Structure of a prehandover mammalian ribosomal SRP·SRP receptor targeting complex

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Abstract

Signal recognition particle (SRP) targets proteins to the endoplasmic reticulum (ER). SRP recognizes the ribosome synthesizing a signal sequence and delivers it to the SRP receptor (SR) on the ER membrane followed by the transfer of the signal sequence to the translocon. Here, we present the cryo-EM structure of the mammalian translating ribosome in complex with SRP and SR in a conformation preceding signal sequence handover. The structure visualizes all eukaryotic-specific SRP and SR proteins and reveals their roles in stabilizing this conformation by forming a large protein assembly at the distal site of SRP RNA. We provide biochemical evidence that the GTP hydrolysis of SRP•SR is delayed at this stage, possibly to provide a time window for signal sequence handover to the translocon.

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The Ras-RasGAP complex: structural basis for GTPase activation and its loss in oncogenic Ras mutants.

K Scheffzek, M R Ahmadian, W Kabsch … (1997)

The three-dimensional structure of the complex between human H-Ras bound to guanosine diphosphate and the guanosine triphosphatase (GTPase)-activating domain of the human GTPase-activating protein p120GAP (GAP-334) in the presence of aluminum fluoride was solved at a resolution of 2.5 angstroms. The structure shows the partly hydrophilic and partly hydrophobic nature of the communication between the two molecules, which explains the sensitivity of the interaction toward both salts and lipids. An arginine side chain (arginine-789) of GAP-334 is supplied into the active site of Ras to neutralize developing charges in the transition state. The switch II region of Ras is stabilized by GAP-334, thus allowing glutamine-61 of Ras, mutation of which activates the oncogenic potential, to participate in catalysis. The structural arrangement in the active site is consistent with a mostly associative mechanism of phosphoryl transfer and provides an explanation for the activation of Ras by glycine-12 and glutamine-61 mutations. Glycine-12 in the transition state mimic is within van der Waals distance of both arginine-789 of GAP-334 and glutamine-61 of Ras, and even its mutation to alanine would disturb the arrangements of residues in the transition state.

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Sampling the conformational space of the catalytic subunit of human γ-secretase

Xiao-chen Bai, Eeson Rajendra, Guanghui Yang … (2015)

Human γ-secretase is an intra-membrane protease that cleaves many different substrates. Aberrant cleavage of Notch is implicated in cancer, while abnormalities in cutting amyloid precursor protein lead to Alzheimer's disease. Our previous cryo-EM structure of γ-secretase revealed considerable disorder in its catalytic subunit presenilin. Here, we describe an image classification procedure that characterizes molecular plasticity at the secondary structure level, and apply this method to identify three distinct conformations in our previous sample. In one of these conformations, an additional transmembrane helix is visible that cannot be attributed to the known components of γ-secretase. In addition, we present a γ-secretase structure in complex with the dipeptidic inhibitor N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT). Our results reveal how conformational mobility in the second and sixth transmembrane helices of presenilin is greatly reduced upon binding of DAPT or the additional helix, and form the basis for a new model of how substrate enters the transmembrane domain. DOI: http://dx.doi.org/10.7554/eLife.11182.001

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Purification of a membrane-associated protein complex required for protein translocation across the endoplasmic reticulum.

P. Walter, G Blobel (1980)

The capacity of microsomal membranes to translocate nascent presecretory proteins across their lipid bilayer can be largely abolished by extracting them with high ionic strength buffers. It can be reconstituted by adding the salt extract back to the depleted membranes [Warren, G. & Doberstein, B. (1978) Nature (London) 273, 569-571]. Utilizing hydrophobic chromatography, we purified to homogeneity a protein component of the salt extract that reconstitutes the translocation activity of the extracted membranes. This component behaves as a homogeneous species upon gel filtration, ion-exchange chromatography, adsorption chromatography, and sucrose-gradient centrifugation. When examined by polyacrylamide gel electrophoresis in NaDodSO4, six polypeptides with apparent Mr of 72,000, 68,000, 54,000, 19,000, 14,000, and 9000 are observed in about equal and constant stoichiometry, suggesting that they are subunits of a complex. The sedimentation coefficient of 11S is in good agreement with the sum of the Mr of the subunits. The Mr 68,000 and 9000 subunits label intensely with N-[3H]ethylmaleimide. Thus, the reported sulfhydryl group requirement of the translocation activity in the unfractionated extract [Jackson, R. C., Walter, P. & Blobel, G. (1980) Nature (London), 286, 174-176] may be localized to either or both the Mr 68,000 and 9000 subunits of the purified complex.