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

ABSTRACT: Inherited mutations in VPS35 and the kinase LRRK2 lead to hyperphosphorylation of Rab GTPases and promote the formation of phospho-Rab signalling complexes. A subset of RH2 domain-containing proteins from the RILP-homology family, including RILP, RILPL1, RILPL2, JIP3, and JIP4 are Rab effectors that recognize the LRRK2-phosphorylated switch 2 threonine of phospho-Rab8a and phospho-Rab10. More recently, phospho-Rabs have been found on lysosomal membranes within multi-protein assemblies involving TMEM55B and RILPL1. TMEM55B is a 284-residue lysosomal membrane protein with no homology to known proteins. It comprises a 218-residue cytosolic N-terminal region and two predicted transmembrane α-helices. Residues 80–160, which face the cytosol, mediate binding to a C-terminal motif of RILPL1, formed after RILPL1 associates with phospho-Rab8A. Here, we report the crystal structures of TMEM55B alone and in complex with a C-terminal RILPL1 peptide, encompassing the TMEM55B interaction region, which we define as the TMEM55B Binding Motif (TBM). The cytosolic domain of TMEM55B adopts a rigid architecture of two tandem RING-like domains, each forming a Zn²⁺-stabilized 40-residue β-sandwich. TBM binding is mediated primarily by backbone hydrogen bonding and anchored by two glutamate residues from RILPL1. These findings support a model in which RILPL1 is recruited to phospho-Rab8A–positive lysosomes prior to TMEM55B engagement. Further co-immunoprecipitation and mutational analyses indicate that TMEM55B forms complexes independently of phospho-Rabs with proteins containing a conserved TBM motif, like that of RILPL1, including JIP3, JIP4, OCRL, WDR81, and TBC1D9B. Together, these findings uncover previously unrecognized regulatory networks associated with TMEM55B and lysosomal function and suggest that TMEM55B serves as a central hub for adaptor recruitment at the lysosomal membrane.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Hek-293et Cell, Early Embryonic Cell, Kidney

DISEASE(S): Parkinson's Disease

SUBMITTER: Raja Sekhar Nirujogi  

LAB HEAD: Dario R. Alessi

PROVIDER: PXD067414 | Pride | 2025-11-30

REPOSITORIES: Pride