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1.  Mechanism of farnesylated CAAX protein processing by the integral membrane protease Rce1 
Nature  2013;504(7479):10.1038/nature12754.
CAAX proteins play essential roles in multiple signalling pathways, controlling processes such as proliferation, differentiation and carcinogenesis 1. The ~120 mammalian CAAX proteins function at cellular membranes and include the Ras superfamily of small GTPases, nuclear lamins, the γ-subunit of heterotrimeric GTPases, and several protein kinases and phosphatases 2. Proper localization of CAAX proteins to cell membranes is orchestrated by a series of post-translational modifications of their C-terminal CAAX motifs 3 (where C is cysteine, A is an aliphatic amino acid and X is any amino acid). These reactions involve cysteine prenylation, -AAX tripeptide cleavage, and methylation of the carboxyl prenylated Cys residue. The major CAAX protease activity is mediated by the Ras and a-factor converting enzyme 1 (Rce1), an integral membrane protease of the endoplasmic reticulum 4,5. Information on the architecture and proteolytic mechanism of Rce1 has been lacking. Here, we report the crystal structure of a Methanococcus maripaludis homolog of Rce1, whose endopeptidase specificity for farnesylated peptides mimics that of eukaryotic Rce1. Its structure, comprising eight transmembrane α-helices, and catalytic site, are distinct from other intramembrane proteases (IMPs). Catalytic residues are located ~10 Å into the membrane and are exposed to the cytoplasm and membrane through a conical cavity that accommodates the prenylated CAAX substrate. The farnesyl lipid is proposed to bind to a site at the opening of two transmembrane α-helices, which then positions the scissile bond adjacent to a glutamate-activated nucleophilic water molecule. This study suggests that Rce1 is the founding member of a novel IMP family, the glutamate IMPs.
doi:10.1038/nature12754
PMCID: PMC3864837  PMID: 24291792
2.  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  2013;425(22):4236-4248.
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.
Graphical Abstract
Highlights
•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.
doi:10.1016/j.jmb.2013.04.004
PMCID: PMC3898896  PMID: 23583778
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
3.  Building a pseudo-atomic model of the anaphase-promoting complex 
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.
doi:10.1107/S0907444913018593
PMCID: PMC3817697  PMID: 24189235
anaphase-promoting complex; single-particle electron microscopy; pseudo-atomic model
4.  Crystallization and preliminary X-ray crystallographic analysis of a galactose-specific lectin from Dolichos lablab  
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.
doi:10.1107/S1744309106001448
PMCID: PMC2150945  PMID: 16511291
Dolichos lablab; galactose-specific lectins; legume lectins

Results 1-4 (4)