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.

Keywords: chemical cross-linking; conformational changes; mass spectrometry; nanobody; phosphoinositide 3-kinase (PI3K).

Fig. 1.

Preparation and characterization of PI3Kα-specific nanobodies. (A) Overview of the nanobody generation process. (B and C) SDS-PAGE/Coomassie blue stain and size exclusion chromatography of the purified nanobodies Nb3-126, Nb3-142, and Nb3-159. (D–F) SPR sensorgrams demonstrate that nanobodies Nb3-126, Nb3-142, and Nb3-159 bind to PI3Kα with affinities of 12.90, 24.30, and 7.90 nM, respectively. (G) HTRF PI 3-kinase assay of purified PI3Kα with indicated nanobodies and inhibitors. Data are from triplicate experiments and reported as median values of three independent measurements.

Fig. 2.

Cryo-EM analysis and modeling of PI3Kα-nanobody complexes. (A) Nb3-126, (B) Nb3-142, and (C) Nb3-159 are shown in three different ways: (Left) cryo-EM density, (Middle) model, and (Right) local resolution plotted on 1% FDR confidence interval. (D) Domain structure of p110α and p85α.

Fig. 3.

Cryo-EM of PI3Kα-nanobody complexes shows previously unreported features. (A) Nb3-126 leads to a high-resolution cryo-EM electron density map as illustrated by a representative α-helix, residues 808 to 826 of p110α. (B) Nb3-142 has stabilized extra density as compared with other cryo-EM data. (C and D) Nb3-159 has an altered position of the ABD and iSH2 domains in comparison with other PI3Kα structures.

Fig. 4.

Three-dimensional variability analysis of the complex of PI3Kα with Nb3-159 shows extreme variability of the ABD and iSH2 domains. (A) The first component of the Nb3-159 3DVA resolves a motion from a structure similar to the consensus PI3Kα structure (blue) toward a structure with a radically deflected ABD and iSH2 domain (red), and overlay of these structures shows the degree of deflection (Middle). (B) Models of the two states of component 1 demonstrate a discontinuity in the iSH2 domain that cannot be explained without flexibility or kinking of the coiled-coil domain. (C) rmsd values calculated for the Cα positions show that the near edge of the iSH2 is deflected downward, while the far edge is less deflected. There are also changes in the kinase domain N lobes (773 to 777) and C lobes (864 to 874) as well as 803 to 811.

Fig. 5.

Cryo-EM of DSG cross-linked PI3Kα Nb3-142 complex. Two views of the DSG cross-linked PI3Kα complex with Nb3-142 are shown as (Left) electron density, (Center) modeling, and (Right) 1% FDR confidence surface with local resolution data. Chemical cross-linking allows some previously unresolved regions such as the RBD domain to be stabilized but leads to a decrease in resolution of the unresolved SH3, BH, and cSH2 domain density compared with the un–cross-linked structure. Resolution colors are identical to Fig. 2.

Fig. 6.

Structural models of PI3Kα bound by (A) Nb3-126, (B) Nb3-142, and (C) Nb3-159. CXMS-derived restraints allow for docking of unresolved domains with the core of PI3Kα in the Nb3-142 stabilized structure. DSG and BS³ chemical cross-linking mass spectrometry data were used to construct distance restraints which were used along with existing structural data for the SH3, BH, and cSH2 domains. The cSH2 domain is positioned in front of the kinase domain and blocks access to this site. The BH domain is positioned below the kinase, iSH2, and C2 domains. The position of the SH3 domain is flexible, and docking simulations do not give a consistent position for this domain.