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Chemical crosslinking and mass spectrometry of the human Rix1 complex

ABSTRACT: PELP1 is a scaffold protein with diverse cellular functions including its role as the central scaffold for the human Rix1 complex (PELP1, WDR18, TEX10, and SENP3). We have reconstituted the mammalian Rix1 complex and identified a sub-complex made of the conserved Rix1 domain of PELP1 and the WDR18 protein. Moreover, using Cryo-electron microscopy (cryo-EM) and aided by chemical crosslinking and mass spectrometry, we determined the structure of this PELP1 Rix1 domain complexed with WDR18.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Jason Williams

LAB HEAD: Jason G. Williams

PROVIDER: PXD037729 | Pride | 2023-03-11

REPOSITORIES: Pride

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			Action	DRS
	QE1021122_Gordon_01.mgf	Mgf
	QE1021122_Gordon_01.raw	Raw
	QE1021122_Gordon_04.mgf	Mgf
	QE1021122_Gordon_04.raw	Raw
	QE1120921_Gordon_01_control.mgf	Mgf

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Publications

Cryo-EM reveals the architecture of the PELP1-WDR18 molecular scaffold.

Gordon Jacob J Chapus Fleur L FL Viverette Elizabeth G EG Williams Jason G JG Deterding Leesa J LJ Krahn Juno M JM Borgnia Mario J MJ Rodriguez Joseph J Warren Alan J AJ Stanley Robin E RE

Nature communications 20221109 1

PELP1 (Proline-, Glutamic acid-, Leucine-rich protein 1) is a large scaffolding protein that functions in many cellular pathways including steroid receptor (SR) coactivation, heterochromatin maintenance, and ribosome biogenesis. PELP1 is a proto-oncogene whose expression is upregulated in many human cancers, but how the PELP1 scaffold coordinates its diverse cellular functions is poorly understood. Here we show that PELP1 serves as the central scaffold for the human Rix1 complex whose members in ...[more]

PMID: 36351913

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MC4 crosslinking of rhodopsin and GRK1 complex

Project description:G protein-coupled receptor (GPCR) kinases (GRKs) selectively phosphorylate agonist bound GPCRs, thereby switching the signal from G protein-dependent to arrestin-dependent pathways, including receptor internalization and downregulation. There are currently no high-resolution details known about how a GRK recognizes an activated receptor, but most functional studies indicate that its unique N-terminus is key to agonist-dependent phosphorylation. Herein, we report the 7.2 Å cryo-electron microscopy (EM) single particle reconstruction of the rhodopsin-rhodopsin kinase (GRK1) complex. The structure reveals a 1:1 assembly with multiple contact points between the GRK1 kinase domain and rhodopsin, the most prominent being the insertion of the GRK1 N-terminal helix directly into the cytoplasmic cleft formed by rhodopsin in its active state. Cross-linking coupled with mass spectrometry, along with functional studies, confirmed the observed interface between the proteins and revealed regions of the complex that are dynamic.

2021-07-15 | PXD019215 | Pride

BS3 crosslinking of GRC3 and Las1

Project description:Ribosome assembly is a complex process involving the coordination of multiple enzymes. A recently discovered endoribonuclease (RNase), Las1, and the poly-nucleotide kinase (PNK), Grc3, assemble into a complex. To attempt to understand the coordination of the two enzymes in this complex, we performed chemical crosslinking and mass spectrometry and also solved a series of cryo-electron microscopy structures of RNase PNK in multiple conformational states.

2019-09-11 | PXD014738 | Pride

Conformational rearrangements upon start codon recognition in human 48S translation initiation complex

Project description:Selection of the translation start codon is a key step during protein synthesis in human cells. We obtained cryo-EM structures of human 48S initiation complexes and characterized the intermediates of codon recognition by kinetic methods using eIF1A as a reporter. Both approaches capture two distinct ribosome populations formed on an mRNA with a cognate AUG codon in the presence of eIF1A, eIF1, eIF2–GTP–Met-tRNAiMet, eIF3, eIF4A and eIF4B. The ‘open’ 40S subunit conformation differs from the human 48S scanning complex and represents an intermediate preceding the codon recognition step. The ‘closed’ form is similar to reported structures of complexes from yeast and mammals formed upon codon recognition, except for the orientation of eIF1A, which is unique in our structure. Kinetic experiments show how various initiation factors mediate the population distribution of open and closed conformations until 60S subunit docking. Our results provide insights into the timing and structure of human translation initiation intermediates and suggest the differences in the mechanisms of start codon selection between mammals and yeast.

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AOCM-CPC - Kinetochores are macromolecular protein complexes that ensure accurate chromosome segregation by linking chromosomes to spindle microtubules and integrating safeguard mechanisms

Project description:Kinetochores are macromolecular protein complexes that ensure accurate chromosome segregation by linking chromosomes to spindle microtubules and integrating safeguard mechanisms. In yeast, the inner kinetochore, also known as Constitutive Centromere Associated Network (CCAN), is specifically established at point centromeres and has been implicated in contributing to Aurora-BIpl1 function. In an attempt to gain a more detailed picture of the budding yeast kinetochore architecture, crosslink-guided in vitro reconstitution revealed novel direct interactions of the inner kinetochore assembled on Cse4CENP-A nucleosomes. The Ame1/Okp1CENP-U/Q heterodimer selectively bound Cse4CENP-A nucleosomes through the Cse4 N-terminus, providing an explanation for the essential role of the COMA complex in budding yeast. Moreover, the Sli15/Ipl1 core chromosomal passenger complex was found to directly interact with COMA in vitro, suggesting a hitherto unknown role of the COMA complex in establishing biorientation. In line with this finding, in vivo artificial tethering of Sli15 to inner kinetochore proteins rescued synthetically lethal subunit deletion phenotypes in a Sli15 centromere targeting deficient mutant. This study reveals characteristics of the inner kinetochore architecture assembled at point centromeres and its implications on chromosomal passenger complex function.

2019-04-12 | PXD011235 | Pride

The COMA complex plays a role in positioning Ipl1 activity proximal to Cse4 nucleosomes

2019-04-12 | PXD011236 | Pride

XL-MS of 5S RNP preribosomal complexes

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2023-06-13 | PXD040087 | Pride

Early Scanning of Nascent Polypeptides inside the Ribosomal Tunnel by NAC

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2019-07-25 | PXD011995 | Pride

Structure of Saccharomyces cerevisiae Mediator-RNA polymerase II pre-initiation complex for divergent transcription

Project description:This study has determined structure of transcription initiation complexes including a DNA-bound activator, RNA polymerase II (Pol II), and Mediator on a divergent promoter GAL80/SUT719 using a combination of cryo-EM and XL-MS analyses. Our cryo-EM single-particle analysis reveals a dimeric form of Med-PIC through the Mediator Tail module induced by the activator protein. Density of the upstream DNA bound to the Gal4-VP16 was identifiable along the Mediator Tail module, while XL-MS localized flexible regions that were not visible by cryo-EM analysis, such as activator-binding domains (ABDs and KIX).

2024-05-22 | PXD036149 | Pride

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2020-09-30 | PXD011775 | Pride

Identification of small molecule activators of the ubiquitin ligase E6AP/UBE3A and Angelman syndrome-derived E6AP/UBE3A variants

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