Mapping the architecture of the initiating phosphoglycosyl transferase from S. enterica O-antigen biosynthesis in a liponanoparticle.

Bacterial cell surface glycoconjugates are critical for cell survival and for interactions between bacteria and their hosts. Consequently, the pathways responsible for their biosynthesis have untapped potential as therapeutic targets. The localization of many glycoconjugate biosynthesis enzymes to the membrane represents a significant challenge for expressing, purifying, and characterizing these enzymes. Here, we leverage cutting-edge detergent-free methods to stabilize, purify, and structurally characterize WbaP, a phosphoglycosyl transferase (PGT) from the Salmonella enterica (LT2) O-antigen biosynthesis. From a functional perspective, these studies establish WbaP as a homodimer, reveal the structural elements responsible for dimerization, shed light on the regulatory role of a domain of unknown function embedded within WbaP, and identify conserved structural motifs between PGTs and functionally unrelated UDP-sugar dehydratases. From a technological perspective, the strategy developed here is generalizable and provides a toolkit for studying other classes of small membrane proteins embedded in liponanoparticles beyond PGTs.

Mapping the architecture of the initiating phosphoglycosyl transferase from S. enterica O-antigen biosynthesis in a liponanoparticle.

Bacterial cell surface glycoconjugates are critical for cell survival and for interactions between bacteria and their hosts. Consequently, the pathways responsible for their biosynthesis have untapped potential as therapeutic targets. The localization of many glycoconjugate biosynthesis enzymes to the membrane represents a significant challenge for expressing, purifying, and characterizing these enzymes. Here, we leverage cutting-edge methods to stabilize, purify, and structurally characterize WbaP, a phosphoglycosyl transferase (PGT) from Salmonella enterica (LT2) O-antigen biosynthesis without detergent solubilization from the lipid bilayer. From a functional perspective, these studies establish WbaP as a homodimer, reveal the structural elements responsible for oligomerization, shed light on the regulatory role of a domain of unknown function embedded within WbaP, and identify conserved structural motifs between PGTs and functionally unrelated UDP-sugar dehydratases. From a technological perspective, the strategy developed here is generalizable and provides a toolkit for studying small membrane proteins embedded in liponanoparticles beyond PGTs.

Co-conserved sequence motifs are predictive of substrate specificity in a family of monotopic phosphoglycosyl transferases.

Monotopic phosphoglycosyl transferases (monoPGTs) are an expansive superfamily of enzymes that catalyze the first membrane-committed step in the biosynthesis of bacterial glycoconjugates. MonoPGTs show a strong preference for their cognate nucleotide diphospho-sugar (NDP-sugar) substrates. However, despite extensive characterization of the monoPGT superfamily through previous development of a sequence similarity network comprising >38,000 nonredundant sequences, the connection between monoPGT sequence and NDP-sugar substrate specificity has remained elusive. In this work, we structurally characterize the C-terminus of a prototypic monoPGT for the first time and show that 19 C-terminal residues play a significant structural role in a subset of monoPGTs. This new structural information facilitated the identification of co-conserved sequence "fingerprints" that predict NDP-sugar substrate specificity for this subset of monoPGTs. A Hidden Markov model was generated that correctly assigned the substrate of previously unannotated monoPGTs. Together, these structural, sequence, and biochemical analyses have delivered new insight into the determinants guiding substrate specificity of monoPGTs and have provided a strategy for assigning the NDP-sugar substrate of a subset of enzymes in the superfamily that use UDP-di-N-acetyl bacillosamine. Moving forward, this approach may be applied to identify additional sequence motifs that serve as fingerprints for monoPGTs of differing UDP-sugar substrate specificity.

Probing Monotopic Phosphoglycosyl Transferases from Complex Cellular Milieu.

Monotopic phosphoglycosyl transferase enzymes (monoPGTs) initiate the assembly of prokaryotic glycoconjugates essential for bacterial survival and proliferation. MonoPGTs belong to an expansive superfamily with a diverse and richly annotated sequence space; however, the biochemical roles of most monoPGTs in glycoconjugate biosynthesis pathways remain elusive. To better understand these critical enzymes, we have implemented activity-based protein profiling (ABPP) probes as protein-centric, membrane protein compatible tools that lay the groundwork for understanding the activity and regulation of the monoPGT superfamily from a cellular proteome. With straightforward gel-based readouts, we demonstrate robust, covalent labeling at the active site of various representative monoPGTs from cell membrane fractions using 3-phenyl-2H-azirine probes.

Drosophila emerins control LINC complex localization and transcription to regulate myonuclear position.

Mispositioned nuclei are a hallmark of skeletal muscle disease. Many of the genes that are linked to Emery-Dreifuss muscular dystrophy (EDMD) encode proteins that are critical for nuclear movement in various cells, suggesting that disruptions in nuclear movement and position may contribute to disease progression. However, how these genes are coordinated to move nuclei is not known. Here, we focussed on two different emerin proteins in Drosophila, Bocksbeutel and Otefin, and their effects on nuclear movement. Although nuclear position was dependent on both, elimination of either Bocksbeutel or Otefin produced distinct phenotypes that were based in differential effects on the KASH-domain protein Klarsicht. Specifically, loss of Bocksbeutel reduced Klarsicht localization to the nucleus and resulted in a disruption in nuclear separation. Loss of Otefin increased the transcription of Klarsicht and led to premature separation of nuclei and their positioning closer to the edge of the muscle. Consistent with opposing functions, nuclear position is normal in otefin; bocksbeutel double mutants. These data indicate emerin-dependent regulation of Klarsicht levels in the nuclear envelope is a critical determinant of nuclear position.

Arg-513 and Leu-531 Are Key Residues Governing Time-Dependent Inhibition of Cyclooxygenase-2 by Aspirin and Celebrex.

Aspirin and Celebrex are well-known time-dependent inhibitors of the cyclooxygenases (COX). Molecular dynamics simulations suggest that Arg-513 and Leu-531 contribute to the structural mechanisms of COX inhibition. We used mutagenesis and functional analyses to characterize how substitutions at these positions influence time-dependent inhibition by aspirin and Celebrex. We show that substitutions of Leu-531 with asparagine and phenylalanine significantly attenuate time-dependent inhibition of COX-2 by these drugs. The introduction of side chain bulk, rigidity, and charge would disrupt the formation of the initial noncovalent complex, in the case of aspirin, and the "high-affinity" binding state, in the case of Celebrex. Substitution of Arg-513 with histidine (the equivalent residue in COX-1) resulted in a 2-fold potentiation of aspirin inhibition, in support of the hypothesis that the presence of histidine in COX-1 lowers the activation barrier associated with the formation of the initial noncovalent enzyme-inhibitor complex. As a corollary, we previously hypothesized that the flexibility associated with Leu-531 contributes to the binding of arachidonic acid (AA) to acetylated COX-2 to generate 15R-hydroxyeicosatetraenoic acid (15R-HETE). We determined the X-ray crystal structure of AA bound to Co3+-protoporphyrin IX-reconstituted V349I murine COX-2 (muCOX-2). V349I muCOX-2 was utilized as a surrogate to trap AA in a conformation leading to 15R-HETE. AA binds in a C-shaped pose, facilitated by the rotation of the Leu-531 side chain. Ile-349 is positioned to sterically shield antarafacial oxygen addition at carbon-15 in a manner similar to that proposed for the acetylated Ser-530 side chain.

Emery-Dreifuss muscular dystrophy-linked genes and centronuclear myopathy-linked genes regulate myonuclear movement by distinct mechanisms.

Muscle cells are a syncytium in which the many nuclei are positioned to maximize the distance between adjacent nuclei. Although mispositioned nuclei are correlated with many muscle disorders, it is not known whether this common phenotype is the result of a common mechanism. To answer this question, we disrupted the expression of genes linked to Emery-Dreifuss muscular dystrophy (EDMD) and centronuclear myopathy (CNM) in Drosophila and evaluated the position of the nuclei. We found that the genes linked to EDMD and CNM were each necessary to properly position nuclei. However, the specific phenotypes were different. EDMD-linked genes were necessary for the initial separation of nuclei into distinct clusters, suggesting that these factors relieve interactions between nuclei. CNM-linked genes were necessary to maintain the nuclei within clusters as they moved toward the muscle ends, suggesting that these factors were necessary to maintain interactions between nuclei. Together these data suggest that nuclear position is disrupted by distinct mechanisms in EDMD and CNM.