GroEL provides a folding pathway with lower apparent activation energy compared to spontaneous refolding of human carbonic anhydrase II

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/117184/1/feb2s0014579397006637.pd

Structural basis of ubiquitin-independent PP1 complex disassembly by p97

The AAA+-ATPase p97 (also called VCP or Cdc48) unfolds proteins and disassembles protein complexes in numerous cellular processes, but how substrate complexes are loaded onto p97 and disassembled is unclear. Here, we present cryo-EM structures of p97 in the process of disassembling a protein phosphatase-1 (PP1) complex by extracting an inhibitory subunit from PP1. We show that PP1 and its partners SDS22 and inhibitor-3 (I3) are loaded tightly onto p97, surprisingly via a direct contact of SDS22 with the p97 N-domain. Loading is assisted by the p37 adapter that bridges two adjacent p97 N-domains underneath the substrate complex. A stretch of I3 is threaded into the central channel of the spiral-shaped p97 hexamer, while other elements of I3 are still attached to PP1. Thus, our data show how p97 arranges a protein complex between the p97 N-domain and central channel, suggesting a hold-and-extract mechanism for p97-mediated disassembly

Structure of an Hsp90-Cdc37-Cdk4 complex

Activation of many protein kinases depends on their interaction with the Hsp90 molecular chaperone system. Recruitment of protein kinase clients to the Hsp90 chaperone system is mediated by the cochaperone adaptor protein Cdc37, which acts as a scaffold, simultaneously binding protein kinases and Hsp90. We have now expressed and purified an Hsp90-Cdc37-Cdk4 complex, defined its stoichiometry, and determined its 3D structure by single-particle electron microscopy. Comparison with the crystal structure of Hsp90 allows us to identify the locations of Cdc37 and Cdk4 in the complex and suggests a mechanism by which conformational changes in the kinase are coupled to the Hsp90 ATPase cycle

Structure of the poly-C9 component of the complement membrane attack complex

The membrane attack complex (MAC)/perforin-like protein complement component 9 (C9) is the major component of the MAC, a multi-protein complex that forms pores in the membrane of target pathogens. In contrast to homologous proteins such as perforin and the cholesterol-dependent cytolysins (CDCs), all of which require the membrane for oligomerisation, C9 assembles directly onto the nascent MAC from solution. However, the molecular mechanism of MAC assembly remains to be understood. Here we present the 8 Å cryo-EM structure of a soluble form of the poly-C9 component of the MAC. These data reveal a 22-fold symmetrical arrangement of C9 molecules that yield an 88-strand pore-forming β-barrel. The N-terminal thrombospondin-1 (TSP1) domain forms an unexpectedly extensive part of the oligomerisation interface, thus likely facilitating solution-based assembly. These TSP1 interactions may also explain how additional C9 subunits can be recruited to the growing MAC subsequent to membrane insertion

Multi-Scale Simulations Provide Supporting Evidence for the Hypothesis of Intramolecular Protein Translocation in GroEL/GroES Complexes

The biological function of chaperone complexes is to assist the folding of non-native proteins. The widely studied GroEL chaperonin is a double-barreled complex that can trap non-native proteins in one of its two barrels. The ATP-driven binding of a GroES cap then results in a major structural change of the chamber where the substrate is trapped and initiates a refolding attempt. The two barrels operate anti-synchronously. The central region between the two barrels contains a high concentration of disordered protein chains, the role of which was thus far unclear. In this work we report a combination of atomistic and coarse-grained simulations that probe the structure and dynamics of the equatorial region of the GroEL/GroES chaperonin complex. Surprisingly, our simulations show that the equatorial region provides a translocation channel that will block the passage of folded proteins but allows the passage of secondary units with the diameter of an alpha-helix. We compute the free-energy barrier that has to be overcome during translocation and find that it can easily be crossed under the influence of thermal fluctuations. Hence, strongly non-native proteins can be squeezed like toothpaste from one barrel to the next where they will refold. Proteins that are already fairly close to the native state will not translocate but can refold in the chamber where they were trapped. Several experimental results are compatible with this scenario, and in the case of the experiments of Martin and Hartl, intra chaperonin translocation could explain why under physiological crowding conditions the chaperonin does not release the substrate protein

Perforin proteostasis is regulated through its C2 domain: supra-physiological cell death mediated by T431D-perforin

The pore forming, Ca2+-dependent protein, perforin, is essential for the function of cytotoxic lymphocytes, which are at the frontline of immune defence against pathogens and cancer. Perforin is a glycoprotein stored in the secretory granules prior to release into the immune synapse. Congenital perforin deficiency causes fatal immune dysregulation, and is associated with various haematological malignancies. At least 50% of pathological missense mutations in perforin result in protein misfolding and retention in the endoplasmic reticulum. However, the regulation of perforin proteostasis remains unexplored. Using a variety of biochemical assays that assess protein stability and acquisition of complex glycosylation, we demonstrated that the binding of Ca2+ to the C2 domain stabilises perforin and regulates its export from the endoplasmic reticulum to the secretory granules. As perforin is a thermo-labile protein, we hypothesised that by altering its C2 domain it may be possible to improve protein stability. On the basis of the X-ray crystal structure of the perforin C2 domain, we designed a mutation (T431D) in the Ca2+ binding loop. Mutant perforin displayed markedly enhanced thermal stability and lytic function, despite its trafficking from the endoplasmic reticulum remaining unchanged. Furthermore, by introducing the T431D mutation into A90V perforin, a pathogenic mutation, which results in protein misfolding, we corrected the A90V folding defect and completely restored perforin’s cytotoxic function. These results revealed an unexpected role for the Ca2+-dependent C2 domain in maintaining perforin proteostasis and demonstrated the possibility of designing perforin with supra-physiological cytotoxic function through stabilisation of the C2 domain

Phase-plate electron microscopy: a novel imaging tool to reveal close-to-life nano-structures

After slow progress in the efforts to develop phase plates for electron microscopes, functional phase plate using thin carbon film has been reported recently. It permits collecting high-contrast images of close-to-life biological structures with cryo-fixation and without staining. This report reviews the state of the art for phase plates and what is innovated with them in biological electron microscopy. The extension of thin-film phase plates to the material-less type using electrostatic field or magnetic field is also addressed

The Celiac Disease and Diabetes-Dietary Intervention and Evaluation Trial (CD-DIET) protocol: A randomised controlled study to evaluate treatment of asymptomatic coeliac disease in type 1 diabetes

Introduction: Coeliac disease (CD) is an autoimmune condition characterised by gluten-induced intestinal inflammation, and observed at a 5-10 fold greater prevalence in type 1 diabetes. While universal screening for CD in patients with diabetes is frequently advocated, objective data is limited as to benefits on diabetes control, bone health or quality of life related to the adoption of a gluten-free diet (GFD) in the large proportion of patients with diabetes with asymptomatic CD. The Celiac Disease and Diabetes-Dietary Intervention and Evaluation Trial (CD-DIET) study is a multicenter, randomised controlled trial to evaluate the efficacy and safety of a GFD in patients with type 1 diabetes with asymptomatic CD. Methods and analysis: Children and adults (8-45 years) with type 1 diabetes will be screened for asymptomatic CD. Eligible patients with biopsy-proven CD will be randomly assigned in a 1:1 ratio to treatment with a GFD for 1 year, or continue with a gluten-containing diet. The primary outcome will evaluate the impact of the GFD on change in glycated haemoglobin. Secondary outcomes will evaluate changes in bone mineral density, blood glucose variability and health-related quality of life between GFD-treated and the regular diet group over a 1-year period. The study was initiated in 2012 and has subsequently expanded to multiple paediatric and adult centres in Ontario, Canada. Ethics and dissemination: The findings from this study will provide high-quality evidence as to the impact of GFD treatment on glycaemic control and complications in asymptomatic children and adults with CD and type 1 diabetes. Trial registration number: NCT01566110