Accumulation of Mad2–Cdc20 complex during spindle checkpoint activation requires binding of open and closed conformers of Mad2 in Saccharomyces cerevisiae

The spindle assembly checkpoint (SAC) coordinates mitotic progression with sister chromatid alignment. In mitosis, the checkpoint machinery accumulates at kinetochores, which are scaffolds devoted to microtubule capture. The checkpoint protein Mad2 (mitotic arrest deficient 2) adopts two conformations: open (O-Mad2) and closed (C-Mad2). C-Mad2 forms when Mad2 binds its checkpoint target Cdc20 or its kinetochore receptor Mad1. When unbound to these ligands, Mad2 folds as O-Mad2. In HeLa cells, an essential interaction between C- and O-Mad2 conformers allows Mad1-bound C-Mad2 to recruit cytosolic O-Mad2 to kinetochores. In this study, we show that the interaction of the O and C conformers of Mad2 is conserved in Saccharomyces cerevisiae. MAD2 mutant alleles impaired in this interaction fail to restore the SAC in a mad2 deletion strain. The corresponding mutant proteins bind Mad1 normally, but their ability to bind Cdc20 is dramatically impaired in vivo. Our biochemical and genetic evidence shows that the interaction of O- and C-Mad2 is essential for the SAC and is conserved in evolution

Molecular Basis for the Dual Function of Eps8 on Actin Dynamics: Bundling and Capping

The unusual dual functions of the actin-binding protein EPS8 as an actin capping and actin bundling factor are mapped to distinct structural features of the protein and to distinct physiological activities in vivo

Structure of a ubiquitin-loaded HECT ligase reveals the molecular basis for catalytic priming

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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)

Etudes structurales de Arf6 et Rab11a (deux petites protéines G impliquées dans la régulation du trafic intracellulaire)

Le transport de protéines et membranes entre les différents compartiments de la cellule est régulé par les petites protéines G des familles Arf et Rab. Comme les autres protéines G, elles basculent entre une forme inactive liée au GDP et une active liée au GTP, et fonctionnent ainsi comme des interrupteurs moléculaires. Les diverses protéines Arf et Rab sont spécifiquement associées à des voies cellulaires distinctes et se localisent sur des compartiments cellulaires spécifiques. Nous avons étudié les bases de ces spécificités d'action grâce à la résolution des cycles structuraux GDP/GTP de deux de ces protéines, Arf6 et Rab11a. Les fonctions cellulaires de Arf6 sont différentes de celles de Arf1, malgré le fait que ces deux protéines partagent 67% d'identité de séquence. Nous avons résolu la structure cristalline de la forme active de Arf6. De façon surprenante, alors que les formes inactives de Arf1 et Arf6 peuvent être distinguées en structure, leurs formes actives apparaissent remarquablement similaires. En particulier, leurs régions switch sont non seulement presque identiques en séquence, mais présentent aussi la même structure. Nous proposons que la spécificité d'action des protéines Arf soit basée sur des interactions (i) de leurs formes liées au GDP avec leurs partenaires spécifiques et/ou (ii) qui impliquent outre aux régions switch classiques (qui portent l'information sur le nucléotide lié) des régions spécifiques aux différentes protéines Arf. Les structures de Rab11a dans sa forme active et inactive montrent des particularités distinctives de cette protéine G. Rab11aGDP cristallise comme un dimère. Nous suggérons que ce dimère puisse exister in vivo, et représenter ainsi un pool associé à la membrane sous forme GDP. D'autre part, la structure de Rab11aGTP permet l'identification d'une surface formée de résidus hautement variables entre protéines Rab. Nous proposons que cette surface puisse être une région de reconnaissance spécifique des protéines Rab.Proteins and membranes are transported between the different compartments of the cell through processes that are regulated by small GTP-binding proteins of the Arf and Rab families. Like other small G proteins, they function as molecular switches, cycling between an inactive GDP-bound form and an active GTP-bound form, which differ in structure in their 'switch regions'. The diverse Arf and Rab proteins are specifically associated with distinct cellular pathways and localize to precise cellular compartments. We have investigated the bases of these specificities of action by elucidating the GDP/GTP structural cycle of two of these proteins, Arf6 and Rab11a. We have solved the crystal structure of the active form of full length human Arf6, which displays cellular functions unrelated to that of well-characterized Arfl, despite sharing with it ~670% sequence identity. We round that, while Arfl and Arf6 GDP-bound forms are structurally recognizable, their active forms are remarkably similar in structure. Moreover, their switch regions are not only almost identical in sequence, but also fold in the same structure. Therefore, we propose that specificity of action of Arf proteins is based on (i) interactions of their GDP-bound forms with specific partners and/or (ii) interactions that involve both the classical switch regions (that provide information on the bound nucleotide) and Arf-specific regions. Rab11a is a member of a previously structurally uncharacterized Rab protein subfamily. The structures of human Rab11a in its inactive and active forms reveal features peculiar to this Rab subfamily. Rab11a-GDP crystallizes as a dimer. We suggest that this dimer could exist in vivo, and represent a membrane-associated GDP-bound pool. On the other hand Rab11a-GTP structure allows the identification of a surface shaped by residues highly variable between Rab proteins. We propose that this area could be a putative region for Rab protein specific recognition.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Arf, Arl, Arp and Sar proteins: a family of GTP-binding proteins with a structural device for 'front–back' communication

Arf proteins are important regulators of cellular traffic and the founding members of an expanding family of homologous proteins and genomic sequences. They depart from other small GTP-binding proteins by a unique structural device, which we call the 'interswitch toggle', that implements front–back communication from the N-terminus to the nucleotide binding site. Here we define the sequence and structural determinants that propagate information across the protein and identify them in all of the Arf family proteins other than Arl6 and Arl4/Arl7. The positions of these determinants lead us to propose that Arf family members with the interswitch toggle device are activated by a bipartite mechanism acting on opposite sides of the protein. The presence of this communication device might provide a more useful basis for unifying Arf homologs as a family than do the cellular functions of these proteins, which are mostly unrelated. We review available genomic sequences and functional data from this perspective, and identify a novel subfamily that we call Arl8

Purification and characterization of a DNA-binding recombinant PREP1:PBX1 complex.

Human PREP1 and PBX1 are homeodomain transcriptional factors, whose biochemical and structural characterization has not yet been fully described. Expression of full-length recombinant PREP1 (47.6 kDa) and PBX1 (46.6 kDa) in E. coli is difficult because of poor yield, high instability and insufficient purity, in particular for structural studies. We cloned the cDNA of both proteins into a dicistronic vector containing an N-terminal glutathione S-transferase (GST) tag and co-expressed and co-purified a stable PBX1:PREP1 complex. For structural studies, we produced two C-terminally truncated complexes that retain their ability to bind DNA and are more stable than the full-length proteins through various purification steps. Here we report the production of large amounts of soluble and pure recombinant human PBX1:PREP1 complex in an active form capable of binding DNA

Mechanism of Domain Closure of Sec7 Domains and Role in BFA Sensitivity

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