Cryo-EM of Mitotic Checkpoint Complex-Bound APC/C Reveals Reciprocal and Conformational Regulation of Ubiquitin Ligation.

The mitotic checkpoint complex (MCC) coordinates proper chromosome biorientation on the spindle with ubiquitination activities of CDC20-activated anaphase-promoting complex/cyclosome (APC/C(CDC20)). APC/C(CDC20) and two E2s, UBE2C and UBE2S, catalyze ubiquitination through distinct architectures for linking ubiquitin (UB) to substrates and elongating polyUB chains, respectively. MCC, which contains a second molecule of CDC20, blocks APC/C(CDC20)-UBE2C-dependent ubiquitination of Securin and Cyclins, while differentially determining or inhibiting CDC20 ubiquitination to regulate spindle surveillance, checkpoint activation, and checkpoint termination. Here electron microscopy reveals conformational variation of APC/C(CDC20)-MCC underlying this multifaceted regulation. MCC binds APC/C-bound CDC20 to inhibit substrate access. However, rotation about the CDC20-MCC assembly and conformational variability of APC/C modulate UBE2C-catalyzed ubiquitination of MCC's CDC20 molecule. Access of UBE2C is limiting for subsequent polyubiquitination by UBE2S. We propose that conformational dynamics of APC/C(CDC20)-MCC modulate E2 activation and determine distinctive ubiquitination activities as part of a response mechanism ensuring accurate sister chromatid segregation.

ATPγS stalls splicing after B complex formation but prior to spliceosome activation.

The ATP analog ATPγS inhibits pre-mRNA splicing in vitro, but there have been conflicting reports as to which step of splicing is inhibited by this small molecule and its inhibitory mechanism remains unclear. Here we have dissected the effect of ATPγS on pre-mRNA splicing in vitro. Addition of ATPγS to splicing extracts depleted of ATP inhibited both catalytic steps of splicing. At ATPγS concentrations ≥0.5 mM, precatalytic B complexes accumulate, demonstrating a block prior to or during the spliceosome activation stage. Affinity purification of the ATPγS-stalled B complexes (B(ATPγS)) and subsequent characterization of their abundant protein components by 2D gel electrophoresis revealed that B(ATPγS) complexes are compositionally more homogeneous than B complexes previously isolated in the presence of ATP. In particular, they contain little or no Prp19/CDC5L complex proteins, indicating that these proteins are recruited after assembly of the precatalytic spliceosome. Under the electron microscope, B(ATPγS) complexes exhibit a morphology highly similar to B complexes, indicating that the ATPγS-induced block in the transformation of the B to B(act) complex is not due to a major structural defect. Likely mechanisms whereby ATPγS blocks spliceosome assembly at the activation stage, including inhibition of the RNA helicase Brr2, are discussed. Given their more homogeneous composition, B complexes stalled by ATPγS may prove highly useful for both functional and structural analyses of the precatalytic spliceosome and its conversion into an activated B(act) spliceosomal complex.

Dual RING E3 Architectures Regulate Multiubiquitination and Ubiquitin Chain Elongation by APC/C.

Protein ubiquitination involves E1, E2, and E3 trienzyme cascades. E2 and RING E3 enzymes often collaborate to first prime a substrate with a single ubiquitin (UB) and then achieve different forms of polyubiquitination: multiubiquitination of several sites and elongation of linkage-specific UB chains. Here, cryo-EM and biochemistry show that the human E3 anaphase-promoting complex/cyclosome (APC/C) and its two partner E2s, UBE2C (aka UBCH10) and UBE2S, adopt specialized catalytic architectures for these two distinct forms of polyubiquitination. The APC/C RING constrains UBE2C proximal to a substrate and simultaneously binds a substrate-linked UB to drive processive multiubiquitination. Alternatively, during UB chain elongation, the RING does not bind UBE2S but rather lures an evolving substrate-linked UB to UBE2S positioned through a cullin interaction to generate a Lys11-linked chain. Our findings define mechanisms of APC/C regulation, and establish principles by which specialized E3-E2-substrate-UB architectures control different forms of polyubiquitination.

RING E3 mechanism for ubiquitin ligation to a disordered substrate visualized for human anaphase-promoting complex.

For many E3 ligases, a mobile RING (Really Interesting New Gene) domain stimulates ubiquitin (Ub) transfer from a thioester-linked E2∼Ub intermediate to a lysine on a remotely bound disordered substrate. One such E3 is the gigantic, multisubunit 1.2-MDa anaphase-promoting complex/cyclosome (APC), which controls cell division by ubiquitinating cell cycle regulators to drive their timely degradation. Intrinsically disordered substrates are typically recruited via their KEN-box, D-box, and/or other motifs binding to APC and a coactivator such as CDH1. On the opposite side of the APC, the dynamic catalytic core contains the cullin-like subunit APC2 and its RING partner APC11, which collaborates with the E2 UBCH10 (UBE2C) to ubiquitinate substrates. However, how dynamic RING-E2∼Ub catalytic modules such as APC11-UBCH10∼Ub collide with distally tethered disordered substrates remains poorly understood. We report structural mechanisms of UBCH10 recruitment to APC(CDH1) and substrate ubiquitination. Unexpectedly, in addition to binding APC11's RING, UBCH10 is corecruited via interactions with APC2, which we visualized in a trapped complex representing an APC(CDH1)-UBCH10∼Ub-substrate intermediate by cryo-electron microscopy, and in isolation by X-ray crystallography. To our knowledge, this is the first structural view of APC, or any cullin-RING E3, with E2 and substrate juxtaposed, and it reveals how tripartite cullin-RING-E2 interactions establish APC's specificity for UBCH10 and harness a flexible catalytic module to drive ubiquitination of lysines within an accessible zone. We propose that multisite interactions reduce the degrees of freedom available to dynamic RING E3-E2∼Ub catalytic modules, condense the search radius for target lysines, increase the chance of active-site collision with conformationally fluctuating substrates, and enable regulation.

Mechanism of polyubiquitination by human anaphase-promoting complex: RING repurposing for ubiquitin chain assembly.

Polyubiquitination by E2 and E3 enzymes is a predominant mechanism regulating protein function. Some RING E3s, including anaphase-promoting complex/cyclosome (APC), catalyze polyubiquitination by sequential reactions with two different E2s. An initiating E2 ligates ubiquitin to an E3-bound substrate. Another E2 grows a polyubiquitin chain on the ubiquitin-primed substrate through poorly defined mechanisms. Here we show that human APC's RING domain is repurposed for dual functions in polyubiquitination. The canonical RING surface activates an initiating E2-ubiquitin intermediate for substrate modification. However, APC engages and activates its specialized ubiquitin chain-elongating E2 UBE2S in ways that differ from current paradigms. During chain assembly, a distinct APC11 RING surface helps deliver a substrate-linked ubiquitin to accept another ubiquitin from UBE2S. Our data define mechanisms of APC/UBE2S-mediated polyubiquitination, reveal diverse functions of RING E3s and E2s, and provide a framework for understanding distinctive RING E3 features specifying ubiquitin chain elongation.

Substrate binding on the APC/C occurs between the coactivator Cdh1 and the processivity factor Doc1.

The anaphase-promoting complex/cyclosome (APC/C) is a 22S ubiquitin ligase complex that initiates chromosome segregation and mitotic exit. We have used biochemical and electron microscopic analyses of Saccharomyces cerevisiae and human APC/C to address how the APC/C subunit Doc1 contributes to recruitment and processive ubiquitylation of APC/C substrates, and to understand how APC/C monomers interact to form a 36S dimeric form. We show that Doc1 interacts with Cdc27, Cdc16 and Apc1 and is located in the vicinity of the cullin-RING module Apc2-Apc11 in the inner cavity of the APC/C. Substrate proteins also bind in the inner cavity, in close proximity to Doc1 and the coactivator Cdh1, and induce conformational changes in Apc2-Apc11. Our results suggest that substrates are recruited to the APC/C by binding to a bipartite substrate receptor composed of a coactivator protein and Doc1.

The MIS12 complex is a protein interaction hub for outer kinetochore assembly.

Kinetochores are nucleoprotein assemblies responsible for the attachment of chromosomes to spindle microtubules during mitosis. The KMN network, a crucial constituent of the outer kinetochore, creates an interface that connects microtubules to centromeric chromatin. The NDC80, MIS12, and KNL1 complexes form the core of the KMN network. We recently reported the structural organization of the human NDC80 complex. In this study, we extend our analysis to the human MIS12 complex and show that it has an elongated structure with a long axis of approximately 22 nm. Through biochemical analysis, cross-linking-based methods, and negative-stain electron microscopy, we investigated the reciprocal organization of the subunits of the MIS12 complex and their contacts with the rest of the KMN network. A highlight of our findings is the identification of the NSL1 subunit as a scaffold supporting interactions of the MIS12 complex with the NDC80 and KNL1 complexes. Our analysis has important implications for understanding kinetochore organization in different organisms.

The evolutionarily conserved core design of the catalytic activation step of the yeast spliceosome.

Metazoan spliceosomes exhibit an elaborate protein composition required for canonical and alternative splicing. Thus, the minimal set of proteins essential for activation and catalysis remains elusive. We therefore purified in vitro assembled, precatalytic spliceosomal complex B, activated B(act), and step 1 complex C from the simple eukaryote Saccharomyces cerevisiae. Mass spectrometry revealed that yeast spliceosomes contain fewer proteins than metazoans and that each functional stage is very homogeneous. Dramatic compositional changes convert B to B(act), which is composed of approximately 40 evolutionarily conserved proteins that organize the catalytic core. Additional remodeling occurs concomitant with step 1, during which nine proteins are recruited to form complex C. The moderate number of proteins recruited to complex C will allow investigations of the chemical reactions in a fully defined system. Electron microscopy reveals high-quality images of yeast spliceosomes at defined functional stages, indicating that they are well-suited for three-dimensional structure analyses.

Structure of the anaphase-promoting complex/cyclosome interacting with a mitotic checkpoint complex.

Once all chromosomes are connected to the mitotic spindle (bioriented), anaphase is initiated by the protein ubiquitylation activity of the anaphase-promoting complex/cyclosome (APC/C) and its coactivator Cdc20 (APC/C(Cdc20)). Before chromosome biorientation, anaphase is delayed by a mitotic checkpoint complex (MCC) that inhibits APC/C(Cdc20). We used single-particle electron microscopy to obtain three-dimensional models of human APC/C in various functional states: bound to MCC, to Cdc20, or to neither (apo-APC/C). These experiments revealed that MCC associates with the Cdc20 binding site on APC/C, locks the otherwise flexible APC/C in a "closed" state, and prevents binding and ubiquitylation of a wide range of different APC/C substrates. These observations clarify the structural basis for the inhibition of APC/C by spindle checkpoint proteins.

Structure of yeast U6 snRNPs: arrangement of Prp24p and the LSm complex as revealed by electron microscopy.

Protein components of the U6 snRNP (Prp24p and LSm2-8) are thought to act cooperatively in facilitating the annealing of U6 and U4 snRNAs during U4/U6 di-snRNP formation. To learn more about the spatial arrangement of these proteins in S. cerevisiae U6 snRNPs, we investigated the structure of this particle by electron microscopy. U6 snRNPs, purified by affinity chromatography and gradient centrifugation, and then immediately adsorbed to the carbon film support, revealed an open form in which the Prp24 protein and the ring formed by the LSm proteins were visible as two separate morphological domains, while particles stabilized by chemical cross-linking in solution under mild conditions before binding to the carbon film exhibited a compact form, with the two domains in close proximity to one another. In the open form, individual LSm proteins were located by a novel approach employing C-terminal genetic tagging of the LSm proteins with yECitrine. These studies show the Prp24 protein at defined distances from each subunit of the LSm ring, which in turn suggests that the LSm ring is positioned in a consistent manner on the U6 RNA. Furthermore, in agreement with the EM observations, UV cross-linking revealed U6 RNA in contact with the LSm2 protein at the interface between Prp24p and the LSm ring. Further, LSmp-Prp24p interactions may be restricted to the closed form, which appears to represent the solution structure of the U6 snRNP particle.

GraFix: sample preparation for single-particle electron cryomicroscopy.

We developed a method, named GraFix, that considerably improves sample quality for structure determination by single-particle electron cryomicroscopy (cryo-EM). GraFix uses a glycerol gradient centrifugation step in which the complexes are centrifuged into an increasing concentration of a chemical fixation reagent to prevent aggregation and to stabilize individual macromolecules. The method can be used to prepare samples for negative-stain, cryo-negative-stain and, particularly, unstained cryo-EM.

Composition and three-dimensional EM structure of double affinity-purified, human prespliceosomal A complexes.

Little is known about the higher-order structure of prespliceosomal A complexes, in which pairing of the pre-mRNA's splice sites occurs. Here, human A complexes were isolated under physiological conditions by double-affinity selection. Purified complexes contained stoichiometric amounts of U1, U2 and pre-mRNA, and crosslinking studies indicated that these form concomitant base pairing interactions with one another. A complexes contained nearly all U1 and U2 proteins plus approximately 50 non-snRNP proteins. Unexpectedly, proteins of the hPrp19/CDC5 complex were also detected, even when A complexes were formed in the absence of U4/U6 snRNPs, demonstrating that they associate independent of the tri-snRNP. Double-affinity purification yielded structurally homogeneous A complexes as evidenced by electron microscopy, and allowed for the first time the generation of a three-dimensional structure. A complexes possess an asymmetric shape (approximately 260 x 200 x 195 angstroms) and contain a main body with various protruding elements, including a head-like domain and foot-like protrusions. Complexes isolated here are well suited for in vitro assembly studies to determine factor requirements for the A to B complex transition.

Localization of the coactivator Cdh1 and the cullin subunit Apc2 in a cryo-electron microscopy model of vertebrate APC/C.

The anaphase-promoting complex/cyclosome (APC/C) is a ubiquitin ligase with essential functions in mitosis, meiosis, and G1 phase of the cell cycle. APC/C recognizes substrates via coactivator proteins such as Cdh1, and bound substrates are ubiquitinated by E2 enzymes that interact with a hetero-dimer of the RING subunit Apc11 and the cullin Apc2. We have obtained three-dimensional (3D) models of human and Xenopus APC/C by angular reconstitution and random conical tilt (RCT) analyses of negatively stained cryo-electron microscopy (cryo-EM) preparations, have determined the masses of these particles by scanning transmission electron microscopy (STEM), and have mapped the locations of Cdh1 and Apc2. These proteins are located on the same side of the asymmetric APC/C, implying that this is where substrates are ubiquitinated. We have further identified a large flexible domain in APC/C that adopts a different orientation upon Cdh1 binding. Cdh1 may thus activate APC/C both by recruiting substrates and by inducing conformational changes.