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Escherichia coli contains operons called "addiction modules," encoding toxin and antitoxin, which are responsible for growth arrest and cell death. Here, we demonstrate that MazF toxin encoded by "mazEF addiction module" is a sequence-specific (ACA) endoribonuclease functional only for single-stranded RNA. MazF works as a ribonuclease independent of ribosomes, and is, therefore, functionally distinct from RelE, another E. coli toxin, which assists mRNA cleavage at the A site on ribosomes. Upon induction, MazF cleaves whole cellular mRNAs to efficiently block protein synthesis. Purified MazF inhibited protein synthesis in both prokaryotic and eukaryotic cell-free systems. This inhibition was released by MazE, the labile antitoxin against MazF. Thus, MazF functions as a toxic endoribonuclease to interfere with the function of cellular mRNAs by cleaving them at specific sequences leading to rapid cell growth arrest and cell death. The role of such endoribonucleases may have broad implication in cell physiology under various growth conditions.

Stromal interaction molecule-1 (STIM1) activates store-operated Ca2+ entry (SOCE) in response to diminished luminal Ca2+ levels. Here, we present the atomic structure of the Ca2+-sensing region of STIM1 consisting of the EF-hand and sterile alpha motif (SAM) domains (EF-SAM). The canonical EF-hand is paired with a previously unidentified EF-hand. Together, the EF-hand pair mediates mutually indispensable hydrophobic interactions between the EF-hand and SAM domains. Structurally critical mutations in the canonical EF-hand, "hidden" EF-hand, or SAM domain disrupt Ca2+ sensitivity in oligomerization via destabilization of the entire EF-SAM entity. In mammalian cells, EF-SAM destabilization mutations within full-length STIM1 induce punctae formation and activate SOCE independent of luminal Ca2+. We provide atomic resolution insight into the molecular basis for STIM1-mediated SOCE initiation and show that the folded/unfolded state of the Ca2+-sensing region of STIM is crucial to SOCE regulation.

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In a variety of cells, the Ca2+ signalling process is mediated by the endoplasmic-reticulum-membrane-associated Ca2+ release channel, inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R). Being ubiquitous and present in organisms ranging from humans to Caenorhabditis elegans, InsP3R has a vital role in the control of cellular and physiological processes as diverse as cell division, cell proliferation, apoptosis, fertilization, development, behaviour, memory and learning. Mouse type I InsP3R (InsP3R1), found in high abundance in cerebellar Purkinje cells, is a polypeptide with three major functionally distinct regions: the amino-terminal InsP3-binding region, the central modulatory region and the carboxy-terminal channel region. Here we present a 2.2-A crystal structure of the InsP3-binding core of mouse InsP3R1 in complex with InsP3. The asymmetric, boomerang-like structure consists of an N-terminal beta-trefoil domain and a C-terminal alpha-helical domain containing an 'armadillo repeat'-like fold. The cleft formed by the two domains exposes a cluster of arginine and lysine residues that coordinate the three phosphoryl groups of InsP3. Putative Ca2+-binding sites are identified in two separate locations within the InsP3-binding core.

CRYSTALLOGRAPHY OF BIOLOGICAL MACROMOLECULES these conserved regions suggests that the folding of CHM and GDI may be similar. Site-directed mutagenesis of a region defined by two of the SCRs which fmms a compact structure at the apex of domain I reveals a critical role for this region in the binding of Rab proteins. The crystal structure of GD I has been deten11ined using MIRAS method and refined to crys1:c1.llographic R-value of 19.8% and R-free 27.5% for a total of 4 30 residues of the molecule in ti1e asymmetlic unit. MS04.07b.03 COMPLEX THE STRUCTURE OF EF-Tu-EF-Ts SUGGESTS A MECHANISM FOR NUCLEOTIDE RELEASE.

The association of p120 catenin (p120) with the juxtamembrane domain (JMD) of the cadherin cytoplasmic tail is critical for the surface stability of cadherin-catenin cell-cell adhesion complexes. Here, we present the crystal structure of p120 isoform 4A in complex with the JMD core region (JMD(core)) of E-cadherin. The p120 armadillo repeat domain contains modular binding pockets that are complementary to electrostatic and hydrophobic properties of the JMD(core). Single-residue mutations within the JMD(core)-binding site of p120 abolished its interaction with E- and N-cadherins in vitro and in cultured cells. These mutations of p120 enabled us to clearly differentiate between N-cadherin-dependent and -independent steps of neuronal dendritic spine morphogenesis crucial for synapse development. NMR studies revealed that p120 regulates the stability of cadherin-mediated cell-cell adhesion by associating with the majority of the JMD, including residues implicated in clathrin-mediated endocytosis and Hakai-dependent ubiquitination of E-cadherin, through its discrete "dynamic" and "static" binding sites.