Data publication with the structural biology data grid supports live analysis

Sponges of the porifera family harbor some of the evolutionary most ancient orthologs of the B-cell lymphoma-2 (Bcl-2) family, a protein family critical to regulation of apoptosis. The genome of the sponge Geodia cydonium contains the putative pro-survival Bcl-2 homolog BHP2, which protects sponge tissue as well as mammalian Hek-293 and NIH-3T3 cells against diverse apoptotic stimuli. The Lake Baikal demosponge Lubomirskia baicalensis has been shown to encode both putative pro-survival Bcl-2 (LB-Bcl-2) and pro-apoptotic Bcl-2 members (LB-Bak-2), which have been implied in axis formation (branches) in L. baicalensis. However, the molecular mechanism of action of sponge-encoded orthologs of Bcl-2 remains to be clarified. Here, we report that the pro-survival Bcl-2 ortholog BHP2 from G. cydonium is able to bind the BH3 motif of a pro-apoptotic Bcl-2 protein, LB-Bak-2 of the sponge L. baicalensis. Furthermore, we determined the crystal structure of BHP2 bound to LB-Bak-2, which revealed that using a binding groove conserved across all pro-survival Bcl-2 proteins, BHP2 binds multi-motif Bax-like proteins through their BH3-binding regions. However, BHP2 discriminates against BH3-only bearing proteins by blocking access to a hydrophobic pocket that is critical for BH3 motif binding in pro-survival Bcl-2 proteins from higher organisms. This differential binding mode is reflected in a structure-based phylogenetic comparison of BHP2 with other Bcl-2 family members, which revealed that BHP2 does not cluster with either Bcl-2 members of higher organisms or pathogen-encoded homologs, and assumes a discrete position. Our findings suggest that the molecular machinery and mechanisms for executing Bcl-2-mediated apoptosis as observed in mammals are evolutionary ancient, with early regulation of apoptotic machineries closely resembling their modern counterparts in mammals rather than Caenorhabditis elegans or drosophila.

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“…Raw diffraction images were deposited in the SBGrid Data Bank 42 using the accession code doi:10.15785/SBGRID/38810.15785/SBGRID/388.…”

Section: Methodsmentioning

confidence: 99%

Structural insight into an evolutionarily ancient programmed cell death regulator – the crystal structure of marine sponge BHP2 bound to LB-Bak-2

2017

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“…The crystallographic data and refined model statistics are presented in Table 1. The raw crystal diffraction images have been uploaded to the Structural Biology Data Grid (60) and are available for download at https://doi.org/10.15785/SBGRID/364.…”

Section: Methodsmentioning

confidence: 99%

Conserved ABC Transport System Regulated by the General Stress Response Pathways of Alpha- and Gammaproteobacteria

et al. 2017

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Brucella abortus E1 is an EcfG family sigma factor that regulates the transcription of dozens of genes in response to diverse stress conditions and is required for maintenance of chronic infection in a mouse model. A putative ATPbinding cassette transporter operon, bab1_0223-bab1_0226, is among the most highly activated gene sets in the E1 regulon. The proteins encoded by the operon resemble quaternary ammonium-compatible solute importers but are most similar in sequence to the broadly conserved YehZYXW system, which remains largely uncharacterized. Transcription of yehZYXW is activated by the general stress sigma factor S in Enterobacteriaceae, which suggests a functional role for this transport system in bacterial stress response across the classes Alphaproteobacteria and Gammaproteobacteria. We present evidence that B. abortus YehZYXW does not function as an importer of known compatible solutes under physiological conditions and does not contribute to the virulence defect of a E1 -null strain. The sole in vitro phenotype associated with genetic disruption of this putative transport system is reduced growth in the presence of high Li ϩ ion concentrations. A crystal structure of B. abortus YehZ revealed a class II periplasmic binding protein fold with significant structural homology to Archaeoglobus fulgidus ProX, which binds glycine betaine. However, the structure of the YehZ ligand-binding pocket is incompatible with high-affinity binding to glycine betaine. This is consistent with weak measured binding of YehZ to glycine betaine and related compatible solutes. We conclude that YehZYXW is a conserved, stress-regulated transport system that is phylogenetically and functionally distinct from quaternary ammonium-compatible solute importers.IMPORTANCE Brucella abortus E1 regulates transcription in response to stressors encountered in its mammalian host and is necessary for maintenance of chronic infection in a mouse model. The functions of the majority of genes regulated by E1 remain undefined. We present a functional/structural analysis of a conserved putative membrane transport system (YehZYXW) whose expression is strongly activated by E1 . Though annotated as a quaternary ammonium osmolyte uptake system, experimental physiological studies and measured ligand-binding properties of the periplasmic binding protein (PBP), YehZ, are inconsistent with this function. A crystal structure of B. abortus YehZ provides molecular insight into differences between bona fide quaternary ammonium osmolyte importers and YehZ-related proteins, which form a distinct phylogenetic and functional group of PBPs.

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“…Crystallographic data and refined model statistics are presented in Table S3 in the supplemental material. The diffraction data set for this crystal form has been uploaded to the Structural Biology Data Grid (62) and is available for download at http://dx.doi.org/10.15785/SBGRID/203.…”

Section: Methodsmentioning

confidence: 99%

WrpA Is an Atypical Flavodoxin Family Protein under Regulatory Control of the Brucella abortus General Stress Response System

et al. 2016

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The general stress response (GSR) system of the intracellular pathogen Brucella abortus controls the transcription of approximately 100 genes in response to a range of stress cues. The core genetic regulatory components of the GSR are required for B. abortus survival under nonoptimal growth conditions in vitro and for maintenance of chronic infection in an in vivo mouse model. The functions of the majority of the genes in the GSR transcriptional regulon remain undefined. bab1_1070 is among the most highly regulated genes in this regulon: its transcription is activated 20-to 30-fold by the GSR system under oxidative conditions in vitro. We have solved crystal structures of Bab1_1070 and demonstrate that it forms a homotetrameric complex that resembles those of WrbA-type NADH:quinone oxidoreductases, which are members of the flavodoxin protein family. However, IMPORTANCEBrucella abortus is an etiological agent of brucellosis, which is among the most common zoonotic diseases worldwide. The general stress response (GSR) regulatory system of B. abortus controls the transcription of approximately 100 genes and is required for maintenance of chronic infection in a murine model; the majority of GSR-regulated genes remain uncharacterized. We present in vitro and in vivo functional and structural analyses of WrpA, whose expression is strongly induced by GSR under oxidative conditions. Though WrpA is structurally related to NADH:quinone oxidoreductases, it does not bind redox cofactors in solution, nor does it exhibit oxidoreductase activity in vitro. However, WrpA does affect spleen inflammation in a murine infection model. Our data provide evidence that WrpA forms a new functional class of WrbA/flavodoxin family proteins. Bacterial pathogens face fluctuating chemical and physical challenges in the host environment, including low pH, antimicrobial peptides, nutrient sequestration, and oxidative burst. These conditions can damage essential cellular components, including nucleic acids, proteins, and lipids. As such, bacteria encode a variety of protection mechanisms, including antioxidant enzymes, DNA damage repair systems, and efflux systems (1-4), which enable the cell to resist attacks by the host immune system (5-7).The diverse flavodoxin family, which is composed of enzymes that noncovalently bind a flavin cofactor and transfer electrons between a variety of substrates, can help to maintain cellular redox balance and confer resistance to oxidative stress (8-10). Flavodoxins and flavodoxin-like proteins are also known to promote pathogen virulence and to play a role in host infection and colonization (8,(11)(12)(13)(14)(15)(16)(17)(18), in addition to important roles in enzyme activation and metabolism (15). The flavodoxin-like protein WrbA (tryptophan [W] repressor binding protein) has been described for several microbial species, in which it is proposed to facilitate adaptation to varied chemical changes in the environment (13,(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)

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Data publication with the structural biology data grid supports live analysis

Cited by 123 publications

References 61 publications

Structural insight into an evolutionarily ancient programmed cell death regulator – the crystal structure of marine sponge BHP2 bound to LB-Bak-2

Structural insight into an evolutionarily ancient programmed cell death regulator – the crystal structure of marine sponge BHP2 bound to LB-Bak-2

Conserved ABC Transport System Regulated by the General Stress Response Pathways of Alpha- and Gammaproteobacteria

WrpA Is an Atypical Flavodoxin Family Protein under Regulatory Control of the Brucella abortus General Stress Response System

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