All auxiliary ␣2␦ subunits of voltage-gated Ca 2؉ (CaV) channels contain an extracellular Von Willebrand factor-A (VWA) domain that, in ␣2␦-1 and -2, has a perfect metal-ion-dependent adhesion site (MIDAS). Modeling of the ␣2␦-2 VWA domain shows it to be highly likely to bind a divalent cation. Mutating the three key MIDAS residues responsible for divalent cation binding resulted in a MIDAS mutant ␣2␦-2 subunit that was still processed and trafficked normally when it was expressed alone. However, unlike WT ␣2␦-2, the MIDAS mutant ␣2␦-2 subunit did not enhance and, in some cases, further diminished Ca V1.2, -2.1, and -2.2 currents coexpressed with ␤1b by using either Ba 2؉ or Na ؉ as a permeant ion. Furthermore, expression of the MIDAS mutant ␣2␦-2 reduced surface expression and strongly increased the perinuclear retention of Ca V␣1 subunits at the earliest time at which expression was observed in both Cos-7 and NG108 -15 cells. Despite the presence of endogenous ␣2␦ subunits, heterologous expression of ␣2␦-2 in differentiated NG108 -15 cells further enhanced the endogenous high-threshold Ca 2؉ currents, whereas this enhancement was prevented by the MIDAS mutations. Our results indicate that ␣2␦ subunits normally interact with the CaV␣1 subunit early in their maturation, before the appearance of functional plasma membrane channels, and an intact MIDAS motif in the ␣2␦ subunit is required to promote trafficking of the ␣1 subunit to the plasma membrane by an integrin-like switch. This finding provides evidence for a primary role of a VWA domain in intracellular trafficking of a multimeric complex, in contrast to the more usual roles in binding extracellular ligands in other exofacial VWA domains.integrin ͉ neuron ͉ motif ͉ expression V oltage-gated Ca 2ϩ (Ca V ) channels are composed of a poreforming ␣1 subunit that determines the main biophysical properties of the channel. For the Ca V 1 and -2 subfamilies, this subunit is associated with an intracellular ␤ subunit (for review, see refs. 1 and 2) and a membrane-anchored, predominantly extracellular ␣ 2 ␦ subunit (for review, see ref.3). Mammalian genes encoding 10 ␣1, 4 ␤, and 4 ␣ 2 ␦ subunits have been identified (for reviews, see refs. 2 and 4). The topology of the ␣ 2 ␦ protein has been determined in detail only for ␣ 2 ␦-1 but is thought to generalize to all 4 ␣ 2 ␦ subunits (for review, see ref.3). All ␣ 2 ␦ subunits have predicted N-terminal signal sequences, indicating that the N terminus is extracellular. In early studies of ␣ 2 ␦-1 purified from skeletal and cardiac muscle, it was determined that the ␣ 2 subunit is disulfide-bonded to a transmembrane ␦ subunit, and both subunits are the products of a single gene, encoding the ␣ 2 ␦ protein, that is posttranslationally cleaved into ␣ 2 and ␦ (5).Subsequent to the identification of ␣ 2 ␦ subunits as stoichiometric components of skeletal muscle Ca 2ϩ channels, ␣ 2 ␦ subunits have also been shown to be associated with native cardiac (L-type) (6) and neuronal N-and P͞Q-type channels (7,8). In coexpression stud...

Aurora A phosphorylation-induced interaction of TACC3 and clathrin coordinates adjacent domains in each protein to create a microtubule-binding interface, whereas a distinct site in TACC3 recruits ch-TOG to mitotic spindles.

No abstract

SummaryMitosis is controlled by multiple protein kinases, many of which are abnormally expressed in human cancers. Nek2, Nek6, Nek7, and Nek9 are NIMA-related kinases essential for proper mitotic progression. We determined the atomic structure of Nek7 and discovered an autoinhibited conformation that suggests a regulatory mechanism not previously described in kinases. Additionally, Nek2 adopts the same conformation when bound to a drug-like molecule. In both structures, a tyrosine side chain points into the active site, interacts with the activation loop, and blocks the αC helix. Tyrosine mutants of Nek7 and the related kinase Nek6 are constitutively active. The activity of Nek6 and Nek7, but not the tyrosine mutant, is increased by interaction with the Nek9 noncatalytic C-terminal domain, suggesting a mechanism in which the tyrosine is released from its autoinhibitory position. The autoinhibitory conformation is common to three Neks and provides a potential target for selective kinase inhibitors.

Proteins of the echinoderm microtubule-associated protein (EMAP)-like (EML) family contribute to formation of the mitotic spindle and interphase microtubule network. They contain a unique hydrophobic EML protein (HELP) motif and a variable number of WD40 repeats. Recurrent gene rearrangements in nonsmall cell lung cancer fuse EML4 to anaplastic lymphoma kinase (ALK), causing expression of several fusion oncoprotein variants. We have determined a 2.6-Å crystal structure of the representative ∼70-kDa core of EML1, revealing an intimately associated pair of β-propellers, which we term a TAPE (tandem atypical propeller in EMLs) domain. One propeller is highly atypical, having a discontinuous subdomain unrelated to a WD40 motif in place of one of its blades. This unexpected feature shows how a propeller structure can be assembled from subdomains with distinct folds. The HELP motif is not an independent domain but forms part of the hydrophobic core that joins the two β-propellers. The TAPE domain binds α/β-tubulin via its conserved, concave surface, including part of the atypical blade. Mapping the characteristic breakpoints of each EML4-ALK variant onto our structure indicates that the EML4 TAPE domain is truncated in many variants in a manner likely to make the fusion protein structurally unstable. We found that the heat shock protein 90 (Hsp90) inhibitor ganetespib induced degradation of these variants whereas others lacking a partial TAPE domain were resistant in both overexpression models and patient-derived cell lines. The Hsp90-sensitive EML4-ALK variants are exceptions to the rule that oncogenic fusion proteins involve breakpoints in disordered regions of both partners. structural biology | stratified medicine

The Ca V ␤ subunits of voltage-gated calcium channels regulate these channels in several ways. Here we investigate the role of these auxiliary subunits in the expression of functional N-type channels at the plasma membrane and in the modulation by G-protein-coupled receptors of this neuronal channel. To do so, we mutated tryptophan 391 to an alanine within the ␣-interacting domain (AID) in the I-II linker of Ca V 2.2. We showed that the mutation W391 virtually abolishes the binding of Ca V ␤1b and Ca V ␤2a to the Ca V 2.2 I-II linker and strongly reduced current density and cell surface expression of both Ca V 2.2/␣2␦-2/␤1b and/␤2a channels. When associated with Ca V ␤1b, the W391A mutation also prevented the Ca V ␤1b-mediated hyperpolarization of Ca V 2.2 channel activation and steady-state inactivation. However, the mutated Ca V 2.2W391A/␤1b channels were still inhibited to a similar extent by activation of the D 2 dopamine receptor with the agonist quinpirole. Nevertheless, key hallmarks of G-protein modulation of N-type currents, such as slowed activation kinetics and prepulse facilitation, were not observed for the mutated channel. In contrast, Ca V ␤2a was still able to completely modulate the biophysical properties of Ca V 2.2W391A channel and allow voltage-dependent G-protein modulation of Ca V 2.2W391A. Additional data suggest that the concentration of Ca V ␤2a in the proximity of the channel is enhanced independently of its binding to the AID by its palmitoylation. This is essentially sufficient for all of the functional effects of Ca V ␤2a, which may occur via a second lower-affinity binding site, except trafficking the channel to the plasma membrane, which requires interaction with the AID region.

Myc family proteins promote cancer by inducing widespread changes in gene expression. Their rapid turnover by the ubiquitin–proteasome pathway is regulated through phosphorylation of Myc Box I and ubiquitination by the E3 ubiquitin ligase SCFFbxW7. However, N-Myc protein (the product of theMYCNoncogene) is stabilized in neuroblastoma by the protein kinase Aurora-A in a manner that is sensitive to certain Aurora-A–selective inhibitors. Here we identify a direct interaction between the catalytic domain of Aurora-A and a site flanking Myc Box I that also binds SCFFbxW7. We determined the crystal structure of the complex between Aurora-A and this region of N-Myc to 1.72-Å resolution. The structure indicates that the conformation of Aurora-A induced by compounds such as alisertib and CD532 is not compatible with the binding of N-Myc, explaining the activity of these compounds in neuroblastoma cells and providing a rational basis for the design of cancer therapeutics optimized for destabilization of the complex. We also propose a model for the stabilization mechanism in which binding to Aurora-A alters how N-Myc interacts with SCFFbxW7to disfavor the generation of Lys48-linked polyubiquitin chains.

The structural co-ordinates reported for EML2 11-60 and EML4 6-64 I38M appear in the PDB under codes 4CGB and 4CGC respectively. Page heading: EML trimerization domain drives MT-bindingKeywords: EML1, EML2, EML4-ALK, crystal structure, coiled-coil, microtubule 2 SUMMARY STATEMENTWe present crystal structures of a trimeric coiled-coil domain found in EML proteins. This trimerization domain mediates self-association and interactions between a subset of EML proteins. Microtubule-association of EML proteins requires the trimerization domain and an adjacent basic region. ABSTRACTProteins of the echinoderm microtubule associated protein-like (EML) family contribute to formation of the mitotic spindle and interphase microtubule (MT) network. EML1-4 consist of WD40 repeats and an N-terminal region containing a putative coiled-coil. Recurrent gene rearrangements in nonsmall cell lung cancer (NSCLC) fuse EML4 to anaplastic lymphoma kinase (ALK) causing expression of several oncogenic fusion variants. The fusions have constitutive ALK activity due to self-association through the EML4 coiled-coil. We have determined crystal structures of the coiledcoils from EML2 and EML4, which describe the structural basis of both EML self-association and oncogenic EML4-ALK activation. The structures reveal a trimeric oligomerization state directed by a conserved pattern of hydrophobic residues and salt bridges. We show that the trimerization domain (TD) of EML1 is necessary and sufficient for self-association. The TD is also essential for MT binding, however this property requires an adjacent basic region. These observations prompted us to investigate MT association of EML4-ALK and EML1-ABL1 fusions in which variable portions of the EML component are present. Uniquely, EML4-ALK variant 3, which includes the TD and basic region of EML4 but none of the WD40 repeats, was localized to MTs, both when expressed recombinantly and in a patient-derived NSCLC cell line (H2228). This raises the question of whether the mislocalization of ALK activity to MTs might influence downstream signalling and malignant properties of cells. Furthermore, the structure of EML4 TD may enable the development of protein-protein interaction inhibitors targeting the trimerization interface, providing a possible avenue towards therapeutic intervention in EML4-ALK NSCLC.