Molecular architecture of polycomb repressive complexes.

The polycomb group (PcG) proteins are a large and diverse family that epigenetically repress the transcription of key developmental genes. They form three broad groups of polycomb repressive complexes (PRCs) known as PRC1, PRC2 and Polycomb Repressive DeUBiquitinase, each of which modifies and/or remodels chromatin by distinct mechanisms that are tuned by having variable compositions of core and accessory subunits. Until recently, relatively little was known about how the various PcG proteins assemble to form the PRCs; however, studies by several groups have now allowed us to start piecing together the PcG puzzle. Here, we discuss some highlights of recent PcG structures and the insights they have given us into how these complexes regulate transcription through chromatin.

Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin.

Protein interactions with peptides generally have low thermodynamic and mechanical stability. Streptococcus pyogenes fibronectin-binding protein FbaB contains a domain with a spontaneous isopeptide bond between Lys and Asp. By splitting this domain and rational engineering of the fragments, we obtained a peptide (SpyTag) which formed an amide bond to its protein partner (SpyCatcher) in minutes. Reaction occurred in high yield simply upon mixing and amidst diverse conditions of pH, temperature, and buffer. SpyTag could be fused at either terminus or internally and reacted specifically at the mammalian cell surface. Peptide binding was not reversed by boiling or competing peptide. Single-molecule dynamic force spectroscopy showed that SpyTag did not separate from SpyCatcher until the force exceeded 1 nN, where covalent bonds snap. The robust reaction conditions and irreversible linkage of SpyTag shed light on spontaneous isopeptide bond formation and should provide a targetable lock in cells and a stable module for new protein architectures.

Structural Basis for the Activation of the Deubiquitinase Calypso by the Polycomb Protein ASX.

Ubiquitin C-terminal hydrolase deubiquitinase BAP1 is an essential tumor suppressor involved in cell growth control, DNA damage response, and transcriptional regulation. As part of the Polycomb repression machinery, BAP1 is activated by the deubiquitinase adaptor domain of ASXL1 mediating gene repression by cleaving ubiquitin (Ub) from histone H2A in nucleosomes. The molecular mechanism of BAP1 activation by ASXL1 remains elusive, as no structures are available for either BAP1 or ASXL1. Here, we present the crystal structure of the BAP1 ortholog from Drosophila melanogaster, named Calypso, bound to its activator, ASX, homolog of ASXL1. Based on comparative structural and functional analysis, we propose a model for Ub binding by Calypso/ASX, uncover decisive structural elements responsible for ASX-mediated Calypso activation, and characterize the interaction with ubiquitinated nucleosomes. Our results give molecular insight into Calypso function and its regulation by ASX and provide the opportunity for the rational design of mechanism-based therapeutics to treat human BAP1/ASXL1-related tumors.

Competition between antagonistic complement factors for a single protein on N. meningitidis rules disease susceptibility.

Genome-wide association studies have found variation within the complement factor H gene family links to host susceptibility to meningococcal disease caused by infection with Neisseria meningitidis (Davila et al., 2010). Mechanistic insights have been challenging since variation within this locus is complex and biological roles of the factor H-related proteins, unlike factor H, are incompletely understood. N. meningitidis subverts immune responses by hijacking a host-immune regulator, complement factor H (CFH), to the bacterial surface (Schneider et al., 2006; Madico et al., 2007; Schneider et al., 2009). We demonstrate that complement factor-H related 3 (CFHR3) promotes immune activation by acting as an antagonist of CFH. Conserved sequences between CFH and CFHR3 mean that the bacterium cannot sufficiently distinguish between these two serum proteins to allow it to hijack the regulator alone. The level of protection from complement attack achieved by circulating N. meningitidis therefore depends on the relative levels of CFH and CFHR3 in serum. These data may explain the association between genetic variation in both CFH and CFHR3 and susceptibility to meningococcal disease.