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Title | HDX-MS-optimized approach to characterize nanobodies as tools for biochemical and structural studies of class IB phosphoinositide 3-kinases. |
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Journal, issue, pages | Structure, Vol. 29, Issue 12, Page 1371-11381.e6, Year 2021 |
Publish date | Dec 2, 2021 |
Authors | Manoj K Rathinaswamy / Kaelin D Fleming / Udit Dalwadi / Els Pardon / Noah J Harris / Calvin K Yip / Jan Steyaert / John E Burke / |
PubMed Abstract | There is considerable interest in developing antibodies as modulators of signaling pathways. One of the most important signaling pathways in higher eukaryotes is the phosphoinositide 3-kinase (PI3K) ...There is considerable interest in developing antibodies as modulators of signaling pathways. One of the most important signaling pathways in higher eukaryotes is the phosphoinositide 3-kinase (PI3K) pathway, which plays fundamental roles in growth, metabolism, and immunity. The class IB PI3K, PI3Kγ, is a heterodimeric complex composed of a catalytic p110γ subunit bound to a p101 or p84 regulatory subunit. PI3Kγ is a critical component in multiple immune signaling processes and is dependent on activation by Ras and G protein-coupled receptors (GPCRs) to mediate its cellular roles. Here we describe the rapid and efficient characterization of multiple PI3Kγ binding single-chain camelid nanobodies using hydrogen-deuterium exchange (HDX) mass spectrometry (MS) for structural and biochemical studies. We identify nanobodies that stimulated lipid kinase activity, block Ras activation, and specifically inhibited p101-mediated GPCR activation. Overall, our work reveals insight into PI3Kγ regulation and identifies sites that may be exploited for therapeutic development. |
External links | Structure / PubMed:34348129 |
Methods | EM (single particle) |
Resolution | 2.89 Å |
Structure data | EMDB-23808, PDB-7mez: |
Source |
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Keywords | TRANSFERASE / PI3K / p110 / PIK3CG / PIK3R5 / p101 / phosphoinositide 3-kinase / PIP3 / IMMUNE SYSTEM |