An excited state underlies gene regulation of a transcriptional riboswitch

Riboswitches control gene expression through ligand-dependent structural rearrangements of the sensing aptamer domain. However, we found that the Bacillus cereus fluoride riboswitch aptamer adopts identical tertiary structures in solution with and without ligand. Using chemical exchange saturation transfer (CEST) NMR spectroscopy, we revealed that the structured ligand-free aptamer transiently accesses a low-populated (~1%) and short-lived (~3 ms) excited conformational state that unravels a conserved ‘linchpin’ base pair to signal transcription termination. Upon fluoride binding, this highly localized fleeting process is allosterically suppressed to activate transcription. We demonstrated that this mechanism confers effective fluoride-dependent gene activation over a wide range of transcription rates, which is essential for robust toxicity response across diverse cellular conditions. These results unveil a novel switching mechanism that employs ligand-dependent suppression of an aptamer excited state to coordinate regulatory conformational transitions rather than adopting distinct aptamer ground-state tertiary architectures, exemplifying a new mode of ligand-dependent RNA regulation

Structure modeling of RNA using sparse NMR constraints

RNAs fold into distinct molecular conformations that are often essential for their functions. Accurate structure modeling of complex RNA motifs, including ubiquitous non-canonical base pairs and pseudoknots, remains a challenge. Here, we present an NMR-guided all-atom discrete molecular dynamics (DMD) platform, iFoldNMR, for rapid and accurate structure modeling of complex RNAs. We show that sparse distance constraints from imino resonances, which can be readily obtained from routine NMR experiments and easier to compile than laborious assignments of non-solvent-exchangeable protons, are sufficient to direct a DMD search for low-energy RNA conformers. Benchmarking on a set of RNAs with complex folds spanning up to 56 nucleotides in length yields structural models that recapitulate experimentally determined structures with all-heavy-atom RMSDs ranging from 2.4 to 6.5 Å. This platform represents an efficient approach for high-throughput RNA structure modeling and will facilitate analysis of diverse, newly discovered functional RNAs

The structure of Apolipoprotein E isoforms and their role in Alzheimer's disease

Alzheimer’s disease (AD) is a chronic, incurable neurodegenerative disorder. AD is the most common form of dementia. The strongest genetic risk factor for AD is the expression of the E4 isoform of Apolipoprotein E (ApoE4), found in ~15% of the population. The other two common variants of this protein, namely ApoE2 and ApoE3, are respectively protective and neutral isoforms in regards to the development of AD. The three ApoE variants are strikingly similar in sequence, differing only at two locations. However, biophysical studies reveal that while functionally comparable to the other variants, ApoE4 is less thermally stable and shows a misfolded intermediate state, which has been associated with the onset of AD. Despite the strong connection to AD and extensive research in lipid metabolism, the ApoE4 structural features responsible for its pathogenic role in AD remain unresolved. In this work, we explore the unique conformational landscape of ApoE isoforms to identify specific structural features responsible for AD onset. We perform a series of molecular dynamics simulations for all three ApoE isoforms, and we find that ApoE4 has access to a unique folding intermediate conformation with inter-domain interactions not seen in ApoE2 or ApoE3. We generate a structural model of an ApoE4-specific misfolded state to predict mutations that can affect the stability of key contacts in this specific conformation. In addition to identifying interacting residues in the misfolded intermediate state, we create models with C-terminal truncations to narrow in on the most vital inter-domain contacts. To determine the inherent isoform differences, we study residue communication networks using dynamic cross- correlations to show that even in the native conformation, ApoE4 exhibits unique dynamic properties that ultimately lead to its distinctive conformational landscape. Our findings suggest that the underlying role of ApoE4 in the development of AD can be linked to its isoform-specific structural and conformational dynamics.Doctor of Philosoph

Free energy landscapes of ApoE isoforms.

<p>ApoE isoforms’ conformational landscapes derived from PMF as a function of RMSD and Rg of ApoE variants’ N-terminal domains. C-terminal domains are excluded from the analysis to reduce the degeneracy of protein conformational states. (A-C) The free energy landscapes from REX/DMD simulations at T1 (~275 K for all three ApoE isoforms) are isolated in the lowest range of RMSD and Rg suggesting the majority of conformations are close to the native N-terminal domain state. (D-F) At T2 (~321 K, ~318 K, and ~309 K for ApoE2, ApoE3 and ApoE4 respectively) all three variants explore a larger area of the conformational landscape as denoted by the larger RMSD and Rg values. (G-I) At T3 (~340 K, ~338 K, and ~328 K for ApoE2, ApoE3 and ApoE4 respectively) the isoforms transition to their intermediate states. ApoE3 is characterized by both the native and alternate N-terminal domain conformations, while ApoE2 visits only the latter. ApoE4 exhibits a unique, more compact intermediate conformational state as denoted by the smaller range of RMSD and Rg values compared to the two other variants. (J-L) At T4 (~355 K, ~365 K, and ~342 K for ApoE2, ApoE3, and ApoE4, respectively) all three isoforms undergo complete unfolding as inferred by their extended landscapes in the high range of RMSD and Rg values, although ApoE4 also visits the previous conformational states identified at temperature T3. (Note the different scale on x- and y-axes; representative structures are reported in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004359#pcbi.1004359.s002" target="_blank">S2 Fig</a>). The color bar represents the relative Helmholtz free energy in kcal/mol.</p

ApoE4-specific Misfolded Intermediate Identified by Molecular Dynamics Simulations

<div><p>The increased risk of developing Alzheimer’s disease (AD) is associated with the <i>APOE</i> gene, which encodes for three variants of Apolipoprotein E, namely E2, E3, E4, differing only by two amino acids at positions 112 and 158. ApoE4 is known to be the strongest risk factor for AD onset, while ApoE3 and ApoE2 are considered to be the AD-neutral and AD-protective isoforms, respectively. It has been hypothesized that the ApoE isoforms may contribute to the development of AD by modifying the homeostasis of ApoE physiological partners and AD-related proteins in an isoform-specific fashion. Here we find that, despite the high sequence similarity among the three ApoE variants, only ApoE4 exhibits a misfolded intermediate state characterized by isoform-specific domain-domain interactions in molecular dynamics simulations. The existence of an ApoE4-specific intermediate state can contribute to the onset of AD by altering multiple cellular pathways involved in ApoE-dependent lipid transport efficiency or in AD-related protein aggregation and clearance. We present what we believe to be the first structural model of an ApoE4 misfolded intermediate state, which may serve to elucidate the molecular mechanism underlying the role of ApoE4 in AD pathogenesis. The knowledge of the structure for the ApoE4 folding intermediate provides a new platform for the rational design of alternative therapeutic strategies to fight AD.</p></div

Temperature-dependent pair-wise inter-residue distances of ApoE isoforms.

<p>(A-C) At T1 (~275 K for all three ApoE isoforms) and (D-F) at T2 (~321 K, ~318 K, and ~309 K for ApoE2, ApoE3 and ApoE4 respectively), all three isoforms exhibits the highest level of inter-residue contacts observed in the REX/DMD simulations, with ApoE4 having the highest density contacts. (G-I) At T3 (~340 K, ~338 K, and ~328 K for ApoE2, ApoE3 and ApoE4 respectively), all three isoforms exhibit a dramatic decrease in density of inter-residue contacts. ApoE4 displays a unique series of contacts (outlined in red) mediating the domain-domain interaction as discussed in the main text. (J-L) At T4 (~355 K, ~365 K, and ~342 K for ApoE2, ApoE3, and ApoE4, respectively), the majority of inter-residue contacts have been lost besides some transient contacts involving the N-terminal helix–4. The upper and lower triangular matrices represent respectively the average and the standard deviation of the pair-wise inter-residue distance in Å. The color bar represents the distance between the centroid computed over the residues’ side chains in Å.</p

Representative structures of ApoE isoforms.

<p>(A) ApoE3 representative structure (<i>i</i>.<i>e</i>., centroid of the most populated cluster) from clustering analysis of the protein conformations extracted from the free energy basin at T1 (~275 K, see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004359#pcbi.1004359.g002" target="_blank">Fig 2B</a>). The same compact, native state of the N-terminal helices is observed in all three ApoE isoforms (see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004359#pcbi.1004359.s002" target="_blank">S2A–S2C Fig</a>). At T3 (~340 K, ~338 K, and ~328 K for ApoE2, ApoE3 and ApoE4 respectively) the representative structure of the intermediate state for: (B) ApoE2 exhibits an expanded volume of the N-terminal domain due to an increase of the average inter-helical distances; (C) ApoE3 exhibits a pairing of N-terminal helix–1 and helix–4 which separate from helix–2 and helix–3; (D) ApoE4 exhibits a separation of helix–1 from the other three helices. Such conformation represents the identified isoform-specific misfolded intermediate state (inter-residue contacts shown in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004359#pcbi.1004359.s006" target="_blank">S6A Fig</a>) The size of the most populated cluster is reported in each panel. For all structures, helix–1 (H1), helix–2 (H2), helix–3 (H3), and helix–4 (H4) are represented in purple, green, blue, and red, cartoon respectively. The rest of the protein is represented in grey cartoon. (The sequence numbers for helices is reported in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004359#pcbi.1004359.s001" target="_blank">S1 Fig</a>).</p

ApoE isoform-specific mutations.

<p>ApoE isoform-specific mutations.</p

Heat capacity curves of ApoE isoforms.

<p>(A) The heat capacity (Cv) curves computed using WHAM on REX/DMD trajectories for ApoE2 (black), ApoE3 (red) and ApoE4 (blue) in the range of 275 to 400 K show intermediates states that appear at different temperatures for each isoform. The position of the first peak (<i>i</i>.<i>e</i>., unfolding of the hydrophobic core of the protein) suggests that ApoE4 is less thermally stable than ApoE2 and ApoE3. (B-D) Cv curves of individual ApoE isoforms including the error bars (shaded grey area). The shaded grey area in panels B-D represents the statistical uncertainty (<i>i</i>.<i>e</i>., the square root of the variance of the specific heat) in the WHAM estimation of heat capacity. Local minima in the curves at temperatures T1, T2, T3, and T4 represent different conformational states of the protein for each ApoE variant.</p