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1. Visualizing a two-state conformational ensemble in stem–loop 3 of the transcriptional regulator 7SK RNA

   https://doi.org/10.1093/nar/gkad1159  

   Camara, Momodou B.; Lange, Bret; Yesselman, Joseph D.; Eichhorn, Catherine D. (December 2023, Nucleic Acids Research)

   Abstract Structural plasticity is integral to RNA function; however, there are currently few methods to quantitatively resolve RNAs that have multiple structural states. NMR spectroscopy is a powerful approach for resolving conformational ensembles but is size-limited. Chemical probing is well-suited for large RNAs but provides limited structural and kinetics information. Here, we integrate the two approaches to visualize a two-state conformational ensemble for the central stem–loop 3 (SL3) of 7SK RNA, a critical element for 7SK RNA function in transcription regulation. We find that the SL3 distal end exchanges between two equally populated yet structurally distinct states in both isolated SL3 constructs and full-length 7SK RNA. We rationally designed constructs that lock SL3 into a single state and demonstrate that both chemical probing and NMR data fit to a linear combination of the two states. Comparison of vertebrate 7SK RNA sequences shows either or both states are highly conserved. These results provide new insights into 7SK RNA structural dynamics and demonstrate the utility of integrating chemical probing with NMR spectroscopy to gain quantitative insights into RNA conformational ensembles. 

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2. Illuminating an invisible state of the HIV-1 capsid protein CTD dimer using ¹⁹ F NMR and weighted ensemble simulations

   https://doi.org/10.1073/pnas.2420371122  

   Yang, Darian T; Chong, Lillian T; Gronenborn, Angela M (February 2025, Proceedings of the National Academy of Sciences)

   The HIV-1 capsid protein (CA) assembles into a conical shell during viral maturation, encasing and protecting the viral RNA genome. The C-terminal domain (CTD) of the two-domain capsid protein dimerizes, and this dimer connects individual chains in the mature capsid lattice. Previous NMR studies have shown that different dimer arrangements can be formed by isolated capsid protein chains and in assembled capsid lattices; however, the dynamics and functional relevance of these alternate dimers are unknown. To explore the conformational landscape of the CA-CTD dimer, we carried out atomistic molecular dynamics simulations using the weighted ensemble path sampling strategy, generating an ensemble of conformations. Focusing on the two dimer forms previously observed via solution NMR, we refined the conformational ensemble to highlight two metastable states using a Markov state model. Experimentally, we measured the interconversion rates between the two alternate dimers using¹⁹F NMR, and these rates showed good agreement with the interconversion rates derived from the simulations. After identifying the key interactions that distinguish the dimer states, the alternate dimer was further experimentally verified through disulfide crosslinking. Our results demonstrate the advantages of pairing weighted ensemble path sampling with¹⁹F NMR to gain atomistic insights into the hidden dimer state of the HIV-1 capsid protein. 

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3. Reaction Coordinate and Thermodynamics of Base Flipping in RNA

   https://doi.org/10.1021/acs.jctc.0c01199  

   Levintov, Lev; Paul, Sanjib; Vashisth, Harish (February 2021, Journal of Chemical Theory and Computation)

   null (Ed.)

   Base flipping is a key biophysical event involved in recognition of various ligands by ribonucleic acid (RNA) molecules. However, the mechanism of base flipping in RNA remains poorly understood, in part due to the lack of atomistic details on complex rearrangements in neighboring bases. In this work, we applied transition path sampling (TPS) methods to study base flipping in a double-stranded RNA (dsRNA) molecule that is known to interact with RNA-editing enzymes through this mechanism. We obtained an ensemble of 1000 transition trajectories to describe the base-flipping process. We used the likelihood maximization method to determine the refined reaction coordinate (RC) consisting of two collective variables (CVs), a distance and a dihedral angle between nucleotides that form stacking interactions with the flipping base. The free energy profile projected along the refined RC revealed three minima, two corresponding to the initial and final states and one for a metastable state. We suggest that the metastable state likely represents a wobbled conformation of nucleobases observed in NMR studies that is often characterized as the flipped state. The analyses of reactive trajectories further revealed that the base flipping is coupled to a global conformational change in a stem-loop of dsRNA. 

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4. Oxidation of phenylalanyl-tRNA synthetase positively regulates translational quality control

   https://doi.org/10.1073/pnas.1901634116  

   Steiner, Rebecca E.; Kyle, Amanda M.; Ibba, Michael (May 2019, Proceedings of the National Academy of Sciences)

   Accurate translation of the genetic code is maintained in part by aminoacyl-tRNA synthetases (aaRS) proofreading mechanisms that ensure correct attachment of a cognate amino acid to a transfer RNA (tRNA). During environmental stress, such as oxidative stress, demands on aaRS proofreading are altered by changes in the availability of cytoplasmic amino acids. For example, oxidative stress increases levels of cytotoxic tyrosine isomers, noncognate amino acids normally excluded from translation by the proofreading activity of phenylalanyl-tRNA synthetase (PheRS). Here we show that oxidation of PheRS induces a conformational change, generating a partially unstructured protein. This conformational change does not affect Phe or Tyr activation or the aminoacylation activity of PheRS. However, in vitro and ex vivo analyses reveal that proofreading activity to hydrolyze Tyr-tRNA Phe is increased during oxidative stress, while the cognate Phe-tRNA Phe aminoacylation activity is unchanged. In HPX − , Escherichia coli that lack reactive oxygen-scavenging enzymes and accumulate intracellular H 2 O 2 , we found that PheRS proofreading is increased by 11%, thereby providing potential protection against hazardous cytoplasmic m -Tyr accumulation. These findings show that in response to oxidative stress, PheRS proofreading is positively regulated without negative effects on the enzyme’s housekeeping activity in translation. Our findings also illustrate that while the loss of quality control and mistranslation may be beneficial under some conditions, increased proofreading provides a mechanism for the cell to appropriately respond to environmental changes during oxidative stress. 

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5. Distinct conformational states enable transglutaminase 2 to promote cancer cell survival versus cell death

   https://doi.org/10.1038/s42003-024-06672-x  

   Aplin, Cody; Zielinski, Kara_A; Pabit, Suzette; Ogunribido, Deborah; Katt, William_P; Pollack, Lois; Cerione, Richard_A; Milano, Shawn_K (August 2024, Communications Biology)

   Abstract Transglutaminase 2 (TG2) is a GTP-binding, protein-crosslinking enzyme that has been investigated as a therapeutic target for Celiac disease, neurological disorders, and aggressive cancers. TG2 has been suggested to adopt two conformational states that regulate its functions: a GTP-bound, closed conformation, and a calcium-bound, crosslinking-active open conformation. TG2 mutants that constitutively adopt an open conformation are cytotoxic to cancer cells. Thus, small molecules that bind and stabilize the open conformation of TG2 could offer a new therapeutic strategy. Here, we investigate TG2, using static and time-resolved small-angle X-ray scattering (SAXS) and single-particle cryoelectron microscopy (cryo-EM), to determine the conformational states responsible for conferring its biological effects. We also describe a newly developed TG2 inhibitor, LM11, that potently kills glioblastoma cells and use SAXS to investigate how LM11 affects the conformational states of TG2. Using SAXS and cryo-EM, we show that guanine nucleotides bind and stabilize a monomeric closed conformation while calcium binds to an open state that can form higher order oligomers. SAXS analysis suggests how a TG2 mutant that constitutively adopts the open state binds nucleotides through an alternative mechanism to wildtype TG2. Furthermore, we use time resolved SAXS to show that LM11 increases the ability of calcium to bind and stabilize an open conformation, which is not reversible by guanine nucleotides and is cytotoxic to cancer cells. Taken together, our findings demonstrate that the conformational dynamics of TG2 are more complex than previously suggested and highlight how conformational stabilization of TG2 by LM11 maintains TG2 in a cytotoxic conformational state. 

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