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

Abstract

Monoclonal antibodies are widely used to detect and characterize membrane proteins, yet for most such reagents, the molecular basis of target recognition remains structurally undefined. Here, we report the structural and biochemical characterization of 37E5, a monoclonal antibody that recognizes MLC1, an astrocytic membrane protein implicated in gliovascular homeostasis and megalencephalic leukoencephalopathy with subcortical cysts. 37E5 specifically detects endogenous MLC1 across multiple experimental systems, including brain lysates and mouse and human brain tissues, and binds purified human MLC1 with nanomolar affinity as determined by biolayer interferometry. To define the molecular basis of recognition, we determined 3.2 and 3.0 Å cryo-EM structures of the 37E5 Fab in apo and antigen-bound states, respectively. The antigen-bound structure revealed continuous density for an MLC1-derived peptide within the paratope, stabilized by coordinated hydrogen-bonding, ionic, and hydrophobic interactions. Structural comparison between apo and bound states showed localized conformational changes confined to the complementarity-determining regions, consistent with a restrained, local induced-fit adjustment of the paratope; because these backbone deviations are small, they are interpreted only in the context of the coordinate uncertainty expected at this resolution. Comparative analyses further explained species-dependent recognition through sequence variation at key epitope residues, corroborated by binding assays across four MLC1 orthologs. These findings define the structural basis of selective MLC1 recognition by 37E5 and illustrate, for this antibody-peptide pair, how experimental cryo-EM structures complement AI-based prediction methods by resolving peptide orientation and interaction geometry at the antibody-antigen interface.

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