Structural And Catalytic Insights Into A Novel O-GlcNAc Transferase Complex GtfA-GtfB From Streptococcus Pneumoniae

Protein glycosylation catalyzed by the O-G1cNAc transferase (OGT) plays a critical role in various biological processes in not only eukaryotes but also prokaryotes. In human, aberrant O-GlcNAcylations are correlated with many challenging diseases, including diabetes, cardiovascular disease, neurodegenerative diseases and cancer. Whereas, the O-GlcNAcylation of glycoprotein in bacteria is closely related with the physiology and pathogenesis, especially for the pathogenic bacterial strains.Notably, serine-rich repeat glycoproteins (SRRPs) that exclusively exist in Gram-positive pathogens are a large family of surface adhesins, which are important for bacterial adhesion, immune evasion, colonization, biofilm formation and virulence. It was identified an enzyme complex, O-GlcNAc transferase (termed OGT) GtfA and its partner GtfB from Streptococcus pneumoniae, is required for the initial step of glycosylation of the serine-rich repeat protein (PsrP) by transferring the GlcNAc residue from UDP-GlcNAc to PsrP.In this manuscript, we determined the crystal structure of GtfA in complex with GlcNAc and UDP at2.0A resolution. The GtfA structure comprises a core structure of GT-B fold and a β-meander "add-on" DUF1975domain, which reveals a novel structure of a twisted10-stranded antiparallel β-sheet for the first time. Despite of the distinct structure from the all-a tetratricopeptide repeat (TPR) units of only two structure-known OGTs (hOGT and XcOGT), DUF1975also plays a role in acceptor binding or recognition. The in vitro and in vivo O-GlcNAcylation assays proved that DUF1975domain of GtfA involves the recognition of the acceptor PsrP and its co-activator GtfB; thus is critical for the intact OGT activity. Further glycoproteomic analyses revealed that the intact GtfA-GtfB complex was specific to modify the serine residues of PrsP, and all serine could be O-GlcNAcylated. Furthermore, in order to elucidate the mechanism of PrsP O-GlcNAcylation, we also crystallized Apo GtfB as well as GtfA-GtfB complex in UDP and GlcNAc binding form which diffracted to7.0and4.5respectivly. At the meantime, a docking model of GtfA with the N-terminal18-residue peptide of the first serine-rich repeat region (SRR1) of PsrP was performed, suggesting a rational retaining catalytic mechanism that is distinguished from two structure-known inverting OGTs. Take together, our structural and biochemical study provides insights into a novel retaining O-GlcNAc transferase complex GtfA-GtfB. We are firstly unraveled the catalytic subunit GtfA structurally, and identified a novel β-meander "add-on" DUF1975domain, which is a mediator essential to protein interaction or recognition. In addition, we are firstly elucidated GtfA-GtfB is a serine-specific OGT, despite adopting a core structure of GtfA similar to that in the GT4family, GtfA belongs to EC2.4.1.255according to the catalytic activity. Our results provide a structural basis for the rational design of therapeutic drugs and new vaccine against pneumococcal related diseases.