Browsing by Author "Roy, Susmita"

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    A magnesium-induced triplex pre-organizes the SAM-II riboswitch
    (Public Library of Science, 2017) Roy, Susmita; Lammert, Heiko; Hayes, Ryan L.; Chen, Bin; LeBlanc, Regan; Dayie, T.Kwaku; Onuchic, José Nelson; Sanbonmatsu, Karissa Y.; Center for Theoretical Biological Physics
    Our 13C- and 1H-chemical exchange saturation transfer (CEST) experiments previously revealed a dynamic exchange between partially closed and open conformations of the SAM-II riboswitch in the absence of ligand. Here, all-atom structure-based molecular simulations, with the electrostatic effects of Manning counter-ion condensation and explicit magnesium ions are employed to calculate the folding free energy landscape of the SAM-II riboswitch. We use this analysis to predict that magnesium ions remodel the landscape, shifting the equilibrium away from the extended, partially unfolded state towards a compact, pre-organized conformation that resembles the ligand-bound state. Our CEST and SAXS experiments, at different magnesium ion concentrations, quantitatively confirm our simulation results, demonstrating that magnesium ions induce collapse and pre-organization. Agreement between theory and experiment bolsters microscopic interpretation of our simulations, which shows that triplex formation between helix P2b and loop L1 is highly sensitive to magnesium and plays a key role in pre-organization. Pre-organization of the SAM-II riboswitch allows rapid detection of ligand with high selectivity, which is important for biological function.
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    A VDAC1-mediated NEET protein chain transfers [2Fe-2S] clusters between the mitochondria and the cytosol and impacts mitochondrial dynamics
    (National Academy of Sciences, 2022) Karmi, Ola; Marjault, Henri-Baptiste; Bai, Fang; Roy, Susmita; Sohn, Yang-Sung; Yahana, Merav Darash; Morcos, Faruck; Ioannidis, Konstantinos; Nahmias, Yaakov; Jennings, Patricia A.; Mittler, Ron; Onuchic, José N.; Nechushtai, Rachel; Center for Theoretical Biological Physics
    Mitochondrial inner NEET (MiNT) and the outer mitochondrial membrane (OMM) mitoNEET (mNT) proteins belong to the NEET protein family. This family plays a key role in mitochondrial labile iron and reactive oxygen species (ROS) homeostasis. NEET proteins contain labile [2Fe-2S] clusters which can be transferred to apo-acceptor proteins. In eukaryotes, the biogenesis of [2Fe-2S] clusters occurs within the mitochondria by the iron–sulfur cluster (ISC) system; the clusters are then transferred to [2Fe-2S] proteins within the mitochondria or exported to cytosolic proteins and the cytosolic iron–sulfur cluster assembly (CIA) system. The last step of export of the [2Fe-2S] is not yet fully characterized. Here we show that MiNT interacts with voltage-dependent anion channel 1 (VDAC1), a major OMM protein that connects the intermembrane space with the cytosol and participates in regulating the levels of different ions including mitochondrial labile iron (mLI). We further show that VDAC1 is mediating the interaction between MiNT and mNT, in which MiNT transfers its [2Fe-2S] clusters from inside the mitochondria to mNT that is facing the cytosol. This MiNT–VDAC1–mNT interaction is shown both experimentally and by computational calculations. Additionally, we show that modifying MiNT expression in breast cancer cells affects the dynamics of mitochondrial structure and morphology, mitochondrial function, and breast cancer tumor growth. Our findings reveal a pathway for the transfer of [2Fe-2S] clusters, which are assembled inside the mitochondria, to the cytosol.
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    Magnesium controls aptamer-expression platform switching in the SAM-I riboswitch
    (Oxford University Press, 2019) Roy, Susmita; Hennelly, Scott P.; Lammert, Heiko; Onuchic, José Nelson; Sanbonmatsu, Karissa Y.
    Investigations of most riboswitches remain confined to the ligand-binding aptamer domain. However, during the riboswitch mediated transcription regulation process, the aptamer domain and the expression platform compete for a shared strand. If the expression platform dominates, an anti-terminator helix is formed, and the transcription process is active (ON state). When the aptamer dominates, transcription is terminated (OFF state). Here, we use an expression platform switching experimental assay and structure-based electrostatic simulations to investigate this ON-OFF transition of the full length SAM-I riboswitch and its magnesium concentration dependence. Interestingly, we find the ratio of the OFF population to the ON population to vary non-monotonically as magnesium concentration increases. Upon addition of magnesium, the aptamer domain pre-organizes, populating the OFF state, but only up to an intermediate magnesium concentration level. Higher magnesium concentration preferentially stabilizes the anti-terminator helix, populating the ON state, relatively destabilizing the OFF state. Magnesium mediated aptamer-expression platform domain closure explains this relative destabilization of the OFF state at higher magnesium concentration. Our study reveals the functional potential of magnesium in controlling transcription of its downstream genes and underscores the importance of a narrow concentration regime near the physiological magnesium concentration ranges, striking a balance between the OFF and ON states in bacterial gene regulation.
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    Redox-dependent gating of VDAC by mitoNEET
    (National Academy of Sciences, 2019) Lipper, Colin H.; Stofleth, Jason T.; Bai, Fang; Sohn, Yang-Sung; Roy, Susmita; Mittler, Ron; Nechushtai, Rachel; Onuchic, José Nelson; Jennings, Patricia A.
    MitoNEET is an outer mitochondrial membrane protein essential for sensing and regulation of iron and reactive oxygen species (ROS) homeostasis. It is a key player in multiple human maladies including diabetes, cancer, neurodegeneration, and Parkinson’s diseases. In healthy cells, mitoNEET receives its clusters from the mitochondrion and transfers them to acceptor proteins in a process that could be altered by drugs or during illness. Here, we report that mitoNEET regulates the outer-mitochondrial membrane (OMM) protein voltage-dependent anion channel 1 (VDAC1). VDAC1 is a crucial player in the cross talk between the mitochondria and the cytosol. VDAC proteins function to regulate metabolites, ions, ROS, and fatty acid transport, as well as function as a “governator” sentry for the transport of metabolites and ions between the cytosol and the mitochondria. We find that the redox-sensitive [2Fe-2S] cluster protein mitoNEET gates VDAC1 when mitoNEET is oxidized. Addition of the VDAC inhibitor 4,4′-diisothiocyanatostilbene-2,2′-disulfonate (DIDS) prevents both mitoNEET binding in vitro and mitoNEET-dependent mitochondrial iron accumulation in situ. We find that the DIDS inhibitor does not alter the redox state of MitoNEET. Taken together, our data indicate that mitoNEET regulates VDAC in a redox-dependent manner in cells, closing the pore and likely disrupting VDAC’s flow of metabolites.
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    Structural and Dynamical Order of a Disordered Protein: Molecular Insights into Conformational Switching of PAGE4 at the Systems Level
    (MDPI, 2019) Lin, Xingcheng; Kulkarni, Prakash; Bocci, Federico; Schafer, Nicholas P.; Roy, Susmita; Tsai, Min-Yeh; He, Yanan; Chen, Yihong; Rajagopalan, Krithika; Mooney, Steven M.; Zeng, Yu; Weninger, Keith; Grishaev, Alex; Onuchic, José Nelson; Levine, Herbert; Wolynes, Peter G.; Salgia, Ravi; Rangarajan, Govindan; Uversky, Vladimir; Orban, John; Jolly, Mohit Kumar
    Folded proteins show a high degree of structural order and undergo (fairly constrained) collective motions related to their functions. On the other hand, intrinsically disordered proteins (IDPs), while lacking a well-defined three-dimensional structure, do exhibit some structural and dynamical ordering, but are less constrained in their motions than folded proteins. The larger structural plasticity of IDPs emphasizes the importance of entropically driven motions. Many IDPs undergo function-related disorder-to-order transitions driven by their interaction with specific binding partners. As experimental techniques become more sensitive and become better integrated with computational simulations, we are beginning to see how the modest structural ordering and large amplitude collective motions of IDPs endow them with an ability to mediate multiple interactions with different partners in the cell. To illustrate these points, here, we use Prostate-associated gene 4 (PAGE4), an IDP implicated in prostate cancer (PCa) as an example. We first review our previous efforts using molecular dynamics simulations based on atomistic AWSEM to study the conformational dynamics of PAGE4 and how its motions change in its different physiologically relevant phosphorylated forms. Our simulations quantitatively reproduced experimental observations and revealed how structural and dynamical ordering are encoded in the sequence of PAGE4 and can be modulated by different extents of phosphorylation by the kinases HIPK1 and CLK2. This ordering is reflected in changing populations of certain secondary structural elements as well as in the regularity of its collective motions. These ordered features are directly correlated with the functional interactions of WT-PAGE4, HIPK1-PAGE4 and CLK2-PAGE4 with the AP-1 signaling axis. These interactions give rise to repeated transitions between (high HIPK1-PAGE4, low CLK2-PAGE4) and (low HIPK1-PAGE4, high CLK2-PAGE4) cell phenotypes, which possess differing sensitivities to the standard PCa therapies, such as androgen deprivation therapy (ADT). We argue that, although the structural plasticity of an IDP is important in promoting promiscuous interactions, the modulation of the structural ordering is important for sculpting its interactions so as to rewire with agility biomolecular interaction networks with significant functional consequences.