The crystal structure of annexin VI indicates relative rotation of the two lobes upon membrane binding

AbstractThe crystal structure of bovine liver annexin VI has been determined to low resolution by molecular replacement. The first lobe (domains 1–4) is rotated about 90° relative to the second lobe (domains 5–8). Since the same crystal form (P43, 68 × 68 × 205 Å) grew from (NH4)2SO4, polyethylene glycol, and sodium acetate with and without added calcium, this probably reflects the structure in solution. When bound to a lipid monolayer both lobes of annexin VI are coplanar. This implies a significant change in conformation upon binding to membranes

Expression of bovine annexin A4 in E. coli rescues cytokinesis blocked by beta-lactam antibiotics

Treatment of bacteria with beta-lactam antibiotics can impair the process of cytokinesis, the final step in cell division, leading to the formation of a filamentous form of the bacteria. The expression of a mammalian calcium-dependent, membrane-binding protein, bovine annexin A4, in E. coli was found to reverse the inhibitory effects on cytokinesis of the beta-lactam antibiotics ampicillin, piperacillin, and cephalexin. This novel activity of the annexin was blocked by mutation of calcium binding sites in the annexin, indicating roles for calcium binding to the annexin and the binding of the annexin to membranes in restoring cytokinesis. The filamentous form of the bacteria has been reported to be more resistant to phagocytosis by cells of the immune system in eukaryotic hosts. Therefore, expression of annexins in pathogenic bacteria, by promoting the breakdown of the bacterial filaments, might serve as an adjuvant to enhance the efficacy of beta-lactam antibiotics

Domain Formation in a Fluid Mixed Lipid Bilayer Modulated through Binding of the C2 Protein Motif

The role and mechanism of formation of lipid domains in a functional membrane have generally received limited attention. Our approach, based on the hypothesis that thermodynamic coupling between lipid−lipid and protein−lipid interactions can lead to domain formation, uses a combination of an experimental lipid bilayer model system and Monte Carlo computer simulations of a simple model of that system. The experimental system is a fluid bilayer composed of a binary mixture of phosphatidylcholine (PC) and phosphatidylserine (PS), containing 4% of a pyrene-labeled anionic phospholipid. Addition of the C2 protein motif (a structural domain found in proteins implicated in eukaryotic signal transduction and cellular trafficking processes) to the bilayer first increases and then decreases the excimer/monomer ratio of the pyrene fluorescence. We interpret this to mean that protein binding induces anionic lipid domain formation until the anionic lipid becomes saturated with protein. Monte Carlo simulations were performed on a lattice representing the lipid bilayer to which proteins were added. The important parameters are an unlike lipid−lipid interaction term and an experimentally derived preferential protein−lipid interaction term. The simulations support the experimental conclusion and indicate the existence of a maximum in PS domain size as a function of protein concentration. Thus, lipid−protein coupling is a possible mechanism for both lipid and protein clustering on a fluid bilayer. Such domains could be precursors of larger lipid−protein clusters (‘rafts'), which could be important in various biological processes such as signal transduction at the level of the cell membrane

Calcium-dependent regulation of tumour necrosis factor-alpha receptor signalling by copine.

The role of copines in regulating signalling from the TNF-alpha (tumour necrosis factor-alpha) receptor was probed by the expression of a copine dominant-negative construct in HEK293 (human embryonic kidney 293) cells. The construct was found to reduce activation of the transcription factor NF-kappaB (nuclear factor-kappaB) by TNF-alpha. The introduction of calcium into HEK293 cells either through the activation of muscarinic cholinergic receptors or through the application of the ionophore A23187 was found to enhance TNF-alpha-dependent activation of NF-kappaB. This effect of calcium was completely blocked by the copine dominant-negative construct. TNF-alpha was found to greatly enhance the expression of endogenous copine I, and the responsiveness of the TNF-alpha signalling pathway to muscarinic stimulation increased in parallel with the increased copine I expression. The copine dominant-negative construct also inhibited the TNF-alpha-dependent degradation of IkappaB, a regulator of NF-kappaB. All of the effects of the dominant-negative construct could be reversed by overexpression of full-length copine I, suggesting that the construct acts specifically through competitive inhibition of copine. One of the identified targets of copine I is the NEDD8-conjugating enzyme UBC12 (ubiquitin C12), that promotes the degradation of IkappaB through the ubiquitin ligase enzyme complex SCF(betaTrCP). Therefore the copine dominant-negative construct might inhibit TNF-alpha signalling by dysregulation or mislocalization of UBC12. Based on these results, a hypothesis is presented for possible roles of copines in regulating other signalling pathways in animals, plants and protozoa