Journal: Nature Communications

Article Title: Structural insights into isoform-specific RAS-PI3Kα interactions and the role of RAS in PI3Kα activation

doi: 10.1038/s41467-024-55766-x

Figure Lengend Snippet: A Domain architecture for the catalytic (p110α) and regulatory (p85α) subunits of PI3Kα, alongside RAS GTPases used in this study. Domains or subunits shown in white were not included in the crystallization constructs utilized in this study. B Bar graph comparing the binding affinities calculated from two technical replicates of PI3Kα-RBD with wild-type and point mutants of KRAS, RRAS2, and MRAS, showing that the mutants of KRAS and RRAS2 exhibit enhanced affinity for forming stable complexes with p110α. The data range is shown as error bars, with two replicates shown as circles. C Overall structure of the RRAS2-p110α complex shown in cartoon and surface representations. D Overall structure of the MRAS-p110α complex depicted in cartoon and surface representations. E Chemical structure of the glue compound D927 and an ITC profile showing a significant increase in the binding affinity between KRAS (GMPPNP) and p110α-RBD in the presence of D927. K D values are presented as the mean ± standard deviation derived from two replicates. F Overall structure of the KRAS-p110α complex in the presence of glue D927 is shown in cartoon and surface representations. The p110α RBD, C2, helical, and kinase domains are colored blue, pale green, orange, and cyan, respectively, and the linker regions between these domains are shown in light gray. The ABD domain, depicted in white in panel A, was excluded from the crystallization constructs and is therefore absent in panels ( C – F ). RRAS2, MRAS, and KRAS are colored pink, purple, and wheat, respectively, with the switch-I and switch-II regions highlighted in red and green, respectively. The nucleotide GMPPNP is depicted as sticks, glue compound D927 is shown as magenta-colored sticks or spheres, and Mg 2+ ions are represented as gray spheres.

Article Snippet: The PIK3R1 (p85) elements for all three constructs originated from an entry clone with a polyhedrin promoter upstream of an untagged p85 (optimized for insect cell expression by ATUM, Inc.).

Techniques: Crystallization Assay, Construct, Binding Assay, Standard Deviation, Derivative Assay

Journal: Nature Communications

Article Title: Structural insights into isoform-specific RAS-PI3Kα interactions and the role of RAS in PI3Kα activation

doi: 10.1038/s41467-024-55766-x

Figure Lengend Snippet: A Structural superposition of p110α in the RAS-p110α complex and the p110α-p85 (niSH2) complex (PDB: 4OVU) containing nSH2 domains reveals conformational differences in the activation loop and similarities in kα11 and the U-motif. B Enlarged view of kα11, U-motif, and the activation loop with the inhibitory nSH2 domain as shown in panel A. C Structural superposition of p110α in the RAS-p110α complex and the p110α-p85 (iSH2) complex (PDB: 5DXH) without the inhibitory nSH2 domain reveals conformational similarities in the activation loop and differences in kα11 and the U-motif. D Enlarged view of kα11, U-motif, and the activation loop without the inhibitory nSH2 domain as shown in panel C.

Article Snippet: The PIK3R1 (p85) elements for all three constructs originated from an entry clone with a polyhedrin promoter upstream of an untagged p85 (optimized for insect cell expression by ATUM, Inc.).

Techniques: Activation Assay

Journal: Nature Communications

Article Title: Structural insights into isoform-specific RAS-PI3Kα interactions and the role of RAS in PI3Kα activation

doi: 10.1038/s41467-024-55766-x

Figure Lengend Snippet: A Model of RAS bound to PI3Kα, generated by aligning the RRAS2-p110α complex on the apo-structure of the p110α-p85(iSH2) complex (PDB: 4OVV) and incorporating the structures of nSH2 and cSH2 domains of p85 solved in complex with the pY motifs of receptor tyrosine kinases (PDB: 2IUI and 5AUL). The binding of the nSH2 and cSH2 domains to the pY motifs of RTK relieves the inhibitory effect of p85 on p110α. The membrane-anchored RAS (pink), upon binding to p110α-RBD, positions PI3Kα at the membrane, thereby facilitating access of membrane-bound PIP2 to the active site for the catalytic reaction. B Schematic illustration depicting the activation mechanism of wild-type PI3Kα and its variants with helical and kinase domain mutations. Created in BioRender. Simanshu, D. (2024) https://BioRender.com/v62l361 .

Article Snippet: The PIK3R1 (p85) elements for all three constructs originated from an entry clone with a polyhedrin promoter upstream of an untagged p85 (optimized for insect cell expression by ATUM, Inc.).

Techniques: Generated, Binding Assay, Membrane, Activation Assay