Publications

In mitosis, the spindle assembly checkpoint (SAC) ensures genome stability by delaying chromosome segregation until all sister chromatids have achieved bipolar attachment to the mitotic spindle. The SAC is imposed by the mitotic checkpoint complex (MCC), whose assembly is catalysed by unattached chromosomes and which binds and inhibits the anaphase-promoting complex/cyclosome (APC/C), the E3 ubiquitin ligase that initiates chromosome segregation. Here, using the crystal structure of Schizosaccharomyces pombe MCC (a complex of mitotic spindle assembly checkpoint proteins Mad2, Mad3 and APC/C co-activator protein Cdc20), we reveal the molecular basis of MCC-mediated APC/C inhibition and the regulation of MCC assembly. The MCC inhibits the APC/C by obstructing degron recognition sites on Cdc20 (the substrate recruitment subunit of the APC/C) and displacing Cdc20 to disrupt formation of a bipartite D-box receptor with the APC/C subunit Apc10. Mad2, in the closed conformation (C-Mad2), stabilizes the complex by optimally positioning the Mad3 KEN-box degron to bind Cdc20. Mad3 and p31(comet) (also known as MAD2L1-binding protein) compete for the same C-Mad2 interface, which explains how p31(comet) disrupts MCC assembly to antagonize the SAC. This study shows how APC/C inhibition is coupled to degron recognition by co-activators.

Protein tyrosine phosphatase 1B (PTP1B) plays important roles in downregulation of insulin and leptin signaling and is an established therapeutic target for diabetes and obesity. PTP1B is regulated by reactive oxygen species (ROS) produced in response to various stimuli, including insulin. The reversibly oxidized form of the enzyme (PTP1B-OX) is inactive and undergoes profound conformational changes at the active site. We generated conformation-sensor antibodies, in the form of single-chain variable fragments (scFvs), that stabilize PTP1B-OX and thereby inhibit its phosphatase function. Expression of conformation-sensor scFvs as intracellular antibodies (intrabodies) enhanced insulin-induced tyrosyl phosphorylation of the β subunit of the insulin receptor and its substrate IRS-1 and increased insulin-induced phosphorylation of PKB/AKT. Our data suggest that stabilization of the oxidized, inactive form of PTP1B with appropriate therapeutic molecules may offer a paradigm for phosphatase drug development.

The posttranslational modification of C-terminal CAAX motifs in proteins such as Ras, most Rho GTPases, and G protein γ subunits, plays an essential role in determining their subcellular localization and correct biological function. An integral membrane methyltransferase, isoprenylcysteine carboxyl methyltransferase (ICMT), catalyzes the final step of CAAX processing after prenylation of the cysteine residue and endoproteolysis of the -AAX motif. We have determined the crystal structure of a prokaryotic ICMT ortholog, revealing a markedly different architecture from conventional methyltransferases that utilize S-adenosyl-L-methionine (SAM) as a cofactor. ICMT comprises a core of five transmembrane α helices and a cofactor-binding pocket enclosed within a highly conserved C-terminal catalytic subdomain. A tunnel linking the reactive methyl group of SAM to the inner membrane provides access for the prenyl lipid substrate. This study explains how an integral membrane methyltransferase achieves recognition of both a hydrophilic cofactor and a lipophilic prenyl group attached to a polar protein substrate.

In metazoans, the Ras-Raf-MEK (mitogen-activated protein-kinase kinase)-ERK (extracellular signal-regulated kinase) signalling pathway relays extracellular stimuli to elicit changes in cellular function and gene expression. Aberrant activation of this pathway through oncogenic mutations is responsible for a large proportion of human cancer. Kinase suppressor of Ras (KSR) functions as an essential scaffolding protein to coordinate the assembly of Raf-MEK-ERK complexes. Here we integrate structural and biochemical studies to understand how KSR promotes stimulatory Raf phosphorylation of MEK (refs 6, 7). We show, from the crystal structure of the kinase domain of human KSR2 (KSR2(KD)) in complex with rabbit MEK1, that interactions between KSR2(KD) and MEK1 are mediated by their respective activation segments and C-lobe αG helices. Analogous to BRAF (refs 8, 9), KSR2 self-associates through a side-to-side interface involving Arg 718, a residue identified in a genetic screen as a suppressor of Ras signalling. ATP is bound to the KSR2(KD) catalytic site, and we demonstrate KSR2 kinase activity towards MEK1 by in vitro assays and chemical genetics. In the KSR2(KD)-MEK1 complex, the activation segments of both kinases are mutually constrained, and KSR2 adopts an inactive conformation. BRAF allosterically stimulates the kinase activity of KSR2, which is dependent on formation of a side-to-side KSR2-BRAF heterodimer. Furthermore, KSR2-BRAF heterodimerization results in an increase of BRAF-induced MEK phosphorylation via the KSR2-mediated relay of a signal from BRAF to release the activation segment of MEK for phosphorylation. We propose that KSR interacts with a regulatory Raf molecule in cis to induce a conformational switch of MEK, facilitating MEK's phosphorylation by a separate catalytic Raf molecule in trans.

The ubiquitylation of cell-cycle regulatory proteins by the large multimeric anaphase-promoting complex (APC/C) controls sister chromatid segregation and the exit from mitosis. Selection of APC/C targets is achieved through recognition of destruction motifs, predominantly the destruction (D)-box and KEN (Lys-Glu-Asn)-box. Although this process is known to involve a co-activator protein (either Cdc20 or Cdh1) together with core APC/C subunits, the structural basis for substrate recognition and ubiquitylation is not understood. Here we investigate budding yeast APC/C using single-particle electron microscopy and determine a cryo-electron microscopy map of APC/C in complex with the Cdh1 co-activator protein (APC/C(Cdh1)) bound to a D-box peptide at ∼10 Å resolution. We find that a combined catalytic and substrate-recognition module is located within the central cavity of the APC/C assembled from Cdh1, Apc10--a core APC/C subunit previously implicated in substrate recognition--and the cullin domain of Apc2. Cdh1 and Apc10, identified from difference maps, create a co-receptor for the D-box following repositioning of Cdh1 towards Apc10. Using NMR spectroscopy we demonstrate specific D-box-Apc10 interactions, consistent with a role for Apc10 in directly contributing towards D-box recognition by the APC/C(Cdh1) complex. Our results rationalize the contribution of both co-activator and core APC/C subunits to D-box recognition and provide a structural framework for understanding mechanisms of substrate recognition and catalysis by the APC/C.

The anaphase-promoting complex or cyclosome (APC/C) is an unusually large E3 ubiquitin ligase responsible for regulating defined cell cycle transitions. Information on how its 13 constituent proteins are assembled, and how they interact with co-activators, substrates and regulatory proteins is limited. Here, we describe a recombinant expression system that allows the reconstitution of holo APC/C and its sub-complexes that, when combined with electron microscopy, mass spectrometry and docking of crystallographic and homology-derived coordinates, provides a precise definition of the organization and structure of all essential APC/C subunits, resulting in a pseudo-atomic model for 70% of the APC/C. A lattice-like appearance of the APC/C is generated by multiple repeat motifs of most APC/C subunits. Three conserved tetratricopeptide repeat (TPR) subunits (Cdc16, Cdc23 and Cdc27) share related superhelical homo-dimeric architectures that assemble to generate a quasi-symmetrical structure. Our structure explains how this TPR sub-complex, together with additional scaffolding subunits (Apc1, Apc4 and Apc5), coordinate the juxtaposition of the catalytic and substrate recognition module (Apc2, Apc11 and Apc10 (also known as Doc1)), and TPR-phosphorylation sites, relative to co-activator, regulatory proteins and substrates.

The anaphase-promoting complex/cyclosome (APC/C), an E3 ubiquitin ligase responsible for controlling cell cycle transitions, is a multisubunit complex assembled from 13 different proteins. Numerous APC/C subunits incorporate multiple copies of the tetratricopeptide repeat (TPR). Here, we report the crystal structure of Schizosaccharomyces pombe Cut9 (Cdc16/Apc6) in complex with Hcn1 (Cdc26), showing that Cdc16/Cut9 is a contiguous TPR superhelix of 14 TPR units. A C-terminal block of TPR motifs interacts with Hcn1, whereas an N-terminal TPR block mediates Cdc16/Cut9 self-association through a homotypic interface. This dimer interface is structurally related to the N-terminal dimerization domain of Cdc27, demonstrating that both Cdc16/Cut9 and Cdc27 form homo-dimers through a conserved mechanism. The acetylated N-terminal Met residue of Hcn1 is enclosed within a chamber created from the Cut9 TPR superhelix. Thus, in complex with Cdc16/Cut9, the N-acetyl-Met residue of Hcn1, a putative degron for the Doa10 E3 ubiquitin ligase, is inaccessible for Doa10 recognition, protecting Hcn1/Cdc26 from ubiquitin-dependent degradation. This finding may provide a structural explanation for a mechanism to control the stoichiometry of proteins participating in multisubunit complexes.

Target recognition in RNA silencing is governed by the "seed sequence" of a guide RNA strand associated with the PIWI/MID domain of an Argonaute protein in RISC. Using a reconstituted in vitro target recognition system, we show that a model PIWI/MID domain protein confers position-dependent tightening and loosening of guide-strand-target interactions. Over the seed sequence, the interaction affinity is enhanced up to approximately 300-fold. Enhancement is achieved through a reduced entropy penalty for the interaction. In contrast, interactions 3' of the seed are inhibited. We quantified mismatched target recognition inside and outside the seed, revealing amplified discrimination at the third position in the seed mediated by the PIWI/MID domain. Thus, association of the guide strand with the PIWI/MID domain generates an enhanced affinity anchor site over the seed that can promote fidelity in target recognition and stabilize and guide the assembly of the active silencing complex.

At least eight types of ubiquitin chain exist, and individual linkages affect distinct cellular processes. The only distinguishing feature of differently linked ubiquitin chains is their structure, as polymers of the same unit are chemically identical. Here, we have crystallized Lys 63-linked and linear ubiquitin dimers, revealing that both adopt equivalent open conformations, forming no contacts between ubiquitin molecules and thereby differing significantly from Lys 48-linked ubiquitin chains. We also examined the specificity of various deubiquitinases (DUBs) and ubiquitin-binding domains (UBDs). All analysed DUBs, except CYLD, cleave linear chains less efficiently compared with other chain types, or not at all. Likewise, UBDs can show chain specificity, and are able to select distinct linkages from a ubiquitin chain mixture. We found that the UBAN (ubiquitin binding in ABIN and NEMO) motif of NEMO (NF-kappaB essential modifier) binds to linear chains exclusively, whereas the NZF (Npl4 zinc finger) domain of TAB2 (TAK1 binding protein 2) is Lys 63 specific. Our results highlight remarkable specificity determinants within the ubiquitin system.

Activation of Rho guanosine triphosphatases (GTPases) to the guanine triphosphate (GTP)-bound state is a critical event in their regulation of the cytoskeleton and cell signaling. Members of the DOCK family of guanine nucleotide exchange factors (GEFs) are important activators of Rho GTPases, but the mechanism of activation by their catalytic DHR2 domain is unknown. Through structural analysis of DOCK9-Cdc42 complexes, we identify a nucleotide sensor within the alpha10 helix of the DHR2 domain that contributes to release of guanine diphosphate (GDP) and then to discharge of the activated GTP-bound Cdc42. Magnesium exclusion, a critical factor in promoting GDP release, is mediated by a conserved valine residue within this sensor, whereas binding of GTP-Mg2+ to the nucleotide-free complex results in magnesium-inducing displacement of the sensor to stimulate discharge of Cdc42-GTP. These studies identify an unusual mechanism of GDP release and define the complete GEF catalytic cycle from GDP dissociation followed by GTP binding and discharge of the activated GTPase.