Molecular Basis of Lipid-linked Sugar Transfer

Project Summary The majority of synthesized proteins undergo glycosylation, a process by which a carbohydrate, or glycan, is attached onto the protein surface serving both structural and functional roles. Proper glycosylation in the lumen of the endoplasmic reticulum requires membrane accessible sugars, primarily mannose, in order to synthesize complex, branched oligosaccharides for transfer onto nascent peptides. To traffic sugars along the membrane, they must be transferred onto lipid polyprenyl carriers, namely dolichol phosphate. The synthesis of these mannose glycosyl carriers by the Dolichol Phosphate Mannose 1 (DPM1) represents the primarily mechanism of glycosyl donor biosynthesis, a pathway that is subsequently disrupted in severe types of Continental Disorders of Glycosylation (CDGs). Since sugars play a critical role in folding, stability, and affect the function of numerous proteins across several organ systems, DPM1 causing CDGs result in severe development delays, seizures, liver disease, and neuromuscular impairment. The molecule mechanism of DPM1 remains unsolved, largely due to a lack of structural information about its interaction with both the sugar and polyprenyl substrate which need to be mediated towards an active site that lies just outside the membrane. Existing structural studies have focused on a bacterial enzyme homolog, GtrB, which provides a model for understanding DPM1 and CDG. Although GtrB has been structurally characterized, significant questions remain about the binding sites and conformational changes required for its enzymatic activity. Specifically, we lack a model by which a hydrophobic polyprenyl carrier is brought into proximity to the cytosolic sugar substrate and if a conformational change is needed within the membrane region of GtrB. Here, we propose a time-resolved cryo-EM approach to study the enzymatic states of GtrB catalysis. The approach involves a 'flash and freeze' system using chemically-caged substrates to rapidly initiate enzyme activity during sample preparation with UV light. This method is designed to overcome the limitations of traditional cryo-EM in capturing transient and high-energy states. By applying this technique, the project will identify and analyze various structural states of GtrB, including its holo, intermediate, and product-bound forms, thereby providing a detailed model of glycolipid biosynthesis. The collected model will be integrated with molecular dynamics to provide a holistic model of catalysis that is experimentally and computationally validated. We observe that to mediate polyprenyl coordination, there is a major shift in two peripheral membrane helices in order to lower the lipid carrier into the cytosolic active site. Several mutations in DPM1 that cause CDG can be mapped to this region, indicating a conserved mechanism critical for product formation. We will investigate the kinetic effects of these mutations by monitoring the rate of product formation in a native-liposome environment. Through these investigations, the project aims to enhance our understanding of glycosylation and its disruption in genetic disorders.