Shape–function insights into bifunctional O-GlcNActransferase of Listeria monocytogenes EGD-e

Abstract

O-GlcNAcylation is an important post-translational modification of proteins. O-GlcNAcylated proteins have crucial roles in several cellular contexts both in eukaryotes and in bacteria. O-GlcNActransferase (OGT) is the enzyme instrumental in O-GlcNAcylation of proteins. OGT is conserved across eukaryotes. The first bacterial OGT discovered is GmaR in Listeria monocytogenes. GmaR is a GT-2 family bifunctional protein that catalyzes glycosylation of the flagellin protein FlaA and controls transcription of flagellar motility genes in a temperature-dependent manner. Here, we provide methods for heterologous expression and purification of recombinant GmaR and FlaA, in vivo/in vitro glycosylation assays, analysis of the molecular form of recombinant GmaR and detailed enzyme kinetics. We study the structure and functional dynamics of GmaR. Using solution small-angle X-ray scattering and molecular modeling, we show that GmaR adopts an extended shape with two distinctly spaced structural units in the presence of cofactor Mg2+ and with donor UDP-GlcNAc and cofactor combined. Comparisons of restored structures revealed that in-solution binding of Mg2+ ions brings about shape rearrangements and induces structural-rigidity in hyper-variable (HV) regions at the N-terminus of GmaR protein. Taking function and shape data together, we describe that Mg2+ binding enables GmaR to adopt a shape that can bind the substrate. The manuscript provides the first 3D solution structure of a bacterial OGT of GT-2 family and detailed biochemical characterization of GmaR to facilitate its future applications.

In Vitro Characterization of an O-Specific Glycosyltransferase Involved in Flagellin Glycosylation

“…The GT-A and TPR domains of FlgGT1 share a high degree of sequence identity with GmaR (31.9%), but the CTD has a lower degree of identity (15.5%). Solution small-angle X-ray scattering experiments have demonstrated that the C-terminal domain of GmaR undergoes conformational changes, adopting more extended structures upon interacting with Mg 2+ and UDP-GlcNAc . This structural rearrangement might be involved in binding of the acceptor flagellin substrate.…”

“…To ascertain the acceptor sequence preference of FlgGT1, sequence logos were generated for both the glycosylated and nonglycosylated residues within the D1 domain (Figure 3B). 28 Contrary to the preference for glycine residues at positions −1 and +1 adjacent to the glycosylation sites seen in glycocin glycosyltransferases, no such specificity was observed for FlgGT1, indicating its unique acceptor preference. To gain structural insights into the glycosylation sites, we calculated the accessible surface areas of nonglycosylated and glycosylated residues.…”

In Vitro Characterization of an O-Specific Glycosyltransferase Involved in Flagellin Glycosylation

“…The GT-A and TPR domains of FlgGT1 share a high degree of sequence identity with GmaR (31.9%), but the CTD has a lower degree of identity (15.5%). Solution small-angle X-ray scattering experiments have demonstrated that the C-terminal domain of GmaR undergoes conformational changes, adopting more extended structures upon interacting with Mg 2+ and UDP-GlcNAc . This structural rearrangement might be involved in binding of the acceptor flagellin substrate.…”

“…To ascertain the acceptor sequence preference of FlgGT1, sequence logos were generated for both the glycosylated and nonglycosylated residues within the D1 domain (Figure 3B). 28 Contrary to the preference for glycine residues at positions −1 and +1 adjacent to the glycosylation sites seen in glycocin glycosyltransferases, no such specificity was observed for FlgGT1, indicating its unique acceptor preference. To gain structural insights into the glycosylation sites, we calculated the accessible surface areas of nonglycosylated and glycosylated residues.…”

Demystifying the O-GlcNAc Code: A Systems View

“…Another close homologue of OGT (SeOGT) was identified from the cyanobacterium Synechococcus elongatus PCC 7942, showing active-site homology and similar domain structure to eukaryotic OGTs . Besides these, relatively distant OGT homologues have been identified, including the Gtf1-Gtf2 complex in Streptococcus parasanguinis , the GtfA-GtfB complex in Streptococcus pneumonia and Streptococcus gordonii , , the SdgA-SdgB complex in Staphylococcus aureus , and GmaR in Listeria monocytogenes . − As one of the first OGTs identified and characterized in prokaryotes, GmaR can specifically O-GlcNAcylates a prokaryotic protein. Of note, the distant homologues of eukaryotic OGT often have different structural and enzymatic properties.…”

“…Of note, the distant homologues of eukaryotic OGT often have different structural and enzymatic properties. For example, it has been found that GmaR adopts an extended shape with two distinctly spaced structural units and is a bifunctional protein (working as an OGT enzyme and an antirepressor). , TpeL (for toxin C. perfringens large cytotoxin), which is produced by several C. perfringens types (including types A–C), shows the capacity to use UDP-GlcNAc to modify several proteins. − In addition, there are bacteria that do not seem to have OGT homologues (or catalytic domains). One example is Escherichia coli , in which no putative OGTs have been identified.…”

Structural basis of flagellar motility regulation by the MogR repressor and the GmaR antirepressor in Listeria monocytogenes

“…GmaR is a dual-function protein that consists of 637 residues (Figure 1B and Supplementary Figure S2 ). The N-terminal part of GmaR (∼180 residues) contains a glycosyltransferase domain that catalyzes the transfer of N-acetyl glucosamine to the flagellar filament protein flagellin ( 14 , 19 ). The C-terminal part of GmaR (∼460 residues) is required to exert an antagonistic effect on MogR-mediated transcriptional repression ( 18 ).…”

In Vitro Characterization of an O-Specific Glycosyltransferase Involved in Flagellin Glycosylation

“…The GT-A and TPR domains of FlgGT1 share a high degree of sequence identity with GmaR (31.9%), but the CTD has a lower degree of identity (15.5%). Solution small-angle X-ray scattering experiments have demonstrated that the C-terminal domain of GmaR undergoes conformational changes, adopting more extended structures upon interacting with Mg 2+ and UDP-GlcNAc . This structural rearrangement might be involved in binding of the acceptor flagellin substrate.…”

“…To ascertain the acceptor sequence preference of FlgGT1, sequence logos were generated for both the glycosylated and nonglycosylated residues within the D1 domain (Figure 3B). 28 Contrary to the preference for glycine residues at positions −1 and +1 adjacent to the glycosylation sites seen in glycocin glycosyltransferases, no such specificity was observed for FlgGT1, indicating its unique acceptor preference. To gain structural insights into the glycosylation sites, we calculated the accessible surface areas of nonglycosylated and glycosylated residues.…”

Demystifying the O-GlcNAc Code: A Systems View

“…Another close homologue of OGT (SeOGT) was identified from the cyanobacterium Synechococcus elongatus PCC 7942, showing active-site homology and similar domain structure to eukaryotic OGTs . Besides these, relatively distant OGT homologues have been identified, including the Gtf1-Gtf2 complex in Streptococcus parasanguinis , the GtfA-GtfB complex in Streptococcus pneumonia and Streptococcus gordonii , , the SdgA-SdgB complex in Staphylococcus aureus , and GmaR in Listeria monocytogenes . − As one of the first OGTs identified and characterized in prokaryotes, GmaR can specifically O-GlcNAcylates a prokaryotic protein. Of note, the distant homologues of eukaryotic OGT often have different structural and enzymatic properties.…”

“…Of note, the distant homologues of eukaryotic OGT often have different structural and enzymatic properties. For example, it has been found that GmaR adopts an extended shape with two distinctly spaced structural units and is a bifunctional protein (working as an OGT enzyme and an antirepressor). , TpeL (for toxin C. perfringens large cytotoxin), which is produced by several C. perfringens types (including types A–C), shows the capacity to use UDP-GlcNAc to modify several proteins. − In addition, there are bacteria that do not seem to have OGT homologues (or catalytic domains). One example is Escherichia coli , in which no putative OGTs have been identified.…”

Structural basis of flagellar motility regulation by the MogR repressor and the GmaR antirepressor in Listeria monocytogenes

“…GmaR is a dual-function protein that consists of 637 residues (Figure 1B and Supplementary Figure S2 ). The N-terminal part of GmaR (∼180 residues) contains a glycosyltransferase domain that catalyzes the transfer of N-acetyl glucosamine to the flagellar filament protein flagellin ( 14 , 19 ). The C-terminal part of GmaR (∼460 residues) is required to exert an antagonistic effect on MogR-mediated transcriptional repression ( 18 ).…”

In Vitro Characterization of an O-Specific Glycosyltransferase Involved in Flagellin Glycosylation

“…The GT-A and TPR domains of FlgGT1 share a high degree of sequence identity with GmaR (31.9%), but the CTD has a lower degree of identity (15.5%). Solution small-angle X-ray scattering experiments have demonstrated that the C-terminal domain of GmaR undergoes conformational changes, adopting more extended structures upon interacting with Mg 2+ and UDP-GlcNAc . This structural rearrangement might be involved in binding of the acceptor flagellin substrate.…”

“…To ascertain the acceptor sequence preference of FlgGT1, sequence logos were generated for both the glycosylated and nonglycosylated residues within the D1 domain (Figure 3B). 28 Contrary to the preference for glycine residues at positions −1 and +1 adjacent to the glycosylation sites seen in glycocin glycosyltransferases, no such specificity was observed for FlgGT1, indicating its unique acceptor preference. To gain structural insights into the glycosylation sites, we calculated the accessible surface areas of nonglycosylated and glycosylated residues.…”

Demystifying the O-GlcNAc Code: A Systems View

“…Another close homologue of OGT (SeOGT) was identified from the cyanobacterium Synechococcus elongatus PCC 7942, showing active-site homology and similar domain structure to eukaryotic OGTs . Besides these, relatively distant OGT homologues have been identified, including the Gtf1-Gtf2 complex in Streptococcus parasanguinis , the GtfA-GtfB complex in Streptococcus pneumonia and Streptococcus gordonii , , the SdgA-SdgB complex in Staphylococcus aureus , and GmaR in Listeria monocytogenes . − As one of the first OGTs identified and characterized in prokaryotes, GmaR can specifically O-GlcNAcylates a prokaryotic protein. Of note, the distant homologues of eukaryotic OGT often have different structural and enzymatic properties.…”

“…Of note, the distant homologues of eukaryotic OGT often have different structural and enzymatic properties. For example, it has been found that GmaR adopts an extended shape with two distinctly spaced structural units and is a bifunctional protein (working as an OGT enzyme and an antirepressor). , TpeL (for toxin C. perfringens large cytotoxin), which is produced by several C. perfringens types (including types A–C), shows the capacity to use UDP-GlcNAc to modify several proteins. − In addition, there are bacteria that do not seem to have OGT homologues (or catalytic domains). One example is Escherichia coli , in which no putative OGTs have been identified.…”

Structural basis of flagellar motility regulation by the MogR repressor and the GmaR antirepressor in Listeria monocytogenes

“…GmaR is a dual-function protein that consists of 637 residues (Figure 1B and Supplementary Figure S2 ). The N-terminal part of GmaR (∼180 residues) contains a glycosyltransferase domain that catalyzes the transfer of N-acetyl glucosamine to the flagellar filament protein flagellin ( 14 , 19 ). The C-terminal part of GmaR (∼460 residues) is required to exert an antagonistic effect on MogR-mediated transcriptional repression ( 18 ).…”