Cryo-EM structures of an anti-MLC1 Fab in apo and peptide-bound states reveal the structural basis of antigen recognition

Abstract

Monoclonal antibodies are indispensable tools in structural biology and biomedical research, but defining the molecular basis of their specificity remains challenging. Here, we developed a novel monoclonal antibody (37E5) against the astrocytic membrane protein MLC1, a component of gliovascular signaling implicated in megalencephalic leukoencephalopathy with subcortical cysts. 37E5 demonstrated high specificity and versatility across biochemical, cellular, and histological assays, enabling reliable detection of MLC1 in both human and mouse tissue. Using single-particle cryo-EM, we determined [~]3 [A] resolution structures of the 37E5 Fab in apo and antigen-bound states, despite the small molecular mass ([~]50 kDa), close to the lower size limit of cryo-EM. The antigen-bound structure revealed continuous density for an MLC1-derived peptide and enabled atomic mapping of polar and non-polar interaction networks. Conformational changes in CDR-L1 and CDR-L2 indicated an induced-fit mechanism of recognition. Comparison with AlphaFold-predicted models underscored the accuracy of Fab backbone prediction but revealed major limitations in modeling epitope-paratope geometry. These findings establish 37E5 as a versatile antibody for mechanistic studies of gliovascular biology and MLC disease, while demonstrating that cryo-EM can achieve atomic-level characterization of small Fab-antigen complexes, thereby expanding the methodological frontier of antibody-antigen structural biology. SignificanceThis study defines the molecular basis of MLC1 recognition by a novel monoclonal antibody, establishes 37E5 as a versatile reagent for mechanistic and translational research, and demonstrates the feasibility of cryo-EM to resolve dynamic features of small Fab-antigen complexes.