SWI/SNF subunit BAF155 N-terminus structure informs the impact of cancer-associated mutations and reveals a potential drug binding site.

SWI/SNF (BAF) chromatin remodelling complexes are key regulators of gene expression programs, and attractive drug targets for cancer therapies. Here we show that the N-terminus of the BAF155/SMARCC1 subunit contains a putative DNA-binding MarR-like domain, a chromodomain and a BRCT domain that are interconnected to each other to form a distinct module. In this structure the chromodomain makes interdomain interactions and has lost its canonical function to bind to methylated lysines. The structure provides new insights into the missense mutations that target this module in cancer. This study also reveals two adjacent, highly-conserved pockets in a cleft between the domains that form a potential binding site, which can be targeted with small molecules, offering a new strategy to target SWI/SNF complexes

SWI/SNF subunit BAF155 N-terminus structure informs the impact of cancer-associated mutations and reveals a potential drug binding site.

SWI/SNF (BAF) chromatin remodelling complexes are key regulators of gene expression programs, and attractive drug targets for cancer therapies. Here we show that the N-terminus of the BAF155/SMARCC1 subunit contains a putative DNA-binding MarR-like domain, a chromodomain and a BRCT domain that are interconnected to each other to form a distinct module. In this structure the chromodomain makes interdomain interactions and has lost its canonical function to bind to methylated lysines. The structure provides new insights into the missense mutations that target this module in cancer. This study also reveals two adjacent, highly-conserved pockets in a cleft between the domains that form a potential binding site, which can be targeted with small molecules, offering a new strategy to target SWI/SNF complexes

A high-resolution structure of the EF-hand domain of human polycystin-2.

Autosomal dominant polycystic kidney disease (ADPKD) affects over 1:1000 of the worldwide population and is caused by mutations in two genes, PKD1 and PKD2. PKD2 encodes a 968-amino acid membrane spanning protein, Polycystin-2 (PC-2), which is a member of the TRP ion channel family. The C-terminal cytoplasmic tail contains an EF-hand motif followed by a short coiled-coil domain. We have determined the structure of the EF-hand region of PC-2 using NMR spectroscopy. The use of different boundaries, compared with those used in previous studies, have enabled us to determine a high resolution structure and show that the EF hand motif forms a standard calcium-binding pocket. The affinity of this pocket for calcium has been measured and mutants that both decrease and increase its affinity for the metal ion have been created

The Autophagy Receptor TAX1BP1 and the Molecular Motor Myosin VI Are Required for Clearance of Salmonella Typhimurium by Autophagy.

Autophagy plays a key role during Salmonella infection, by eliminating these pathogens following escape into the cytosol. In this process, selective autophagy receptors, including the myosin VI adaptor proteins optineurin and NDP52, have been shown to recognize cytosolic pathogens. Here, we demonstrate that myosin VI and TAX1BP1 are recruited to ubiquitylated Salmonella and play a key role in xenophagy. The absence of TAX1BP1 causes an accumulation of ubiquitin-positive Salmonella, whereas loss of myosin VI leads to an increase in ubiquitylated and LC3-positive bacteria. Our structural studies demonstrate that the ubiquitin-binding site of TAX1BP1 overlaps with the myosin VI binding site and point mutations in the TAX1BP1 zinc finger domains that affect ubiquitin binding also ablate binding to myosin VI. This mutually exclusive binding and the association of TAX1BP1 with LC3 on the outer limiting membrane of autophagosomes may suggest a molecular mechanism for recruitment of this motor to autophagosomes. The predominant role of TAX1BP1, a paralogue of NDP52, in xenophagy is supported by our evolutionary analysis, which demonstrates that functionally intact NDP52 is missing in Xenopus and mice, whereas TAX1BP1 is expressed in all vertebrates analysed. In summary, this work highlights the importance of TAX1BP1 as a novel autophagy receptor in myosin VI-mediated xenophagy. Our study identifies essential new machinery for the autophagy-dependent clearance of Salmonella typhimurium and suggests modulation of myosin VI motor activity as a potential therapeutic target in cellular immunity.FB and DAT thank the Wellcome Trust (www.wellcome.ac.uk) for funding of a University Award to FB (086743), the CIMR Strategic Award (100140) and an equipment grant [093026]. FB also thanks the Medical Research Council UK (www.mrc.ac.uk) for funding of a project grant (MR/K000888/1). JKJ, MA and MB were supported by the Medical Research Council UK (www.mrc.ac.uk) (U105184325).This is the final published version. It first appeared at http://dx.doi.org/10.1371/journal.ppat.100517

Structural consequences of nucleophosmin mutations in acute myeloid leukemia.

Mutations affecting NPM1 (nucleophosmin) are the most common genetic lesions found in acute myeloid leukemia (AML). NPM1 is one of the most abundant proteins found in the nucleolus and has links to the MDM2/p53 tumor suppressor pathway. A distinctive feature of NPM1 mutants in AML is their aberrant localization to the cytoplasm of leukemic cells. This mutant phenotype is the result of the substitution of several C-terminal residues, including one or two conserved tryptophan residues, with a leucine-rich nuclear export signal. The exact molecular mechanism underlying the loss of nucleolar retention, and the role of the tryptophans, remains unknown. In this study we have determined the structure of an independently folded globular domain in the C terminus of NPM1 using NMR spectroscopy, and we report that the conserved tryptophans are critical for structure. This domain is necessary for the nucleolar targeting of NPM1 and is disrupted by mutations in AML with cytoplasmic NPM1. Furthermore, we identify conserved surface-exposed lysine residues that are functionally rather than structurally important for nucleolar localization. This study provides new focus for efforts to understand the pathogenesis of AML with cytoplasmic NPM1 and may be used to aid the design of small molecules that target the C-terminal domain of NPM1 to act as novel anti-proliferative and anti-leukemia therapeutics

The autophagy receptor TAX1BP1 and the molecular motor myosin VI are required for clearance of Salmonella typhimurium by autophagy

Autophagy plays a key role during Salmonella infection, by eliminating these pathogens following escape into the cytosol. In this process, selective autophagy receptors, including the myosin VI adaptor proteins optineurin and NDP52, have been shown to recognize cytosolic pathogens. Here, we demonstrate that myosin VI and TAX1BP1 are recruited to ubiquitylated Salmonella and play a key role in xenophagy. The absence of TAX1BP1 causes an accumulation of ubiquitin-positive Salmonella, whereas loss of myosin VI leads to an increase in ubiquitylated and LC3-positive bacteria. Our structural studies demonstrate that the ubiquitin-binding site of TAX1BP1 overlaps with the myosin VI binding site and point mutations in the TAX1BP1 zinc finger domains that affect ubiquitin binding also ablate binding to myosin VI. This mutually exclusive binding and the association of TAX1BP1 with LC3 on the outer limiting membrane of autophagosomes may suggest a molecular mechanism for recruitment of this motor to autophagosomes. The predominant role of TAX1BP1, a paralogue of NDP52, in xenophagy is supported by our evolutionary analysis, which demonstrates that functionally intact NDP52 is missing in Xenopus and mice, whereas TAX1BP1 is expressed in all vertebrates analysed. In summary, this work highlights the importance of TAX1BP1 as a novel autophagy receptor in myosin VI-mediated xenophagy. Our study identifies essential new machinery for the autophagy-dependent clearance of Salmonella typhimurium and suggests modulation of myosin VI motor activity as a potential therapeutic target in cellular immunity

Identification of the barstar binding site of barnase by NMR spectroscopy and hydrogen-deuterium exchange

AbstractThe extracellular ribonuclease from Bacillus amyloliquifaciens, barnase, forms a tightly-bound one-to-one complex with its intracellular inhibitor barstar. The barstar binding site on barnase was characterised by comparing the differences in the chemical shift and hydrogen-deuterium exchange rates between free and bound barnase. Chemical shift assignments of barnase in the complex with barstar were determined from 3D NOESY-HMQC and TOCSY-HMQC spectra of a complex that had been prepared with uniformly 15N-labelled barnase and unlabelled barstar. Hydrogen exchange rates were obtained from an analysis of a series of [15N]HMQC spectra of a sample prepared in the same manner exchanged into D2O. The largest changes in either chemical shift or hydrogen-deuterium exchange rate are observed for residues located in the active-site and substrate binding loops indicating that barstar inhibits barnase activity by sterically blocking the active site

Recruitment of TBK

Mammalian cells deploy autophagy to defend their cytosol against bacterial invaders. Anti-bacterial autophagy relies on the core autophagy machinery, cargo receptors, and "eat-me" signals such as galectin-8 and ubiquitin that label bacteria as autophagy cargo. Anti-bacterial autophagy also requires the kinase TBK1, whose role in autophagy has remained enigmatic. Here we show that recruitment of WIPI2, itself essential for anti-bacterial autophagy, is dependent on the localization of catalytically active TBK1 to the vicinity of cytosolic bacteria. Experimental manipulation of TBK1 recruitment revealed that engagement of TBK1 with any of a variety of Salmonella-associated "eat-me" signals, including host-derived glycans and K48- and K63-linked ubiquitin chains, suffices to restrict bacterial proliferation. Promiscuity in recruiting TBK1 via independent signals may buffer TBK1 functionality from potential bacterial antagonism and thus be of evolutionary advantage to the host