Components of the chromatin remodelling switch/sucrose nonfermentable (SWI/SNF) complex are recurrently mutated in tumors, suggesting that altering the activity of the complex plays a role in oncogenesis. However, the role that the individual subunits play in this process is not clear. We set out to develop an inhibitor compound targeting the bromodomain of BRD9 in order to evaluate its function within the SWI/SNF complex. Here, we present the discovery and development of a potent and selective BRD9 bromodomain inhibitor series based on a new pyridinone-like scaffold. Crystallographic information on the inhibitors bound to BRD9 guided their development with respect to potency for BRD9 and selectivity against BRD4. These compounds modulate BRD9 bromodomain cellular function and display antitumor activity in an AML xenograft model. Two chemical probes, BI-7273 (1) and BI-9564 (2), were identified that should prove to be useful in further exploring BRD9 bromodomain biology in both in vitro and in vivo settings.

The specific and rapid formation of protein complexes is essential for diverse cellular processes such as remodeling of actin filaments in response to the interaction between Rho GTPases and the Wiskott-Aldrich syndrome proteins (WASp and N-WASp). Although Cdc42, TC10, and other members of the Rho family have been implicated in binding to and activating the WAS proteins, the exact nature of such a protein-protein recognition process has remained obscure. Here, we describe a mechanism that ensures rapid and selective long-range Cdc42-WASp recognition. The crystal structure of TC10, together with mutational and bioinformatic analyses, proved that the basic region of WASp and two unique glutamates in Cdc42 generate favorable electrostatic steering forces that control the accelerated WASp-Cdc42 association reaction. This process is a prerequisite for WASp activation and a critical step in temporal regulation and integration of WASp-mediated cellular responses.

Rac1b was recently identified in malignant colorectal tumors as an alternative splice variant of Rac1 containing a 19-amino acid insertion next to the switch II region. The structures of Rac1b in the GDP-and the GppNHp-bound forms, determined at a resolution of 1.75 Å, reveal that the insertion induces an open switch I conformation and a highly mobile switch II. As a consequence, Rac1b has an accelerated GEF-independent GDP/GTP exchange and an impaired GTP hydrolysis, which is restored partially by GTPase-activating proteins. Interestingly, Rac1b is able to bind the GTPasebinding domain of PAK but not full-length PAK in a GTP-dependent manner, suggesting that the insertion does not completely abolish effector interaction. The presented study provides insights into the structural and biochemical mechanism of a self-activating GTPase.

Roles of NapF, NapG and NapH, subunits of the Escherichia coli periplasmic nitrate reductase, in ubiquinol oxidation that NapG and H, but not NapF, are essential for electron transfer from ubiquinol to NapAB. The decreased yield of biomass resulting from loss of NapF in a Ubi + Men+ strain implicates NapF in an energyconserving role coupled to the oxidation of ubiquinol. We propose that NapG and H form an energyconserving quinol dehydrogenase functioning as either components of a proton pump or in a Q cycle, as electrons are transferred from ubiquinol to NapC. IntroductionEnergy-conserving electron transfer pathways in enteric bacteria are usually depicted as a series of substratespecific dehydrogenases feeding electrons into a common quinone pool, from which they are transferred via specific quinol dehydrogenases to cytochrome oxidases during aerobic growth or terminal reductases during anaerobic growth. This is clearly an oversimplification, however, because there are three types of functional quinone in Escherichia coli, ubiquinone 8 (UQ) and the naphthoquinones demethylmenaquinone (DMK) and menaquinone (MK). UQ is generally regarded as the 'aerobic' quinone in the sense that ubiquinone is far more abundant than MK and DMK during aerobic growth (Wallace and Young, 1977;Wissenbach et al., 1992;Soballe and Poole, 1999). Furthermore, UQ is essential for succinoxidase activity. Conversely, the naphthoquinone pool is essential for anaerobic respiration using nitrite, fumarate, dimethyl sulphoxide (DMSO) or trimethylamine N-oxide (TMAO) (Wissenbach et al., 1990;1992;Tyson et al., 1997). The selectivity of quinones for specific electron donors or acceptors can be explained by the difference in mid-point redox potential between the UQ/UQH 2 couple (E m,7 = +113 mV) and the MK/MKH 2 couple (E m,7 = -74 mV) (Soballe and Poole, 1999). In addition, there may also be structural constraints that limit the enzymes of the respiratory chain to binding a specific quinone.Nitrate respiration in E. coli has a unique position as electrons from both UQH 2 and MKH 2 , but not DMKH 2 , can be used for nitrate reduction (Wissenbach et al., 1990;1992;Tyson et al., 1997). E. coli expresses three nitrate reductases. Two of them, nitrate reductases A and Z, are membrane bound and reduce nitrate in the cytoplasm. SummaryThe nap operon of Escherichia coli K-12, encoding a periplasmic nitrate reductase (Nap), encodes seven proteins. The catalytic complex in the periplasm, NapA-NapB, is assumed to receive electrons from the quinol pool via the membrane-bound cytochrome NapC. Like NapA, B and C, a fourth polypeptide, NapD, is also essential for Nap activity. However, none of the remaining three polypeptides, NapF, G and H, which are predicted to encode non-haem, iron-sulphur proteins, are essential for Nap activity, and their function is currently unknown. The relative rates of growth and electron transfer from physiological substrates to Nap have been investigated using strains defective in the two membrane-bound nitrate reductases, and als...

As an alternative pathway of controlled cell death, necroptosis can be triggered by tumor necrosis factor via the kinases RIPK1/RIPK3 and the effector protein mixed-lineage kinase domain-like protein (MLKL). Upon activation, MLKL oligomerizes and integrates into the plasma membrane via its executioner domain. Here, we present the X-ray and NMR costructures of the human MLKL executioner domain covalently bound via Cys86 to a xanthine class inhibitor. The structures reveal that the compound stabilizes the interaction between the auto-inhibitory brace helix α6 and the four-helix bundle by stacking to Phe148. An NMR-based functional assay observing the conformation of this helix showed that the F148A mutant is unresponsive to the compound, providing further evidence for the importance of this interaction. Real-time and diffusion NMR studies demonstrate that xanthine derivatives inhibit MLKL oligomerization. Finally, we show that the other well-known MLKL inhibitor Necrosulfonamide, which also covalently modifies Cys86, must employ a different mode of action.