Computationally guided insights into substrate recognition and specificity of Alg1

Abstract

AbstractThe assembly of N-linked glycans for the N-glycosylation, which is an important protein modification process, starts with the consecutive synthesis of the dolichol-linked oligosaccharide (DLO) precursor in the endoplasmic reticulum (ER). Asparagine-linked glycosylation protein 1 (Alg1) is the first mannosyltransferase (MTase) in DLO synthesis, which transfers a mannose residue from the sugar donor GDP-mannose (GDP-Man) to the substrate dolichol pyrophosphate-GlcNAc2 (Dol-PP-GlcNAc2) to form a β-1,4 glycosidic bond. However, little is known about its process of substrate recognition and the mechanism for its substrate specificity. Here, the models of large-scale systems in solution and membrane environments were built, revealing the substrate recognition and specificity mechanism of Alg1 based on extensive all-atom microsecond molecular dynamics (MD) simulations. In this study, the unique non-natural donor recognition ofScAlg1 and its mechanisms was reported. Further, the cooperation of the hydrophobic region and lysine in substrate recognition was revealed for bothScAlg1 andHsAlg1. Moreover, the functional roles of these sites were confirmed via mutant activity assays invitro. Additionally, the substrate binding process of Alg1 was simulated, elucidating the effect of dolichol on substrate specificity. This work reports the substrate recognition of Alg1 and reveals mechanisms for its sugar donor and substrate selectivity, which could further aid in mechanistic studies of the DLO biosynthetic process and drug development targeting Alg1.