Features critical for membrane binding revealed by DivIVA crystal structure

Abstract

Poster Sessions C217 spacer-and RPEL-actins, which bind weakly in solution. Cytoplasmic MRTF-A localization in resting fibroblasts requires spacer-actin binding. The bipartite importin {alpha-beta} binding site is buried in the pentameric assembly, explaining how elevated G-actin concentrations prevent MRTF-A nuclear import. The structures show how actin loads onto the RPEL domain, and reveal a molecular mechanism by which actin can control the activity of one of its ligands. The diazotrophic soil bacterium Azotobacter vinelandii utilizes a FeMo-cofactor containing nitrogenase to accomplish nitrogen fixation. As nitrogenase is required in large amounts, the cell extracts a lot of molybdenum (Mo) from the environment and stores it in a special Molybdenum Storage Protein (MoSto)[1] as polyoxometalate clusters [2]. MoSto crystallizes in a needle-like shape. Best crystals diffract to 1.6Å resolution. An X-ray crystal analysis of the Mo-loaded MoSto revealed different types of Mo-oxide based clusters some being covalently bound while others are not. These clusters are synthesized in an ATP-dependent process whose mechanism is not yet known. Invitro experiments showed that it is possible to entirely deplete MoSto from its metal clusters and subsequently reload it again. The depletion is a pH-driven triphasic process which can be varied with temperature and time of incubation [1]. MoSto can load up to 120 atoms per protein molecule. Further research is necessary to determine the mechanism how the clusters are built from monomolybdates and how their selective release from MoSto is organized.[1] J. Schemberg et al., ChemBioChem 2008, 9, 595-602 DivIVA is a highly conserved protein in Gram-positive bacteria that localizes at the poles and division sites, presumably through direct sensing of membrane curvature. The correct localization of the protein is essential for its biological function, which involve both the correct placement of the septum at midcell and also the segregation of the DNA into the pre-spore during sporulation. DivIVA deletion causes filamentous growth in Bacillus subtilis, whereas overexpression causes hyphal branching in Streptomyces coelicolor. The mechanism of subcellular DivIVA targeting and function remain unknown. We present data that demonstrates that DivIVA binds directly to bacteria membrane and explains how this protein shows a multifunctional behavior.We prove that DivIVA is a two independent domains protein linked by a flexible tag and both domains together are essential to fulfill protein localization and function. The crystal structure determination shows that both domains share a parallel coiled-coil folding. Surprisingly the N-terminal domain also includes crossed and intertwined loops between the two coiled-coil molecules, which is completely new and is involved in protein targeting in combination with the presence of a key hydrophobic residue surrounded by positively charged residues. To clarify N-terminal domain function in DivIVA membrane targeting we have used an in vivo and in vitr...

The Ser/Thr protein kinase AfsK regulates polar growth and hyphal branching in the filamentous bacteria Streptomyces

“…The function of B. subtilis DivIVA depends on direct interactions with MinJ and the nucleoid-associated protein RacA (15,43), and the function of S. coelicolor DivIVA is also likely to depend on the direct recruitment of other proteins to the cell poles (5,6). Further, crystal structures of B. subtilis DivIVA show how the oligomers may interact with the membrane via an exposed phenylalanine residue in the highly conserved N-terminal part of the protein (40), and the polar and septal targeting of the B. subtilis DivIVA appears to be explained by a preference of the oligomers for negatively curved membrane surfaces (15,44).…”

“…2). Although this C-terminal domain is not conserved outside of Streptomyces orthologues (10), it lies just downstream of the conserved second coiled-coil segment, which is known to be important in the oligomerization of B. subtilis DivIVA (40). It is therefore possible that the AfsK-mediated phosphorylation influences oligomerization, acting as a means to control the assembly or disassembly of multimeric complexes or higher-order structures formed by DivIVA in the cell.…”

“…No concomitant degradation or decrease in cellular DivIVA content is observed, suggesting that high AfsK activity causes disassembly of the DivIVA-containing apical polarisome. DivIVA is a self-assembling coiled-coil protein that forms oligomers and higher-order complexes and is involved in polar targeting in a range of Gram-positive bacteria (15,30,(38)(39)(40). The AfsK-mediated phosphorylation of DivIVA in Streptomyces occurs on two trypsin-generated fragments in the C-terminal domain (Fig.…”

Coiled-coil protein Scy is a key component of a multiprotein assembly controlling polarized growth inStreptomyces

“…Although most of the new branch points appear to be from convex hyphal curvatures (14), there are many more negative hyphal curvatures than emerging tips; thus, although membrane curvature might facilitate DivIVA recruitment, Streptomyces must have a mechanism to restrict DivIVA to selected sites only. We propose that it is the Scy assembly, which, by sequestering multiple DivIVA tetramers (42), regulates the number of DivIVA foci, and therefore controls the number of polarity centers in Streptomyces.…”

Caulobacter PopZ forms an intrinsically disordered hub in organizing bacterial cell poles

“…The C-terminal region of PopZ is analogous to the C-terminal region of DivIVA, in that both form homo-oligomers that undergo higher-order assembly into interconnected scaffold lattices (8,9). The middle region of PopZ is analogous to a cytoplasmic portion of HubP, in that both are acidic and proline-rich.…”