Melissa G. Chambers

Molecular Mechanism of V(D)J Recombination from Synaptic RAG1-RAG2 Complex Structures

Diverse repertoires of antigen-receptor genes that result from combinatorial splicing of coding segments by V(D)J recombination are hallmarks of vertebrate immunity. The (RAG1-RAG2)2 recombinase (RAG) recognizes recombination signal sequences (RSSs) containing a heptamer, a spacer of 12 or 23 base pairs, and a nonamer (12-RSS or 23-RSS) and introduces precise breaks at RSS-coding segment junctions. RAG forms synaptic complexes only with one 12-RSS and one 23-RSS, a dogma known as the 12/23 rule that governs the recombination fidelity. We report cryo-electron microscopy structures of synaptic RAG complexes at up to 3.4 Å resolution, which reveal a closed conformation with base flipping and base-specific recognition of RSSs. Distortion at RSS-coding segment junctions and base flipping in coding segments uncover the two-metal-ion catalytic mechanism. Induced asymmetry involving tilting of the nonamer-binding domain dimer of RAG1 upon binding of HMGB1-bent 12-RSS or 23-RSS underlies the molecular mechanism for the 12/23 rule.

Cryo-EM structure of the protein-conducting ERAD channel Hrd1 in complex with Hrd3

Misfolded endoplasmic reticulum proteins are retro-translocated through the membrane into the cytosol, where they are poly-ubiquitinated, extracted from the membrane, and degraded by the proteasome—a pathway termed endoplasmic reticulum-associated protein degradation (ERAD). Proteins with misfolded domains in the endoplasmic reticulum lumen or membrane are discarded through the ERAD-L and ERAD-M pathways, respectively. In Saccharomyces cerevisiae, both pathways require the ubiquitin ligase Hrd1, a multi-spanning membrane protein with a cytosolic RING finger domain. Hrd1 is the crucial membrane component for retro-translocation, but it is unclear whether it forms a protein-conducting channel. Here we present a cryo-electron microscopy structure of S. cerevisiae Hrd1 in complex with its endoplasmic reticulum luminal binding partner, Hrd3. Hrd1 forms a dimer within the membrane with one or two Hrd3 molecules associated at its luminal side. Each Hrd1 molecule has eight transmembrane segments, five of which form an aqueous cavity extending from the cytosol almost to the endoplasmic reticulum lumen, while a segment of the neighbouring Hrd1 molecule forms a lateral seal. The aqueous cavity and lateral gate are reminiscent of features of protein-conducting conduits that facilitate polypeptide movement in the opposite direction—from the cytosol into or across membranes. Our results suggest that Hrd1 forms a retro-translocation channel for the movement of misfolded polypeptides through the endoplasmic reticulum membrane.

CATCHR, HOPS and CORVET tethering complexes share a similar architecture

We show here that the Saccharomyces cerevisiae GARP complex and the Cog1–4 subcomplex of the COG complex, both members of the complexes associated with tethering containing helical rods (CATCHR) family of multisubunit tethering complexes, share the same subunit organization. We also show that HOPS, a tethering complex acting in the endolysosomal pathway, shares a similar architecture, thus suggesting that multisubunit tethering complexes use related structural frameworks.

High-resolution cryo-EM analysis of the yeast ATP synthase in a lipid membrane.

Protons find a path Adenosine triphosphate (ATP) synthases are dynamos that interconvert rotational and chemical energy. Capturing the complete structure of these multisubunit membrane-bound complexes has been hindered by their inherent ability to adopt multiple conformations. Srivastava et al. used protein engineering to freeze mitochondrial ATP synthase from yeast in a single conformation and obtained a structure with the inhibitor oligomycin, which binds to the rotating c-ring within the membrane. Hahn et al. show that chloroplast ATP synthase contains a built-in inhibitor triggered by oxidizing conditions in the dark chloroplast. The mechanisms by which these machines are powered are remarkably similar: Protons are shuttled through a channel to the membrane-embedded c-ring, where they drive nearly a full rotation of the rotor before exiting through another channel on the opposite side of the membrane (see the Perspective by Kane). Science, this issue p. eaas9699, p. eaat4318; see also p. 600 The structure of an intact ATP synthase provides insight into how the motor and catalytic components are coupled. INTRODUCTION The mitochondrial adenosine triphosphate (ATP) synthase is the enzyme responsible for the synthesis of more than 90% of the ATP produced by mammalian cells under aerobic conditions. The chemiosmotic mechanism, proposed by Peter Mitchell, states that the enzyme transduces the energy of a proton gradient, generated by the electron transport chain, into the major energy currency of the cell, ATP. The enzyme is a large (about 600,000 Da, in the monomer state) multisubunit complex, with a water soluble complex (F1) that contains three active sites and a membrane complex (Fo) that contains the proton translocation pathway, principally comprised of the a subunit and a ring of 10 c subunits, the c10-ring (10 in yeast, 8 in mammals). F1 has a central rotor that, at one end, is within the core of F1 and, at the other end, is connected to the c10-ring of Fo. During ATP synthesis, the c10-ring rotates, driven by the movement of protons from the cytosol to the mitochondrion, and in turn, the rotor rotates within F1 in steps of 120o. The rotation of the rotor causes conformational changes in the catalytic sites, which provides the energy for the phosphorylation of adenosine diphosphate (ADP), as first proposed in the binding-change hypothesis by Paul Boyer. The peripheral stalk acts as a stator connecting F1 with Fo and prevents the futile rotation of F1 as the rotor spins within it. RATIONALE Structural studies of the ATP synthase have made steady progress since the structure of the F1 complex was described in pioneering work by John Walker. However, obtaining a high-resolution structure of the intact ATP synthase is challenging because it is inherently dynamic. To overcome this conformational heterogeneity, we locked the yeast mitochondrial rotor in a single conformation by fusing a subunit of the stator with a subunit of the rotor, also called the central stalk. The engineered ATP synthase was expressed in yeast and reconstituted into nanodiscs. This facilitated structure determination by cryo–electron microscopy (cryo-EM) under near native conditions. RESULTS Single-particle cryo-EM enabled us to determine the structures of the membrane-embedded monomeric yeast ATP synthase in the presence and absence of the inhibitor oligomycin at 3.8- and 3.6-Å resolution, respectively. The fusion between the rotor and stator caused a twisting of the rotor and a 9° rotation of the c10-ring, in the direction of ATP synthesis, relative to the putative resting state. This twisted conformation likely represents an intermediate state in the ATP synthesis reaction cycle. The structure also shows two proton half-channels formed largely by the a subunit that abut the c10-ring and suggests a mechanism that couples transmembrane proton movement to c10-ring rotation. The cryo-EM density map indicates that oligomycin is bound to at least four sites on the surface of the Fo c10-ring that is exposed to the lipid bilayer; this is supported by binding free-energy molecular dynamics calculations. The sites of oligomycin-resistant mutations in the a subunit suggest that changes in the side-chain configuration of the c subunits at the a-c subunit interface are transmitted through the entire c10-ring. CONCLUSION Our results provide a high-resolution structure of the complete monomeric form of the mitochondrial ATP synthase. The structure provides an understanding of the mechanism of inhibition by oligomycin and suggests how extragenic mutations can cause resistance to this inhibitor. The approach presented in this study paves the way for structural characterization of other functional states of the ATP synthase, which is essential for understanding its functions in physiology and disease. Structure of the monomeric yeast ATP synthase, as determined by cryo-EM, shown as a ribbon diagram. The subunits are shown in different colors. The F1 complex is located at the top center and is composed of six subunits forming a nearly spherical structure and three subunits comprising the central stalk, or rotor. The Fo complex is located at the bottom, with the identity of the c10-ring clearly seen. The peripheral stalk, or stator, is on the left, and the rotor is in the center of the molecule, extending into F1. Mitochondrial adenosine triphosphate (ATP) synthase comprises a membrane embedded Fo motor that rotates to drive ATP synthesis in the F1 subunit. We used single-particle cryo–electron microscopy (cryo-EM) to obtain structures of the full complex in a lipid bilayer in the absence or presence of the inhibitor oligomycin at 3.6- and 3.8-angstrom resolution, respectively. To limit conformational heterogeneity, we locked the rotor in a single conformation by fusing the F6 subunit of the stator with the δ subunit of the rotor. Assembly of the enzyme with the F6-δ fusion caused a twisting of the rotor and a 9° rotation of the Fo c10-ring in the direction of ATP synthesis, relative to the structure of isolated Fo. Our cryo-EM structures show how F1 and Fo are coupled, give insight into the proton translocation pathway, and show how oligomycin blocks ATP synthesis.

Best practices for managing large CryoEM facilities

This paper provides an overview of the discussion and presentations from the Workshop on the Management of Large CryoEM Facilities held at the New York Structural Biology Center, New York, NY on February 6-7, 2017. A major objective of the workshop was to discuss best practices for managing cryoEM facilities. The discussions were largely focused on supporting single-particle methods for cryoEM and topics included: user access, assessing projects, workflow, sample handling, microscopy, data management and processing, and user training.

The structural basis for the functional comparability of factor VIII and the long-acting variant recombinant factor VIII Fc fusion protein.

Essentials Recombinant factor VIII (rFVIII) Fc fusion protein has a 1.5‐fold longer half‐life than rFVIII. Five orthogonal methods were used to characterize the structure of rFVIIIFc compared to rFVIII. The C‐terminal Fc fusion does not perturb the structure of FVIII in rFVIIIFc. The FVIII and Fc components of rFVIIIFc are flexibly tethered and functionally independent.

Abstract B073: Structural basis of the 12-23 rule in V(D)J recombination

The V, D and J segment recombination (V(D)J recombination) is the key event in the development and maturation of T and B lymphocytes, which generates a diverse repertoire of T and B cell clones for the further selection of useful specificities. V(D)J recombination is also the basis for antibody diversity. The initiation step of V(D)J recombination is carried out by a pair of lymphocyte specific enzymes, which are called recombination-activating gene 1 and 2 (RAG1 and RAG2). RAG1 and RAG2 associate with each other and with two recognition signal sequences (RSS, 12RSS and 23RSS) adjacent to the coding ends of the V, D and J segments to catalyze the double stranded breaks required for joining. The reaction goes through several catalytic steps that result in formation of the single recombination complex (SC), the paired complex (PC), the cleaved signal complex (CSC) as well as the signal end complex (SEC). In order to understand the mechanisms of how RAG complex functions on DNA, we solved the structures PC and CSC by cryo-electron microscopy near the atomic resolution. Compared with the Apo-RAG complex that constitutes an open conformation, the PC and CSC reveal a closed conformation with RAG1 in one protomer interacts with RAG2 in another protomer. Furthermore, the first nucleotide of the heptamer in the non-transfer strand is flipped out to avoid the steric hindrance of the 39-OH to attack the phosphodiester bond in the transfer strand. Upon nucleophilic attack and hairpin formation, the nucleotide adjacent to the 59-position of the attacked phosphate is also flipped out to accommodate the tensile force in the hairpin DNA. In addition, we found that the conformation changes in the nonamer-binding domain (NBD), which is distorted toward to the 12RSS, and the distorsion requires two turns for the nonamer DNA in the 23RSS to reach the NBD. Finally, we found that two HMGB1 molecules, rather than one, are required to bind and bend both 12RSS and 23RSS DNA to adjust the conformational change of NBD on each side. In summary, our structural models not only uncover the mechanisms of how RAG protein catalyzes the hairpin formation, but also provide the structural basis of the 12-23 rule in V(D)J recombination. Citation Format: Heng Ru, Melissa G. Chambers, Maofu Liao, Hao Wu. Structural basis of the 12-23 rule in V(D)J recombination. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B073.

Abstract B122: Molecular mechanism of V(D)J recombination from synaptic RAG1-RAG2 complex structures

A hallmark of vertebrate immunity is the diverse repertoire of antigen-receptor genes that results from combinatorial splicing of gene coding segments by V(D)J recombination. The dimeric (RAG1-RAG2)2 recombinase (RAG) recognizes specific recombination signal sequences (RSSs), each containing a heptamer, a spacer of 12 or 23 base pairs, and a nonamer (12-RSS or 23-RSS). RAG only combines one 12-RSS and one 23-RSS, a dogma known as the 12/23 rule that governs the fidelity of V(D)J recombination. RAG introduces precise breaks at RSS-coding segment junctions to generate cleaved RSSs and hairpin coding segments. Here we report cryo-electron microscopy structures of multiple RAG complexes reconstituted with 12-RSS and 23-RSS intermediates and HMGB1 at up to 3.4 angstrom resolution. These structures reveal a closed conformation of RAG upon RSS synapsis, with base flipping and base-specific recognition of RSSs. Distortion at the RSS-coding segment junction and base flipping in the coding segment uncovers the geometry for a two-metal-ion catalytic mechanism. Both 12-RSS and 23-RSS are exceedingly bent by the bound HMGB1. The nonamer-binding domain dimer of RAG1 is flexibly attached to the active site dimer of RAG and is tilted towards the bent 12-RSS but away from the bent 23-RSS in the synaptic complexes, which provides an induced fit mechanism for the 12/23 rule. Collectively, our structures illustrate the elegant mechanisms of RAG-catalyzed V(D)J recombination at the molecular level. Citation Format: Heng Ru, Melissa G. Chambers, Tian-Min Fu, Alexander B. Tong, Maofu Liao, Hao Wu. Molecular mechanism of V(D)J recombination from synaptic RAG1-RAG2 complex structures [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B122.