Genetic interaction mapping informs integrative structure determination of protein complexes
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ABSTRACT:
PROVIDER: PRJNA672982 | ENA |
REPOSITORIES: ENA
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Project description:Determining structures of protein complexes is crucial for understanding cellular functions. Here, we describe an integrative structure determination approach that relies on in vivo measurements of genetic interactions. We construct phenotypic profiles for point mutations crossed against gene deletions or exposed to environmental perturbations, followed by converting similarities between two profiles into an upper bound on the distance between the mutated residues. We determine the structure of the yeast histone H3/4 complex based on ~500,000 genetic interactions of 350 mutants. We then apply the method to subunits Rpb1-Rpb2 of yeast RNA polymerase II, and subunits RpoB-RpoC of bacterial RNA polymerase. The accuracy is comparable to that based on chemical cross-links; using restraints from both genetic interactions and cross-links further improves model accuracy and precision. The approach provides an efficient means to augment integrative structure determination with in vivo observations.
2020-12-10 | GSE160350 | GEO
Project description: Not available
| S-EPMC7946025 | biostudies-literature
Project description:Objective: HIV-1 Nef suppresses immune surveillance mechanisms to promote viral pathogenesis. Cellular receptor CD4 is essential for HIV entry into host cells, though becomes problematic at later stages in the virus replication cycle. CD4 at the plasma membrane is targeted by Nef for endocytosis and lysosomal degradation via recruitment of clathrin adaptor complex 2 (AP2 complex). Our goal is to use cross-linking mass spectrometry and integrative modeling to aid in structure determination of flexible portions of the Nef-CD4-AP2 complex. Methods: We use disuccinimidyl sulfoxide (DSSO), a MS-cleavable, bifunctional amine-reactive small molecule, to cross-link proximal Lys residues or N-termini of a purified reconstituted Nef-CD4 C-terminal domain fusion protein bound to the AP2Δμ2-CTD complex. Cross-linked proteins were separated by SDS-PAGE, excised from the gel and digested with trypsin. The resulting peptides were analyzed by specialized LC-MS3 experiments for identification of cross-linked residues using MSconvert (to obtain individual MS2 and MS3 level mgf files), ProteinProspector (to obtain peptide identification results files), and XLTools (to identify cross-linked peptides from results, MS2, and MS3 files). Additional scripts were used to summarize cross-linked results. Results: We find that an intricate combination of conformational changes occurs in both Nef and AP2 to enable CD4 binding and downregulation. A pocket on Nef previously identified as crucial for recruiting class I MHC is also responsible for recruiting CD4, revealing a potential approach to inhibit two of Nef’s activities and sensitize the virus to immune clearance.
2021-09-09 | PXD019338 | Pride
Project description: Not available
| S-EPMC3519461 | biostudies-literature
Project description:Peripheral light harvesting (LH) antenna complexes have been studied extensively in the purple nonsulfur bacterium Rhodopseudomonas palustris because it produces different types of LH complexes under high light intensities (LH2 complex) and low light intensities (LH3 and LH4 complex). The ability of R. palustris to alter its peripheral LH complexes in response to changes in light intensity is attributed to the multiple operons that encode the a and b peptides that make up these complexes, whose expression is affected by light intensity, light quality, and oxygen tension. However, low resolution structures, amino acid similarities between the complexes, and a lack of transcriptional analysis made it difficult to determine the LH complexes composition and functions under different light intensities. It was also unclear how much diversity of the R. palustris LH complexes exists in nature.Results: To gain insight into the composition of the LH complexes, their function under high light intensities and low light intensities, and their prevalence in the environment we undertook an integrative genomics approach using 15 closely related R. palustris strains isolated from the environment and 5 R. palustris ecotypes whose genomes have been sequenced. We sequenced the genomes for the 15 closely related strains and using RNA-seq carried out transcriptomic analysis on all 20 strains grown under high light intensity and low light intensity. We were able to determine that even closely related R. palustris strains had differences in their pucBA gene content and expression, even under the same growth conditions. We also found that the LH2 complex could compensate for the lack of an LH4 complex under LL intensities but not under extremely LL intensities. Conclusions: This is the first time an integrative genomics approach has been used to study light harvesting in the environment. The variation observed in LH gene composition and expression in environmental isolates of R. palustris likely reflects how these strains have adapted to specific light conditions in the environment. We have also shown that there is redundancy between some of the LH complexes under certain light intensities, which may partially explain why multiple operons encoding LH complexes have evolved and been maintained in R. palustris. Examing the variation observed in LH gene composition and expression in various environmental isolates
2015-03-01 | E-GEOD-59544 | biostudies-arrayexpress
Project description:Background: Complex biological networks control fundamental processes such as reproduction. The relationship of network structure to biological function was investigated in a global gene interaction network developed from ovary tissues of the model fish, fathead minnow (Pimephales promelas). Results: A global ovary gene interaction network was inferred from gene expression in ovaries of fathead minnow representing 288 different exposure conditions and 1,472 microarrays. More than 74 percent of the interactions in two subnetworks and 37% of the connections in a third subnetwork were also present as known connections found in curated databases and the literature. Subnetworks within the network were enriched in specific pathways and key ovarian functions. The network location of differentially expressed genes from different ovary stages was consistent with known functional changes at those stages. The global network provide additional insight into gene function when gene directly linked to differentially expressed genes are considered in enrichment analysis. Analysis of the impact of bisphenol A on ovarian gene expression demonstrated that the network provides an informative frame work in which to investigate mechanisms by which chemical effects occur. Conclusions: Our results suggest that the ovary transcriptional network is composed of several connected subnetworks where each is enriched in specific functions. Construction of a global gene regulatory network from multiple experiments provides valuable insight into the function of genes and the impact of chemicals on biological systems. total RNA from the ovary tissue of treated or control fish labeled in single color was hybridized to Agilent fathead minnow microarray (design 019597)
2016-07-06 | E-GEOD-70936 | biostudies-arrayexpress
Project description:Background: Complex biological networks control fundamental processes such as reproduction. The relationship of network structure to biological function was investigated in a global gene interaction network developed from ovary tissues of the model fish, fathead minnow (Pimephales promelas). Results: A global ovary gene interaction network was inferred from gene expression in ovaries of fathead minnow representing 288 different exposure conditions and 1,472 microarrays. More than 74 percent of the interactions in two subnetworks and 37% of the connections in a third subnetwork were also present as known connections found in curated databases and the literature. Subnetworks within the network were enriched in specific pathways and key ovarian functions. The network location of differentially expressed genes from different ovary stages was consistent with known functional changes at those stages. The global network provide additional insight into gene function when gene directly linked to differentially expressed genes are considered in enrichment analysis. Analysis of the impact of bisphenol A on ovarian gene expression demonstrated that the network provides an informative frame work in which to investigate mechanisms by which chemical effects occur. Conclusions: Our results suggest that the ovary transcriptional network is composed of several connected subnetworks where each is enriched in specific functions. Construction of a global gene regulatory network from multiple experiments provides valuable insight into the function of genes and the impact of chemicals on biological systems.
2016-07-06 | GSE70936 | GEO
Project description:This SuperSeries is composed of the following subset Series: GSE30558: Bisulphite-sequencing of chromatin immunoprecipitated DNA (BisChiP-seq) directly informs methylation status GSE34340: Bisulphite sequencing of native LNCaP and PrEC DNA [methylation array] Refer to individual Series
2012-01-18 | E-GEOD-34403 | biostudies-arrayexpress
Project description:Peripheral light harvesting (LH) antenna complexes have been studied extensively in the purple nonsulfur bacterium Rhodopseudomonas palustris because it produces different types of LH complexes under high light intensities (LH2 complex) and low light intensities (LH3 and LH4 complex). The ability of R. palustris to alter its peripheral LH complexes in response to changes in light intensity is attributed to the multiple operons that encode the a and b peptides that make up these complexes, whose expression is affected by light intensity, light quality, and oxygen tension. However, low resolution structures, amino acid similarities between the complexes, and a lack of transcriptional analysis made it difficult to determine the LH complexes composition and functions under different light intensities. It was also unclear how much diversity of the R. palustris LH complexes exists in nature.Results: To gain insight into the composition of the LH complexes, their function under high light intensities and low light intensities, and their prevalence in the environment we undertook an integrative genomics approach using 15 closely related R. palustris strains isolated from the environment and 5 R. palustris ecotypes whose genomes have been sequenced. We sequenced the genomes for the 15 closely related strains and using RNA-seq carried out transcriptomic analysis on all 20 strains grown under high light intensity and low light intensity. We were able to determine that even closely related R. palustris strains had differences in their pucBA gene content and expression, even under the same growth conditions. We also found that the LH2 complex could compensate for the lack of an LH4 complex under LL intensities but not under extremely LL intensities. Conclusions: This is the first time an integrative genomics approach has been used to study light harvesting in the environment. The variation observed in LH gene composition and expression in environmental isolates of R. palustris likely reflects how these strains have adapted to specific light conditions in the environment. We have also shown that there is redundancy between some of the LH complexes under certain light intensities, which may partially explain why multiple operons encoding LH complexes have evolved and been maintained in R. palustris.
2015-03-01 | GSE59544 | GEO
Project description:The COP9 signalosome (CSN) is an evolutionarily conserved protein complex that functions as a deneddylase to inactivate Cullin-RING E3 ligases for controlling protein ubiquitination. CSN possesses structural flexibility that is important for its activation upon binding to a diverse array of CRLs. The canonical and non-canonical CSN complexes consist of 8 (CSN1-8) and 9 (CSN1-9) subunits, respectively. Although CSN9 is not essential for CSN assembly and function, it appears to be important in non-catalytic regulation of CRLs by CSN. Here we employed a combinatory cross-linking mass spectrometry (XL-MS) approach to generate the largest PPI maps of human CSN complexes, which significantly enhanced the precision of integrative structural modeling. The resulting integrative structures allowed us not only to elucidate architectures of both complexes, but also to assess CSN structural dynamics that was not described in the crystal structure. In addition, we have determined CSN9 docking sites and its impact on the CSN structure. While CSN9 binding did not induce global conformational changes, it triggered subunit local reorientations that may be associated with CSN9 involvement in CSN-mediated steric regulation of CRLs.
2020-02-04 | PXD014673 | Pride