Structural basis for binding of RILPL1 to TMEM55B reveals a lysosomal platform for adaptor assembly through a conserved peptide motif

Inherited mutations in VPS35 and LRRK2 kinase lead to hyperphosphorylation of Rab GTPases. RH2 domain-containing proteins from the RILP homology family, such as RILPL1, are Rab effectors that recognize the LRRK2-phosphorylated switch 2 threonine of phospho-Rab8A and phospho-Rab10. Phospho-Rabs are also seen on lysosomal membranes in complex with RILPL1 and TMEM55B, a 284-residue lysosomal membrane protein lacking homology to known proteins. Here, we report crystal structures of the cytosolic region 80-166 of TMEM55B alone and in complex with a C-terminal RILPL1 peptide, which we define as the TMEM55B-binding motif (TBM). The RILPL1 TBM sits in a shallow groove across two tandem RING-like domains of TMEM55B, each forming a Zn²⁺-stabilized 40-residue β-sandwich. Co-immunoprecipitation and mass spectrometry studies indicate that TMEM55B forms complexes independently of phospho-Rabs with conserved TBMs found in JIP3, JIP4, OCRL, WDR81, and TBC1D9B. These studies suggest that TMEM55B acts as a central hub for adaptor recruitment on lysosomes.