Fig. 2: Detailed interactions and mutational effects at the LRRK2:14-3-3₂ binding interfaces.

a Overview of the LRRK2:14-3-3₂ contact regions in LRRK2:14-3-3₂ complex. Insets detail the primary and secondary interaction sites, supported by the corresponding cryo-EM densities. b Close-up view of the primary interactions, where LRRK2 phosphorylation sites pS910 and pS935 engage with the 14-3-3 substrate binding grooves. Assignment of this interaction is supported by integration of structural fitting, mass spectrometry, and biochemical validation. c Close-up view of the secondary interactions, showing LRRK2 COR-A and COR-B subdomain residues contacting the α−9 helices of the 14-3-3 dimer. d Quantitative analysis of LRRK2/14-3-3 interactions through Co-IP experiments of LRRK2 with endogenous 14-3-3, comparing WT LRRK2 with mutants at the secondary interface, as well as primary interface mutants (S910A/S935A). Data illustrate the impact of mutations on the interaction strength. Refer to Supplementary Fig. 16 for representative membrane images and source data for complete membrane images. Data are mean ± SEM (n  =  3 independent experiments), significance of difference was quantified using one-way Brown–Forsythe and Welch ANOVA test and reported with the exact p values in the source data file. e Binding affinity between (WT or mutant) LRRK2 and (WT or mutant) 14-3-3 proteins was determined by MST. Mutations at the primary (left) and secondary (right) binding sites were analyzed. Data are mean ± SEM (n  =  3 independent experiments), significance of difference was quantified using one-way Brown–Forsythe and Welch ANOVA test and reported with the exact p values in the source data file. Refer to Supplementary Fig. 11 for full binding curves.