Necroptosis is considered to be complementary to the classical caspase-dependent programmed cell death pathway, apoptosis. The pseudokinase Mixed Lineage Kinase Domain-Like (MLKL) is an essential effector protein in the necroptotic cell death pathway downstream of the protein kinase Receptor Interacting Protein . How MLKL causes cell death is unclear, however RIPK3-mediated phosphorylation of the activation loop in MLKL trips a molecular switch to induce necroptotic cell death. Here, we show that the MLKL pseudokinase domain acts as a latch to restrain the N-terminal four-helix bundle (4HB) domain and that unleashing this domain results in formation of a high-molecularweight, membrane-localized complex and cell death. Using alaninescanning mutagenesis, we identified two clusters of residues on opposing faces of the 4HB domain that were required for the 4HB domain to kill cells. The integrity of one cluster was essential for membrane localization, whereas MLKL mutations in the other cluster did not prevent membrane translocation but prevented killing; this demonstrates that membrane localization is necessary, but insufficient, to induce cell death. Finally, we identified a small molecule that binds the nucleotide binding site within the MLKL pseudokinase domain and retards MLKL translocation to membranes, thereby preventing necroptosis. This inhibitor provides a novel tool to investigate necroptosis and demonstrates the feasibility of using small molecules to target the nucleotide binding site of pseudokinases to modulate signal transduction.pseudoenzyme | RIP kinase | ATP mimetic | programmed necrosis P rogrammed necrosis or "necroptosis" has emerged in the past 5 years as a cell death mechanism that complements the conventional cell death pathway, apoptosis, in multicellular organisms. In contrast to apoptosis, necroptosis does not appear to serve an important role in multicellular organism development (1-3) but participates in the defense against pathogens and is a likely culprit in destructive inflammatory conditions (4-7). Receptor Interacting Protein Kinase-3 (RIPK3) was identified as a key effector of necroptosis in 2009 (4, 5) and its substrate, the pseudokinase Mixed Lineage Kinase Domain-Like (MLKL), in 2012 (8, 9), but the molecular events following RIPK3-mediated phosphorylation of MLKL required to induce cell death are unclear. The RIPK1/ RIPK3/MLKL necrosome has been proposed to activate PGAM5 (phosphoglycerate mutase 5) and Drp1 (Dynamin-related protein 1) to cause mitochondrial fragmentation and cell death (10), but the requirement for PGAM5, Drp1, and mitochondria for necroptosis has been questioned (1, 11-13).We described the structure of mouse MLKL revealing that MLKL contains a C-terminal pseudokinase domain and an N-terminal four-helix bundle (4HB) domain connected by a two-helix linker (the "brace" helices) (1). Based on our mutational and biochemical analyses, we proposed that the catalytically inactive pseudokinase domain functions as a molecular switch and that RIPK3-mediated phosphorylat...

During chromosome synthesis in Escherichia coli, replication forks are blocked by Tus bound Ter sites on approach from one direction but not the other. To study the basis of this polarity, we measured the rates of dissociation of Tus from forked TerB oligonucleotides, such as would be produced by the replicative DnaB helicase at both the fork-blocking (nonpermissive) and permissive ends of the Ter site. Strand separation of a few nucleotides at the permissive end was sufficient to force rapid dissociation of Tus to allow fork progression. In contrast, strand separation extending to and including the strictly conserved G-C(6) base pair at the nonpermissive end led to formation of a stable locked complex. Lock formation specifically requires the cytosine residue, C(6). The crystal structure of the locked complex showed that C(6) moves 14 A from its normal position to bind in a cytosine-specific pocket on the surface of Tus.

The thiol-disulfide oxidoreductase enzyme DsbA catalyzes the formation of disulfide bonds in the periplasm of Gram-negative bacteria. DsbA substrates include proteins involved in bacterial virulence. In the absence of DsbA, many of these proteins do not fold correctly, which renders the bacteria avirulent. Thus DsbA is a critical mediator of virulence and inhibitors may act as antivirulence agents. Biophysical screening has been employed to identify fragments that bind to DsbA from Escherichia coli. Elaboration of one of these fragments produced compounds that inhibit DsbA activity in vitro. In cell-based assays, the compounds inhibit bacterial motility, but have no effect on growth in liquid culture, which is consistent with selective inhibition of DsbA. Crystal structures of inhibitors bound to DsbA indicate that they bind adjacent to the active site. Together, the data suggest that DsbA may be amenable to the development of novel antibacterial compounds that act by inhibiting bacterial virulence.

We have identified a class of molecules, known as 2-aminothiazoles (2-ATs), as frequent-hitting fragments in biophysical binding assays. This was exemplified by 4-phenylthiazol-2-amine being identified as a hit in 14/14 screens against a diverse range of protein targets, suggesting that this scaffold is a poor starting point for fragment-based drug discovery. This prompted us to analyze this scaffold in the context of an academic fragment library used for fragment-based drug discovery (FBDD) and two larger compound libraries used for high-throughput screening (HTS). This analysis revealed that such "promiscuous 2-aminothiazoles" (PrATs) behaved as frequent hitters under both FBDD and HTS settings, although the problem was more pronounced in the fragment-based studies. As 2-ATs are present in known drugs, they cannot necessarily be deemed undesirable, but the combination of their promiscuity and difficulties associated with optimizing them into a lead compound makes them, in our opinion, poor scaffolds for fragment libraries.

The solution structures of three related peptides (A1, A2, and A9) corresponding to the Thr 671 -Leu 690 region of the skeletal muscle dihydropyridine receptor II-III loop have been investigated using nuclear magnetic resonance spectroscopy. Peptide A1, the native sequence, is less effective in activating ryanodine receptor calcium release channels than A2 (Ser 687 to Ala substitution and Asp 1112 has also been reported (8). The peptide A1 region may be necessary for the DHPR II-III loop to bind to the RyR, whereas other regions of the loop are necessary for the transmission of the ECC signal from the S4 part of the DHPR to the II-III loop/RyR binding region (7). Evidence for this hypothesis is that the cardiac DHPR can support skeletal type ECC when the peptide A region contains the cardiac sequence, provided that residues 725-742 contain the skeletal sequence (9), suggesting that (a) both cardiac and skeletal peptide A1 regions bind to the RyR, and (b) residues 725-742 are essential for transmission of the ECC (7). If this hypothesis is correct, activation of RyR channels by peptide A1 or by the II-III loop may reflect binding to the RyR, rather than activation equivalent to ECC. The hypothesis is supported by the fact that the full cardiac II-III loop activated skeletal RyRs (3, 4).Two regions of peptide A1 are important in its interaction with RyRs. The highly charged 681 RKRRK 685 sequence is essential for RyR activation by peptide A1 (6, 10), and Ser 687 is important, although its precise role is not clear. The II-III loop does not activate RyRs when Ser 687 is either phosphorylated or replaced by alanine (4). In contrast, replacement of Ser 687 with Ala increases the ability of peptide A1 to activate RyRs (7), whereas a similar replacement in a 25-amino acid peptide (Glu 666 -Pro 690 ) reduces [ 3 H]ryanodine binding (11). The different effects of Ser 687 to Ala substitution are likely to reflect structural differences between peptides of different lengths. The structure of the protein-protein interaction sites on the II-III loop and on the RyR must be understood in order to comprehend these functional observations and the structural changes that could occur during ECC. Neither structure has been determined.Here, we address the structural basis for Ca 2ϩ release from the SR by the native DHPR sequence from Thr 671 -Leu 690 (peptide A1) and the enhanced ability of peptide A2 (S687A substitution) to release Ca 2ϩ from skeletal SR and to activate skeletal RyRs. We determined the solution structures for three peptides using NMR spectroscopy. These peptides are a small, sevenresidue peptide encompassing the basic region essential for RyR activation (A9), peptide A1 (containing Ser 687 ), and peptide A2 (containing a S687A substitution). The results show that the smaller A9 peptide is unstructured, whereas both peptides A1 and A2 have a propensity to form helical structures at their N-terminal end. The C-terminal part of A1 is highly mobile, whereas the C-terminal part of A2 is more constrained. Because we find...