Browsing by Author "Galaz-Davison, Pablo"

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    A contact-based analysis of local energetic frustration dynamics identifies key residues enabling RfaH fold-switch
    (2024) Gonzalez-Higueras, Jorge; Freiberger, Maria Ines; Galaz-Davison, Pablo; Parra, R. Gonzalo; Ramirez-Sarmiento, Cesar A.
    Fold-switching enables metamorphic proteins to reversibly interconvert between two highly dissimilar native states to regulate their protein functions. While about 100 proteins have been identified to undergo fold-switching, unveiling the key residues behind this mechanism for each protein remains challenging. Reasoning that fold-switching in proteins is driven by dynamic changes in local energetic frustration, we combined fold-switching simulations generated using simplified structure-based models with frustration analysis to identify key residues involved in this process based on the change in the density of minimally frustrated contacts during refolding. Using this approach to analyze the fold-switch of the bacterial transcription factor RfaH, we identified 20 residues that significantly change their frustration during its fold-switch, some of which have been experimentally and computationally reported in previous works. Our approach, which we developed as an additional module for the FrustratometeR package, highlights the role of local frustration dynamics in protein fold-switching and offers a robust tool to enhance our understanding of other proteins with significant conformational shifts.
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    Allosteric couplings upon binding of RfaH to transcription elongation complexes
    (2022) Alejandro Molina, Jose; Galaz-Davison, Pablo; Komives, Elizabeth A.; Artsimovitch, Irina; Ramirez-Sarmiento, Cesar A.
    In every domain of life, NusG-like proteins bind to the elongating RNA polymerase (RNAP) to support processive RNA synthesis and to couple transcription to ongoing cellular processes. Structures of factor-bound transcription elongation complexes (TECs) reveal similar contacts to RNAP, consistent with a shared mechanism of action. However, NusG homologs differ in their regulatory roles, modes of recruitment, and effects on RNA synthesis. Some of these differences could be due to conformational changes in RNAP and NusG-like proteins, which cannot be captured in static structures. Here, we employed hydrogen-deuterium exchange mass spectrometry to investigate changes in local and non-local structural dynamics of Escherichia coli NusG and its paralog RfaH, which have opposite effects on expression of xenogenes, upon binding to TEC. We found that NusG and RfaH regions that bind RNAP became solvent-protected in factor-bound TECs, whereas RNAP regions that interact with both factors showed opposite deuterium uptake changes when bound to NusG or RfaH. Additional changes far from the factor-binding site were observed only with RfaH. Our results provide insights into differences in structural dynamics exerted by NusG and RfaH during binding to TEC, which may explain their different functional outcomes and allosteric regulation of transcriptional pausing by RfaH.
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    Coevolution-derived native and non-native contacts determine the emergence of a novel fold in a universally conserved family of transcription factors
    (2022) Galaz-Davison, Pablo; Ferreiro, Diego U.; Ramirez-Sarmiento, Cesar A.
    The NusG protein family is structurally and functionally conserved in all domains of life. Its members directly bind RNA polymerases and regulate transcription processivity and termination. RfaH, a divergent sub-family in its evolutionary history, is known for displaying distinct features than those in NusG proteins, which allows them to regulate the expression of virulence factors in enterobacteria in a DNA sequence-dependent manner. A striking feature is its structural interconversion between an active fold, which is the canonical NusG three-dimensional structure, and an autoinhibited fold, which is distinctively novel. How this novel fold is encoded within RfaH sequence to encode a metamorphic protein remains elusive. In this work, we used publicly available genomic RfaH protein sequences to construct a complete multiple sequence alignment, which was further augmented with metagenomic sequences and curated by predicting their secondary structure propensities using JPred. Coevolving pairs of residues were calculated from these sequences using plmDCA and GREMLIN, which allowed us to detect the enrichment of key metamorphic contacts after sequence filtering. Finally, we combined our coevolutionary predictions with molecular dynamics to demonstrate that these interactions are sufficient to predict the structures of both native folds, where coevolutionary-derived non-native contacts may play a key role in achieving the compact RfaH novel fold. All in all, emergent coevolutionary signals found within RfaH sequences encode the autoinhibited and active folds of this protein, shedding light on the key interactions responsible for the action of this metamorphic protein.
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    Developing and Implementing Cloud-Based Tutorials That Combine Bioinformatics Software, Interactive Coding, and Visualization Exercises for Distance Learning on Structural Bioinformatics
    (2021) Engelberger, Felipe; Galaz-Davison, Pablo; Bravo, Graciela; Rivera, Maira; Ramirez-Sarmiento, Cesar A.
    The COVID-19 pandemic has swiftly forced a change in learning strategies across educational institutions, from extensively relying on in-person activities toward online teaching. It is particularly difficult to adapt courses that depend on physical equipment to be now carried out remotely. This is the case for bioinformatics, which typically requires dedicated computer classrooms, as the logistics of granting remote access to a workstation or relying on the computational resources of each student is not trivial. A possible workaround is using cloud server-based computing resources, such as Google Colaboratory, a free web browser application that allows the writing and execution of Python programming through Jupyter notebooks, integrating text, images, and code cells. Following a cloud-based approach, we migrated the practical activities of a course on molecular modeling and simulation into the Google Colaboratory environment resulting in 12 tutorials that introduce students to topics such as phylogenetic analysis, molecular modeling, molecular docking, several flavors of molecular dynamics, and coevolutionary analysis. Each of these notebooks includes a brief introduction to the topic, software installation, execution of the required tools, and analysis of results, with each step properly described. Using a Likert scale questionnaire, a pool of students positively evaluated these tutorials in terms of the time required for their completion, their ability to understand the content and exercises developed in each session, and the practical significance and impact that these computational tools have on scientific research. All tutorials are freely available at https: //github.com/pb3lab/ibm3202.
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    Dimer dissociation is a key energetic event in the fold-switch pathway of KaiB
    (2022) Rivera, Maira; Galaz-Davison, Pablo; Retamal-Farfan, Ignacio; Komives, Elizabeth A.; Ramirez-Sarmiento, Cesar A.
    Cyanobacteria possesses the simplest circadian clock, composed of three proteins that act as a phosphorylation oscillator: KaiA, KaiB, and KaiC. The timing of this oscillator is determined by the fold-switch of KaiB, a structural rearrangement of its C-terminal half that is accompanied by a change in the oligomerization state. During the day, KaiB forms a stable tetramer (gsKaiB), whereas it adopts a monomeric thioredoxin-like fold during the night (fsKaiB). Although the structures and functions of both native states are well studied, little is known about the sequence and structure determinants that control their structural interconversion. Here, we used confinement molecular dynamics (CCR-MD) and folding simulations using structure-based models to show that the dissociation of the gsKaiB dimer is a key energetic event for the fold-switch. Hydrogen-deuterium ex-change mass spectrometry (HDXMS) recapitulates the local stability of protein regions reported by CCR-MD, with both ap-proaches consistently indicating that the energy and backbone flexibility changes are solely associated with the region that fold-switches between gsKaiB and fsKaiB and that the localized regions that differentially stabilize gsKaiB also involve regions outside the dimer interface. Moreover, two mutants (R23C and R75C) previously reported to be relevant for altering the rhyth-micity of the Kai clock were also studied by HDXMS. Particularly, R75C populates dimeric and monomeric states with a deute-rium incorporation profile comparable to the one observed for fsKaiB, emphasizing the importance of the oligomerization state of KaiB for the fold-switch. These findings suggest that the information necessary to control the rhythmicity of the cyanobacterial biological clock is, to a great extent, encoded within the KaiB sequence.
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    Dissecting the structural and functional consequences of the evolutionary proline-glycine deletion in the wing 1 region of the forkhead domain of human FoxP1
    (2024) Tamarin, Stephanie; Galaz-Davison, Pablo; Ramirez-Sarmiento, Cesar A.; Babul, Jorge; Medina, Exequiel
    The human FoxP transcription factors dimerize via three-dimensional domain swapping, a unique feature among the human Fox family, as result of evolutionary sequence adaptations in the forkhead domain. This is the case for the conserved glycine and proline residues in the wing 1 region, which are absent in FoxP proteins but present in most of the Fox family. In this work, we engineered both glycine (G) and proline-glycine (PG) insertion mutants to evaluate the deletion events in FoxP proteins in their dimerization, stability, flexibility, and DNA-binding ability. We show that the PG insertion only increases protein stability, whereas the single glycine insertion decreases the association rate and protein stability and promotes affinity to the DNA ligand.
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    DNA facilitates heterodimerization between human transcription factors FoxP1 and FoxP2 by increasing their conformational flexibility
    (2023) Conuecar, Ricardo; Asela, Isabel; Rivera, Maira; Galaz-Davison, Pablo; Gonzalez-Higueras, Jorge; Hamilton, George L.; Engelberger, Felipe; Ramirez-Sarmiento, Cesar A.; Babul, Jorge; Sanabria, Hugo; Medina, Exequiel
    Transcription factors regulate gene expression by binding to DNA. They have disordered regions and specific DNA-binding domains. Binding to DNA causes structural changes, including folding and interactions with other molecules. The FoxP subfamily of transcription factors in humans is unique because they can form heterotypic interactions without DNA. However, it is unclear how they form heterodimers and how DNA binding affects their function. We used computational and experimental methods to study the structural changes in FoxP1's DNA-binding domain when it forms a heterodimer with FoxP2. We found that FoxP1 has complex and diverse conformational dynamics, transitioning between compact and extended states. Surprisingly, DNA binding increases the flexibility of FoxP1, contrary to the typical folding-upon-binding mechanism. In addition, we observed a 3-fold increase in the rate of heterodimerization after FoxP1 binds to DNA. These findings emphasize the importance of structural flexibility in promoting heterodimerization to form transcriptional complexes.
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    Local energetic frustration conservation in protein families and superfamilies
    (2023) Freiberger, Maria I.; Ruiz-Serra, Victoria; Pontes, Camila; Romero-Durana, Miguel; Galaz-Davison, Pablo; Ramirez-Sarmiento, Cesar A.; Schuster, Claudio D.; Marti, Marcelo A.; Wolynes, Peter G.; Ferreiro, Diego U.; Parra, R. Gonzalo; Valencia, Alfonso
    Energetic local frustration offers a biophysical perspective to interpret the effects of sequence variability on protein families. Here we present a methodology to analyze local frustration patterns within protein families and superfamilies that allows us to uncover constraints related to stability and function, and identify differential frustration patterns in families with a common ancestry. We analyze these signals in very well studied protein families such as PDZ, SH3, alpha and beta globins and RAS families. Recent advances in protein structure prediction make it possible to analyze a vast majority of the protein space. An automatic and unsupervised proteome-wide analysis on the SARS-CoV-2 virus demonstrates the potential of our approach to enhance our understanding of the natural phenotypic diversity of protein families beyond single protein instances. We apply our method to modify biophysical properties of natural proteins based on their family properties, as well as perform unsupervised analysis of large datasets to shed light on the physicochemical signatures of poorly characterized proteins such as the ones belonging to emergent pathogens.
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    Palmitic and Stearic Acids Inhibit Chaperone-Mediated Autophagy (CMA) in POMC-like Neurons In Vitro
    (2022) Espinosa, Rodrigo; Gutierrez, Karla; Rios, Javiera; Ormeno, Fernando; Yanten, Liliana; Galaz-Davison, Pablo; Ramirez-Sarmiento, Cesar A.; Parra, Valentina; Albornoz, Amelina; Alfaro, Ivan E.; Burgos, Patricia, V; Morselli, Eugenia; Criollo, Alfredo; Budini, Mauricio
    The intake of food with high levels of saturated fatty acids (SatFAs) is associated with the development of obesity and insulin resistance. SatFAs, such as palmitic (PA) and stearic (SA) acids, have been shown to accumulate in the hypothalamus, causing several pathological consequences. Autophagy is a lysosomal-degrading pathway that can be divided into macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA). Previous studies showed that PA impairs macroautophagy function and insulin response in hypothalamic proopiomelanocortin (POMC) neurons. Here, we show in vitro that the exposure of POMC neurons to PA or SA also inhibits CMA, possibly by decreasing the total and lysosomal LAMP2A protein levels. Proteomics of lysosomes from PA- and SA-treated cells showed that the inhibition of CMA could impact vesicle formation and trafficking, mitochondrial components, and insulin response, among others. Finally, we show that CMA activity is important for regulating the insulin response in POMC hypothalamic neurons. These in vitro results demonstrate that CMA is inhibited by PA and SA in POMC-like neurons, giving an overview of the CMA-dependent cellular pathways that could be affected by such inhibition and opening a door for in vivo studies of CMA in the context of the hypothalamus and obesity.
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    The N-terminal domain of RfaH plays an active role in protein fold-switching
    (2021) Galaz-Davison, Pablo; Roman, Ernesto A.; Ramirez-Sarmiento, Cesar A.; Wallqvist, Anders; Elofsson, Arne; Wallqvist, Anders; Elofsson, Arne; Wallqvist, Anders; Elofsson, Arne; Wallqvist, Anders; Elofsson, Arne
    Author summary Proteins commonly adopt a single three-dimensional structure that is required for biological function. Nevertheless, proteins are not isolated in the cell, and the presence of binding partners can give rise to alternate structural configurations. Metamorphic proteins represent an extreme case of the latter, by folding into at least two well-defined configurations that are both structurally and functionally different. For RfaH, a virulence factor in enterobacteria, two distinct folds are found: an autoinhibited state in which its two protein domains strongly interact, and an active state in which these domains dissociate due to a specific DNA signal on RNA polymerases. This activation is accompanied by the refolding of the C-terminal domain (CTD) from an alpha-helical structure to a beta-barrel. Our work employs computational simulations to explore the role of the N-terminal domain (NTD) in regulating the metamorphic behavior of RfaH, determining that this domain has a major part in orienting and binding to the CTD in its alpha-helical fold, and in stabilizing an intermediate state instead of the fully folded beta-barrel. These results suggest that the NTD not only participates in stabilizing the autoinhibited state, but also aids in fold-switching back to it after active RfaH is released from RNA polymerase.