Prakash Dube

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.

Arrangement of RNA and proteins in the spliceosomal U1 small nuclear ribonucleoprotein particle

In eukaryotic cells, freshly synthesized messenger RNA (pre-mRNA) contains stretches of non-coding RNA that must be excised before the RNA can be translated into protein. Their removal is catalysed by the spliceosome, a large complex formed when a number of small nuclear ribonucleoprotein particles (snRNPs) bind sequentially to the pre-mRNA. The first snRNP to bind is called U1; other snRNPs (U2, U4/U6 and U5) follow. Here we describe the three-dimensional structure of human U1 snRNP, determined by single-particle electron cryomicroscopy at 10 Å resolution. The reconstruction reveals a doughnut-shaped central element that accommodates the seven Sm proteins common to all snRNPs, supporting a proposed model of circular Sm protein arrangement. By taking earlier biochemical results into account, we were able to assign the remaining density of the map to the other known components of U1 snRNP, deriving a structural model that describes the three-dimensional arrangement of proteins and RNA in U1 snRNP.

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/CCdc20). Before chromosome biorientation, anaphase is delayed by a mitotic checkpoint complex (MCC) that inhibits APC/CCdc20. 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.

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‐mRNAs 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 ∼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 (∼260 × 200 × 195 Å) 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.

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

The NSL1 subunit structures interactions between the MIS12, NDC80, and KNL1 kinetochore complexes (see also a related paper by Maskell et al. in this issue).

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.

Three-Dimensional Structure of the Anaphase-Promoting Complex

The anaphase-promoting complex (APC) is a cell cycle-regulated ubiquitin-protein ligase, composed of at least 11 subunits, that controls progression through mitosis and G1. Using cryo-electron microscopy and angular reconstitution, we have obtained a three-dimensional model of the human APC at a resolution of 24 A. The APC has a complex asymmetric structure 140 A x 140 A x 135 A in size, in which an outer protein wall surrounds a large inner cavity. We discuss the possibility that this cavity represents a reaction chamber in which ubiquitination reactions take place, analogous to the inner cavities formed by other protein machines such as the 26S proteasome and chaperone complexes. This cage hypothesis could help to explain the great subunit complexity of the APC.

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 APCs 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.

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.