AlphaFold2 assists in providing novel mechanistic insights into the interactions among the LUBAC subunits

• AlphaFold2 predictions reveal that the LTM motif modulates the HOIP-UBA structure, affecting LUBAC stability. • GeoPPI analysis shows that the LTM motif reduces binding affinity between UBL domains and HOIP, decreasing complex stability. • HOIP (629‒695) and HOIP-UBA cooperatively bind to HOIL-1L-UBL, forming a stable elongated domain (HOIP 466‒695). • Molecular dynamics, SPR, and ITC confirm the structural and functional significance of the HOIP-HOIL-1L interaction in LUBAC.