Unliganded and CMP-Neu5Ac bound structures of human α-2,6-sialyltransferase ST6Gal I at high resolution

Abstract

Sialic acid residues found as terminal monosaccharides in various types of glycan chains in cell surface glycoproteins and glycolipids have been identified as important contributors of cell-cell interactions in normal vs. abnormal cellular behavior and are pivotal in diseases such as cancers. In vertebrates, sialic acids are attached to glycan chains by a conserved subset of sialyltransferases with different enzymatic and substrate specificities. ST6Gal I is a sialyltransferase using activated CMP-sialic acids as donor substrates to catalyze the formation of a α2,6-glycosidic bond between the sialic acid residue and the acceptor disaccharide LacNAc. Understanding sialyltransferases at the molecular and structural level shed light into the function. We present here two human ST6Gal I structures, which show for the first time the enzyme in the unliganded state and with the full donor substrate CMP-Neu5Ac bound. Comparison of these structures reveal flexibility of the catalytic loop, since in the unliganded structure Tyr354 adopts a conformation seen also as an alternate conformation in the substrate bound structure. CMP-Neu5Ac is bound with the side chain at C-5 of the sugar residue directed towards empty space at the surface of the protein. Furthermore, the exact binding mode of the sialic acid moiety of the substrate directly involves sialylmotifs L, S and III and positions the sialylmotif VS in the immediate vicinity. .

Sialyltransferase Inhibitors Suppress Breast Cancer Metastasis

“…To further study the binding mode of the designed ST inhibitors, such as 2aA – 2eA , 3cB – 3dB , and 4cC – 4dC with respect to that of the reference inhibitors AL10, LCAG, and Lith- O -ASP in the ST6GALI structure (PDB 6QVT), we built a conformation of two asymmetric polypeptide chains (A/B) of human ST6GALI structure for molecular docking, minimization, and binding energy calculations. Except ST6GALI, the crystal structure of porcine ST3GALI (2WNF) was also used to construct the donor–acceptor conformation, but it was not successful during the minimization process .…”

Structural Insights in Mammalian Sialyltransferases and Fucosyltransferases: We Have Come a Long Way, but It Is Still a Long Way Down

“…Human ST sequences have low sequence similarity but share 10 invariant residues-five in motif L, two in motif S and VS, and one in motif III [23,27]. Motif L is mainly engaged in binding donor substrates, while sialylmotifs S, III, and VS are involved in binding acceptor substrates or both substrates [23,27,30,31]. Both L and S contain an invariant cysteine residue and participate in the formation of an intramolecular disulfide linkage essential for the active conformation of the enzyme [32,33].…”

“…The sequence consensuses of motifs III and VS in human STs are ((H/y)-Y-(Y/W/F/h)-(D/E/q/g)) and H-x4-E (where lowercase/capital letters imply low/strong occurrence of the amino acid), respectively [28,34]. Multiple sequence alignment of STs in vertebrates revealed the presence of family motifs containing four to twenty amino acids specific to each ST family, thus implicating another level of amino acid conservation among STs [29,31,35]. Except for ST6GALNAC, all STs contain four common amino acid sequences located eight amino acids downstream of the 3 -end of sialylmotif L, termed motif "a".…”

“…FUT3, FUT5, and FUT6 can transfer L-fucose (L-Fuc) to both non-sialylated and sialylated acceptors. FUT4 and FUT9 prefer non-sialylated type II acceptors, while FUT7 is specific for the sialylated type II acceptor [31,41,42]. FUT10 and FUT11 add L-Fuc onto the innermost GlcNAc residue of the chitobiose unit of biantennary N-glycans and are clearly distinguishable from the classical α1,3 FUTs (i.e., FUT3, FUT4, FUT5, FUT6, and FUT7), which utilize short and linear lactosamine-related acceptors [25,43].…”