Figure 2.
Syd binds active Rab2 via the RH2 domain and also binds kinesin-1 and dynein motors. Co-IP experiments performed on lysates from HEK cells transfected with constructs encoding epitope-tagged Drosophila proteins and MD simulation of the Syd:Rab2 interaction. (A) Myc-tagged full-length Syd and truncated Syd-N2 (Syd1-529) co-immunoprecipitate HA-tagged GTP-locked, constitutively active Rab2Q65L. In comparison, coprecipitation of HA-Rab2Q65L by myc-tagged dNischarin and RUFY is near background levels. (B) WT HA-Rab2 and HA-Rab2Q65L co-immunoprecipitate myc-Syd more efficiently than GDP-locked, inactive HA-Rab2S20N. (C) Structure of Syd. Top, expected structure of Syd homodimer assembled from three separate AlphaFold predictions. The WD40 domain of only one Syd monomer is shown. Bottom, schematic representation of the domain architecture of full-length Syd (isoform A, UniProt Q9GQF1) and of truncated Syd variants. (D and E) Co-IP of myc-tagged Syd fragments C1 and N1–N3 (shown in C) by HA-Rab2Q65L. Only myc-Syd-N2 and myc-Syd-N3, which contain the RH2 domain, coprecipitate with Rab2. (F) Co-IP of myc-Rab2Q65L by HA-tagged Syd fragments N2, N4, and N5. (G) FLAG-tagged Klc co-immunoprecipitates in complex with myc-Syd-N2 and HA-Rab2Q65L but not with HA-Rab2Q65L alone. (H) AlphaFold prediction of Syd LZII-RH2 (Syd359–526) dimer in complex with two copies of Rab2. Inset, R468 in Syd RH2 helix 1 is predicted to engage in ionic interactions with E42 in the Rab2 switch I region and E467 in the other RH2 monomer helix 1. L465 is predicted to engage in a hydrophobic interaction with Rab2 switch II I71. (I) Comparison of Co-IP of WT myc-Syd-N2 and mutant myc-Syd-N2L465A,R468A with HA-Rab2Q65L. (J) MD simulation. The Syd-RH2 dimer and one Rab2 moiety from the AlphaFold prediction in H were isolated in silico and pulled apart (the red arrow represents the pulling force direction) to estimate the free energy of the Rab2:Syd-RH2 binding. Free energy curves (mean and standard error, n = 10 for each curve) as a function of Rab2:RH2 center-of-mass distance were calculated for WT LZII-RH2 (blue), RH2L465A,R468A (green), RH2R468A (red), and RH2ΔHelix-2 (yellow), which was truncated after V504, removing the entire helix 2. (K) Co-IP of V5-tagged DLIC with HA-tagged Syd fragments N2, N4, and N5. Note the higher Co-IP efficiency for Syd-N4, where the C-terminal half of the RH2 domain (see Fig. S2 C) is absent, compared with Syd-N2, which has an intact RH2 domain. Source data are available for this figure: SourceData F2.

Syd binds active Rab2 via the RH2 domain and also binds kinesin-1 and dynein motors. Co-IP experiments performed on lysates from HEK cells transfected with constructs encoding epitope-tagged Drosophila proteins and MD simulation of the Syd:Rab2 interaction. (A) Myc-tagged full-length Syd and truncated Syd-N2 (Syd1-529) co-immunoprecipitate HA-tagged GTP-locked, constitutively active Rab2Q65L. In comparison, coprecipitation of HA-Rab2Q65L by myc-tagged dNischarin and RUFY is near background levels. (B) WT HA-Rab2 and HA-Rab2Q65L co-immunoprecipitate myc-Syd more efficiently than GDP-locked, inactive HA-Rab2S20N. (C) Structure of Syd. Top, expected structure of Syd homodimer assembled from three separate AlphaFold predictions. The WD40 domain of only one Syd monomer is shown. Bottom, schematic representation of the domain architecture of full-length Syd (isoform A, UniProt Q9GQF1) and of truncated Syd variants. (D and E) Co-IP of myc-tagged Syd fragments C1 and N1–N3 (shown in C) by HA-Rab2Q65L. Only myc-Syd-N2 and myc-Syd-N3, which contain the RH2 domain, coprecipitate with Rab2. (F) Co-IP of myc-Rab2Q65L by HA-tagged Syd fragments N2, N4, and N5. (G) FLAG-tagged Klc co-immunoprecipitates in complex with myc-Syd-N2 and HA-Rab2Q65L but not with HA-Rab2Q65L alone. (H) AlphaFold prediction of Syd LZII-RH2 (Syd359–526) dimer in complex with two copies of Rab2. Inset, R468 in Syd RH2 helix 1 is predicted to engage in ionic interactions with E42 in the Rab2 switch I region and E467 in the other RH2 monomer helix 1. L465 is predicted to engage in a hydrophobic interaction with Rab2 switch II I71. (I) Comparison of Co-IP of WT myc-Syd-N2 and mutant myc-Syd-N2L465A,R468A with HA-Rab2Q65L. (J) MD simulation. The Syd-RH2 dimer and one Rab2 moiety from the AlphaFold prediction in H were isolated in silico and pulled apart (the red arrow represents the pulling force direction) to estimate the free energy of the Rab2:Syd-RH2 binding. Free energy curves (mean and standard error, n = 10 for each curve) as a function of Rab2:RH2 center-of-mass distance were calculated for WT LZII-RH2 (blue), RH2L465A,R468A (green), RH2R468A (red), and RH2ΔHelix-2 (yellow), which was truncated after V504, removing the entire helix 2. (K) Co-IP of V5-tagged DLIC with HA-tagged Syd fragments N2, N4, and N5. Note the higher Co-IP efficiency for Syd-N4, where the C-terminal half of the RH2 domain (see Fig. S2 C) is absent, compared with Syd-N2, which has an intact RH2 domain. Source data are available for this figure: SourceData F2.

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