Molecular determinants for interaction of SHEP1 with Cas localize to a highly solvent‐protected region in the complex

Derunes, Céline; Burgess, Rosemary; Iraheta, Emilio; Kellerer, Robert; Becherer, Kathleen; Gessner, Chris R.; Li, Sheng; Hewitt, Krissi M.; Vuori, Kristiina; Pasquale, Elena B.; Woods, Virgil L.; Ely, Kathryn R.

doi:10.1016/j.febslet.2005.11.070

Cited by 12 publications

(14 citation statements)

References 24 publications

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“…This surface is in good agreement with a solventprotected region on SHEP1 αI in the context of the SHEP1 362-702 -Cas 836-968 complex, carried out by Derunes et al using deuterium-exchange mass spectroscopy. 38 To experimentally confirm the binding surface on BCAR3, we mutated residues on helix αI. Indeed, the R743A and E733A/R743A mutants of BCAR3 failed to bind to HEF1 in immunoprecipitates of transiently transfected MCF-7 cells.…”

Section: Structure Determination By Saxsmentioning

confidence: 99%

Structural Insights into the Association between BCAR3 and Cas Family Members, an Atypical Complex Implicated in Anti-Oestrogen Resistance

Garron¹,

Arsenieva²,

Zhong³

et al. 2009

Journal of Molecular Biology

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Section: Structure Determination By Saxsmentioning

confidence: 99%

Structural Insights into the Association between BCAR3 and Cas Family Members, an Atypical Complex Implicated in Anti-Oestrogen Resistance

Garron¹,

Arsenieva²,

Zhong³

et al. 2009

Journal of Molecular Biology

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“…HDX mass spectrometry has been broadly applied to study protein dynamics and structure, particularly for the protein binding with ligands, substrates, DNA and other molecules [4,7-10]. Such analysis has enabled the illustration of mechanisms for enzyme substrate interaction and the molecular determinants during protein binding [11,12]. The fundamental concept of HDX mass spectrometry analysis is based on the mass increase of a protein when the protein protons exchanged with solvent deuterium [6].…”

Section: Introductionmentioning

confidence: 99%

HDX-Analyzer: a novel package for statistical analysis of protein structure dynamics

Liu

Uzuner

et al. 2011

BMC Bioinformatics

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BackgroundHDX mass spectrometry is a powerful platform to probe protein structure dynamics during ligand binding, protein folding, enzyme catalysis, and such. HDX mass spectrometry analysis derives the protein structure dynamics based on the mass increase of a protein of which the backbone protons exchanged with solvent deuterium. Coupled with enzyme digestion and MS/MS analysis, HDX mass spectrometry can be used to study the regional dynamics of protein based on the m/z value or percentage of deuterium incorporation for the digested peptides in the HDX experiments. Various software packages have been developed to analyze HDX mass spectrometry data. Despite the progresses, proper and explicit statistical treatment is still lacking in most of the current HDX mass spectrometry software. In order to address this issue, we have developed the HDXanalyzer for the statistical analysis of HDX mass spectrometry data using R, Python, and RPY2.Implementation and resultsHDXanalyzer package contains three major modules, the data processing module, the statistical analysis module, and the user interface. RPY2 is employed to enable the connection of these three components, where the data processing module is implemented using Python and the statistical analysis module is implemented with R. RPY2 creates a low-level interface for R and allows the effective integration of statistical module for data processing. The data processing module generates the centroid for the peptides in form of m/z value, and the differences of centroids between the peptides derived from apo and ligand-bound protein allow us to evaluate whether the regions have significant changes in structure dynamics or not. Another option of the software is to calculate the deuterium incorporation rate for the comparison. The two types of statistical analyses are Paired Student’s t-test and the linear combination of the intercept for multiple regression and ANCOVA model. The user interface is implemented with wxpython to facilitate the data visualization in graphs and the statistical analysis output presentation. In order to evaluate the software, a previously published xylanase HDX mass spectrometry analysis dataset is processed and presented. The results from the different statistical analysis methods are compared and shown to be similar. The statistical analysis results are overlaid with the three dimensional structure of the protein to highlight the regional structure dynamics changes in the xylanase enzyme.ConclusionStatistical analysis provides crucial evaluation of whether a protein region is significantly protected or unprotected during the HDX mass spectrometry studies. Although there are several other available software programs to process HDX experimental data, HDXanalyzer is the first software program to offer multiple statistical methods to evaluate the changes in protein structure dynamics based on HDX mass spectrometry analysis. Moreover, the statistical analysis can be carried out for both m/z value and deuterium incorporation rate. In addition, the s...

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“…The development of an automated hydrogen exchange-liquid chromatography-mass spectrometry-based technology, referred to as enhanced deuterium exchangemass spectrometry (DXMS), has further significantly improved the throughput, comprehensiveness, and resolution (24). DXMS is now a mature platform for studying protein structure (25)(26)(27)(28)(29), protein dynamics (30 -38), proteinligand (39 -42), and protein-protein interactions (43)(44)(45)(46)(47)(48) at the submolecular level. Since the atomic structure of cAMPbound Epac in its active state is not currently available, the molecular mechanism of Epac activation is not known.…”

mentioning

confidence: 99%

Conformational Analysis of Epac Activation Using Amide Hydrogen/Deuterium Exchange Mass Spectrometry

Brock

Fan

Mei

et al. 2007

Journal of Biological Chemistry

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Exchange proteins directly activated by cAMP (Epac) play important roles in mediating the effects of cAMP through the activation of downstream small GTPases, Rap. To delineate the mechanism of Epac activation, we probed the conformation and structural dynamics of Epac using amide hydrogen/deuterium exchange and structural modeling. Our studies show that cAMP induces significant conformational changes that lead to a spatial rearrangement of the regulatory components of Epac and allows the exposure of the catalytic core for effector binding without imposing significant conformational change on the catalytic core. Homology modeling and comparative structural analyses of the cAMP binding domains of Epac and cAMP-dependent protein kinase (PKA) lead to a model of Epac activation, in which Epac and PKA activation by cAMP employs the same underlying principle, although the detailed structural and conformational changes associated with Epac and PKA activation are significantly different.Exchange proteins directly activated by cAMP (Epac) 4 are a family of novel guanine nucleotide exchange factors regulated by the second messenger cAMP (1, 2). Epac proteins have been shown to be involved in a myriad of cAMP-related cellular functions in parallel to the classic intracellular cAMP receptor, cAMP-dependent protein kinase (PKA) (3-12). There are two major isoforms of Epac, Epac1 and Epac2, each encoded by an independent gene and with distinctive tissue distribution in mammalian cells. Epac1 and Epac2 share extensive sequence homology, and both contain an N-terminal regulatory region and a C-terminal catalytic region. The catalytic region of Epac consists of a RAS exchange motif (REM) domain, RAS association (RA) domain, and a classic CDC25 homology domain (CDC25HD) responsible for nucleotide exchange activity. Whereas the regulatory region of Epac1 and Epac2 shares a Dishevelled/Egl-10/pleckstrin (DEP) domain followed by a cAMP-binding domain (CBD), an additional CBD N-terminal to the DEP domain is presented in Epac2. The function of this extra CBD domain (CBD-A) is not clear, since it binds cAMP with low affinity and does not seem to be essential for Epac regulation by cAMP (13).Biochemical and structural analyses by Bos and Wittinghofer's groups have revealed a general scheme of Epac regulation (13-16). In the absence of cAMP, the N-terminal regulatory region of Epac acts as an autoinhibitor of the C-terminal guanine nucleotide exchange factor activity by sterically blocking the access of the downstream effector, Rap. Binding of cAMP to the high affinity CBD leads to a conformational change that releases the CDC25HD from the interdomain inhibition imposed by the CBD. This regulatory scheme is further supported by a recently solved crystal structure of the full-length Epac2 in the absence of cAMP (17). In this autoinhibited Epac2 structure, the regulatory and catalytic regions form two distinctive structural lobes. The smaller regulatory lobe is anchored to the larger catalytic lobe by the so-called "switchboard" structure...

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Molecular determinants for interaction of SHEP1 with Cas localize to a highly solvent‐protected region in the complex

Cited by 12 publications

References 24 publications

Structural Insights into the Association between BCAR3 and Cas Family Members, an Atypical Complex Implicated in Anti-Oestrogen Resistance

Structural Insights into the Association between BCAR3 and Cas Family Members, an Atypical Complex Implicated in Anti-Oestrogen Resistance

HDX-Analyzer: a novel package for statistical analysis of protein structure dynamics

Conformational Analysis of Epac Activation Using Amide Hydrogen/Deuterium Exchange Mass Spectrometry

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