Dissecting the Black Box of AlphaFold in Protein–Protein Complex Assembly

AlphaFold achieves unprecedented accuracy in modeling protein–protein complexes, yet the principles governing complex assembly remain unclear. Here, we develop a unified interpretability framework for AlphaFold-Multimer and AlphaFold3 to dissect the mechanisms underlying complex formation. We demonstrate that inter-protein coevolution is not a major determinant of assembly. Instead, complex structures are primarily driven by monomer geometry together with interface-level pattern matching between backbone complementarity and residue identities. By visualizing the iterative propagation of distance constraints during inference, we uncover a hierarchical process in which monomer-level constraints are established prior to cross-chain interactions, directly demonstrating that inter-chain geometry is inferred from monomer geometries rather than being encoded by coevolutionary signals. Application to antigen–antibody complexes further reveals that reduced prediction accuracy arises from the non-canonical and structurally plastic nature of immune interfaces, identifying accurate modeling of interface conformations and recognition of atypical antigen–antibody interaction patterns as key bottlenecks for improving immune complex prediction.