Structural Based Bioinformatics for Identification of UBDs
We hypothesized that there are hidden UBDs within the Protein Data Bank (PDB). To identify these UBDs in-silico, we developed a novel structural based search algorithm. Superimposing UBD:Ub complexes from different UBDs families, highlighted a minimal atomic signature (cluster) sufficient for Ub binding (red and gray mesh balls in E2-25k). We then searched the PDB with this signature. As expected, known UBDs earned high scores. Interestingly, within these high scores some unknown potential UBDs were found including ALIX-V domain (the projection of the physico-chemical Ub binding properties of E2-25k on ALIX-V is shown as red and gray balls).
ALIX links between Apoptosis-Linked Gene-2 (ALG-2), the Endosomal Sorting Complexes Required for Transport (ESCRT) complexes, and neuronal death. Morever, ALIX plays a pivotal role in ubiquitylated-cargo trafficking at the MVB, in cytokinesis and retrovirus (like HIV) budding.
We then demonstrated that ALIX-V is a bona-fide UBD. We used Micro Scale Thermophoresis (MST) to quantify the affinity of wild-type and mutant ALIX:Ub complexes designed based on the structural model.
We finally set-up functional assays with the wild-type and the mutants of ALIX-V and demonstrated the importance of Ub-binding for the budding of retrovirus (Keren-Kaplan, Attali et al. EMBO J 2013).
Currently, we utilize the developed algorithm to characterize the Ub-binding sites of several other protein structures in including the vWA domain of Rpn10 as we recently published in Nature communication.