Structural and mechanistic themes in glycoconjugate biosynthesis at membrane interfaces

Abstract

Peripheral and integral membrane proteins feature in stepwise assembly of complex glycans and glycoconjugates. Catalysis on membrane-bound substrates features challenges with substrate solubility and active-site accessibility. However, advantages in enzyme and substrate orientation and control of lateral membrane diffusion provide order to the multistep processes. Recent glycosyltransferase (GT) studies show that substrate diversity is met by the selection of folds which do not converge upon a common mechanism. Examples of polyprenol phosphate phosphoglycosyl transferases (PGTs) highlight that divergent fold families catalyze the same reaction with different mechanisms. Lipid A biosynthesis enzymes illustrate that variations on the robust Rossmann fold allow substrate diversity. Improved understanding of GT and PGT structure and function holds promise for better function prediction and improvement of therapeutic inhibitory ligands.

Figure 1

Structures and transformations of enzymes in glycan and glycoconjugate biosynthesis (approximate location with respect to membrane shown with yellow lines). (a) Polyprenol phosphate glycosyl transferase exemplified by Dol-P-Man synthase. Glycosyl transfer by from a GDP-Man donor to Dol-P by a GT-A fold transferase on the cytoplasmic face of the ER membrane, followed the action of a flippase to translocate the Dol-P-Man to the ER lumen. Ribbon diagram of Dol-PMan and GDP bound to P. furiosus DPMS (5MM1) with Mg²⁺ superimposed from a related structure (5MLZ). (b) Monotopic and polytopic phosphoglycosyl transferases. Left: The monotopic PGT PglC (5W7L) from C. concisus transfers a C1-phosphosugar from UDP-diNAcBac to Und-P at the inception of the pgl pathway for N-linked protein glycosylation in Campylobacter species. Right: The polytopic PGT MraY from A. aeolicus (4J72) transfers MurNAc-pentapeptide to Und-P. All PGTs use a common pool of Und-P. (c) Cellulose synthase bound to nascent cellulose polymer and Mg²⁺. Cellulose synthase from Rhodobacter sphaeroides (5EJZ) includes a BcsA subunit with both a cytosolic GT-A fold domain and a transmembrane domain supporting channel function. The essential periplasmic BcsB domain caps and contributes a single helix to the transmembrane domain. (d) Elaboration of lipid A intermediate by WaaC. WaaC from Escherichia coli (6DFE) is a GT-B fold GT that catalyzes heptose transfer to the Kdo₂-lipid A intermediate. The transfer occurs at a distance above the membrane to accommodate the Kdo_2-lipid A structure.

Figure 2

Topologies of Pren-P glycosyltransferases. (a) Topologies of Type I, Type II and Type III DPMS enzymes from diverse organisms and GtrB from Synechocystis. The number of amino acids in each soluble domain is denoted. (b) Secondary structure schematic of the P. furiosus Type III DPMS highlighting transmembrane, juxtamembrane, and soluble domain helices (5MM1).

Figure 3. Comparison of MraY with Mg²⁺ cofactor and complexed to muraymycin D2 (MD2) shows plasticity of the binding site.

(a) Close up of MraY with Mg²⁺ cofactor shown as green sphere with essential acidic residues shown as sticks (4J72). Image shows van der Waals surfaces on cartoon rendition. (b) Close up of MraY complexed with muraymycin D2 (5CKR) in exact alignment with (a). Image shows van der Waals surfaces on cartoon rendition and illustrates conformity of MraY to antibiotic shape and lack of Mg²⁺ occupancy. (c) Chemical structure of MD2. (d) Analysis of MD2 binding to MraY (prepared using LigPlot⁺ [31])

Figure 4. Model of cellulose biosynthesis

Rhodobacter sphaeroides BscA and BscB subunits (5EIY) are shown as ribbon diagrams (periplasmic domain, grey; transmembrane domain, blue; glycosyltransferase domain, light blue; bent helix, dark blue). The motion of a gating loop (red) and finger helix (orange) allow coordination of bond formation (elongation) and translocation, effecting polymer secretion (a-d). [Note that in all panels the gating loop and helix are schematized to emphasize the conformational cycle and shown with respect to the 5EIY structure]. Cellulose synthase from crystals incubated with non-hydrolyzable substrate analogue (UDPCH₂-Glc) plus a galactose-capped polymer yielded the substrate-bound complex with finger helix up and gating loop contacting substrate UDP-glucose (depicted a purple and green hexagons) (a). The product complex after glycosyl transfer obtained by first elongating with 2-fluoro-glucose then incubating with UDP/Mg²⁺ shows the finger helix and gating loop remain in position (b). The pre-translocation complex observed from crystals incubated with UDP-Glc in the absence of Mg²⁺ allowing addition of a single glycan unit shows UDP release and retraction of the gating loop (c). The finger helix moves to the up position in its post translocation conformation (d) and the gating loop moves in either concomitant with or following UDP-Glc binding. During translocation, hydrophobic residues lining the channel enforce planarity, causing rotation of the newly added glycan, creating the “dimeric” chemical structure of the repeating unit of cellulose (interactions analyzed in [42]).

Figure 5. Enzymes of lipid A modification and biosynthesis

(a) Ribbon depiction of Cupriavidus metallidurans ArnT (5F15) with bound Und-P (green sticks). The approximate location of the UndP phosphoryl group with respect to membrane (shown in yellow lines) highlights the active-site location at the membrane interface. Comparison of ribbon diagrams of (b) Aquifex aeolicus WaaA (2XCU) and (c) Raoultella terrigena WbbB (5FA1) in complex with CMP (green sticks) shows the insertions into the two GT-B fold Rossmann domains (blue and orange) in WbbB that afford distinct substrate specificity.