Abstract
The control of cell movement through manipulation of cytoskeletal structure has therapeutic prospects notably in the development of novel anti-metastatic drugs. In this study, we determine the structure of Ras-binding domain (RBD) of ELMO1, a protein involved in cytoskeletal regulation, both alone and in complex with the activator RhoG and verify its targetability through computational nanobody design. Using our dock-and-design approach optimized with native-like initial pose selection, we obtain Nb01, a detectable binder from scratch in the first-round design. An affinity maturation step guided by structure-activity relationship at the interface generates 23 Nb01 sequence variants and 17 of them show enhanced binding to ELMO1-RBD and are modeled to form major spatial overlaps with RhoG. The best binder, Nb29, inhibited ELMO1-RBD/RhoG interaction. Molecular dynamics simulation of the flexibility of CDR2 and CDR3 of Nb29 reveal the design of stabilizing mutations at the CDR-framework junctions potentially confers the affinity enhancement.