Target selection of soluble protein complexes for structural proteomics studies

BACKGROUND: Protein expression in E. coli is the most commonly used system to produce protein for structural studies, because it is fast and inexpensive and can produce large quantity of proteins. However, when proteins from other species such as mammalian are produced in this system, problems of protein expression and solubility arise [1]. Structural genomics project are currently investigating proteomics pipelines that would produce sufficient quantities of recombinant proteins for structural studies of protein complexes. To investigate how the E. coli protein expression system could be used for this purpose, we purified apoptotic binary protein complexes formed between members of the Caspase Associated Recruitment Domain (CARD) family. RESULTS: A combinatorial approach to the generation of protein complexes was performed between members of the CARD domain protein family that have the ability to form hetero-dimers between each other. In our method, each gene coding for a specific protein partner is cloned in pET-28b (Novagen) and PGEX2T (Amersham) expression vectors. All combinations of protein complexes are then obtained by reconstituting complexes from purified components in native conditions, after denaturation-renaturation or co-expression. Our study applied to 14 soluble CARD domain proteins revealed that co-expression studies perform better than native and denaturation-renaturation methods. In this study, we confirm existing interactions obtained in vivoin mammalian cells and also predict new interactions. CONCLUSION: The simplicity of this screening method could be easily scaled up to identify soluble protein complexes for structural genomic projects. This study reports informative statistics on the solubility of human protein complexes expressed in E.coli belonging to the human CARD protein family

SecYEG activates GTPases to drive the completion of cotranslational protein targeting

Signal recognition particle (SRP) and its receptor (SR) comprise a highly conserved cellular machine that cotranslationally targets proteins to a protein-conducting channel, the bacterial SecYEG or eukaryotic Sec61p complex, at the target membrane. Whether SecYEG is a passive recipient of the translating ribosome or actively regulates this targeting machinery remains unclear. Here we show that SecYEG drives conformational changes in the cargo-loaded SRP–SR targeting complex that activate it for GTP hydrolysis and for handover of the translating ribosome. These results provide the first evidence that SecYEG actively drives the efficient delivery and unloading of translating ribosomes at the target membrane

Streamlining T cell engager development with a diverse panel of fully human CD3-binding antibodies, bispecific engineering technology, and an integrated discovery engine

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From nanoliter to large-scale bioreactors: How integrated technologies bring antibody treatments to patients faster

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The PAAD domain OF IFI16 reveals a novel ssDNA binding function for the death domain super family

The PAAD Death domain, involved in apoptosis and inflammation, shares a 6-helix bundle fold similar to other apoptotic sub-family members (DD/DED/CARD), but with a disordered region in helix 3. To investigate the structural basis for this difference I measured and compared thermodynamic folding parameters between PAAD and CARD members and show that PAAD domains have low stability and can undergo conformational changes when induced by mutagenesis or secondary structure promoting agents. The structural plasticity of the PAAD domain is consistent with an induced fit mechanism of ligand binding that may confer different protein-ligand interactions, contrasting with the common assumption that the Death domain is a protein-protein interaction domain. Finally, I challenged the above assumption by showing that the PAAD domain from the HIN-200 family member, IFI16, has all the characteristics of a single stranded nucleic acid binding protein, motivating further studies for the discovery of new PAAD-ligand interactions and functions

On the quaternary structure and gating of the bacterial protein translocation channel

The SecY protein-conducting channel associates with different cytosolic partners to drive the translocation of preprotein substrates across the bacterial inner membrane. In this thesis, several outstanding questions regarding the structure and function of the SecY channel are addressed. Our first study is motivated by the poorly defined interactions between the channel and its binding partners. We characterize the binding mode and stoichiometry of two SecY interacting proteins, the SecA ATPase and Syd, which each form 1:1 complexes with the channel. In the second study, we isolate the SecY dimer (i.e. two SecY channels), which is shown to be essential to activate the SecA ATPase activity and support protein transport. Analysis of SecY dimers in vivo further demonstrates that each constituent SecY copy has a different role in the translocation reaction. Finally, we discover that the SecY channel, in addition to transporting preprotein substrates, is also highly specific for monovalent anions. This selective conductance explains why translocation does cause a general membrane permeability and cell death. Our findings are discussed in the broader context of genetic, biochemical and structural information on the SecY channel and other translocation components.Medicine, Faculty ofBiochemistry and Molecular Biology, Department ofGraduat

Targeting Alternative Sites on the Androgen Receptor to Treat Castration-Resistant Prostate Cancer

Recurrent, metastatic prostate cancer continues to be a leading cause of cancer-death in men. The androgen receptor (AR) is a modular, ligand-inducible transcription factor that regulates the expression of genes that can drive the progression of this disease, and as a consequence, this receptor is a key therapeutic target for controlling prostate cancer. The current drugs designed to directly inhibit the AR are called anti-androgens, and all act by competing with androgens for binding to the androgen/ligand binding site. Unfortunately, with the inevitable progression of the cancer to castration resistance, many of these drugs become ineffective. However, there are numerous other regulatory sites on this protein that have not been exploited therapeutically. The regulation of AR activity involves a cascade of complex interactions with numerous chaperones, co-factors and co-regulatory proteins, leading ultimately to direct binding of AR dimers to specific DNA androgen response elements within the promoter and enhancers of androgen-regulated genes. As part of the family of nuclear receptors, the AR is organized into modular structural and functional domains with specialized roles in facilitating their inter-molecular interactions. These regions of the AR present attractive, yet largely unexploited, drug target sites for reducing or eliminating androgen signaling in prostate cancers. The design of small molecule inhibitors targeting these specific AR domains is only now being realized and is the culmination of decades of work, including crystallographic and biochemistry approaches to map the shape and accessibility of the AR surfaces and cavities. Here, we review the structure of the AR protein and describe recent advancements in inhibiting its activity with small molecules specifically designed to target areas distinct from the receptor’s androgen binding site. It is anticipated that these new classes of anti-AR drugs will provide an additional arsenal to treat castration-resistant prostate cancer.Other UBCReviewedFacult

The maltose ABC transporter: Action of membrane lipids on the transporter stability, coupling and ATPase activity

AbstractThe coupling between ATP hydrolysis and substrate transport remains a key question in the understanding of ABC-mediated transport. We show using the MalFGK2 complex reconstituted into nanodiscs, that membrane lipids participate directly to the coupling reaction by stabilizing the transporter in a low energy conformation. When surrounded by short acyl chain phospholipids, the transporter is unstable and hydrolyzes large amounts of ATP without inducing maltose. The presence of long acyl chain phospholipids stabilizes the conformational dynamics of the transporter, reduces its ATPase activity and restores dependence on maltose. Membrane lipids therefore play an essential allosteric function, they restrict the transporter ATPase activity to increase coupling to the substrate. In support to the notion, we show that increasing the conformational dynamics of MalFGK2 with mutations in MalF increases the transporter ATPase activity but decreases the maltose transport efficiency