Abstract
In all living things, complex carbohydrates play a vital role in numerous biological processes, from protein quality control to cell recognition, energy storage, cell wall formation and pathogenesis. Many of these processes are performed in topologically extracellular compartments or on the cell surface. From viruses to bacteria and higher vertebrates, a wide array of structurally diverse polysaccharides are exported through distinct secretion systems. These systems evolved to transport often long and charged sugar chains across hydrophobic membranes to their respective sites of action. During my doctoral work, I investigated two distinct and mechanistically unique polysaccharide secretion systems, hyaluronan synthase (HAS) and the bacterial capsular polysaccharides (CPS) ABC transporter KpsMT-KpsE. Both are involved in the export of hyaluronan (HA), an acidic heteropolysaccharide of alternating N-acetylglucosamine and glucuronic acid sugars. HA is ubiquitously found in the vertebrate extracellular matrix but also in some bacteria, playing crucial yet distinct roles in both. In higher organisms, HA modulates essential physiological processes in health and disease, including cell differentiation, tissue homeostasis, and angiogenesis, whereas in microbes, HA aids in capsule and biofilm formation, serves as nutrient storage and enhances virulence.
HAS is a remarkable small enzyme that combines bifunctional glycosyl-transferase activity with nascent polymer export. In Chapter 2, I present five cryo-electron microscopy structures of a viral HAS homolog at different states during substrate binding and initiation of polymer synthesis. Combined with biochemical analyses and molecular dynamics simulations, our data reveal how HAS selects its substrates, hydrolyzes the first substrate to prime the synthesis reaction, opens an HA-conducting transmembrane (TM) channel, ensures alternating substrate polymerization, and coordinates HA inside its TM pore. Our work proposes a detailed model for the formation of an acidic extracellular heteropolysaccharide and sheds unprecedented light on the multitasking enzyme responsible for the biosynthesis of an abundant and essential glycosaminoglycan in the human body.
In contrast, Gram-negative bacteria utilize a more complicated, multienzyme machinery to first synthesize and then export CPS. CPS are synthesized on a phospholipid anchor that facilitates its biosynthesis and secretion. In Chapter 3, I present my work on ABC transporter mediated CPS translocation, wherein I elucidate crucial steps of CPS recognition and secretion in Gram-negative bacteria. Through in vivo studies of HA encapsulation, I demonstrate that KpsMT exhibits broad substrate specificity, reveal that a KpsMT-KpsE complex is sufficient for CPS translocation across the inner bacterial membrane, and determine the distribution of CPS and its secretion system on the cell surface. Cryogenic electron microscopy analyses of a KpsMT-KpsE complex in six different states reveal a KpsE-encased ABC transporter, conformational rearrangements of KpsMT during ATP hydrolysis, and substrate recognition inside a lipid-exposed electropositive canyon. Our work provides the first atomistic model for CPS secretion.
