Browsing by Author "Komives, Elizabeth A."

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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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    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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    Hydrogen-deuterium exchange mass spectrometry reveals folding and allostery in protein-protein interactions
    (2018) Ramírez Sarmiento, Cesar Antonio; Komives, Elizabeth A.
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    The Folding Unit of Phosphofructokinase-2 as Defined by the Biophysical Properties of a Monomeric Mutant
    (2015) Ramírez Sarmiento, Cesar Antonio; Báez, Mauricio; Zamora, Ricardo A.; Balasubramaniam, Deepa; Babul, Jorge; Komives, Elizabeth A.; Guixé, Victoria
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    The Light Chain Allosterically Enhances the Protease Activity of Murine Urokinase-Type Plasminogen Activator
    (2024) Torres-Paris, Constanza; Song, Harriet J.; Engelberger, Felipe; Ramirez-Sarmiento, Cesar A.; Komives, Elizabeth A.
    The active form of the murine urokinase-type plasminogen activator (muPA) is formed by a 27-residue disordered light chain connecting the amino-terminal fragment (ATF) with the serine protease domain. The two chains are tethered by a disulfide bond between C1(CT) in the disordered light chain and C122(CT) in the protease domain. Previous work showed that the presence of the disordered light chain affected the inhibition of the protease domain by antibodies. Here we show that the disordered light chain induced a 3.7-fold increase in k cat of the protease domain of muPA. In addition, hydrogen-deuterium exchange mass spectrometry (HDX-MS) and accelerated molecular dynamics (AMD) were performed to identify the interactions between the disordered light chain and the protease domain. HDX-MS revealed that the light chain is contacting the 110s, the turn between the beta 10- and beta 11-strand, and the beta 7-strand. A reduction in deuterium uptake was also observed in the activation loop, the 140s loop and the 220s loop, which forms the S1-specificty pocket where the substrate binds. These loops are further away from where the light chain seems to be interacting with the protease domain. Our results suggest that the light chain most likely increases the activity of muPA by allosterically favoring conformations in which the specificity pocket is formed. We propose a model by which the allostery would be transmitted through the beta-strands of the beta-barrels to the loops on the other side of the protease domain.
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    Three-Dimensional Domain Swapping Changes the Folding Mechanism of the Forkhead Domain of FoxP1
    (2016) Medina, Exequiel; Córdova, Cristóbal; Villalobos, Pablo; Reyes, Javiera; Komives, Elizabeth A.; Ramirez Sarmiento, Cesar Antonio; Jorge Babul
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    Unusual dimerization of a BcCsp mutant leads to reduced conformational dynamics
    (2017) Carvajal, Alonso I.; Vallejos, Gabriel; Komives, Elizabeth A.; Castro Fernández, Víctor; Leonardo, Diego A.; Garratt, Richard C.; Ramírez Sarmiento, Cesar Antonio; Babul, Jorge