T-cell immunoglobulin and mucin-domain containing protein (TIM) structural dynamics revealed by molecular dynamics simulations.

Abstract

Mucin-domain glycoproteins are characterized by extremely dense O-glycosylation that contributes to a unique, bottle-brush secondary structure which can extend away from the cell surface or form extracellular gel-like secretions. Mucin-type O-glycans are characterized by an initiating α-N-acetylgalactosamine (α-GalNAc) that is further elaborated into several core structures containing sialylation, fucosylation and/or ABO blood group antigens. As a result, mucin domains serve as highly heterogeneous swaths of glycosylation that exert both biophysical and biochemical influence on the cellular milieu. In particular, the T-cell immunoglobulin and mucin-domain containing family of proteins (TIM-1, 3, 4) decorate immune cells and act as key checkpoint inhibitors in cancer. However, their dense O-glycosylation remains enigmatic both in glycoproteomic landscape and structural dynamics, primarily due to the challenges associated with studying intrinsically disordered mucin domains. Through the discovery and use of a novel mucinase that selectively cleaves along the mucin glycoprotein backbone, we fully characterize the glycoproteomic landscape and construct the first all-atom computational model of TIM3 and TIM4. We probe how glycosylation, both type and density, affects structural and dynamical features of these proteins. Overall, we present a powerful computational workflow to better understand the detailed molecular structure of the mucinome.