Structural Characterization of an astrocytic membrane protein, MLC1

초록

MLC1 is a membrane protein with multi-transmembrane domains and is mainly expressed in astrocytes. Genetic mutants on Mlc1 are associated with an early-onset rare genetic neurological disease called Megalencephalic Leukoencephalopathy with Subcortical Cysts (MLC), which leads to progressive neurodegeneration. MLC typically presents with infantile macrocephaly, followed by progressive gait disturbances and cognitive impairment. However, despite its clinical importance, the structure and function of MLC1 remain poorly understood. In our previous study, Wild-type MLC1 localized to the plasma membrane, altered cell morphology and motility, and increased filopodia about threefold compared to GFP controls. In contrast, pathogenic mutants such as S280L and P92S failed to induce these effects. And we showed that wild-type MLC1 efficiently localizes to the plasma membrane, where it induces marked changes in cell morphology and motility, in contrast, pathogenic mutants remain trapped within the cell and are functionally inactive. Structural and biochemical analyses, including epitope mapping, crosslinking, electron microscopy, and single-molecule photobleaching, further revealed that MLC1 adopts an eight-transmembrane topology and assembles into oligomers. To gain structural insights, we performed cryo-EM analysis of human MLC1. The protein was expressed in HEK293S GnTI⁻ cells, purified under optimized conditions, and imaged by cryo-EM. Although a ~3.3 Å dataset was obtained, preferred particle orientations limited highresolution reconstruction. To address this issue, we tested strategies to improve orientation distribution, including the addition of trace CTAB to LMNG micelles and reconstitution into lipid nanodiscs, both of which enhanced particle orientation diversity and image quality.