A hydrophobic core stabilizes the residual structure in the RRM2 intermediate state of the ALS-linked protein TDP-43.

Folding intermediates mediate both protein folding and the misfolding and aggregation observed in human diseases, including amyotrophic lateral sclerosis (ALS), and are prime targets for therapeutic interventions. In this study, we identified the core nucleus of structure for a folding intermediate in the second RNA recognition motif (RRM2) of the ALS-linked RNA-binding protein, TDP-43, using a combination of experimental and computational approaches. Urea equilibrium unfolding studies revealed that the RRM2 intermediate state consists of collapsed residual secondary structure localized to the N-terminal half of RRM2, while the C-terminus is largely disordered. Steered molecular dynamics simulations and mutagenesis studies yielded key stabilizing hydrophobic contacts that, when mutated to alanine, severely disrupt the overall fold of RRM2. In combination, these findings suggest a role for this RRM intermediate in normal TDP-43 function as well as serving as a template for misfolding and aggregation through the low stability and non-native secondary structure.

The Role of Substrate Mediated Allostery in the Catalytic Competency of the Bacterial Oligosaccharyltransferase PglB.

The oligosaccharyltransferase of Campylobacter lari (PglB) catalyzes the glycosylation of asparagine in the consensus sequence N-X-S/T, where X is any residue except proline. Molecular dynamics simulations of PglB bound to two different substrates were used to characterize the differences in the structure and dynamics of the substrate-enzyme complexes that can explain the higher catalytic efficiency observed for substrates containing threonine at the +2 position rather than serine. We observed that a threonine-containing substrate is more tightly bound than a serine-containing substrate. Because serine lacks a methyl group relative to threonine, the serine-containing peptide cannot stably form simultaneous van der Waals interactions with T316 and I572 as the threonine-containing substrate can. As a result, the peptide-PglB interaction is destabilized and the allosteric communication between the periplasmic domain and external loop EL5 is disrupted. These changes ultimately lead to the reorientation of the periplasmic domain relative to the transmembrane domain such that the two domains are further apart compared to PglB bound to the threonine-containing peptide. The crystal structure of PglB bound to the peptide and a lipid-linked oligosaccharide analog shows a pronounced closing of the periplasmic domain over the transmembrane domain in comparison to structures of PglB with peptide only, indicating that a closed conformation of the domains is needed for catalysis. The results of our studies suggest that lower enzymatic activity observed for serine versus threonine results from a combination of less stable binding and structural changes in PglB that influence the ability to form a catalytically competent state. This study illustrates a mechanism for substrate specificity via modulation of dynamic allosteric pathways.

ALS-linked PFN1 variants exhibit loss and gain of functions in the context of formin-induced actin polymerization.

Profilin-1 (PFN1) plays important roles in modulating actin dynamics through binding both monomeric actin and proteins enriched with polyproline motifs. Mutations in PFN1 have been linked to the neurodegenerative disease amyotrophic lateral sclerosis (ALS). However, whether ALS-linked mutations affect PFN1 function has remained unclear. To address this question, we employed an unbiased proteomics analysis in mammalian cells to identify proteins that differentially interact with mutant and wild-type (WT) PFN1. These studies uncovered differential binding between two ALS-linked PFN1 variants, G118V and M114T, and select formin proteins. Furthermore, both variants augmented formin-mediated actin assembly relative to PFN1 WT. Molecular dynamics simulations revealed mutation-induced changes in the internal dynamic couplings within an alpha helix of PFN1 that directly contacts both actin and polyproline, as well as structural fluctuations within the actin- and polyproline-binding regions of PFN1. These data indicate that ALS-PFN1 variants have the potential for heightened flexibility in the context of the ternary actin-PFN1-polyproline complex during actin assembly. Conversely, PFN1 C71G was more severely destabilized than the other PFN1 variants, resulting in reduced protein expression in both transfected and ALS patient lymphoblast cell lines. Moreover, this variant exhibited loss-of-function phenotypes in the context of actin assembly. Perturbations in actin dynamics and assembly can therefore result from ALS-linked mutations in PFN1. However, ALS-PFN1 variants may dysregulate actin polymerization through different mechanisms that depend upon the solubility and stability of the mutant protein.

Analysis of Emerging Variants in Structured Regions of the SARS-CoV-2 Genome.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has motivated a widespread effort to understand its epidemiology and pathogenic mechanisms. Modern high-throughput sequencing technology has led to the deposition of vast numbers of SARS-CoV-2 genome sequences in curated repositories, which have been useful in mapping the spread of the virus around the globe. They also provide a unique opportunity to observe virus evolution in real time. Here, we evaluate two sets of SARS-CoV-2 genomic sequences to identify emerging variants within structured cis-regulatory elements of the SARS-CoV-2 genome. Overall, 20 variants are present at a minor allele frequency of at least 0.5%. Several enhance the stability of Stem Loop 1 in the 5' untranslated region (UTR), including a group of co-occurring variants that extend its length. One appears to modulate the stability of the frameshifting pseudoknot between ORF1a and ORF1b, and another perturbs a bi-ss molecular switch in the 3'UTR. Finally, 5 variants destabilize structured elements within the 3'UTR hypervariable region, including the S2M (stem loop 2 m) selfish genetic element, raising questions as to the functional relevance of these structures in viral replication. Two of the most abundant variants appear to be caused by RNA editing, suggesting host-viral defense contributes to SARS-CoV-2 genome heterogeneity. Our analysis has implications for the development of therapeutics that target viral cis-regulatory RNA structures or sequences.

Analysis of Rapidly Emerging Variants in Structured Regions of the SARS-CoV-2 Genome.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has motivated a widespread effort to understand its epidemiology and pathogenic mechanisms. Modern high-throughput sequencing technology has led to the deposition of vast numbers of SARS-CoV-2 genome sequences in curated repositories, which have been useful in mapping the spread of the virus around the globe. They also provide a unique opportunity to observe virus evolution in real time. Here, we evaluate two cohorts of SARS-CoV-2 genomic sequences to identify rapidly emerging variants within structured cis-regulatory elements of the SARS-CoV-2 genome. Overall, twenty variants are present at a minor allele frequency of at least 0.5%. Several enhance the stability of Stem Loop 1 in the 5'UTR, including a set of co-occurring variants that extend its length. One appears to modulate the stability of the frameshifting pseudoknot between ORF1a and ORF1b, and another perturbs a bi-stable molecular switch in the 3'UTR. Finally, five variants destabilize structured elements within the 3'UTR hypervariable region, including the S2M stem loop, raising questions as to the functional relevance of these structures in viral replication. Two of the most abundant variants appear to be caused by RNA editing, suggesting host-viral defense contributes to SARS-CoV-2 genome heterogeneity. This analysis has implications for the development of therapeutics that target viral cis-regulatory RNA structures or sequences, as rapidly emerging variations in these regions could lead to drug resistance.

Structural Rearrangement upon Fragmentation of the Stability Core of the ALS-Linked Protein TDP-43.

Amyotrophic lateral sclerosis (ALS) is the most common adult degenerative motor neuron disease. Experimental evidence indicates a direct role of transactive-response DNA-binding protein 43 (TDP-43) in the pathology of ALS and other neurodegenerative diseases. TDP-43 has been identified as a major component of cytoplasmic inclusions in patients with sporadic ALS; however, the molecular basis of the disease mechanism is not yet fully understood. Fragmentation within the second RNA recognition motif (RRM2) of TDP-43 has been observed in patient tissues and may play a role in the formation of aggregates in disease. To determine the structural and dynamical changes resulting from the truncation that could lead to aggregation and toxicity, we performed molecular dynamics simulations of the full-length RRM2 domain (the stability core of TDP-43) and of a truncated variant (where residues 189-207 are deleted to mimic a site of cleavage within RRM2 found in ALS patients). Our simulations show heterogeneous structural reorganization and decreased stability of the truncated RRM2 domain compared to the full-length domain, consistent with previous experimental results. The decreased stability and structural reorganization in the truncated RRM2 result in a higher probability of protein-protein interactions through altered electrostatic surface charges and increased accessibility of hydrophobic residues (including the nuclear export sequence), providing a rationale for the increased cytoplasmic aggregation of RRM2 fragments seen in sporadic ALS patients.

Probing the structural and dynamical effects of the charged residues of the TZF domain of TIS11d.

A member of the TTP family of proteins, TIS11d binds RNA with high specificity using a pair of CCCH-type tandem zinc fingers separated by a 18 residue long linker. Our previous work showed that the formation of hydrogen bonds between the C-terminal residue E220 and the residues of the linker region stabilized a compact structure of TIS11d in the absence of RNA. To investigate the role of the C-terminal residues in the structure of unbound TIS11d, the E220A mutant and the truncation mutant lacking the last two residues (D219/E220) were studied using molecular dynamics, NMR spectroscopy, and biochemical methods. This study confirmed the importance of the charged residues D219 and E220 in maintaining structural stability in unbound TIS11d and elucidated the underlying physical mechanisms. We observed a greater structural heterogeneity for the residues of the linker in the molecular dynamics trajectories of both mutant proteins relative to the wild-type. This heterogeneity was more pronounced in the D219/E220 deletion mutant than in the E220A mutant, indicating that a greater reduction of the charge of the C-terminus results in greater flexibility. In agreement with the increased flexibility and the reduced number of negatively charged residues of the D219/E220 deletion mutant, we measured more unfavorable entropic and a more favorable enthalpic contribution to the free energy of RNA binding in the mutant than in the wild-type protein. The relative orientation of the zinc fingers was stabilized by the electrostatic interaction between E220 and positively charged residues of the linker in TIS11d. In the E220A mutant, the relative orientation of the zinc fingers was less constrained, whereas in the D219/E220 deletion mutant, little orientational preference was observed. We posit that favorable electrostatic interactions provide a mechanism to promote preferential orientation of separate domains without imposing structural rigidity.

Insight into the allosteric mechanism of Scapharca dimeric hemoglobin.

Allosteric regulation is an essential function of many proteins that control a variety of different processes such as catalysis, signal transduction, and gene regulation. Structural rearrangements have historically been considered the main means of communication between different parts of a protein. Recent studies have highlighted the importance, however, of changes in protein flexibility as an effective way to mediate allosteric communication across a protein. Scapharca dimeric hemoglobin (HbI) is the simplest possible allosteric system, with cooperative ligand binding between two identical subunits. Thermodynamic equilibrium studies of the binding of oxygen to HbI have shown that cooperativity is an entropically driven effect. The change in entropy of the system observed upon ligand binding may arise from changes in the protein, the ligand, or the water of the system. The goal of this study is to determine the contribution of the change in entropy of the protein backbone to HbI cooperative binding. Molecular dynamics simulations and nuclear magnetic resonance relaxation techniques have revealed that the fast internal motions of HbI contribute to the cooperative binding to carbon monoxide in two ways: (1) by contributing favorably to the free energy of the system and (2) by participating in the cooperative mechanism at the HbI subunit interface. The internal dynamics of the weakly cooperative HbI mutant, F97Y, were also investigated with the same methods. The changes in backbone NH dynamics observed for F97Y HbI upon ligand binding are not as large as for the wild type, in agreement with the reduced cooperativity observed for this mutant. The results of this study indicate that interface flexibility and backbone conformational entropy of HbI participate in and are important for the cooperative mechanism of carbon monoxide binding.

A computational study of RNA binding and specificity in the tandem zinc finger domain of TIS11d.

TIS11d is a member of the CCCH-type family of tandem zinc finger (TZF) proteins; the TZF domain of TIS11d (residues 151-220) is sufficient to bind and destabilize its target mRNAs with high specificity. In this study, the TZF domain of TIS11d is simulated in an aqueous environment in both the free and RNA-bound states. Multiple nanosecond timescale molecular dynamics trajectories of TIS11d wild-type and E157R/E195K mutant with different RNA sequences were performed to investigate the molecular basis for RNA binding specificities of this TZF domain. A variety of measures of the protein structure, fluctuations, and dynamics were used to analyze the trajectories. The results of this study support the following conclusions: (1) the structure of the two fingers is maintained in the free state but a global reorientation occurs to yield a more compact structure; (2) mutation of the glutamate residues at positions 157 and 195 to arginine and lysine, respectively, affects the RNA recognition by this TIS11d mutant in agreement with the findings of Pagano et al. (J Biol Chem 2007; 282:8883-8894); and (3) we predict that the E157R/E195K mutant will present a more relaxed RNA binding specificity relative to wild-type TIS11d based on the more favorable nonsequence-specific Coulomb interaction of the two positively charged residues at positions 157 and 195 with the RNA backbone, which compensates for a partial loss of the stacking interaction of aromatic side chains with the RNA bases.

Accurate Estimates of Free Energy Changes in Charge Mutations.

The ability to determine the effect of charge changes on the free energy is necessary for fundamental studies of the electrostatic contribution to protein binding and stability. Currently, calculations of differences in free energy for charge mutations carried out in systems with periodic boundary conditions must include an approximate self-energy correction that can be on the same order of magnitude as the calculated free energy change. Here, a new method for accurately calculating the free energy change associated with any alchemical mutation, regardless of charge, is presented. In this method, paired mutations of opposite charge exactly cancel the self-energy term because of its quadratic charge dependence. Since the self-energy term implicitly cancels within the method, a correction never needs to be applied, and the statistical uncertainty associated is thereby removed. An implementation procedure is described and applied to the free energy of ionic hydration and a charged amino acid mutation.