scholarly journals Peptide-binding dependent conformational changes regulate the transcriptional activity of the quorum-sensor NprR

2013 ◽  
Vol 41 (16) ◽  
pp. 7920-7933 ◽  
Author(s):  
Samira Zouhir ◽  
Stéphane Perchat ◽  
Magali Nicaise ◽  
Javier Perez ◽  
Beatriz Guimaraes ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Transcriptional Activity ◽  
Peptide Binding ◽  
Quorum Sensor

scholarly journals Exploring the conformational landscape of a lanthipeptide synthetase using native mass spectrometry

2020 ◽  
Author(s):  
Nuwani W. Weerasinghe ◽  
Yeganeh Habibi ◽  
Kevin A. Uggowitzer ◽  
Christopher J. Thibodeaux
Keyword(s):  
Mass Spectrometry ◽  
Gas Phase ◽  
Conformational Changes ◽  
Ion Mobility ◽  
Peptide Binding ◽  
Native Mass Spectrometry ◽  
Order Structure ◽  
Conformational Sampling ◽  
Precursor Peptide ◽  
Conformational Landscape

AbstractLanthipeptides are ribosomally-synthesized and post-translationally modified peptide (RiPP) natural products that are biosynthesized in a multistep maturation process by enzymes (lanthipeptide synthetases) that possess relaxed substrate specificity. Recent evidence has suggested that some lanthipeptide synthetases are structurally dynamic enzymes that are allosterically activated by precursor peptide binding, and that conformational sampling of the enzyme-peptide complex may play an important role in defining the efficiency and sequence of biosynthetic events. These “biophysical” processes, while critical for defining the activity and function of the synthetase, remain very challenging to study with existing methodologies. Herein, we show that native nanoelectrospray ionization coupled to ion mobility mass spectrometry (nanoESI-IM-MS) provides a powerful and sensitive means for investigating the conformational landscapes and intermolecular interactions of lanthipeptide synthetases. Namely, we demonstrate that the class II lanthipeptide synthetase (HalM2) and its non-covalent complex with the cognate HalA2 precursor peptide can be delivered into the gas phase in a manner that preserves native structures and intermolecular enzyme-peptide contacts. Moreover, gas phase ion mobility studies of the natively-folded ions demonstrate that peptide binding and mutations to dynamic structural elements of HalM2 alter the conformational landscape of the enzyme, and that the precursor peptide itself exhibits higher order structure in the mass spectrometer. Cumulatively, these data support previous claims that lanthipeptide synthetases are structurally dynamic enzymes that undergo functionally relevant conformational changes in response to precursor peptide binding. This work establishes nanoESI-IM-MS as a versatile approach for unraveling the relationships between protein structure and biochemical function in RiPP biosynthetic systems.

A role for prolyl isomerase PIN1 in the phosphorylation-dependent modulation of PRRXL1 function

2017 ◽  
Vol 474 (5) ◽  
pp. 683-697
Author(s):  
Ricardo Soares-dos-Reis ◽  
Ana Sofia Pessoa ◽  
Ana Filipa Dias ◽  
Miguel Falcão ◽  
Mariana Raimundo Matos ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Spinal Cord ◽  
Transcription Factor ◽  
Conformational Changes ◽  
Dorsal Root ◽  
Transcriptional Activity ◽  
Phosphorylation Sites ◽  
Prolyl Isomerase ◽  
Null Cell

Prrxl1 encodes for a paired-like homeodomain transcription factor essential for the correct establishment of the dorsal root ganglion — spinal cord nociceptive circuitry during development. Prrxl1-null mice display gross anatomical disruption of this circuitry, which translates to a markedly diminished sensitivity to noxious stimuli. Here, by the use of an immunoprecipitation and mass spectrometry approach, we identify five highly conserved phosphorylation sites (T110, S119, S231, S233 and S251) in PRRXL1 primary structure. Four are phospho-S/T-P sites, which suggest a role for the prolyl isomerase PIN1 in regulating PRRXL1. Accordingly, PRRXL1 physically interacts with PIN1 and displays diminished transcriptional activity in a Pin1-null cell line. Additionally, these S/T-P sites seem to be important for PRRXL1 conformation, and their point mutation to alanine or aspartate down-regulates PRRXL1 transcriptional activity. Altogether, our findings provide evidence for a putative novel role of PIN1 in the development of the nociceptive system and indicate phosphorylation-mediated conformational changes as a mechanism for regulating the PRRXL1 role in the process.

scholarly journals Dual control of RegX3 transcriptional activity by SenX3 and PknB

2019 ◽  
Vol 294 (28) ◽  
pp. 11023-11034 ◽  
Author(s):  
Eun-Jin Park ◽  
Yu-Mi Kwon ◽  
Jin-Won Lee ◽  
Ho-Young Kang ◽  
Jeong-Il Oh
Keyword(s):  
Conformational Changes ◽  
Transcriptional Activity ◽  
Response Regulator ◽  
Regulatory System ◽  
Kinase Domain ◽  
Two Component System ◽  
Mobility Shift ◽  
Cellular Replication ◽  
Electrophoretic Mobility Shift Assays

The mycobacterial SenX3–RegX3 two-component system consists of the SenX3 sensor histidine kinase and its cognate RegX3 response regulator. This system is a phosphorelay-based regulatory system involved in sensing environmental Pi levels and induction of genes required for Pi acquisition under Pi-limiting conditions. Here we demonstrate that overexpression of the kinase domain of Mycobacterium tuberculosis PknB (PknB-KDMtb) inhibits the transcriptional activity of RegX3 of both M. tuberculosis and Mycobacterium smegmatis (RegX3Mtb and RegX3Ms, respectively). Mass spectrometry results, along with those of in vitro phosphorylation and complementation analyses, revealed that PknB kinase activity inhibits the transcriptional activity of RegX3Mtb through phosphorylation events at Thr-100, Thr-191, and Thr-217. Electrophoretic mobility shift assays disclosed that phosphorylation of Thr-191 and Thr-217 abolishes the DNA-binding ability of RegX3Mtb and that Thr-100 phosphorylation likely prevents RegX3Mtb from being activated through conformational changes induced by SenX3-mediated phosphorylation. We propose that the convergence of the PknB and SenX3-RegX3 signaling pathways might enable mycobacteria to integrate environmental Pi signals with the cellular replication state to adjust gene expression in response to Pi availability.

scholarly journals PepNN: a deep attention model for the identification of peptide binding sites

2021 ◽  
Author(s):  
Osama Abdin ◽  
Han Wen ◽  
Philip M. Kim
Keyword(s):  
Conformational Changes ◽  
Protein Complex ◽  
Binding Sites ◽  
Structural Information ◽  
Peptide Binding ◽  
Peptide Ligand ◽  
Sequence Information ◽  
Attention Model ◽  
Cellular Processes ◽  
Complex Structural

AbstractProtein-peptide interactions play a fundamental role in facilitating many cellular processes, but remain underexplored experimentally and difficult to model computationally. Here, we introduce PepNN-Struct and PepNN-Seq, structure and sequence-based approaches for the prediction of peptide binding sites on a protein given the sequence of a peptide ligand. The models make use of a novel reciprocal attention module that is able to better reflect biochemical realities of peptides undergoing conformational changes upon binding. To compensate for the scarcity of peptide-protein complex structural information, we make use of available protein-protein complex and protein sequence information through a series of transfer learning steps. PepNN-Struct achieves state-of-the-art performance on the task of identifying peptide binding sites, with a ROC AUC of 0.893 and an MCC of 0.483 on an independent test set. Beyond prediction of binding sites on proteins with a known peptide ligand, we also show that the developed models make reasonable agnostic predictions, allowing for the identification of novel peptide binding proteins.

scholarly journals The Use of in Vitro Peptide Binding Profiles and in Silico Ligand-Receptor Interaction Profiles to Describe Ligand-Induced Conformations of the Retinoid X Receptor α Ligand-Binding Domain

2007 ◽  
Vol 21 (1) ◽  
pp. 30-48 ◽  
Author(s):  
Simon Folkertsma ◽  
Paula I. van Noort ◽  
Arnold de Heer ◽  
Peter Carati ◽  
Ralph Brandt ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Protein Interactions ◽  
Biological Activities ◽  
Peptide Binding ◽  
Retinoid X Receptor ◽  
Receptor Interaction ◽  
Binding Modes ◽  
Automated Method ◽  
Cellular Environment

Abstract It is hypothesized that different ligand-induced conformational changes can explain the different interactions of nuclear receptors with regulatory proteins, resulting in specific biological activities. Understanding the mechanism of how ligands regulate cofactor interaction facilitates drug design. To investigate these ligand-induced conformational changes at the surface of proteins, we performed a time-resolved fluorescence resonance energy transfer assay with 52 different cofactor peptides measuring the ligand-induced cofactor recruitment to the retinoid X receptor-α (RXRα) in the presence of 11 compounds. Simultaneously we analyzed the binding modes of these compounds by molecular docking. An automated method converted the complex three-dimensional data of ligand-protein interactions into two-dimensional fingerprints, the so-called ligand-receptor interaction profiles. For a subset of compounds the conformational changes at the surface, as measured by peptide recruitment, correlate well with the calculated binding modes, suggesting that clustering of ligand-receptor interaction profiles is a very useful tool to discriminate compounds that may induce different conformations and possibly different effects in a cellular environment. In addition, we successfully combined ligand-receptor interaction profiles and peptide recruitment data to reveal structural elements that are possibly involved in the ligand-induced conformations. Interestingly, we could predict a possible binding mode of LG100754, a homodimer antagonist that showed no effect on peptide recruitment. Finally, the extensive analysis of the peptide recruitment profiles provided novel insight in the potential cellular effect of the compound; for the first time, we showed that in addition to the induction of coactivator peptide binding, all well-known RXRα agonists also induce binding of corepressor peptides to RXRα.

Peptide Binding Identifies an ERα Conformation That Generates Selective Activity in Multiple In Vitro Assays

2005 ◽  
Vol 10 (6) ◽  
pp. 590-598 ◽  
Author(s):  
Christopher J. Larson ◽  
Deborah L. Osburn ◽  
Katherine Schmitz ◽  
Leslie Giampa ◽  
Shau-Ming Mong ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Biological Effects ◽  
Peptide Binding ◽  
In Vitro Assays ◽  
Response Elements ◽  
Estrogen Response ◽  
Estrogen Response Elements ◽  
In Vivo Effects

Drugs such as tamoxifen, which act at the estrogen receptor (ER), have very different in vitro and in vivo effects from those of the native hormone. Previous research has established that different ligands induce distinct conformational changes in the ER, thus affecting the interactions of the receptor with cell-specific coactivating or corepressing proteins (cofactors) and estrogen response elements (EREs), thus potentially driving differing biological effects. Affinity-selected peptides have been used to probe the conformational changes that occur within the ER upon binding various ligands. In this study, the authors characterize the ability of several peptides to be recruited to liganded ER under cellular conditions. Approximating ER conformation via recruitment of this peptide to the ER is concluded to be a better predictor of the agonist nature of an ER ligand under these different cellular contexts than is a canonical cotransfection transactivation assay.

Putative functional characteristics of human estrogen receptor-beta isoforms

2003 ◽  
Vol 30 (1) ◽  
pp. 13-29 ◽  
Author(s):  
B Peng ◽  
B Lu ◽  
E Leygue ◽  
LC Murphy
Keyword(s):  
Reporter Gene ◽  
Conformational Changes ◽  
Transcriptional Activity ◽  
Transforming Growth Factor ◽  
Estrogen Receptor Beta ◽  
Estrogen Response ◽  
Protease Digestion ◽  
Human Estrogen Receptor ◽  
Regulated Gene Expression ◽  
And Function

Estrogen receptors (ERalpha and ERbeta) are clearly multifaceted in terms of structure and function. Several relatively abundant ERbeta isoforms have been identified, which can be differentially expressed in various tIssues. In order to provide insight into the possible role of the ERbeta family in breast tIssue a study of the putative functions of the human (h) ERbeta1, hERbeta2 and hERbeta5 isoforms was undertaken. Only hERbeta1 was found to bind ligand, which induced conformational changes as determined by protease digestion assays. All ERbeta isoforms could bind to and bend DNA although the relative efficiency with which they bound DNA differed with hERalpha>hERbeta1>hERbeta2>>hERbeta5. All ERbeta isoforms inhibited ERalpha transcriptional activity on an estrogen-response element (ERE)-reporter gene. The relative activities were hERbeta1>hERbeta2>hERbeta5; however, only hERbeta1 had transcriptional activity of its own. Both LY117018-hERalpha and LY117018-hERbeta1 complexes alone could activate transcription on a TGF-beta3-CAT gene. Although hERbeta2 and hERbeta5 had no activity alone, they inhibited ERalpha but not hERbeta1 transcriptional activity of transforming growth factor (TGF)-beta3-CAT. In marked contrast to activity on an ERE-CAT reporter gene, hERbeta1 did not modulate ERalpha transcriptional activity on a TGF-beta3-CAT reporter gene. These data support promoter-specific differential activities of hERbeta isoforms with respect to models of ERalpha regulated gene expression, and suggest that they may have a role in differentially modulating estrogen action.

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