Structural basis for bacterial energy extraction from atmospheric hydrogen

Diverse aerobic bacteria use atmospheric H2 as an energy source for growth and survival1. This globally significant process regulates the composition of the atmosphere, enhances soil biodiversity and drives primary production in extreme environments2,3. Atmospheric H2 oxidation is attributed to uncharacterized members of the [NiFe] hydrogenase superfamily4,5. However, it remains unresolved how these enzymes overcome the extraordinary catalytic challenge of oxidizing picomolar levels of H2 amid ambient levels of the catalytic poison O2 and how the derived electrons are transferred to the respiratory chain1. Here we determined the cryo-electron microscopy structure of the Mycobacterium smegmatis hydrogenase Huc and investigated its mechanism. Huc is a highly efficient oxygen-insensitive enzyme that couples oxidation of atmospheric H2 to the hydrogenation of the respiratory electron carrier menaquinone. Huc uses narrow hydrophobic gas channels to selectively bind atmospheric H2 at the expense of O2, and 3 [3Fe–4S] clusters modulate the properties of the enzyme so that atmospheric H2 oxidation is energetically feasible. The Huc catalytic subunits form an octameric 833 kDa complex around a membrane-associated stalk, which transports and reduces menaquinone 94 Å from the membrane. These findings provide a mechanistic basis for the biogeochemically and ecologically important process of atmospheric H2 oxidation, uncover a mode of energy coupling dependent on long-range quinone transport, and pave the way for the development of catalysts that oxidize H2 in ambient air

BonA from Acinetobacter baumannii Forms a Divisome-Localized Decamer That Supports Outer Envelope Function

Acinetobacter baumannii is a high-risk pathogen due to the rapid global spread of multidrug-resistant lineages. Its phylogenetic divergence from other ESKAPE pathogens means that determinants of its antimicrobial resistance can be difficult to extrapolate from other widely studied bacteria. A recent study showed that A. baumannii upregulates production of an outer membrane lipoprotein, which we designate BonA, in response to challenge with polymyxins. Here, we show that BonA has limited sequence similarity and distinct structural features compared to lipoproteins from other bacterial species. Analyses through X-ray crystallography, small-angle X-ray scattering, electron microscopy, and multiangle light scattering demonstrate that BonA has a dual BON (Bacterial OsmY and Nodulation) domain architecture and forms a decamer via an unusual oligomerization mechanism. This analysis also indicates this decamer is transient, suggesting dynamic oligomerization plays a role in BonA function. Antisera recognizing BonA shows it is an outer membrane protein localized to the divisome. Loss of BonA modulates the density of the outer membrane, consistent with a change in its structure or link to the peptidoglycan, and prevents motility in a clinical strain (ATCC 17978). Consistent with these findings, the dimensions of the BonA decamer are sufficient to permeate the peptidoglycan layer, with the potential to form a membrane-spanning complex during cell division. IMPORTANCE The pathogen Acinetobacter baumannii is considered an urgent threat to human health. A. baumannii is highly resistant to treatment with antibiotics, in part due to its protective cell envelope. This bacterium is only distantly related to other bacterial pathogens, so its cell envelope has distinct properties and contains components distinct from those of other bacteria that support its function. Here, we report the discovery of BonA, a protein that supports A. baumannii outer envelope function and is required for cell motility. We determine the atomic structure of BonA and show that it forms part of the cell division machinery and functions by forming a complex, features that mirror those of distantly related homologs from other bacteria. By improving our understanding of the A. baumannii cell envelope this work will assist in treating this pathogen

Protein target highlights in CASP15: Analysis of models by structure providers

We present an in-depth analysis of selected CASP15 targets, focusing on their biological and functional significance. The authors of the structures identify and discuss key protein features and evaluate how effectively these aspects were captured in the submitted predictions. While the overall ability to predict three-dimensional protein structures continues to impress, reproducing uncommon features not previously observed in experimental structures is still a challenge. Furthermore, instances with conformational flexibility and large multimeric complexes highlight the need for novel scoring strategies to better emphasize biologically relevant structural regions. Looking ahead, closer integration of computational and experimental techniques will play a key role in determining the next challenges to be unraveled in the field of structural molecular biology

Structural and functional characterisation of the molecular assembly of two pseudokinase scaffolds

© 2019 Ashleigh Kate KroppThe human pseudokinase SgK269, and its structurally related homologue SgK223, are oncogenic interacting scaffolds that promote the assembly of specific tyrosine kinase signalling pathways. SgK223 and SgK269, as well as the recently discovered PEAK3, belong to the PEAK family of protein pseudokinases. They are large, multidomain proteins that are comprised of an N-terminal region of unknown structure and function, a large unstructured PEST region containing tyrosine phosphorylation sites and a C-terminal domain comprised of a pseudokinase domain flanked by regulatory helices. SgK223 and SgK269 have been shown to localise to focal adhesions and their overexpression leads to increased cell migration and changes in cell morphology, hallmarks of cancerous cells. Recent studies from our lab and others have provided structural insight into the C-terminal domain and flanking alpha helices of SgK223 and SgK269. These structures highlighted a conserved mechanism of dimerisation that drives homo- and hetero-association of SgK223 and SgK269 and plays an important role in cell migration. Additionally, SgK223 and SgK269 were demonstrated to undergo homo- and hetero-oligomerisation through their pseudokinase domains. In contrast to the C-terminal domain, little is known about the function of the N-terminal domains of SgK223 and SgK269, although there is sequence conservation between them. In this study, we have begun characterising the N-terminal domains of SgK223 and SgK269 using biophysical and biochemical techniques, initially demonstrating that these domains are monomeric and appear to have no defined secondary structure. To further investigate SgK223 and SgK269 homo- and hetero-association we carried out single site alanine mutagenesis to determine the energetic hotspots at the dimerisation interface of SgK269. Furthermore, we carried out mutagenesis within the N-lobe of SgK223 and SgK269, to investigate the role of this interface in homo- and hetero-oligomerisation. Additionally, we characterised the PEAK family interactions with the critical interacting signalling adaptor protein, CrkII, using biophysical assays and X-ray crystallography. We found that each member of the PEAK family has a proline-rich motif within their PEST linker that interacts with CrkII N-SH3 domain with ~1-3 uM affinity. The crystal structure of the CrkII N-SH3 domain bound to the SgK269 proline-rich motif demonstrated the critical consensus residues for the PEAK family interaction with CrkII. To further investigate the role of SgK223 and SgK269 homo- and hetero-association in cells, these studies were complemented with localisation microscopy techniques. Utilising mutants of SgK223 and SgK269 that can no longer dimerise or oligomerise, we investigated the importance of SgK223 and SgK269 associations for their localisation and thus, role in signalling. Insights into the scaffolding functions of SgK223 and SgK269 will inform how they contribute to the assembly of signalling pathways and hence their role in cancer

Structure of SgK223 pseudokinase reveals novel mechanisms of homotypic and heterotypic association

Pseudokinases lack kinase activity, yet they impact cellular physiology through the regulation of bona fide signaling kinases. Here the authors describe the structure of the SgK223 pseudokinase and its adjacent domains, and identify regulatory interfaces required for self-assembly and downstream signaling

Structural mapping of PEAK pseudokinase interactions identifies 14-3-3 as a molecular switch for PEAK3 signaling

Abstract PEAK pseudokinases regulate cell migration, invasion and proliferation by recruiting key signaling proteins to the cytoskeleton. Despite lacking catalytic activity, alteration in their expression level is associated with several aggressive cancers. Here, we elucidate the molecular details of key PEAK signaling interactions with the adapter proteins CrkII and Grb2 and the scaffold protein 14-3-3. Our findings rationalize why the dimerization of PEAK proteins has a crucial function in signal transduction and provide biophysical and structural data to unravel binding specificity within the PEAK interactome. We identify a conserved high affinity 14-3-3 motif on PEAK3 and demonstrate its role as a molecular switch to regulate CrkII binding and signaling via Grb2. Together, our studies provide a detailed structural snapshot of PEAK interaction networks and further elucidate how PEAK proteins, especially PEAK3, act as dynamic scaffolds that exploit adapter proteins to control signal transduction in cell growth/motility and cancer

Protein target highlights in CASP15: Analysis of models by structure providers

We present an in-depth analysis of selected CASP15 targets, focusing on their biological and functional significance. The authors of the structures identify and discuss key protein features and evaluate how effectively these aspects were captured in the submitted predictions. While the overall ability to predict three-dimensional protein structures continues to impress, reproducing uncommon features not previously observed in experimental structures is still a challenge. Furthermore, instances with conformational flexibility and large multimeric complexes highlight the need for novel scoring strategies to better emphasize biologically relevant structural regions. Looking ahead, closer integration of computational and experimental techniques will play a key role in determining the next challenges to be unraveled in the field of structural molecular biology