Jiansen Jiang

Activation of DegP chaperone-protease via formation of large cage-like oligomers upon binding to substrate proteins

Cells use molecular chaperones and proteases to implement the essential quality control mechanism of proteins. The DegP (HtrA) protein, essential for the survival of Escherichia coli cells at elevated temperatures with homologues found in almost all organisms uniquely has both functions. Here we report a mechanism for DegP to activate both functions via formation of large cage-like 12- and 24-mers after binding to substrate proteins. Cryo-electron microscopic and biochemical studies revealed that both oligomers are consistently assembled by blocks of DegP trimers, via pairwise PDZ1–PDZ2 interactions between neighboring trimers. Such interactions simultaneously eliminate the inhibitory effects of the PDZ2 domain. Additionally, both DegP oligomers were also observed in extracts of E. coli cells, strongly implicating their physiological importance.

Atomic structure of anthrax protective antigen pore elucidates toxin translocation.

Anthrax toxin, comprising protective antigen, lethal factor, and oedema factor, is the major virulence factor of Bacillus anthracis, an agent that causes high mortality in humans and animals. Protective antigen forms oligomeric prepores that undergo conversion to membrane-spanning pores by endosomal acidification, and these pores translocate the enzymes lethal factor and oedema factor into the cytosol of target cells. Protective antigen is not only a vaccine component and therapeutic target for anthrax infections but also an excellent model system for understanding the mechanism of protein translocation. On the basis of biochemical and electrophysiological results, researchers have proposed that a phi (Φ)-clamp composed of phenylalanine (Phe)427 residues of protective antigen catalyses protein translocation via a charge-state-dependent Brownian ratchet. Although atomic structures of protective antigen prepores are available, how protective antigen senses low pH, converts to active pore, and translocates lethal factor and oedema factor are not well defined without an atomic model of its pore. Here, by cryo-electron microscopy with direct electron counting, we determine the protective antigen pore structure at 2.9-Å resolution. The structure reveals the long-sought-after catalytic Φ-clamp and the membrane-spanning translocation channel, and supports the Brownian ratchet model for protein translocation. Comparisons of four structures reveal conformational changes in prepore to pore conversion that support a multi-step mechanism by which low pH is sensed and the membrane-spanning channel is formed.

Conserved SMP domains of the ERMES complex bind phospholipids and mediate tether assembly

Significance Phospholipid exchange between the endoplasmic reticulum (ER) and mitochondria is essential for membrane biogenesis and, ultimately, cell survival. It remains unclear, however, how this exchange is facilitated. Our study investigates a putative involvement of the ER-mitochondrial encounter structure (ERMES), a tethering complex that bridges the ER and mitochondria, in phospholipid transport in yeast. We show that a conserved ERMES domain called the “synaptotagmin-like mitochondrial lipid-binding protein” (SMP) domain preferentially binds phosphatidylcholines and mediates the hierarchical assembly of the tether. The 17-Å-resolution EM structure of the complex formed between the SMP domains present in two ERMES subunits, Mdm12 and Mmm1, reveals an elongated, tubular-shaped heterotetramer traversed by a hydrophobic channel, suggesting a mechanism for lipid transport between the two organelles. Membrane contact sites (MCS) between organelles are proposed as nexuses for the exchange of lipids, small molecules, and other signals crucial to cellular function and homeostasis. Various protein complexes, such as the endoplasmic reticulum-mitochondrial encounter structure (ERMES), function as dynamic molecular tethers between organelles. Here, we report the reconstitution and characterization of subcomplexes formed by the cytoplasm-exposed synaptotagmin-like mitochondrial lipid-binding protein (SMP) domains present in three of the five ERMES subunits—the soluble protein Mdm12, the endoplasmic reticulum (ER)-resident membrane protein Mmm1, and the mitochondrial membrane protein Mdm34. SMP domains are conserved lipid-binding domains found exclusively in proteins at MCS. We show that the SMP domains of Mdm12 and Mmm1 associate into a tight heterotetramer with equimolecular stoichiometry. Our 17-Å-resolution EM structure of the complex reveals an elongated crescent-shaped particle in which two Mdm12 subunits occupy symmetric but distal positions at the opposite ends of a central ER-anchored Mmm1 homodimer. Rigid body fitting of homology models of these SMP domains in the density maps reveals a distinctive extended tubular structure likely traversed by a hydrophobic tunnel. Furthermore, these two SMP domains bind phospholipids and display a strong preference for phosphatidylcholines, a class of phospholipids whose exchange between the ER and mitochondria is essential. Last, we show that the three SMP-containing ERMES subunits form a ternary complex in which Mdm12 bridges Mmm1 to Mdm34. Our findings highlight roles for SMP domains in ERMES assembly and phospholipid binding and suggest a structure-based mechanism for the facilitated transport of phospholipids between organelles.

The architecture of Tetrahymena telomerase holoenzyme

Telomerase adds telomeric repeats to chromosome ends using an internal RNA template and a specialized telomerase reverse transcriptase (TERT), thereby maintaining genome integrity. Little is known about the physical relationships among protein and RNA subunits within a biologically functional holoenzyme. Here we describe the architecture of Tetrahymena thermophila telomerase holoenzyme determined by electron microscopy. Six of the seven proteins and the TERT-binding regions of telomerase RNA (TER) have been localized by affinity labelling. Fitting with high-resolution structures reveals the organization of TERT, TER and p65 in the ribonucleoprotein (RNP) catalytic core. p50 has an unanticipated role as a hub between the RNP catalytic core, p75–p19–p45 subcomplex, and the DNA-binding Teb1. A complete in vitro holoenzyme reconstitution assigns function to these interactions in processive telomeric repeat synthesis. These studies provide the first view of the extensive network of subunit associations necessary for telomerase holoenzyme assembly and physiological function.

Structure of Tetrahymena telomerase reveals previously unknown subunits, functions, and interactions

Chromosome-capping enzyme complex Telomeres cap and protect the ends of our chromosomes. The telomerase complex helps maintain the telomere DNA repeat sequences. Telomerase consists of an RNA and a number of protein subunits. Jiang et al. used cryo–electron microscopy and x-ray crystallography to determine the structure of the Tetrahymena telomerase complex. The telomerase is made up of three subcomplexes, which include two previously unknown protein subunits in addition to the seven known subunits. The structures also reveal the path of the RNA component in the telomerase catalytic core. Science, this issue p. 10.1126/science.aab4070 The structure of a complete telomerase complex reveals two previously unknown subunits and the path of the RNA. INTRODUCTION Telomerase is a ribonucleoprotein complex that extends the telomere DNA at the 3′ ends of linear chromosomes, thereby counteracting the loss of DNA from replication and nucleolytic processing. Although telomerase is largely inactive in somatic cells, it is active in stem cells and highly active in most cancer cell lines, where its activity is necessary for the cells’ immortal phenotype. Thus, telomerase is an important regulator of aging, tumorigenesis, and stem cell renewal. Telomerase uses a template contained within the integral telomerase RNA (TER) and a telomerase reverse transcriptase (TERT) to synthesize multiple copies of the G-strand telomere repeat, which is TTGGGG in Tetrahymena. Telomerase can synthesize telomere repeat DNA in vitro with only TERT and TER, but physiological function requires a variety of other proteins. Telomerase recruitment to telomeres is regulated by the cell cycle, where its activity requires interplay between telomere end-protection and telomerase proteins. Telomere end-maintenance also requires coordinated recruitment of telomerase and DNA polymerase α for synthesis of G and C strands, respectively. RATIONALE A detailed mechanistic description of telomerase and its interaction at telomeres has been hampered by a lack of structural models, due to difficulties in obtaining samples of sufficient quantity and quality, as well as subunit complexity and flexibility and low sequence identity among subunits from different organisms. Unlike yeast and mammalian telomerase, Tetrahymena telomerase is constitutively assembled, making it possible to purify from cells and study all holoenzyme components in a stable complex. In addition to TERT and TER, the Tetrahymena telomerase holoenzyme contains the known proteins p65, p75, p45, p19, p50, and Teb1, a telomeric G-strand binding protein that is paralogous to the large subunit of heterotrimeric replication protein A (RPA). TER contains a template/pseudoknot domain enclosing a template with sequence complementarity to ~1.5 telomere repeats and a separate activating domain. Though TER is essential for activity, the physical arrangement of TER on TERT has remained largely unknown, as have details of protein subunit interactions. RESULTS We report cryo–electron microscopy (cryo-EM) structures at ~9 Å resolution and pseudoatomic modeling of Tetrahymena telomerase, crystal structures of p19 and p45C, and a nuclear magnetic resonance structure of the TER pseudoknot. Two newly identified and functionally distinct RPA-related complexes, Teb1-Teb2-Teb3 (TEB) and p75-p45-p19, are connected to the TERT-TER-p65 catalytic core by p50. The presence of Teb2 and Teb3 is confirmed by mass spectrometry. p19 and p45 are structurally homologous to Ten1 and Stn1, respectively, which are found in the telomere end-binding complex CST (CTC1-STN1-TEN1) that recruits DNA polymerase α. The path of TER on TERT and the location of the TERT essential N-terminal domain (TEN) are revealed, providing mechanistic insights into telomerase function. A network of interactions between TEN, p50, and TEB regulates and enhances activity. p50-Teb1 appears to be structurally and functionally homologous to human TPP1-POT1. Negative-stain electron microscopy of labeled telomeric DNA bound to telomerase reveals the exit path from the template. CONCLUSION The structure of the Tetrahymena telomerase catalytic core and our identification of telomerase holoenzyme subcomplexes that are homologous to those found at mammalian, plant, and yeast telomeres provide new mechanistic insights and suggest commonalities of telomerase interaction, action, and regulation at telomeres. Two views of the Tetrahymena telomerase holoenzyme. The top left image shows a front view of pseudoatomic models as spacefill superimposed on the cryo-EM map. Models of protein domains connected by flexible linkers and not visible in the cryo-EM map are illustrated as ribbons. The bottom right image shows a back-view schematic with the telomerase-bound telomeric G strand connected to a chromosome via double-stranded telomere DNA. Teb1AB domains are presumed to be ordered when DNA bound. Telomerase helps maintain telomeres by processive synthesis of telomere repeat DNA at their 3′-ends, using an integral telomerase RNA (TER) and telomerase reverse transcriptase (TERT). We report the cryo–electron microscopy structure of Tetrahymena telomerase at ~9 angstrom resolution. In addition to seven known holoenzyme proteins, we identify two additional proteins that form a complex (TEB) with single-stranded telomere DNA-binding protein Teb1, paralogous to heterotrimeric replication protein A (RPA). The p75-p45-p19 subcomplex is identified as another RPA-related complex, CST (CTC1-STN1-TEN1). This study reveals the paths of TER in the TERT-TER-p65 catalytic core and single-stranded DNA exit; extensive subunit interactions of the TERT essential N-terminal domain, p50, and TEB; and other subunit identities and structures, including p19 and p45C crystal structures. Our findings provide structural and mechanistic insights into telomerase holoenzyme function.

Atomic Model of CPV Reveals the Mechanism Used by This Single-Shelled Virus to Economically Carry Out Functions Conserved in Multishelled Reoviruses

Unlike the multishelled viruses in the Reoviridae, cytoplasmic polyhedrosis virus (CPV) is single shelled, yet stable and fully capable of carrying out functions conserved within Reoviridae. Here, we report a 3.1 Å resolution cryo electron microscopy structure of CPV and derive its atomic model, consisting of 60 turret proteins (TPs), 120 each of capsid shell proteins (CSPs) and large protrusion proteins (LPPs). Two unique segments of CSP contribute to CPVs stability: an inserted protrusion domain interacting with neighboring proteins, and an N-anchor tying up CSPs together through strong interactions such as β sheet augmentation. Without the need to interact with outer shell proteins, LPP retains only the N-terminal two-third region containing a conserved helix-barrel core and interacts exclusively with CSP. TP is also simplified, containing only domains involved in RNA capping. Our results illustrate how CPV proteins have evolved in a coordinative manner to economically carry out their conserved functions.

Assembly and architecture of the EBV B cell entry triggering complex.

Epstein-Barr Virus (EBV) is an enveloped double-stranded DNA virus of the gammaherpesvirinae sub-family that predominantly infects humans through epithelial cells and B cells. Three EBV glycoproteins, gH, gL and gp42, form a complex that targets EBV infection of B cells. Human leukocyte antigen (HLA) class II molecules expressed on B cells serve as the receptor for gp42, triggering membrane fusion and virus entry. The mechanistic role of gHgL in herpesvirus entry has been largely unresolved, but it is thought to regulate the activation of the virally-encoded gB protein, which acts as the primary fusogen. Here we study the assembly and function of the reconstituted B cell entry complex comprised of gHgL, gp42 and HLA class II. The structure from negative-stain electron microscopy provides a detailed snapshot of an intermediate state in EBV entry and highlights the potential for the triggering complex to bring the two membrane bilayers into proximity. Furthermore, gHgL interacts with a previously identified, functionally important hydrophobic pocket on gp42, defining the overall architecture of the complex and playing a critical role in membrane fusion activation. We propose a macroscopic model of the initiating events in EBV B cell fusion centered on the formation of the triggering complex in the context of both viral and host membranes. This model suggests how the triggering complex may bridge the two membrane bilayers, orienting critical regions of the N- and C- terminal ends of gHgL to promote the activation of gB and efficient membrane fusion.

Atomic structure of the human cytomegalovirus capsid with its securing tegument layer of pp150.

Strong under pressure Human cytomegalovirus (HCMV) is a member of the herpesvirus family that can cause life-threatening infections in those who are immunocompromised. HCMV encodes a genome that is about 50% larger than that of herpes simplex virus 1 (the virus that causes cold sores), but these two viruses have similar-sized capsids. Yu et al. used cryo–electron microscopy to determine the structure of the HCMV capsid to 3.9-Å resolution. This is the first high-resolution capsid structure from the herpesvirus family. It reveals extensive interactions that stabilize the capsid to withstand the high pressure that comes from accommodating such a large genome. Science, this issue p. eaam6892 A structure of human cytomegalovirus shows how the capsid withstands the pressure of containing the largest herpesvirus genome. INTRODUCTION Human cytomegalovirus (HCMV) is a leading cause of congenital defects and a major contributor to life-threatening complications in immunocompromised individuals. HCMV is a β-herpesvirus that more broadly belongs to Herpesviridae, whose members have long been in lockstep with humanity, responsible for ailments from chickenpox (varicella zoster virus, VZV) to the common cold sore (herpes simplex virus 1, HSV-1). Yet HCMV’s ability to establish relatively nontoxic lifelong latency in hosts, its high seroprevalence in human populations, and its large genetic capacity are characteristics shared among herpesviruses that give them desirable advantages over other viral candidates as tools in the development of gene delivery vehicles, oncolytic vectors, and vaccines against not just herpesviruses, but even HIV/AIDS. RATIONALE All human herpesviruses have a highly pressured nucleocapsid (up to tens of atmospheres) thanks to a large genome that packs tightly within a space-constrained capsid. HCMV’s 235-kb genome is by far the largest of any human herpesvirus at twice the size of VZV’s and >50% larger than HSV-1’s, although HCMV has a capsid that is similar in size to those of other herpesviruses. Previous evidence has suggested that the β-herpesvirus–specific tegument protein pp150 contributes to a netlike layer that may stabilize the HCMV capsid, but in the absence of an atomic description of HCMV particles, the exact mechanisms through which capsid stability is achieved have remained unclear. Despite recent advances in high-resolution studies of macromolecular complexes, an atomic structure of a herpesvirus has proved elusive because of the immense challenges posed by their size (more than 2000 Å in diameter) and the associated fragility of such large assemblies. RESULTS By using an improved sample preparation strategy and electron-counting cryo–electron microscopy, we obtained a three-dimensional reconstruction of HCMV at 3.9-Å resolution and derived an atomic structure for the herpesvirus-conserved capsid proteins MCP, Tri1, Tri2, and SCP and the HCMV-specific tegument protein pp150, totaling ~4000 molecules and 62 different conformers. MCPs manifest as a complex of domain insertions around a bacteriophage HK97 gp5–like “Johnson fold” domain, which gives rise to three classes of capsid floor–defining interactions beneath hexons and analogous, though less substantial, interactions beneath pentons. Triplexes, composed of two “embracing” Tri2 conformers and a “third-wheeling” Tri1, fasten the capsid floor. Whereas these stabilization mechanisms are likely conserved across herpesviruses, our structure also reveals HCMV-specific capsid stabilization strategies, including hexon channels that facilitate the packing of DNA and pp150 helix bundles that secure the capsid through a critical cysteine tetrad interaction with SCP, the smallest and least conserved capsid protein across Herpesviridae. CONCLUSION With an exceptionally large genome and high internal capsid pressure, HCMV achieves capsid stability through an extreme form of structural elaboration on a basic Johnson fold topology, relying on not only domain insertions into the major capsid protein and the inclusion of auxiliary heterotrimers, but also the recruitment of a tegumental layer of pp150 to secure its DNA-engorged capsid from without. Beyond providing an organizational blueprint to understand all other herpesviruses, our HCMV atomic structure should inform rational design of therapeutic strategies against HCMV, other herpesviruses, and, in light of recent findings in simian models, potentially HIV/AIDS. A 3.9-Å structure of human cytomegalovirus. One of the 60 asymmetric units that make up HCMV’s 1320-Å-wide capsid is rendered above a colored face of the icosahedral shell. Atomic models of the capsid proteins (SCP, MCP, Tri1, Tri2A, and Tri2B) and capsid-associated pp150 tegument protein (“nt” signifies the N-terminal one-third) reveal a suite of strategies that work in a synergistic manner to stabilize a capsid that is highly pressurized by HCMV’s enormous 235-kb genome. P, peripentonal; C, center; E, edge; Ta to Te, heterotrimeric triplexes composed of Tri1, Tri2A, and Tri2B. Herpesviruses possess a genome-pressurized capsid. The 235-kilobase genome of human cytomegalovirus (HCMV) is by far the largest of any herpesvirus, yet it has been unclear how its capsid, which is similar in size to those of other herpesviruses, is stabilized. Here we report a HCMV atomic structure consisting of the herpesvirus-conserved capsid proteins MCP, Tri1, Tri2, and SCP and the HCMV-specific tegument protein pp150—totaling ~4000 molecules and 62 different conformers. MCPs manifest as a complex of insertions around a bacteriophage HK97 gp5–like domain, which gives rise to three classes of capsid floor–defining interactions; triplexes, composed of two “embracing” Tri2 conformers and a “third-wheeling” Tri1, fasten the capsid floor. HCMV-specific strategies include using hexon channels to accommodate the genome and pp150 helix bundles to secure the capsid via cysteine tetrad–to-SCP interactions. Our structure should inform rational design of countermeasures against HCMV, other herpesviruses, and even HIV/AIDS.

Tetrahymena telomerase holoenzyme assembly, activation, and inhibition by domains of the p50 central hub.

ABSTRACT The eukaryotic reverse transcriptase, telomerase, adds tandem telomeric repeats to chromosome ends to promote genome stability. The fully assembled telomerase holoenzyme contains a ribonucleoprotein (RNP) catalytic core and additional proteins that modulate the ability of the RNP catalytic core to elongate telomeres. Electron microscopy (EM) structures of Tetrahymena telomerase holoenzyme revealed a central location of the relatively uncharacterized p50 subunit. Here we have investigated the biochemical and structural basis for p50 function. We have shown that the p50-bound RNP catalytic core has a relatively low rate of tandem repeat synthesis but high processivity of repeat addition, indicative of high stability of enzyme-product interaction. The rate of tandem repeat synthesis is enhanced by p50-dependent recruitment of the holoenzyme single-stranded DNA binding subunit, Teb1. An N-terminal p50 domain is sufficient to stimulate tandem repeat synthesis and bridge the RNP catalytic core, Teb1, and the p75 subunit of the holoenzyme subcomplex p75/p19/p45. In cells, the N-terminal p50 domain assembles a complete holoenzyme that is functional for telomere maintenance, albeit at shortened telomere lengths. Also, in EM structures of holoenzymes, only the N-terminal domain of p50 is visible. Our findings provide new insights about subunit and domain interactions and functions within the Tetrahymena telomerase holoenzyme.

Single Particle Electron Microscopy Analysis of the Bovine Anion Exchanger 1 Reveals a Flexible Linker Connecting the Cytoplasmic and Membrane Domains

Anion exchanger 1 (AE1) is the major erythrocyte membrane protein that mediates chloride/bicarbonate exchange across the erythrocyte membrane facilitating CO2 transport by the blood, and anchors the plasma membrane to the spectrin-based cytoskeleton. This multi-protein cytoskeletal complex plays an important role in erythrocyte elasticity and membrane stability. An in-frame AE1 deletion of nine amino acids in the cytoplasmic domain in a proximity to the membrane domain results in a marked increase in membrane rigidity and ovalocytic red cells in the disease Southeast Asian Ovalocytosis (SAO). We hypothesized that AE1 has a flexible region connecting the cytoplasmic and membrane domains, which is partially deleted in SAO, thus causing the loss of erythrocyte elasticity. To explore this hypothesis, we developed a new non-denaturing method of AE1 purification from bovine erythrocyte membranes. A three-dimensional (3D) structure of bovine AE1 at 2.4 nm resolution was obtained by negative staining electron microscopy, orthogonal tilt reconstruction and single particle analysis. The cytoplasmic and membrane domains are connected by two parallel linkers. Image classification demonstrated substantial flexibility in the linker region. We propose a mechanism whereby flexibility of the linker region plays a critical role in regulating red cell elasticity.