Peptidoglycan synthesis and turnover in relation to cell growth and division has been studied by using a new labeling method. This method involves the incorporation of fluorescently labeled peptidoglycan precursors into the cell wall by means of the cell-wall recycling pathway. We show that Escherichia coli is able to import exogenous added murein tripeptide labeled with N-7-nitro-2,1,3-benzoxadiazol-4-yl (AeK-NBD) into the cytoplasm where it enters the peptidoglycan biosynthesis route, resulting in fluorescent labels specifically located in the cell wall. When wild-type cells were grown in the presence of the fluorescent peptide, peptidoglycan was uniformly labeled in cells undergoing elongation. Cells in the process of division displayed a lack of labeled peptidoglycan at mid-cell. Analysis of labeling patterns in cell division mutants showed that the occurrence of unlabeled peptidoglycan is dependent on the presence of FtsZ, but independent of FtsQ and FtsI. Accumulation of fluorescence at the division sites of a triple amidase mutant (ΔamiABC) revealed that AeK-NBD is incorporated into septal peptidoglycan. AmiC was shown to be involved in the rapid removal of labeled peptidoglycan side chains at division sites in wild-type cells. Because septal localization of AmiC is dependent on FtsQ and FtsI, this points to the presence of another peptidoglycan hydrolase activity directly dependent on FtsZ.

Members of the CAP superfamily (Cysteine-rich secretory proteins, Antigen 5, and Pathogenesis-related 1 proteins) are characterized by the presence of a CAP domain that is defined by four sequence motifs and a highly conserved tertiary structure. A common structure–function relationship for this domain is hitherto unknown. A characteristic of several CAP proteins is their formation of amyloid-like structures in the presence of lipids. Here we investigate the structural modulation of Golgi-Associated plant Pathogenesis Related protein 1 (GAPR-1) by known interactors of the CAP domain, preceding amyloid-like aggregation. Using isothermal titration calorimetry (ITC), we demonstrate that GAPR-1 binds zinc ions. Zn2+ binding causes a slight but significant conformational change as revealed by CD, tryptophan fluorescence, and trypsin digestion. The Zn2+-induced conformational change was required for the formation of GAPR-1 oligomers and amyloid-like assemblies in the presence of heparin, as shown by ThT fluorescence and TEM. Molecular dynamics simulations show binding of Zn2+ to His54 and His103. Mutation of these two highly conserved residues resulted in strongly diminished amyloid-like aggregation. Finally, we show that proteins from the cysteine-rich secretory protein (CRISP) subfamily are also able to form ThT-positive structures in vitro in a heparin- and Zn2+-dependent manner, suggesting that oligomerization regulated by metal ions could be a common structural property of the CAP domain.

Golgi-Associated Plant Pathogenesis-Related protein 1 (GAPR-1) is a mammalian protein that belongs to the superfamily of plant pathogenesis related proteins group 1 (PR-1). GAPR-1 is a peripheral membrane-binding protein that strongly associates with lipid-enriched microdomains at the cytosolic leaflet of Golgi membranes. Little is known about the mechanism of GAPR-1 interaction with membranes. We previously suggested that dimerization plays a role in the function of GAPR-1 and here we report that phytic acid (inositol hexakisphosphate) induces dimerization of GAPR-1 in solution. Elucidation of the crystal structure of GAPR-1 in the presence of phytic acid revealed that the GAPR-1 dimer differs from the previously published GAPR-1 dimer structure. In this structure, one of the monomeric subunits of the crystallographic dimer is rotated by 28.5°. To study the GAPR-1 dimerization properties, we investigated the interaction with liposomes in a light scattering assay and by flow cytometry. In the presence of negatively charged lipids, GAPR-1 caused a rapid and stable tethering of liposomes. [D81K]GAPR-1, a mutant predicted to stabilize the IP6-induced dimer conformation, also caused tethering of liposomes. [A68K]GAPR-1 however, a mutant predicted to stabilize the non-rotated dimer conformation, is capable of binding to liposomes but did not cause liposome tethering. Our combined data suggest that the charge properties of the lipid bilayer can regulate GAPR-1 dynamics as a potential mechanism to modulate GAPR-1 function.

antibacterial activity against several Gram-positive bacteria but were less active on Gram-10 negative bacteria. The two compounds inhibit the activity of five GTs in the micromolar 11 range. Investigation of the mechanism of action shows that the compounds specifically target 12 peptidoglycan synthesis. Unexpectedly, despite the fact that the compounds were predicted to 13 bind to the GT active site, compound 5b was found to interact with the lipid II substrate via 14 the pyrophosphate motif. In addition, this compound showed a negatively charged 15 phospholipid-dependent membrane depolarization and disruption activity. These small 16 molecules are promising leads for the development of more active and specific compounds to 17 target the essential GT step in cell wall synthesis. 18 19 20

Golgi-Associated plant Pathogenesis Related protein 1 (GAPR-1) is a mammalian protein that is a member of the Cysteine-rich secretory proteins, Antigen 5 and Pathogenesis related proteins group 1 (CAP) superfamily of proteins. A role for the common CAP domain in the function of the diverse superfamily members has not been described so far. Here, we show by a combination of independent techniques including electron microscopy, Thioflavin T fluorescence, and circular dichroism that GAPR-1 has the capability to form amyloid-like fibrils in the presence of liposomes containing negatively charged lipids. Surprisingly, GAPR-1 was also shown to bind the amyloid-oligomer specific antibody A11 in the absence of lipids, indicating that GAPR-1 has an intrinsic tendency to form oligomers. This behavior is characteristic for proteins that interfere with Aβ aggregation and indeed we found that GAPR-1 effectively inhibited aggregation of Aβ(1-40) peptide. Immuno-dot blot analysis revealed that GAPR-1 binds to prefibrillar oligomeric Aβ structures during the early stages of fibril formation. Another CAP domain-containing protein, CRISP2, was also capable of forming fibrils, indicating that oligomerization and fibril formation is a shared characteristic between CAP family members. We suggest that the CAP domain may regulate protein oligomerization in a large variety of proteins that define the CAP superfamily.