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Abstract: The structural basis by which gas-binding heme proteins control their interactions with NO, CO, and O2, is fundamental to enzymology, biotechnology and human health. Cytochromes c´ (cyts c´) are a group of putative NO-binding heme proteins that fall into two families: the well characterised four alpha helix bundle fold (cyts c´-α) and an unrelated family with a largely beta sheet fold (cyts c´-β) resembling that of cytochromes P460. A recent structure of cyt c´-β from Methylococcus capsulatus Bath (McCP-β) revealed two heme pocket phenylalanine residues (Phe 32 and Phe 61) positioned near the distal gas binding site. This feature, dubbed the “Phe cap”, is highly conserved within the sequences of other cyts c´-β, but is absent in their close homologues, the hydroxylamine oxidizing cytochromes P460, although some do contain a single Phe residue. Here we report an integrated structural, spectroscopic, and kinetic characterization of McCP-β complexes with diatomic gases, focusing on the interaction of the Phe cap with NO and CO. Significantly, crystallographic and resonance Raman data show that orientation of the electron rich aromatic ring face of Phe 32 towards distally-bound NO or CO is associated with weakened backbonding and higher off rates. Moreover, we propose that an aromatic quadrupole also contributes to the unusually weak backbonding reported for some heme-based gas sensors, including the mammalian NO-sensor, soluble guanylate cyclase (sGC). Collectively, this study sheds light on the influence of highly conserved distal Phe residues on heme-gas complexes of cytochrome c’−β, including the potential for aromatic quadrupoles to modulate NO and CO binding in other heme proteins.

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Abstract: The linear ubiquitin chain assembly complex synthesises linear Ub chains which constitute a binding and activation platform for components of the TNF signalling pathway. One of the components of LUBAC is the ubiquitin ligase HOIL-1 which has been shown to generate oxyester linkages on several proteins and on linear polysaccharides. We show that HOIL-1 activity requires linear tetra-Ub binding which enables HOIL-1 to mono-ubiquitylate linear Ub chains and polysaccharides. Furthermore, we describe the crystal structure of a C-terminal tandem domain construct of HOIL-1 comprising the IBR and RING2 domains. Interestingly, the structure reveals a unique bi-nuclear Zn-cluster which substitutes the second zinc finger of the canonical RING2 fold. We identify the C-terminal histidine of this bi-nuclear Zn-cluster as the catalytic base required for the ubiquitylation activity of HOIL-1. Our study suggests that the unique zinc-coordinating architecture of RING2 provides a binding platform for ubiquitylation targets.

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Abstract: Staphylococcus aureus and Staphylococcus epidermidis are frequently associated with medical device infections that involve establishment of a bacterial biofilm on the device surface. Staphylococcal surface proteins Aap, SasG and Pls are members of the Periscope Protein class and have been implicated in biofilm formation and host colonisation; they comprise a repetitive region (“B region”) and an N-terminal host colonisation domain within the “A region”, predicted to be a lectin domain. Repetitive E-G5 domains (as found in Aap, SasG and Pls) form elongated ‘stalks’ that would vary in length with repeat number, resulting in projection of the N-terminal A domain variable distances from the bacterial cell surface. Here, we present the structures of the lectin domains within A regions of SasG, Aap and Pls and a structure of the Aap lectin domain attached to contiguous E-G5 repeats, suggesting the lectin domains will sit at the tip of the variable length rod. We demonstrate that these isolated domains (Aap, SasG) are sufficient to bind to human host desquamated nasal epithelial cells. Previously, proteolytic cleavage or a deletion within the A domain have been reported to induce biofilm formation; the structures suggest a potential link between these observations. Intriguingly, whilst the Aap, SasG and Pls lectin domains bind a metal ion, they lack the non-proline cis peptide bond thought to be key for carbohydrate binding by the lectin fold. This suggestion of non-canonical ligand binding should be a key consideration when investigating the host cell interactions of these bacterial surface proteins.

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Abstract: Of the 13 known independent zoonoses of simian immunodeficiency viruses to humans, only one, leading to human immunodeficiency virus (HIV) type 1(M) has become pandemic, causing over 80 million human infections. To understand the specific features associated with pandemic human-to-human HIV spread, we compared replication of HIV-1(M) with non-pandemic HIV-(O) and HIV-2 strains in myeloid cell models. We found that non-pandemic HIV lineages replicate less well than HIV-1(M) owing to activation of cGAS and TRIM5-mediated antiviral responses. We applied phylogenetic and X-ray crystallography structural analyses to identify differences between pandemic and non-pandemic HIV capsids. We found that genetic reversal of two specific amino acid adaptations in HIV-1(M) enables activation of TRIM5, cGAS and innate immune responses. We propose a model in which the parental lineage of pandemic HIV-1(M) evolved a capsid that prevents cGAS and TRIM5 triggering, thereby allowing silent replication in myeloid cells. We hypothesize that this capsid adaptation promotes human-to-human spread through avoidance of innate immune response activation.

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Abstract: Fluorescent proteins have revolutionized cell biology and cell imaging through their use as genetically encoded tags. Structural biology has been pivotal in understanding how their unique fluorescent properties manifest through the formation of the chromophore and how the spectral properties are tuned through interaction networks. This knowledge has in turn led to the construction of novel variants with new and improved properties. Here we describe the process by which fluorescent protein structures are determined, starting from recombinant protein production to structure determination by molecular replacement. We also describe how to incorporate and determine the structures of proteins containing non-natural amino acids. Recent advances in protein engineering have led to reprogramming of the genetic code to allow incorporation of new chemistry at designed residue positions, with fluorescent proteins being at the forefront of structural studies in this area. The impact of such new chemistry on protein structure is still limited; the accumulation of more protein structures will undoubtedly improve our understanding and ability to engineer proteins with new chemical functionality.