期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2022
卷号：119
期号：38
DOI：10.1073/pnas.2210769119
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Significance
PI3Kα is a dimeric lipid kinase consisting of a catalytic subunit p110α and a regulatory subunit p85α. It controls cell proliferation and survival and is an important therapeutic target for cancer. However, the development of effective drugs against PI3Kα requires a level of structural information that is currently unavailable, because the extreme flexibility of PI3Kα interferes with structural analysis. Nanobodies were used in conjunction with chemical cross-linking to generate insights into the identity and the positions of the most flexible domains of PI3Kα and into mechanistic aspects of positional flexibility. The studies also reveal the existence of a previously unreported structural conformation of PI3Kα.
Nanobodies and chemical cross-linking were used to gain information on the identity and positions of flexible domains of PI3Kα. The application of chemical cross-linking mass spectrometry (CXMS) facilitated the identification of the p85 domains BH, cSH2, and SH3 as well as their docking positions on the PI3Kα catalytic core. Binding of individual nanobodies to PI3Kα induced activation or inhibition of enzyme activity and caused conformational changes that could be correlated with enzyme function. Binding of nanobody Nb3-126 to the BH domain of p85α substantially improved resolution for parts of the PI3Kα complex, and binding of nanobody Nb3-159 induced a conformation of PI3Kα that is distinct from known PI3Kα structures. The analysis of CXMS data also provided mechanistic insights into the molecular underpinning of the flexibility of PI3Kα.
