Christy Rani R. Grace

Structure of the N-terminal domain of a type B1 G protein-coupled receptor in complex with a peptide ligand

The corticotropin releasing factor (CRF) family of ligands and their receptors coordinate endocrine, behavioral, autonomic, and metabolic responses to stress and play additional roles within the cardiovascular, gastrointestinal, and other systems. The actions of CRF and the related urocortins are mediated by activation of two receptors, CRF-R1 and CRF-R2, belonging to the B1 family of G protein-coupled receptors. The short-consensus-repeat fold (SCR) within the first extracellular domain (ECD1) of the CRF receptor(s) comprises the major ligand binding site and serves to dock a peptide ligand via its C-terminal segment, thus positioning the N-terminal segment to interact with the receptors juxtamembrane domains to activate the receptor. Here we present the 3D NMR structure of ECD1 of CRF-R2β in complex with astressin, a peptide antagonist. In the structure of the complex the C-terminal segment of astressin forms an amphipathic helix, whose entire hydrophobic face interacts with the short-consensus-repeat motif, covering a large intermolecular interface. In addition, the complex is characterized by intermolecular hydrogen bonds and a salt bridge. These interactions are quantitatively weighted by an analysis of the effects on the full-length receptor affinities using an Ala scan of CRF. These structural studies identify the major determinants for CRF ligand specificity and selectivity and support a two-step model for receptor activation. Furthermore, because of a proposed conservation of the fold for both the ECD1s and ligands, this structure can serve as a model for ligand recognition for the entire B1 receptor family.

BID-induced structural changes in BAK promote apoptosis

The BCL-2–family protein BAK is responsible for mitochondrial outer-membrane permeabilization (MOMP), which leads to apoptosis. The BCL-2 homology 3 (BH3)-only protein BID activates BAK to perform this function. We report the NMR solution structure of the human BID BH3–BAK complex, which identified the activation site at the canonical BH3-binding groove of BAK. Mutating the BAK BH1 in the groove prevented activation and MOMP but not the binding of BID. BAK BH3 mutations allowed BID binding and activation but blunted function by blocking BAK oligomerization. BAK activation follows a hit-and-run mechanism whereby BID dissociates from the trigger site, which allows BAK oligomerization at an overlapping interface. In contrast, the BH3-only proteins NOXA and BAD are predicted to clash with the trigger site and are not activators of BAK. These findings provide insights into the early stages of BAK activation.

PUMA binding induces partial unfolding within BCL-xL to disrupt p53 binding and promote apoptosis

Following DNA damage, nuclear p53 induces the expression of PUMA, a BH3-only protein that binds and inhibits the anti-apoptotic BCL-2 repertoire, including BCL-xL. PUMA, unique amongst BH3-only proteins, disrupts the interaction between cytosolic p53 and BCL-xL, allowing p53 to promote apoptosis via direct activation of the BCL-2 effector molecules, BAX and BAK. Structural investigations using nuclear magnetic resonance spectroscopy and X-ray crystallography revealed that PUMA binding induced partial unfolding of two α-helices within BCL-xL. Wild-type PUMA or a PUMA mutant incapable of causing binding-induced unfolding of BCL-xL equivalently inhibited the anti-apoptotic BCL-2 repertoire to sensitize for death receptor (DR)-activated apoptosis, but only wild-type PUMA promoted p53-dependent, DNA damage-induced apoptosis. Our data suggest that PUMA-induced partial unfolding of BCL-xL disrupts interactions between cytosolic p53 and BCL-xL, releasing the bound p53 to initiate apoptosis. We propose that regulated unfolding of BCL-xL provides a mechanism to promote PUMA-dependent signaling within the apoptotic pathways.

A Dual E3 mechanism for Rub1 ligation to Cdc53

In ubiquitin-like protein (UBL) cascades, a thioester-linked E2∼UBL complex typically interacts with an E3 enzyme for UBL transfer to the target. Here we demonstrate a variant mechanism, whereby the E2 Ubc12 functions with two E3s, Hrt1 and Dcn1, for ligation of the UBL Rub1 to Cdc53s WHB subdomain. Hrt1 functions like a conventional RING E3, with its N terminus recruiting Cdc53 and C-terminal RING activating Ubc12∼Rub1. Dcn1s potentiating neddylation domain (Dcn1(P)) acts as an additional E3, reducing nonspecific Hrt1-mediated Ubc12∼Rub1 discharge and directing Ubc12s active site to Cdc53. Crystal structures of Dcn1(P)-Cdc53(WHB) and Ubc12 allow modeling of a catalytic complex, supported by mutational data. We propose that Dcn1s interactions with both Cdc53 and Ubc12 would restrict the otherwise flexible Hrt1 RING-bound Ubc12∼Rub1 to a catalytically competent orientation. Our data reveal mechanisms by which two E3s function synergistically to promote UBL transfer from one E2 to a target.

Structural polymorphism in the N-terminal oligomerization domain of NPM1

Significance Nucleophosmin (NPM1) is a multifunctional protein with critical roles in ribosome biogenesis, centrosome duplication, and tumor suppression. Despite the established importance of NPM1 as a tumor marker and potential drug target, little is currently known about the molecular mechanisms that govern its various functions. Our manuscript describes that the N-terminal domain of NPM1 (Npm-N) exhibits phosphorylation-dependent structural polymorphism along a broad conformational landscape between two extreme states: a stable, folded pentamer and a globally disordered monomer. We propose that phosphorylation-induced “regulated unfolding” of Npm-N provides a means to modulate NPM1 function and subcellular localization. Our findings will drive future structure-based studies on the roles of regulated unfolding in NPM1 biology and will provide a foundation for NPM1-targeted anticancer drug development. Nucleophosmin (NPM1) is a multifunctional phospho-protein with critical roles in ribosome biogenesis, tumor suppression, and nucleolar stress response. Here we show that the N-terminal oligomerization domain of NPM1 (Npm-N) exhibits structural polymorphism by populating conformational states ranging from a highly ordered, folded pentamer to a highly disordered monomer. The monomer–pentamer equilibrium is modulated by posttranslational modification and protein binding. Phosphorylation drives the equilibrium in favor of monomeric forms, and this effect can be reversed by Npm-N binding to its interaction partners. We have identified a short, arginine-rich linear motif in NPM1 binding partners that mediates Npm-N oligomerization. We propose that the diverse functional repertoire associated with NPM1 is controlled through a regulated unfolding mechanism signaled through posttranslational modifications and intermolecular interactions.

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.

Electron microscopy structure of human APC/C-CDH1-EMI1 reveals multimodal mechanism of E3 ligase shutdown.

The anaphase-promoting complex/cyclosome (APC/C) is a ~1.5-MDa multiprotein E3 ligase enzyme that regulates cell division by promoting timely ubiquitin-mediated proteolysis of key cell-cycle regulatory proteins. Inhibition of human APC/CCDH1 during interphase by early mitotic inhibitor 1 (EMI1) is essential for accurate coordination of DNA synthesis and mitosis. Here, we report a hybrid structural approach involving NMR, electron microscopy and enzymology, which reveal that EMI1s 143-residue C-terminal domain inhibits multiple APC/CCDH1 functions. The intrinsically disordered D-box, linker and tail elements, together with a structured zinc-binding domain, bind distinct regions of APC/CCDH1 to synergistically both block the substrate-binding site and inhibit ubiquitin-chain elongation. The functional importance of intrinsic structural disorder is explained by enabling a small inhibitory domain to bind multiple sites to shut down various functions of a molecular machine nearly 100 times its size.

Sequential Engagement of Distinct MLKL Phosphatidylinositol-Binding Sites Executes Necroptosis

Necroptosis is a cell death pathway regulated by the receptor interacting protein kinase 3 (RIPK3) and the mixed lineage kinase domain-like (MLKL) pseudokinase. How MLKL executes plasma membrane rupture upon phosphorylation by RIPK3 remains controversial. Here, we characterize the hierarchical transduction of structural changes in MLKL that culminate in necroptosis. The MLKL brace, proximal to the N-terminal helix bundle (NB), is involved in oligomerization to facilitate plasma membrane targeting through the low-affinity binding of NB to phosphorylated inositol polar head groups of phosphatidylinositol phosphate (PIP) phospholipids. At the membrane, the NB undergoes a rolling over mechanism to expose additional higher-affinity PIP-binding sites responsible for robust association to the membrane and displacement of the brace from the NB. PI(4,5)P2 is the preferred PIP-binding partner. We investigate the specific association of MLKL with PIPs and subsequent structural changes during necroptosis.

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.

Structural insights into the role of the Smoothened cysteine-rich domain in Hedgehog signalling

Smoothened (Smo) is a member of the Frizzled (FzD) class of G-protein-coupled-receptors (GPCRs), and functions as the key transducer in the Hedgehog (Hh) signalling pathway. Smo has an extracellular cysteine-rich domain (CRD), indispensable for its function and downstream Hh signalling. Despite its essential role, the functional contribution of the CRD to Smo signalling has not been clearly elucidated. However, given that the FzD CRD binds to the endogenous Wnt ligand, it has been proposed that the Smo CRD may bind its own endogenous ligand. Here we present the NMR solution structure of the Drosophila Smo CRD, and describe interactions between the glucocorticoid budesonide (Bud) and the Smo CRDs from both Drosophila and human. Our results highlight a function of the Smo CRD, demonstrating its role in binding to small molecule modulators.