Biology subjects

Schroeder, G. N.

Publications and source records attributed to Schroeder, G. N..

3 recordsLinked to original sources

bioRxiv2026

Actin and AnkJ reciprocally regulate the catalytic state of the Legionella effector Ceg14

Ceg14 is a Legionella pneumophila effector whose enzymatic activity is activated by host actin and inhibited by the cognate metaeffector AnkJ. Recent studies established that Ceg14 cleaves ATP to AMP and pyrophosphate and can transfer AMP to 3-phosphoglycerate, but how actin and AnkJ regulate these catalytic states remained unclear. Here, we show that Ceg14 is selectively activated by G-actin and determine the cryo-EM structure of the Ceg14-actin-AnkJ complex. Structure-guided mutagenesis identifies two actin-binding elements, a C-terminal anchor and an extended sensor region, that are required for Ceg14 activation and toxicity. Structural modelling and molecular dynamics simulations reveal that the catalytic center is assembled from three spatially distinct components: an N-terminal nucleoside-binding region, a phosphate-coordinating region within the actin sensor, and the catalytic triad. G-actin constrains the sensor and thereby organizes the phosphate-binding component of the active site. AnkJ binds at a distinct surface and allosterically repositions the N-terminal domain, disrupting nucleoside coordination while leaving part of the catalytic machinery intact. Consistent with this mechanism, AnkJ strongly suppresses AMP production and 3-phosphoglycerate-stimulated activity, yet the ternary complex retains weak Ceg14-dependent ATP-to-ADP activity. Thus, our results show that actin and AnkJ do not simply switch Ceg14 on and off, but reciprocally remodel its multipart catalytic center to determine its catalytic state and reaction output.

biochemistry↗

bioRxiv2022

A gas phase fractionation acquisition scheme integrating ion mobility for rapid diaPASEF library generation

Data independent acquisition (DIA or DIA/SWATH) mass spectrometry has emerged as a primary measurement strategy in the field of quantitative proteomics. diaPASEF is a recent adaptation that leverages trapped ion mobility spectrometry (TIMS) to improve selectivity and increase sensitivity. The complex fragmentation spectra generated by co-isolation of peptides in DIA mode are most typically analyzed with reference to prior knowledge in the form of spectral libraries. The best-established method for generating libraries uses data dependent acquisition (DDA) mode, or DIA mode if appropriately deconvoluted, often including offline fractionation to increase depth of coverage. More recently strategies for spectral library generation based on gas phase fractionation (GPF), where a representative sample is injected serially using narrow DIA windows that cover different mass ranges of the complete precursor space, have been introduced that performed comparably to deep offline fractionation-based libraries for DIA data analysis. Here, we investigated whether an analogous GPF-based library building approach that accounts for the ion mobility (IM) dimension is useful for the analysis of diaPASEF data and can remove the need for offline fractionation. To enable a rapid library development approach for diaPASEF we designed a GPF acquisition scheme covering the majority of multiply charged precursors in the m/z vs 1/K0 space requiring 7 injections of a representative sample and compared this with libraries generated by direct deconvolution-based analysis of diaPASEF data or by deep offline fractionation and ddaPASEF. We found that library generation by IM-GPF outperformed direct deconvolution of the diaPASEF data and had performance approaching that of a deep offline fractionation library, when analysing diaPASEF data. This establishes the ion mobility integrated GPF scheme as a pragmatic approach to rapid library generation for the analysis of diaPASEF data.

systems biology↗

bioRxiv2018

More than 16,000 effectors in the Legionella genus genome provide multiple, independent combinations for replication in human cells

SignificanceLegionella pneumophila is a bacterial pathogen causing outbreaks of a lethal pneumonia. The genus Legionella comprises 65 species for which aquatic amoebae are the natural reservoirs. Using functional and comparative genomics to deconstruct the entire bacterial genus we reveal the surprising parallel evolutionary trajectories that have led to the emergence of human pathogenic Legionella. An unexpectedly large and unique repository of secreted proteins (>16,000) containing eukaryotic-like proteins acquired from all domains of life (plant, animal, fungal, archaea) is contrasting with a highly conserved type 4 secretion system. This study reveals an unprecedented environmental reservoir of bacterial virulence factors, and provides a new understanding of how reshuffling and gene-acquisition from environmental eukaryotic hosts, may allow for the emergence of human pathogens.\n\nAbstractThe bacterial genus Legionella comprises 65 species among, which Legionella pneumophila is a human pathogen causing severe pneumonia. To understand the evolution of an environmental to an accidental human pathogen, we have functionally analyzed 80 Legionella genomes spanning 58 species. Uniquely, an immense repository of 16,000 secreted proteins encoding 137 different eukaryotic-like domains and more than 200 eukaryotic-like proteins is paired with a highly conserved T4SS. Specifically, we show that eukaryotic Rho and Rab GTPase domains are found nearly exclusively in eukaryotes and Legionella species. Translocation assays for selected Rab-GTPase proteins revealed that they are indeed T4SS secreted substrates. Furthermore, F/U-box and SET domains were present in >70% of all species suggesting that manipulation of host signal transduction, protein turnover and chromatin modification pathways, respectively are fundamental intracellular replication strategies for Legionellae. In contrast, the Sec-7 domain was restricted to L. pneumophila and seven other species, indicating effector repertoire tailoring within different amoebae. Functional screening of 47 species revealed 60% were competent for intracellular replication in THP-1 cells, but interestingly this phenotype was associated with diverse effector assemblages. These data, combined with evolutionary analysis indicate that the capacity to infect eukaryotic cells has been acquired independently many times within the genus and that a highly conserved yet versatile T4SS secretes an exceptional number of different proteins shaped by inter-domain gene transfer. Furthermore we revealed the surprising extent to which legionellae have co-opted genes and thus cellular functions from their eukaryotic hosts and provides a new understanding of how dynamic reshuffling and gene-acquisition has led to the emergence of major human pathogens.

genomics↗