Nanobodies and chemical cross-links advance the structural and functional analysis of PI3Kα

Abstract

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.

Cryo-EM structures of cancer-specific helical and kinase domain mutations of PI3Kα

“…As a result, the activation loop gains greater flexibility and can assume a conformation that favors increased kinase activity ( 29 , 30 ). Additionally, blocking of the active site by the cSH2 domain that is observed with WT PI3Kα ( 54 ) is diminished in PI3Kα-H1047R, suggesting a mobility of the p85α domains that relieves their inhibitory effect on membrane binding of the catalytic core. In 3DVA, the regions that include the amino acid substitution H1047R and the iSH2 domain and ABD show greater mobility than the corresponding domains of the WT protein, and this mobility probably translates into greater enzyme activity.…”

Functional selection in SH3-mediated activation of the PI3 kinase

“…This position is similar to the inhibitory cSH2 position identified in the crystal structure of the PI3β heterocomplex (PDB id 2Y3A) (Zhang et al, 2011). However, unlike the PI3Kβ crystal structure, the cSH2 of the PI3Kα AlphaFold model was not in direct contact with the p110 kinase domain, in line with the lack of density for this domain in experimental PI3Kα structures (Hart et al, 2022). Rather, cSH2 was flexibly anchored through a predicted interaction between the iSH2-cSH2 linker residues Y 607 SLV and the p110α kinase domain, suggestive of a different inhibitory mechanism involving Y 607 SLV ( Supplementary Figure 7C ).…”

“…Our efforts to produce a crystal structure of SH3 domains bound to p85 (and fragments thereof) failed, likely due to the large flexibility of this protein. Atomic structure determination also appeared unfeasible with NMR (due to the observed peak broadening) or cryo-EM (due to the low SH3 affinity and the persistent flexibility of the PR1, BH, PR2 and nSH2 domains even in a cross-linked, nanobody-stabilised complex with p110; (Hart et al, 2022). Therefore, to produce a mechanistic model for the SH3-promoted activation of PI3K, we projected the crosslinks onto the biological p85:p110 heterocomplex ( Figure 4B ).…”

“…Although this interaction with the cSH2 has not been observed in PI3Kα, the p85α cSH2 domain does appear to regulate kinase activation in PI3Kα (Jücker et al, 2002; Yu et al, 1998a) and has been experimentally shown in the complex to reside close to the active site of the p110α kinase domain. However, in PI3Kα the cSH2 domain appears to be more flexibly localised in this position than in PI3Kβ (Hart et al, 2022). Collectively, these structural observations explain how ligands that bind to p85α SH2 domains (i.e.…”

“…Furthermore, cryo-EM structures of p85α bound to p110α lack clear density for the SH3-PR1-BH domains (Hart et al, 2022; Liu et al, 2021), inferring that these domains are not stably associated within the complex formed between p110α and p85α. Hence, it is unknown how SH3 domains can mechanistically activate p110α, and how such an activation based on promiscuous SH3 domain interactions can be selective.…”