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Acta Crystallographica Section D: Biological Crystallography (1)
Acta Crystallographica Section F: Structural Biology and Crystallization Communications (1)
Journal of Molecular Biology (1)
Barford, David (2)
Chang, Leifu (2)
Kulkarni, Kiran (2)
Yang, Jing (2)
Zhang, Ziguo (2)
Conin, Nora (1)
Kulkarni, Kiran A. (1)
Lavanya Latha, V. (1)
Nagender Rao, R. (1)
Siva Kumar, N. (1)
Suguna, K. (1)
da Fonseca, Paula C. A. (1)
Year of Publication
The Four Canonical TPR Subunits of Human APC/C Form Related Homo-Dimeric Structures and Stack in Parallel to Form a TPR Suprahelix☆
Journal of Molecular Biology
The anaphase-promoting complex or cyclosome (APC/C) is a large E3 RING-cullin ubiquitin ligase composed of between 14 and 15 individual proteins. A striking feature of the APC/C is that only four proteins are involved in directly recognizing target proteins and catalyzing the assembly of a polyubiquitin chain. All other subunits, which account for > 80% of the mass of the APC/C, provide scaffolding functions. A major proportion of these scaffolding subunits are structurally related. In metazoans, there are four canonical tetratricopeptide repeat (TPR) proteins that form homo-dimers (Apc3/Cdc27, Apc6/Cdc16, Apc7 and Apc8/Cdc23). Here, we describe the crystal structure of the N-terminal homo-dimerization domain of Schizosaccharomyces pombe Cdc23 (Cdc23Nterm). Cdc23Nterm is composed of seven contiguous TPR motifs that self-associate through a related mechanism to those of Cdc16 and Cdc27. Using the Cdc23Nterm structure, we generated a model of full-length Cdc23. The resultant “V”-shaped molecule docks into the Cdc23-assigned density of the human APC/C structure determined using negative stain electron microscopy (EM). Based on sequence conservation, we propose that Apc7 forms a homo-dimeric structure equivalent to those of Cdc16, Cdc23 and Cdc27. The model is consistent with the Apc7-assigned density of the human APC/C EM structure. The four canonical homo-dimeric TPR proteins of human APC/C stack in parallel on one side of the complex. Remarkably, the uniform relative packing of neighboring TPR proteins generates a novel left-handed suprahelical TPR assembly. This finding has implications for understanding the assembly of other TPR-containing multimeric complexes.
•The paper addresses the structure of the TPR subunits (Cdc23 and Apc7) of the APC/C.•We determine the crystal structure of the N-terminus of Cdc23 showing that Cdc23 homo-dimerizes similar to other canonical TPR subunits of the APC/C Cdc16 and Cdc27.•We generate full-length models of Cdc23 and Apc7 and suggest that the mode of dimerization of Apc7 published previously is incorrect. We propose that Apc7 will homo-dimerize similar to Cdc23, Cdc16 and Cdc27.•We dock Cdc23 and Apc7, as well as Cdc16 and Cdc27, into the negative stain EM structure of the APC/C. This reveals a novel stacking assembly of the four homo-dimeric TPR proteins that forms a left-handed suprahelix.•We show the Cdc23–Cdc16 assemble into a left-handed suprahelix, with implications for TPR assembly and TPR-containing multimeric assemblies.
APC/C, anaphase-promoting complex or cyclosome; TPR, tetratricopeptide repeat; EM, electron microscopy; SAD, single-wavelength anomalous dispersion; PDB, Protein Data Bank; FOM, figure of merit; anaphase-promoting complex; tetratricopeptide repeat (TPR); cell cycle; crystallography; single-particle electron microscopy
Building a pseudo-atomic model of the anaphase-promoting complex
da Fonseca, Paula C. A.
Acta Crystallographica Section D: Biological Crystallography
This article describes an example of molecular replacement in which atomic models are used to interpret electron-density maps determined using single-particle electron-microscopy data.
The anaphase-promoting complex (APC/C) is a large E3 ubiquitin ligase that regulates progression through specific stages of the cell cycle by coordinating the ubiquitin-dependent degradation of cell-cycle regulatory proteins. Depending on the species, the active form of the APC/C consists of 14–15 different proteins that assemble into a 20-subunit complex with a mass of approximately 1.3 MDa. A hybrid approach of single-particle electron microscopy and protein crystallography of individual APC/C subunits has been applied to generate pseudo-atomic models of various functional states of the complex. Three approaches for assigning regions of the EM-derived APC/C density map to specific APC/C subunits are described. This information was used to dock atomic models of APC/C subunits, determined either by protein crystallography or homology modelling, to specific regions of the APC/C EM map, allowing the generation of a pseudo-atomic model corresponding to 80% of the entire complex.
anaphase-promoting complex; single-particle electron microscopy; pseudo-atomic model
Crystallization and preliminary X-ray crystallographic analysis of a galactose-specific lectin from Dolichos lablab
Lavanya Latha, V.
Nagender Rao, R.
Siva Kumar, N.
Acta Crystallographica Section F: Structural Biology and Crystallization Communications
The galactose-specific lectin from the seeds of a leguminous plant, D. lablab, has been crystallized. Molecular-replacement solution using 3.0 Å X-ray diffraction data showed the lectin to be a tetramer.
The galactose-specific lectin from the seeds of Dolichos lablab has been crystallized using the hanging-drop vapour-diffusion technique. The crystals belong to space group P1, with unit-cell parameters a = 73.99, b = 84.13, c = 93.15 Å, α = 89.92, β = 76.01, γ = 76.99°. X-ray diffraction data to a resolution of 3.0 Å have been collected under cryoconditions (100 K) using a MAR imaging-plate detector system mounted on a rotating-anode X-ray generator. Molecular-replacement calculations carried out using the available structures of legume lectins as search models revealed that the galactose-specific lectin from D. lablab forms a tetramer similar to soybean agglutinin; two such tetramers are present in the asymmetric unit.
Dolichos lablab; galactose-specific lectins; legume lectins
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