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Regulatory Role of One Critical Catalytic Loop of Polypeptide N‑Acetyl-Galactosaminyltransferase‑2 in Substrate Binding and Catalysis during Mucin-Type O‑Glycosylation

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journal contribution
posted on 23.10.2019, 17:04 by Jiaqi Tian, Feng Liu, Zhijue Xu, Jingjing Shi, Tao Liang, Yan Zhang, Lin-Tai Da
One of the dominant post-translational modifications in mammals is mucin-type (GalNAc-type) O-glycosylation initiated by polypeptide N-acetyl-galactosaminyltransferases (ppGalNAc-Ts), which is closely associated with many physiological and pathological conditions. An atomic-level understanding of the structural dynamics of one critical catalytic loop in ppGalNAc-Ts from an open to a closed state upon substrate binding, however, is still elusive. Here, by constructing a Markov state model based on extensive all-atom molecular dynamics (MD) simulations with an aggregated simulation time of ∼20 μs, we reveal, at atomistic resolution, the key metastable states of the catalytic loop in ppGalNAc-T2 during its closing/opening dynamics after donor substrate (UDP–GalNAc) binding. The overall catalytic-loop closing motion is estimated to take place at a timescale of ∼tens of μs, with the rate-limiting transition caused by some critical structural rearrangements within the catalytic loop region, coupled with the desolvation process. We find that the presence of UDP–GalNAc facilitates the formation of a stable interaction network with several nonloop residues (i.e., L204, V330, H145, and W331), which in turn promotes the loop closing. Moreover, the functional roles of several critical active-site residues were further evaluated through combining site-directed mutagenesis, high-performance liquid chromatography, MS/MS, and mutant MD simulations. It is intriguing to observe that the UDP–GalNAc binding and the following catalysis can tolerate all the tested loop-residue substitutions, some can even profoundly enhance the enzyme efficacy (i.e., H365A and F377A). More importantly, the catalytic loop is found to play a decisive role in regulating the glycosylation-site preferences. Our work provides the structural basis for the key regulatory factors that dictate the substrate loading and following catalysis during mucin-type O-glycosylation.

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