts preferentially with one strand of oriC. Biochemical and genetic data support the model that DnaD recognizes a new single-stranded DNA (ssDNA) motif located in oriC, the DnaD Recognition Element (DRE). Considered with single particle cryo-electron microscopy (cryo-EM) imaging of DnaD, we propose that the location of the DRE within oriC orchestrates strand-specific recruitment of helicase during DNA replication initiation. These findings significantly advance our mechanistic understanding of bidirectional replication from a bacterial chromosome origin.

Tankyrase Requires SAM Domain-Dependent Polymerization to Support Wnt-ß-Catenin Signaling.

The poly(ADP-ribose) polymerase (PARP) Tankyrase (TNKS and TNKS2) is paramount to Wnt-ß-catenin signaling and a promising therapeutic target in Wnt-dependent cancers. The pool of active ß-catenin is normally limited by destruction complexes, whose assembly depends on the polymeric master scaffolding protein AXIN. Tankyrase, which poly(ADP-ribosyl)ates and thereby destabilizes AXIN, also can polymerize, but the relevance of these polymers has remained unclear. We report crystal structures of the polymerizing TNKS and TNKS2 sterile alpha motif (SAM) domains, revealing versatile head-to-tail interactions. Biochemical studies informed by these structures demonstrate that polymerization is required for Tankyrase to drive ß-catenin-dependent transcription. We show that the polymeric state supports PARP activity and allows Tankyrase to effectively access destruction complexes through enabling avidity-dependent AXIN binding. This study provides an example for regulated signal transduction in non-membrane-enclosed compartments (signalosomes), and it points to novel potential strategies to inhibit Tankyrase function in oncogenic Wnt signaling.

Cryo-EM structure of the Smc5/6 holo-complex.

The Smc5/6 complex plays an essential role in the resolution of recombination intermediates formed during mitosis or meiosis, or as a result of the cellular response to replication stress. It also functions as a restriction factor preventing viral replication. Here, we report the cryogenic EM (cryo-EM) structure of the six-subunit budding yeast Smc5/6 holo-complex, reconstituted from recombinant proteins expressed in insect cells - providing both an architectural overview of the entire complex and an understanding of how the Nse1/3/4 subcomplex binds to the hetero-dimeric SMC protein core. In addition, we demonstrate that a region within the head domain of Smc5, equivalent to the 'W-loop' of Smc4 or 'F-loop' of Smc1, mediates an important interaction with Nse1. Notably, mutations that alter the surface-charge profile of the region of Nse1 which accepts the Smc5-loop, lead to a slow-growth phenotype and a global reduction in the chromatin-associated fraction of the Smc5/6 complex, as judged by single molecule localisation microscopy experiments in live yeast. Moreover, when taken together, our data indicates functional equivalence between the structurally unrelated KITE and HAWK accessory subunits associated with SMC complexes.

Insights into degron recognition by APC/C coactivators from the structure of an Acm1-Cdh1 complex.

The anaphase-promoting complex/cyclosome (APC/C) regulates sister chromatid segregation and the exit from mitosis. Selection of most APC/C substrates is controlled by coactivator subunits (either Cdc20 or Cdh1) that interact with substrate destruction motifs--predominantly the destruction (D) box and KEN box degrons. How coactivators recognize D box degrons and how this is inhibited by APC/C regulatory proteins is not defined at the atomic level. Here, from the crystal structure of S. cerevisiae Cdh1 in complex with its specific inhibitor Acm1, which incorporates D and KEN box pseudosubstrate motifs, we describe the molecular basis for D box recognition. Additional interactions between Acm1 and Cdh1 identify a third protein-binding site on Cdh1 that is likely to confer coactivator-specific protein functions including substrate association. We provide a structural rationalization for D box and KEN box recognition by coactivators and demonstrate that many noncanonical APC/C degrons bind APC/C coactivators at the D box coreceptor.

Structural basis for translocation by AddAB helicase-nuclease and its arrest at χ sites.

In bacterial cells, processing of double-stranded DNA breaks for repair by homologous recombination is dependent upon the recombination hotspot sequence χ (Chi) and is catalysed by either an AddAB- or RecBCD-type helicase-nuclease (reviewed in refs 3, 4). These enzyme complexes unwind and digest the DNA duplex from the broken end until they encounter a χ sequence, whereupon they produce a 3' single-stranded DNA tail onto which they initiate loading of the RecA protein. Consequently, regulation of the AddAB/RecBCD complex by χ is a key control point in DNA repair and other processes involving genetic recombination. Here we report crystal structures of Bacillus subtilis AddAB in complex with different χ-containing DNA substrates either with or without a non-hydrolysable ATP analogue. Comparison of these structures suggests a mechanism for DNA translocation and unwinding, suggests how the enzyme binds specifically to χ sequences, and explains how χ recognition leads to the arrest of AddAB (and RecBCD) translocation that is observed in single-molecule experiments.

Mechanism of farnesylated CAAX protein processing by the intramembrane protease Rce1.

CAAX proteins have essential roles in multiple signalling pathways, controlling processes such as proliferation, differentiation and carcinogenesis. The ∼120 mammalian CAAX proteins function at cellular membranes and include the Ras superfamily of small GTPases, nuclear lamins, the γ-subunit of heterotrimeric GTPases, and several protein kinases and phosphatases. The proper localization of CAAX proteins to cell membranes is orchestrated by a series of post-translational modifications of the carboxy-terminal CAAX motifs (where C is cysteine, A is an aliphatic amino acid and X is any amino acid). These reactions involve prenylation of the cysteine residue, cleavage at the AAX tripeptide and methylation of the carboxyl-prenylated cysteine residue. The major CAAX protease activity is mediated by Rce1 (Ras and a-factor converting enzyme 1), an intramembrane protease (IMP) of the endoplasmic reticulum. Information on the architecture and proteolytic mechanism of Rce1 has been lacking. Here we report the crystal structure of a Methanococcus maripaludis homologue of Rce1, whose endopeptidase specificity for farnesylated peptides mimics that of eukaryotic Rce1. Its structure, comprising eight transmembrane α-helices, and catalytic site are distinct from those of other IMPs. The catalytic residues are located ∼10 Å into the membrane and are exposed to the cytoplasm and membrane through a conical cavity that accommodates the prenylated CAAX substrate. We propose that the farnesyl lipid binds to a site at the opening of two transmembrane α-helices, which results in the scissile bond being positioned adjacent to a glutamate-activated nucleophilic water molecule. This study suggests that Rce1 is the founding member of a novel IMP family, the glutamate IMPs.

The structure and function of an RNA polymerase interaction domain in the PcrA/UvrD helicase.

The PcrA/UvrD helicase functions in multiple pathways that promote bacterial genome stability including the suppression of conflicts between replication and transcription and facilitating the repair of transcribed DNA. The reported ability of PcrA/UvrD to bind and backtrack RNA polymerase (1,2) might be relevant to these functions, but the structural basis for this activity is poorly understood. In this work, we define a minimal RNA polymerase interaction domain in PcrA, and report its crystal structure at 1.5 Å resolution. The domain adopts a Tudor-like fold that is similar to other RNA polymerase interaction domains, including that of the prototype transcription-repair coupling factor Mfd. Removal or mutation of the interaction domain reduces the ability of PcrA/UvrD to interact with and to remodel RNA polymerase complexes in vitro. The implications of this work for our understanding of the role of PcrA/UvrD at the interface of DNA replication, transcription and repair are discussed.

Crystal structure of a vFlip-IKKgamma complex: insights into viral activation of the IKK signalosome.

Key to the pathogenicity of several viruses is activation of the canonical nuclear factor-kappaB (NF-kappaB) transcriptional pathway. Subversion of this tightly regulated mechanism is achieved through the production of host mimetic viral proteins that deregulate the transcription process. One such protein is ks-vFLIP (produced by the Kaposi's sarcoma herpes virus [KSHV]), which associates with IKKgamma, an essential component of the IKK complex or signalosome. This interaction renders the canonical NF-kappaB pathway constitutively active and has been linked to Kaposi's sarcoma and other malignancies. In order to elucidate the molecular basis underpinning ks-vFLIP-induced activation of the IKK signalosome, we have determined the crystal structure of a complex involving a fragment of IKKgamma bound to ks-vFLIP at 3.2 A. In addition to identifying and subsequently probing the ks-vFLIP-IKKgamma interface, we have also investigated the effects of a mutation implicated in the genetic disorder anhydrotic ectodermal dysplasia with immunodeficiency (EDA-ID).

Synthesis of a Ribose-Incorporating Medium Ring Scaffold via a Challenging Ring-Closing Metathesis Reaction.

A practical synthesis of a novel oxabicyclo[6.2.1]undecenetriol useful as a medicinal chemistry scaffold has been developed starting from l-ribose. The sequence involves an oxidation/Grignard addition sequence and a challenging ring-closing metathesis (RCM) reaction as the ring forming step. Exploration of the RCM substrate protecting groups revealed the key factor for successful nine-membered medium ring formation to be conformational bias of the reacting alkenes of the RCM substrate by very bulky silyl ether protecting groups. The synthesis also allowed access to an epimeric triol and saturated and unsaturated variants of the nine-membered ring. The medium ring conformation of the oxabicyclo[6.2.1]undecenetriol was determined by X-ray crystallography and correlated to the solution state conformation by NMR experiments.

A novel route to product specificity in the Suv4-20 family of histone H4K20 methyltransferases.

The delivery of site-specific post-translational modifications to histones generates an epigenetic regulatory network that directs fundamental DNA-mediated processes and governs key stages in development. Methylation of histone H4 lysine-20 has been implicated in DNA repair, transcriptional silencing, genomic stability and regulation of replication. We present the structure of the histone H4K20 methyltransferase Suv4-20h2 in complex with its histone H4 peptide substrate and S-adenosyl methionine cofactor. Analysis of the structure reveals that the Suv4-20h2 active site diverges from the canonical SET domain configuration and generates a high degree of both substrate and product specificity. Together with supporting biochemical data comparing Suv4-20h1 and Suv4-20h2, we demonstrate that the Suv4-20 family enzymes take a previously mono-methylated H4K20 substrate and generate an exclusively di-methylated product. We therefore predict that other enzymes are responsible for the tri-methylation of histone H4K20 that marks silenced heterochromatin.

Structure of the outer membrane complex of a type IV secretion system.

Type IV secretion systems are secretion nanomachines spanning the two membranes of Gram-negative bacteria. Three proteins, VirB7, VirB9 and VirB10, assemble into a 1.05 megadalton (MDa) core spanning the inner and outer membranes. This core consists of 14 copies of each of the proteins and forms two layers, the I and O layers, inserting in the inner and outer membrane, respectively. Here we present the crystal structure of a approximately 0.6 MDa outer-membrane complex containing the entire O layer. This structure is the largest determined for an outer-membrane channel and is unprecedented in being composed of three proteins. Unexpectedly, this structure identifies VirB10 as the outer-membrane channel with a unique hydrophobic double-helical transmembrane region. This structure establishes VirB10 as the only known protein crossing both membranes of Gram-negative bacteria. Comparison of the cryo-electron microscopy (cryo-EM) and crystallographic structures points to conformational changes regulating channel opening and closing.

A neutralizing epitope on the SD1 domain of SARS-CoV-2 spike targeted following infection and vaccination.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike is the target for neutralizing antibodies elicited following both infection and vaccination. While extensive research has shown that the receptor binding domain (RBD) and, to a lesser extent, the N-terminal domain (NTD) are the predominant targets for neutralizing antibodies, identification of neutralizing epitopes beyond these regions is important for informing vaccine development and understanding antibody-mediated immune escape. Here, we identify a class of broadly neutralizing antibodies that bind an epitope on the spike subdomain 1 (SD1) and that have arisen from infection or vaccination. Using cryo-electron microscopy (cryo-EM) and hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS), we show that SD1-specific antibody P008_60 binds an epitope that is not accessible within the canonical prefusion states of the SARS-CoV-2 spike, suggesting a transient conformation of the viral glycoprotein that is vulnerable to neutralization.

The structure of human leucine carboxyl methyltransferase 1 that regulates protein phosphatase PP2A.

Leucine carboxyl methyltransferase 1 (LCMT1) methylates the terminal carboxyl group of the leucine 309 residue of human protein phosphatase 2A (PP2A). PP2A, a key regulator of many cellular processes, has recently generated additional interest as a potential cancer-therapeutic target. The status of PP2A methylation impacts upon the selection of the regulatory subunit by the PP2A core enzyme, thus directing its activity and subcellular localization. An X-ray crystal structure of human LCMT1 protein in complex with the cofactor S-adenosylmethionine (AdoMet) has been solved to a resolution of 2 Å. The structure enables the postulation of a mode of interaction with protein phosphatase PP2A and provides a platform for further functional studies of the regulation of methylation of PP2A.

Structural basis for the inhibition of HTLV-1 integration inferred from cryo-EM deltaretroviral intasome structures.

Between 10 and 20 million people worldwide are infected with the human T-cell lymphotropic virus type 1 (HTLV-1). Despite causing life-threatening pathologies there is no therapeutic regimen for this deltaretrovirus. Here, we screened a library of integrase strand transfer inhibitor (INSTI) candidates built around several chemical scaffolds to determine their effectiveness in limiting HTLV-1 infection. Naphthyridines with substituents in position 6 emerged as the most potent compounds against HTLV-1, with XZ450 having highest efficacy in vitro. Using single-particle cryo-electron microscopy we visualised XZ450 as well as the clinical HIV-1 INSTIs raltegravir and bictegravir bound to the active site of the deltaretroviral intasome. The structures reveal subtle differences in the coordination environment of the Mg2+ ion pair involved in the interaction with the INSTIs. Our results elucidate the binding of INSTIs to the HTLV-1 intasome and support their use for pre-exposure prophylaxis and possibly future treatment of HTLV-1 infection.

Structural basis of Cullin 2 RING E3 ligase regulation by the COP9 signalosome.

Cullin-Ring E3 Ligases (CRLs) regulate a multitude of cellular pathways through specific substrate receptors. The COP9 signalosome (CSN) deactivates CRLs by removing NEDD8 from activated Cullins. Here we present structures of the neddylated and deneddylated CSN-CRL2 complexes by combining single-particle cryo-electron microscopy (cryo-EM) with chemical cross-linking mass spectrometry (XL-MS). These structures suggest a conserved mechanism of CSN activation, consisting of conformational clamping of the CRL2 substrate by CSN2/CSN4, release of the catalytic CSN5/CSN6 heterodimer and finally activation of the CSN5 deneddylation machinery. Using hydrogen-deuterium exchange (HDX)-MS we show that CRL2 activates CSN5/CSN6 in a neddylation-independent manner. The presence of NEDD8 is required to activate the CSN5 active site. Overall, by synergising cryo-EM with MS, we identify sensory regions of the CSN that mediate its stepwise activation and provide a framework for understanding the regulatory mechanism of other Cullin family members.

Tetrameric bacterial sodium channels: characterization of structure, stability, and drug binding.

NaChBac from Bacillus halodurans is a bacterial homologue of mammalian voltage-gated sodium channels. It has been proposed that a NaChBac monomer corresponds to a single domain of the mammalian sodium channel and that, like potassium channels, four monomers form a tetrameric channel. However, to date, although NaChBac has been well-characterized for functional properties by electrophysiological measurements on protein expressed in tissue culture, little information about its structural properties exists because of the difficulties in expressing the protein in large quantities. In this study, we present studies on the overexpression of NaChBac in Escherichia coli, purification of the functional detergent-solubilized channel, its identification as a tetramer, and characterization of its secondary structure, drug binding, and thermal stability. These studies are correlated with a model produced for the protein and provide new insights into the structure-function relationships of this sodium channel.

Introduction of a Methyl Group Curbs Metabolism of Pyrido[3,4- d]pyrimidine Monopolar Spindle 1 (MPS1) Inhibitors and Enables the Discovery of the Phase 1 Clinical Candidate N2-(2-Ethoxy-4-(4-methyl-4 H-1,2,4-triazol-3-yl)phenyl)-6-methyl- N8-neopentylpyrido[3,4- d]pyrimidine-2,8-diamine (BOS172722).

Monopolar spindle 1 (MPS1) occupies a central role in mitosis and is one of the main components of the spindle assembly checkpoint. The MPS1 kinase is an attractive cancer target, and herein, we report the discovery of the clinical candidate BOS172722. The starting point for our work was a series of pyrido[3,4- d]pyrimidine inhibitors that demonstrated excellent potency and kinase selectivity but suffered from rapid turnover in human liver microsomes (HLM). Optimizing HLM stability proved challenging since it was not possible to identify a consistent site of metabolism and lowering lipophilicity proved unsuccessful. Key to overcoming this problem was the finding that introduction of a methyl group at the 6-position of the pyrido[3,4- d]pyrimidine core significantly improved HLM stability. Met ID studies suggested that the methyl group suppressed metabolism at the distant aniline portion of the molecule, likely by blocking the preferred pharmacophore through which P450 recognized the compound. This work ultimately led to the discovery of BOS172722 as a Phase 1 clinical candidate.