Biology subjects

Botzanowski, T.

Publications and source records attributed to Botzanowski, T..

3 recordsLinked to original sources

bioRxiv2024

Deciphering molecular determinants of GPCR-G protein receptor interactions by complementary integrative structural biology methods

Many physiological processes are dependent on G protein-coupled receptors (GPCRs), the biggest family of human membrane proteins and a significant class of therapeutic targets. Once activated by external stimuli, GPCRs use G proteins and arrestins as transducers to generate second messengers and trigger downstream signaling, leading to diverse signaling profiles. The G protein-coupled bile acid receptor 1 (GPBAR1, also known as Takeda G protein-coupled receptor 5, TGR5) is a class A bile acid membrane receptor that regulates energy homeostasis and glucose and lipid metabolism. GPBAR1/G protein interactions are implicated in the prevention of diabetes and the reduction of inflammatory responses, making GPBAR1 a potential therapeutic target for metabolic disorders. Here, we present an integrated structural biology approach combining hydrogen/deuterium exchange mass spectrometry (HDX-MS) and cryo-electron microscopy (cryo-EM) to identify the molecular determinants of GPBAR1 conformational dynamics upon G protein binding. Thanks to extensive optimization of both HDX-MS and cryo-EM workflows, we achieved over 99% sequence coverage along with a 2.5-[A] resolution structure, both of which are state-of-the-art and solely obtained for complete GPCR complexes. Altogether, our results provide information on the under-investigated GPBAR1 binding mode to its cognate G protein, pinpointing the synergic and powerful combination of higher (cryo-EM) and lower (HDX-MS) resolution structural biology techniques to dissect GPCR/G protein binding characteristics. Short statementThis work highlights the utility of integrating cryo-EM and HDX-MS for studying large multiprotein complexes such as GPCR/G protein complexes. Cryo-EM offers high-resolution structural details, while HDX-MS reveals the dynamic conformational changes during assembly, providing a comprehensive structural view of difficult-to-study membrane protein systems.

biophysics↗

bioRxiv2020

Middle-level IM-MS and CIU experiments for improved therapeutic immunoglobulin isotype fingerprinting

Currently approved therapeutic monoclonal antibodies (mAbs) are based on immunoglobulin G (IgG) 1, 2 or 4 isotypes, which differ in their specific inter-chains disulfide bridge connectivities. Different analytical techniques have been reported for mAb isotyping, among which native ion mobility mass spectrometry (IM-MS) and collision induced unfolding (CIU) experiments. However, mAb isotyping by these approaches is based on detection of subtle differences and thus remains challenging at the intactlevel. We report here on middle-level (after IdeS digestion) IM-MS and CIU approaches to afford better differentiation of mAb isotypes. Our method provides simultaneously CIU patterns of F(ab)2 and Fc domains within a single run. Middle-level CIU patterns of F(ab)2 domains enable more reliable classification of mAb isotypes compared to intact level CIU, while CIU fingerprints of Fc domains are overall less informative for mAb isotyping. F(ab)2 regions can thus be considered as diagnostic domains providing specific CIU signatures for mAb isotyping. Benefits of middle-level IM-MS and CIU approaches are further illustrated on the hybrid IgG2/IgG4 eculizumab. While classical analytical techniques led to controversial results, middle-level CIU uniquely allowed to face the challenge of eculizumab << hybridicity >>, highlighting that its F(ab)2 and Fc CIU patterns corresponds to an IgG2 and an IgG4, respectively. Altogether, the middle-level CIU approach is more clear-cut, accurate and straightforward for canonical but also more complex, engineered next generation mAb formats isotyping. Middle-level CIU thus constitutes a real breakthrough for therapeutic protein analysis, paving the way for its implementation in R&D laboratories.

biophysics↗

bioRxiv2019

Aβ(1-42) tetramer and octamer structures reveal edge pores as a mechanism for membrane damage

The formation of amyloid-beta (A{beta}) oligomer pores in the membrane of neurons has been proposed as the means to explain neurotoxicity in Alzheimers disease (AD). It is therefore critical to characterize A{beta} oligomer samples in membrane-mimicking environments. Here we present the first three-dimensional structure of an A{beta} oligomer formed in dodecyl phosphocholine (DPC) micelles, namely an A{beta}(1-42) tetramer. It comprises a {beta}-sheet core made of six {beta}-strands, connected by only two {beta}-turns. The two faces of the {beta}-sheet core are hydrophobic and surrounded by the membrane-mimicking environment. In contrast, the edges of the core are hydrophilic and are solvent-exposed. By increasing the concentration of A{beta}(1-42), we prepared a sample enriched in A{beta}(1-42) octamers, formed by two A{beta}(1-42) tetramers facing each other forming a {beta}-sandwich structure. Notably, samples enriched in A{beta}(1-42) tetramers and octamers are both active in lipid bilayers and exhibit the same types of pore-like behaviour, but they show different occurrence rates. Remarkably, molecular dynamics simulations showed a new mechanism of membrane disruption in which water and ion permeation occurred through lipid-stabilized pores mediated by the hydrophilic residues located on the core {beta}-sheets edges of the A{beta}(1-42) tetramers and octamers.

biophysics↗