Browse
Submit Data
Databases
API
Help

Dataset Information

157 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Interaction of the GEF SH3BP5 and its cognate GTPase Rab11

ABSTRACT: H/D exchange analysis of the GEF SH3BP5 and its interaction with the GTPase Rab11

INSTRUMENT(S): Bruker Daltonics micrOTOF series

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: John Burke

LAB HEAD: John Edmund Burke

PROVIDER: PXD010586 | Pride | 2018-09-18

REPOSITORIES: Pride

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
	01_11_2017_MJHDX30_WT_0.3_0_01_4801.d.zip	Other
	01_11_2017_MJHDX30_WT_0.3_2_0_01_4817.d.zip	Other
	01_11_2017_MJHDX30_WT_300_0_01_4803.d.zip	Other
	01_11_2017_MJHDX30_WT_300_2_0_01_4819.d.zip	Other
	01_11_2017_MJHDX30_p27_0.3_0_01_4813.d.zip	Other

Items per page:

1 - 5 of 144

Publications

Structural determinants of Rab11 activation by the guanine nucleotide exchange factor SH3BP5.

Jenkins Meredith L ML Margaria Jean Piero JP Stariha Jordan T B JTB Hoffmann Reece M RM McPhail Jacob A JA Hamelin David J DJ Boulanger Martin J MJ Hirsch Emilio E Burke John E JE

Nature communications 20180914 1

The GTPase Rab11 plays key roles in receptor recycling, oogenesis, autophagosome formation, and ciliogenesis. However, investigating Rab11 regulation has been hindered by limited molecular detail describing activation by cognate guanine nucleotide exchange factors (GEFs). Here, we present the structure of Rab11 bound to the GEF SH3BP5, along with detailed characterization of Rab-GEF specificity. The structure of SH3BP5 shows a coiled-coil architecture that mediates exchange through a unique Rab- ...[more]

PMID: 30217979

Similar Datasets

Insights into PH1 disease mechanisms from the analyses of the structural stability and dynamics of AGT variants

Project description:Primary hyperoxaluria type I (PH1) is a genetic disease caused by a deficiency in the peroxisomal alanine:glyoxylate aminotransferase (AGT) activity. Mutations in AGT mostly cause protein mistargeting and enhanced aggregation, although the molecular and structural basis of these mechanisms are unknown. In this work, we use hydrogen-deuterium exchange monitored by mass spectrometry (HDX-MS) to provide novel insight into these pathogenic mechanisms. We characterize the wild-type (WT) protein, the LM variant (containing the mutations P11L and I340M, a haplotype more frequent in PH1 patients) and the LM G170R (the most common genotype in PH1, introducing the G170R mutation on the LM background). Our study provides the first experimental analysis of the local stability and dynamics of AGT, showing that stability is heterogeneous in the native state and providing a blueprint for frustrated regions with potentially functional relevance. The LM and LM G170R variants destabilize locally the structure. Enzymatic transamination of the PLP bound to AGT hardly affects stability. Our study thus supports that AGT misfolding is not caused by dramatic effects on the stability and dynamics of the holo-protein.

2024-01-25 | PXD046749 | Pride

Novel modifications of PARP inhibitor veliparib increase PARP1 binding to DNA breaks

Project description:The project is about studying the dynamics of PARP1 when bound to DNA and veliparib derivatives.

2024-03-19 | PXD047072 | Pride

Characterization of m-lipin using HDX-MS

Project description:This project consists of two experiments. The first is mapping the binding interface between the isolated m-lip domain of mouse lipin and liposomes. The second experiments is mapping the binding interface between full length mouse lipin and liposomes. Looking at the isolated m-lip domain, we found that residues 470-490 and 500-550 showed decreases in exchange upon liposome binding. The full-length lipin experiment saw decreases in exchnage in these same regions, as well as in the very C-terminus and very N-terminus regions of the protein. An order-disorder experiment was done on full length lipin where the protein was exposed to a short pulse of deuterium and compared to the fully-deuterated protein. In this instance, we established that the majority of the protein is relatively disordered and does not have secondary structure with high stability

2021-06-29 | PXD022172 | Pride

Simple and Fast Maximally Deuterated Control (maxD) preparation for HDX MS Experiments

Project description:During the analysis steps of hydrogen deuterium exchange (HDX) mass spectrometry (MS) there is an unavoidable loss of deuterons, or back-exchange. Understanding back-exchange is necessary to correct for loss during analysis, to calculate the absolute amount of exchange, and to ensure that deuterium recovery is as high as possible during LC-MS. Back-exchange can be measured and corrected for using a maximally deuterated species (here called maxD) in which the protein is deuterated at all backbone amide positions and analyzed with the same buffer components, %D2O, quenching conditions, and LC-MS parameters used during the analysis of other labeled samples. Here we describe a robust and broadly applicable protocol, using denaturation followed by deuteration, to prepare a maxD control sample in ~40 minutes. The protocol was evaluated with a number of proteins that varied in both size and folded structure. The relative fractional uptake and level of back-exchange with this protocol were both equivalent to those obtained with a previous protocol that requires much more time or one requiring isolation of peptic peptides prior to deuteration. Placing strong denaturation first in the protocol allowed maximum deuteration in a short time (~10 mins) with equal or more deuteration found in other methods. The absence of high temperatures and low pH during the deuteration step limited protein aggregation. This high-performance, fast, and easy to perform protocol should enhance routine preparation of maxD controls for both beginners and for challenging proteins.

2022-07-08 | PXD032924 | Pride

Assessing the dynamics and macromolecular interactions of the intrinsically disordered protein YY1

Project description:YY1 is a ubiquitously expressed, intrinsically disordered transcription factor involved in neural development. The oligomeric state of YY1 varies depending on the environment. These changes may alter its DNA binding ability and hence its transcriptional activity. In addition to its oligomeric state, the interaction of YY1 with proteins such as FOXP2 can impact its role in transcription. The aim of this work is to study the structure and dynamics of YY1 binding to DNA and to determine the influence of oligomerisation and associations with FOXP2 on its DNA binding mechanism. Size exclusion chromatography, fluorescence anisotropy and electrophoretic mobility shift assays were used to study YY1 oligomerisation and interaction with FOXP2. To better understand potential structural changes to YY1 upon DNA binding, hydrogen deuterium exchange mass spectrometry was used. The results indicate that YY1 consists of specific structured regions, while most of the sequence remains disordered. Furthermore, the oligomeric nature of the protein is dependent on ionic strength. DNA affects oligomerisation and the protein undergoes changes in structure and dynamics upon DNA binding. YY1 and FOXP2 were found to interact with each other both in isolation and in the presence of YY1-specific DNA. The heterogeneous, dynamic multimerisation of YY1 identified in this work is, therefore, likely to be important for its ability to make heterologous associations with other proteins such as FOXP2. The interactions that YY1 forms with itself, FOXP2 and DNA form part of an intricate mechanism of transcriptional regulation by YY1, which is vital for appropriate neural development.

2024-01-26 | PXD038431 | Pride

A conservative point mutation in a dynamic antigen-binding loop of human immunoglobulin λ6 light chain promotes pathologic amyloid formation

Project description:Immunoglobulin light chain (LC) amyloidosis (AL) is a life-threatening human disease wherein free monoclonal LCs deposit in vital organs. To determine what makes some LCs amyloidogenic, we explored patient-based amyloidogenic and non-amyloidogenic recombinant LCs from the λ6 subtype prevalent in AL. Hydrogen-deuterium exchange mass spectrometry, structural stability, proteolysis, and amyloid growth studies revealed that the antigen-binding CDR1 loop is the least protected part in the variable domain of λ6 LC, particularly in the AL variant. N32T substitution in CRD1 is identified as a driver of amyloid formation. Substitution N32T increased the amyloidogenic propensity of CDR1 loop, decreased its protection in the native structure, and accelerated amyloid growth in the context of other AL substitutions. The destabilizing effects of N32T propagated across the molecule increasing its dynamics in regions ~30Å away from the substitution site. Such striking long-range effects of a conservative point substitution in a dynamic surface loop may be relevant to Ig function. Comparison of patient-derived and engineered proteins showed that N32T interactions with other substitution sites must contribute to amyloidosis. The results suggest that CDR1 is critical in amyloid formation by other λ6 LCs.

2021-11-25 | PXD026528 | Pride

Dynamics of integrin α5β1, fibronectin, and their complex

Project description:Integrin α5β1 mediates cell adhesion to the extracellular matrix (ECM) by binding fibronectin (Fn). Selectivity for Fn by α5β1 is achieved through recognition of an RGD motif in the 10th type-III Fn domain (Fn10) and the synergy site in the 9th type-III Fn domain (Fn9). However, details of the interaction dynamics are unknown. Here, we compared synergy-site and Fn-truncation mutations for their α5β1-binding affinities and stabilities. We also interrogated binding of the α5β1 ectodomain headpiece fragment to Fn using hydrogen deuterium exchange mass spectrometry (HDX MS) to probe binding sites and sites of integrin conformational change. Our results suggest the synergistic effect of Fn9 requires both specific residues and a folded domain. We found some residues considered important for synergy are required for stability. Additionally, we show decreases in fibronectin HDX are localized to a synergy peptide containing contacting residues in two β-strands, an intervening loop in Fn9, and the RGD-containing loop in Fn10, indicative of binding sites. We also identified binding sites in the α5-subunit β-propeller domain for the Fn9 synergy site and in the β1-subunit βI domain for Fn10 based on decreases in α5β1 HDX. Interestingly, the dominant effect of Fn binding was an increase in α5β1 deuterium exchange distributed over multiple sites that undergo changes in conformation or solvent accessibility and appear to be sites where energy is stored in the higher-energy, open-integrin conformation. Together, our results highlight regions important for α5β1 binding to Fn and dynamics associated with this interaction.

2022-08-09 | PXD031508 | Pride

Detailing the Structural and Dynamic Interactions Between WDR44 and Rab11

Project description:DR44 is a Rab11 effector protein that is thought to regulate ciliogenesis by competing with pro-ciliogenesis factors for Rab11 binding. The structure of WDR44 and the molecular mechanism of its interaction with Rab11 is unknown. To explore the interactions between these two proteins multiple WDR44 constructs were designed and pulled down with Rab11. Using the results of the pulldown assay as a guide we used protein complex prediction software, and hydrogen deuterium exchange mass spectrometry (HDX-MS) to identify the binding interface between WDR44 and Rab11. Mutagenesis was used to make specific mutations in the putative Rab11 binding region of WDR44.

2023-03-11 | PXD035741 | Pride

HDX-MS analysis of PLIN3 membrane binding

Project description:Hydrogen deuterium exchange mass spectrometry of PLIN3 in the presence of three different membrane vesicles to analyze structural changes induced by membrane binding.

2023-04-14 | PXD025717 | Pride

Structural basis of MICAL autoinhibition

Project description:MICAL proteins play a crucial role in cellular dynamics by binding and disassembling actin filaments, impacting processes like axon guidance, cytokinesis, and cell morphology. Their cellular activity is tightly controlled, as dysregulation can lead to detrimental effects on cellular morphology. Although previous studies have suggested that MICALs are autoinhibited, and require Rab proteins to become active, the detailed molecular mechanisms remained unclear. Here, we report the cryo-EM structure of human MICAL1 at a nominal resolution of 3.1 Å. Structural analyses, alongside biochemical and functional studies, show that MICAL1 autoinhibition is mediated by an intramolecular interaction between its N-terminal catalytic and C-terminal coiled-coil domains, blocking F-actin interaction. Moreover, we demonstrate that allosteric changes in the coiled-coil domain and the binding of the tripartite assembly of CH-L2α1-LIM domains to the coiled-coil domain are crucial for MICAL activation and autoinhibition. These mechanisms appear to be evolutionarily conserved, suggesting a potential universality across the MICAL family.

2024-10-30 | PXD057312 | Pride