Desligniere, E. | Search bioRxiv

bioRxiv2025

R2TP-like Quaternary Chaperones: a comprehensive overview to understand the dynamic R2SP complex

The human R2SP complex belongs to the R2TP-like quaternary chaperone family and consists of RUVBL1, RUVBL2, SPAG1 and PIH1D2. R2SP is crucial for the correct assembly of motile cilia (SPAG1 null mutations cause Primary Ciliary Dyskinesia) and the organization of the synaptic zone. RUVBL1/2 ATPases are the powerhouse of this molecular machinery, while SPAG1 and PIH1D2 would be adaptors that interact with specific clients to promote their quaternary assembly. Despite these functional data, little is known about the structure of R2SP and the precise mode of action of these R2TP-like complexes. We have combined biochemical and structural approaches (NMR, structural mass spectrometry and cryo-EM) to investigate the 3D organization of the human R2SP complex, its mode of assembly and ATPase activity. Our study reveals a three-dimensional structure similar to that of the canonical R2TP complex, but also highlights differences in the mode of action of its RUVBL1/2 ATPase core as well as the binding of its adaptors SPAG1 and PIH1D2.

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↗