Modeling allosteric mechanisms of eukaryotic type II topoisomerases.

Type II topoisomerases (TopoIIs) are ubiquitous enzymes that are involved in crucial nuclear processes such as genome organization, chromosome segregation, and other DNA metabolic processes. These enzymes function as large, homodimeric complexes that undergo a complex cycle of binding and hydrolysis of two ATP molecules in their ATPase domains, which regulates the capture and passage of one DNA double-helix through a second, cleaved DNA molecule. This process requires the transmission of information about the state of the bound nucleotide over vast ranges in the TopoII complex. How this information is transmitted at the molecular level to regulate TopoII functions and how protein substitutions disrupt these mechanisms remains largely unknown. Here, we employed extensive microsecond-scale molecular dynamics simulations of the yeast TopoII enzyme in multiple nucleotide-bound states and with amino acid substitutions near both the N and C termini of the complex. Simulation results indicate that the ATPase domains are remarkably flexible on the sub-microsecond timescale and that these dynamics are modulated by the identity of the bound nucleotides and both local and distant amino acid substitutions. Network analyses point toward specific allosteric networks that transmit information about the hydrolysis cycle throughout the complex, which include residues in both the protein and the bound DNA molecule. Amino acid substitutions weaken many of these pathways. Together, our results provide molecular level details on how the TopoII catalytic cycle is controlled through nucleotide binding and hydrolysis and how mutations may disrupt this process.

Modeling Allosteric Mechanisms of Eukaryotic Type II Topoisomerases.

Type II topoisomerases (TopoIIs) are ubiquitous enzymes that are involved in crucial nuclear processes such as genome organization, chromosome segregation, and other DNA metabolic processes. These enzymes function as large, homodimeric complexes that undergo a complex cycle of binding and hydrolysis of two ATP molecules in their ATPase domains, which regulates the capture and passage of one DNA double-helix through a second, cleaved DNA molecule. This process requires the transmission of information about the state of the bound nucleotide over vast ranges in the TopoII complex. How this information is transmitted at the molecular level to regulate TopoII functions and how protein substitutions disrupt these mechanisms remains largely unknown. Here, we employed extensive microsecond scale molecular dynamics simulations of the yeast TopoII enzyme in multiple nucleotide-bound states and with amino acid substitutions near both the N- and C-terminals of the complex. Simulation results indicate that the ATPase domains are remarkably flexible on the sub-microsecond timescale and that these dynamics are modulated by the identity of the bound nucleotides and both local and distant amino acid substitutions. Network analyses point towards specific allosteric networks that transmit information about the hydrolysis cycle throughout the complex, which include residues in both the protein and the bound DNA molecule. Amino acid substitutions weaken many of these pathways. Together, our results provide molecular-level details on how the TopoII catalytic cycle is controlled through nucleotide binding and hydrolysis and how mutations may disrupt this process.

The effects of RNA.DNA-DNA triple helices on nucleosome structures and dynamics.

Noncoding RNAs (ncRNAs) are an emerging epigenetic factor and have been recognized as playing a key role in many gene expression pathways. Structurally, binding of ncRNAs to isolated DNA is strongly dependent on sequence complementary and results in the formation of an RNA.DNA-DNA (RDD) triple helix. However, in vivo DNA is not isolated but is rather packed in chromatin fibers, the fundamental unit of which is the nucleosome. Biochemical experiments have shown that ncRNA binding to nucleosomal DNA is elevated at DNA entry and exit sites and is dependent on the presence of the H3 N-terminal tails. However, the structural and dynamical bases for these mechanisms remain unknown. Here, we have examined the mechanisms and effects of RDD formation in the context of the nucleosome using a series of all-atom molecular dynamics simulations. Results highlight the importance of DNA sequence on complex stability, elucidate the effects of the H3 tails on RDD structures, show how RDD formation impacts the structure and dynamics of the H3 tails, and show how RNA alters the local and global DNA double-helical structure. Together, our results suggest ncRNAs can modify nucleosome, and potentially higher-order chromatin, structures and dynamics as a means of exerting epigenetic control.

Conformational and Thermodynamic Differences Underlying Wild-Type and Mutant Eleven-Nineteen-Leukemia YEATS Domain Specificity for Epigenetic Marks.

Histone post-translational modifications (PTMs) are interpreted by multiple reader domains and proteins to regulate gene expression. The eleven-nineteen-leukemia (ENL) YEATS domain is a prototypical PTM reader that recognizes multiple lysine acetylation marks on the histone H3 tails as a way of recruiting chromatin remodellers. Two ENL YEATS mutations have been identified which have been linked with leukemia, Wilms tumor, and other forms of cancer and result in either an insertion or deletion of residues in the loop connecting beta sheets distant from the protein active site. In vitro experiments have shown that these mutations modulate the selectivities of YEATS domains for various lysine acetylation marks, although different experiments have provided contrasting views on the abilities of the insertion and deletion mutants to discern specific PTMs. Here, we have performed multiple molecular dynamics simulations of wild-type and insertion and deletion mutant YEATS domains free from and in complex with two PTM peptides: one that is acetylated at K9 of H3 and the other that is acetylated at residue K27 of H3. Results show that these two peptides have distinct flexibilities and binding energetics when bound to YEATS domains and that these properties are affected by interactions with residues within and outside of the peptide consensus motif. Furthermore, these properties are modulated by the YEATS insertion and deletion mutants, which results in disparate binding effects in these systems. Together, these results suggest that only the partial exposure of histone tails is sufficient in the context of nucleosomes for YEATS-mediated recognition of acetylation marks on histone tails. They also caution against the overinterpretation of results obtained from experiments on reader domain-histone peptide binding in isolation and not in the full-length nucleosome context.

Directed Inter-domain Motions Enable the IsdH Staphylococcus aureus Receptor to Rapidly Extract Heme from Human Hemoglobin.

Pathogenic Staphylococcus aureus actively acquires iron from human hemoglobin (Hb) using the IsdH surface receptor. Heme extraction is mediated by a tri-domain unit within the receptor that contains its second (N2) and third (N3) NEAT domains joined by a helical linker domain. Extraction occurs within a dynamic complex, in which receptors engage each globin chain; the N2 domain tightly binds to Hb, while substantial inter-domain motions within the receptor enable its N3 domain to transiently distort the globin's heme pocket. Using molecular simulations coupled with Markov modeling, along with stopped-flow experiments to quantitatively measure heme transfer kinetics, we show that directed inter-domain motions within the receptor play a critical role in the extraction process. The directionality of N3 domain motion and the rate of heme extraction is controlled by amino acids within a short, flexible inter-domain tether that connects the N2 and linker domains. In the wild-type receptor directed motions originating from the tether enable the N3 domain to populate configurations capable of distorting Hb's pocket, whereas mutant receptors containing altered tethers are less able to adopt these conformers and capture heme slowly via indirect processes in which Hb first releases heme into the solvent. Thus, our results show inter-domain motions within the IsdH receptor play a critical role in its ability to extract heme from Hb and highlight the importance of directed motions by the short, unstructured, amino acid sequence connecting the domains in controlling the directionality and magnitude of these functionally important motions.

CENP-N promotes the compaction of centromeric chromatin.

The histone variant CENP-A is the epigenetic determinant for the centromere, where it is interspersed with canonical H3 to form a specialized chromatin structure that nucleates the kinetochore. How nucleosomes at the centromere arrange into higher order structures is unknown. Here we demonstrate that the human CENP-A-interacting protein CENP-N promotes the stacking of CENP-A-containing mononucleosomes and nucleosomal arrays through a previously undefined interaction between the α6 helix of CENP-N with the DNA of a neighboring nucleosome. We describe the cryo-EM structures and biophysical characterization of such CENP-N-mediated nucleosome stacks and nucleosomal arrays and demonstrate that this interaction is responsible for the formation of densely packed chromatin at the centromere in the cell. Our results provide first evidence that CENP-A, together with CENP-N, promotes specific chromatin higher order structure at the centromere.

Peptide Dynamics and Metadynamics: Leveraging Enhanced Sampling Molecular Dynamics to Robustly Model Long-Timescale Transitions.

Molecular dynamics simulations can in theory reveal the thermodynamics and kinetics of peptide conformational transitions at atomic-level resolution. However, even with modern computing power, they are limited in the timescales they can sample, which is especially problematic for peptides that are fully or partially disordered. Here, we discuss how the enhanced sampling methods accelerated molecular dynamics (aMD) and metadynamics can be leveraged in a complementary fashion to quickly explore conformational space and then robustly quantify the underlying free energy landscape. We apply these methods to two peptides that have an intrinsically disordered nature, the histone H3 and H4 N-terminal tails, and use metadynamics to compute the free energy landscape along collective variables discerned from aMD simulations. Results show that these peptides are largely disordered, with a slight preference for α-helical structures.

Nucleosome composition regulates the histone H3 tail conformational ensemble and accessibility.

Hexasomes and tetrasomes are intermediates in nucleosome assembly and disassembly. Their formation is promoted by histone chaperones, ATP-dependent remodelers, and RNA polymerase II. In addition, hexasomes are maintained in transcribed genes and could be an important regulatory factor. While nucleosome composition has been shown to affect the structure and accessibility of DNA, its influence on histone tails is largely unknown. Here, we investigate the conformational dynamics of the H3 tail in the hexasome and tetrasome. Using a combination of NMR spectroscopy, MD simulations, and trypsin proteolysis, we find that the conformational ensemble of the H3 tail is regulated by nucleosome composition. As has been found for the nucleosome, the H3 tails bind robustly to DNA within the hexasome and tetrasome, but upon loss of the H2A/H2B dimer, we determined that the adjacent H3 tail has an altered conformational ensemble, increase in dynamics, and increase in accessibility. Similar to observations of DNA dynamics, this is seen to be asymmetric in the hexasome. Our results indicate that nucleosome composition has the potential to regulate chromatin signaling and ultimately help shape the chromatin landscape.

The Dynamic Influence of Linker Histone Saturation within the Poly-Nucleosome Array.

Linker histones bind to nucleosomes and modify chromatin structure and dynamics as a means of epigenetic regulation. Biophysical studies have shown that chromatin fibers can adopt a plethora of conformations with varying levels of compaction. Linker histone condensation, and its specific binding disposition, has been associated with directly tuning this ensemble of states. However, the atomistic dynamics and quantification of this mechanism remains poorly understood. Here, we present molecular dynamics simulations of octa-nucleosome arrays, based on a cryo-EM structure of the 30-nm chromatin fiber, with and without the globular domains of the H1 linker histone to determine how they influence fiber structures and dynamics. Results show that when bound, linker histones inhibit DNA flexibility and stabilize repeating tetra-nucleosomal units, giving rise to increased chromatin compaction. Furthermore, upon the removal of H1, there is a significant destabilization of this compact structure as the fiber adopts less strained and untwisted states. Interestingly, linker DNA sampling in the octa-nucleosome is exaggerated compared to its mono-nucleosome counterparts, suggesting that chromatin architecture plays a significant role in DNA strain even in the absence of linker histones. Moreover, H1-bound states are shown to have increased stiffness within tetra-nucleosomes, but not between them. This increased stiffness leads to stronger long-range correlations within the fiber, which may result in the propagation of epigenetic signals over longer spatial ranges. These simulations highlight the effects of linker histone binding on the internal dynamics and global structure of poly-nucleosome arrays, while providing physical insight into a mechanism of chromatin compaction.

Archaeal chromatin 'slinkies' are inherently dynamic complexes with deflected DNA wrapping pathways.

Eukaryotes and many archaea package their DNA with histones. While the four eukaryotic histones wrap ~147 DNA base pairs into nucleosomes, archaeal histones form 'nucleosome-like' complexes that continuously wind between 60 and 500 base pairs of DNA ('archaeasomes'), suggested by crystal contacts and analysis of cellular chromatin. Solution structures of large archaeasomes (>90 DNA base pairs) have never been directly observed. Here, we utilize molecular dynamics simulations, analytical ultracentrifugation, and cryoEM to structurally characterize the solution state of archaeasomes on longer DNA. Simulations reveal dynamics of increased accessibility without disruption of DNA-binding or tetramerization interfaces. Mg2+ concentration influences compaction, and cryoEM densities illustrate that DNA is wrapped in consecutive substates arranged 90o out-of-plane with one another. Without ATP-dependent remodelers, archaea may leverage these inherent dynamics to balance chromatin packing and accessibility.

All animals, plants and fungi belong to a group of living organisms called eukaryotes. The two other groups are bacteria and archaea, which include unicellular, microscopic organisms. All three groups have genes, which are typically stored on long strands of DNA. Eukaryotes have so much DNA that they use proteins called histones to help package and organize it inside each cell. Archaea also have simplified histones that help store their DNA, and studying these proteins could reveal how eukaryotic histones first evolved. In eukaryotes, groups of eight histones form a short cylinder that organizes a small section of DNA into a structure called a nucleosome. Each cell needs hundreds of thousands of nucleosomes to arrange its DNA. Eukaryotic cells also contain other proteins that release pieces of DNA from histones so that their genetic information can be used. The histones in Archaea don't form discrete nucleosomes, instead, they coil DNA into 'slinky-like' shapes. It's still unclear how DNA packing in archaea works and how it differs from eukaryotes. Bowerman, Wereszczynski and Luger used computer simulations, biochemistry and cryo-electron microscopy to study the histones from archaea. The archaeal 'slinky-like' histone structures are more flexible than nucleosomes, and can open and close like clamshells. This flexibility allows the information in the genomes of Archaea to be easily accessed, so, unlike in eukaryotes, archaeal cells may not need other proteins to release the DNA from the histones. The ability to package DNA allows cells to contain many more genes, so evolving histones was a vital step in the evolution of eukaryotic life, including the appearance of animals. Archaeal histones may reflect early versions of histones in eukaryotes, and can be used to understand how DNA packing has evolved. Furthermore, a greater understanding of Archaea may help better explain their role in health and global ecosystems, and allow their use in industrial applications.

Effects of H2A.B incorporation on nucleosome structures and dynamics.

The H2A.B histone variant is an epigenetic regulator involved in transcriptional upregulation, DNA synthesis, and splicing that functions by replacing the canonical H2A histone in the nucleosome core particle. Introduction of H2A.B results in less compact nucleosome states with increased DNA unwinding and accessibility at the nucleosomal entry and exit sites. Despite being well characterized experimentally, the molecular mechanisms by which H2A.B incorporation alters nucleosome stability and dynamics remain poorly understood. To study the molecular mechanisms of H2A.B, we have performed a series of conventional and enhanced sampling molecular dynamics simulation of H2A.B- and canonical H2A-containing nucleosomes. Results of conventional simulations show that H2A.B weakens protein-protein and protein-DNA interactions at specific locations throughout the nucleosome. These weakened interactions result in significantly more DNA opening from both the entry and exit sites in enhanced sampling simulations. Furthermore, free energy profiles show that H2A.B-containing nucleosomes have significantly broader free wells and that H2A.B allows for sampling of states with increased DNA breathing, which are shown to be stable on the hundreds of nanoseconds timescale with further conventional simulations. Together, our results show the molecular mechanisms by which H2A.B creates less compacted nucleosome states as a means of increasing genetic accessibility and gene transcription.

WASP: a software package for correctly characterizing the topological development of ribbon structures.

We introduce the Writhe Application Software Package (WASP) which can be used to characterisze the topology of ribbon structures, the underlying mathematical model of DNA, Biopolymers, superfluid vorticies, elastic ropes and magnetic flux ropes. This characterization is achieved by the general twist-writhe decomposition of both open and closed ribbons, in particular through a quantity termed the polar writhe. We demonstrate how this decomposition is far more natural and straightforward than artificial closure methods commonly utilized in DNA modelling. In particular, we demonstrate how the decomposition of the polar writhe into local and non-local components distinctly characterizes the local helical structure and knotting/linking of the ribbon. This decomposition provides additional information not given by alternative approaches. As example applications, the WASP routines are used to characterise the evolving topology (writhe) of DNA minicircle and open ended plectoneme formation magnetic/optical tweezer simulations, and it is shown that the decomponsition into local and non-local components is particularly important for the detection of plectonemes. Finally it is demonstrated that a number of well known alternative writhe expressions are actually simplifications of the polar writhe measure.

The molecular basis of selective DNA binding by the BRG1 AT-hook and bromodomain.

The ATP-dependent BAF chromatin remodeling complex plays a critical role in gene regulation by modulating chromatin architecture, and is frequently mutated in cancer. Indeed, subunits of the BAF complex are found to be mutated in >20% of human tumors. The mechanism by which BAF properly navigates chromatin is not fully understood, but is thought to involve a multivalent network of histone and DNA contacts. We previously identified a composite domain in the BRG1 ATPase subunit that is capable of associating with both histones and DNA in a multivalent manner. Mapping the DNA binding pocket revealed that it contains several cancer mutations. Here, we utilize SELEX-seq to investigate the DNA specificity of this composite domain and NMR spectroscopy and molecular modelling to determine the structural basis of DNA binding. Finally, we demonstrate that cancer mutations in this domain alter the mode of DNA association.

Elucidating the influence of linker histone variants on chromatosome dynamics and energetics.

Linker histones are epigenetic regulators that bind to nucleosomes and alter chromatin structures and dynamics. Biophysical studies have revealed two binding modes in the linker histone/nucleosome complex, the chromatosome, where the linker histone is either centered on or askew from the dyad axis. Each has been posited to have distinct effects on chromatin, however the molecular and thermodynamic mechanisms that drive them and their dependence on linker histone compositions remain poorly understood. We present molecular dynamics simulations of chromatosomes with the globular domain of two linker histone variants, generic H1 (genGH1) and H1.0 (GH1.0), to determine how their differences influence chromatosome structures, energetics and dynamics. Results show that both unbound linker histones adopt a single compact conformation. Upon binding, DNA flexibility is reduced, resulting in increased chromatosome compaction. While both variants enthalpically favor on-dyad binding, energetic benefits are significantly higher for GH1.0, suggesting that GH1.0 is more capable than genGH1 of overcoming the large entropic reduction required for on-dyad binding which helps rationalize experiments that have consistently demonstrated GH1.0 in on-dyad states but that show genGH1 in both locations. These simulations highlight the thermodynamic basis for different linker histone binding motifs, and details their physical and chemical effects on chromatosomes.

Lipopolysaccharide Simulations Are Sensitive to Phosphate Charge and Ion Parameterization.

The high proportion of lipopolysaccharide (LPS) molecules in the outer membrane of Gram-negative bacteria makes it a highly effective barrier to small molecules, antibiotic drugs, and other antimicrobial agents. Given this vital role in protecting bacteria from potentially hostile environments, simulations of LPS bilayers and outer membrane systems represent a critical tool for understanding the mechanisms of bacterial resistance and the development of new antibiotic compounds that circumvent these defenses. The basis of these simulations is parameterizations of LPS, which have been developed for all major molecular dynamics force fields. However, these parameterizations differ in both the protonation state of LPS and how LPS membranes behave in the presence of various ion species. To address these discrepancies and understand the effects of phosphate charge on bilayer properties, simulations were performed for multiple distinct LPS chemotypes with different ion parameterizations in both protonated or deprotonated lipid A states. These simulations show that bilayer properties, such as the area per lipid and inter-lipid hydrogen bonding, are highly influenced by the choice of phosphate group charges, cation type, and ion parameterization, with protonated LPS and monovalent cations with modified nonbonded parameters providing the best match to the experiments. Additionally, alchemical free energy simulations were performed to determine theoretical pKa values for LPS and subsequently validated by 31P solid-state nuclear magnetic resonance experiments. Results from these complementary computational and experimental studies demonstrate that the protonated state dominates at physiological pH, contrary to the deprotonated form modeled by many LPS force fields. Overall, these results highlight the sensitivity of LPS simulations to phosphate charge and ion parameters while offering recommendations for how existing models should be updated for consistency between force fields as well as to best match experiments.

The Staphylococcus aureus IsdH Receptor Forms a Dynamic Complex with Human Hemoglobin that Triggers Heme Release via Two Distinct Hot Spots.

Iron is an essential nutrient that is actively acquired by bacterial pathogens during infections. Clinically important Staphylococcus aureus obtains iron by extracting heme from hemoglobin (Hb) using the closely related IsdB and IsdH surface receptors. In IsdH, extraction is mediated by a conserved tridomain unit that contains its second (N2) and third (N3) NEAT domains joined by a helical linker, called IsdHN2N3. Leveraging the crystal structure of the IsdHN2N3:Hb complex, we have probed the mechanism of heme capture using NMR, stopped-flow transfer kinetics measurements, and molecular dynamics (MD) simulations. NMR studies of the 220 kDa IsdHN2N3:Hb complex reveal that it is dynamic, with persistent interdomain motions enabling the linker and N3 domains in the receptor to transiently engage Hb to remove its heme. An alanine mutagenesis analysis reveals that two receptor subsites positioned ~20 Å apart trigger heme release by contacting Hb's F-helix. These subsites are located within the N3 and linker domains and appear to play distinct roles in stabilizing the heme transfer transition state. Linker domain contacts primarily function to destabilize Hb-heme interactions, thereby lowering ΔH‡, while contacts from the N3 subsite play a similar destabilizing role, but also form a bridge through which heme moves from Hb to the receptor. Interestingly, MD simulations suggest that within the transiently forming interface, both the F-helix and receptor bridge are in motion, dynamically sampling conformations that are suitable for heme transfer. Thus, IsdH triggers heme release from Hb via a flexible, low-affinity interface that forms fleetingly in solution.

BEES: Bayesian Ensemble Estimation from SAS.

Many biomolecular complexes exist in a flexible ensemble of states in solution that is necessary to perform their biological function. Small-angle scattering (SAS) measurements are a popular method for characterizing these flexible molecules because of their relative ease of use and their ability to simultaneously probe the full ensemble of states. However, SAS data is typically low dimensional and difficult to interpret without the assistance of additional structural models. In theory, experimental SAS curves can be reconstituted from a linear combination of theoretical models, although this procedure carries a significant risk of overfitting the inherently low-dimensional SAS data. Previously, we developed a Bayesian-based method for fitting ensembles of model structures to experimental SAS data that rigorously avoids overfitting. However, we have found that these methods can be difficult to incorporate into typical SAS modeling workflows, especially for users that are not experts in computational modeling. To this end, we present the Bayesian Ensemble Estimation from SAS (BEES) program. Two forks of BEES are available, the primary one existing as a module for the SASSIE web server and a developmental version that is a stand-alone Python program. BEES allows users to exhaustively sample ensemble models constructed from a library of theoretical states and to interactively analyze and compare each model's performance. The fitting routine also allows for secondary data sets to be supplied, thereby simultaneously fitting models to both SAS data as well as orthogonal information. The flexible ensemble of K63-linked ubiquitin trimers is presented as an example of BEES' capabilities.

Salmonella Membrane Structural Remodeling Increases Resistance to Antimicrobial Peptide LL-37.

Gram-negative bacteria are protected from their environment by an outer membrane that is primarily composed of lipopolysaccharides (LPSs). Under stress, pathogenic serotypes of Salmonella enterica remodel their LPSs through the PhoPQ two-component regulatory system that increases resistance to both conventional antibiotics and antimicrobial peptides (AMPs). Acquired resistance to AMPs is contrary to the established narrative that AMPs circumvent bacterial resistance by targeting the general chemical properties of membrane lipids. However, the specific mechanisms underlying AMP resistance remain elusive. Here we report a 2-fold increase in bacteriostatic concentrations of human AMP LL-37 for S. enterica with modified LPSs. LPSs with and without chemical modifications were isolated and investigated by Langmuir films coupled with grazing-incidence X-ray diffraction (GIXD) and specular X-ray reflectivity (XR). The initial interactions between LL-37 and LPS bilayers were probed using all-atom molecular dynamics simulations. These simulations suggest that initial association is nonspecific to the type of LPS and governed by hydrogen bonding to the LPS outer carbohydrates. GIXD experiments indicate that the interactions of the peptide with monolayers reduce the number of crystalline domains but greatly increase the typical domain size in both LPS isoforms. Electron densities derived from XR experiments corroborate the bacteriostatic values found in vitro and indicate that peptide intercalation is reduced by LPS modification. We hypothesize that defects at the liquid-ordered boundary facilitate LL-37 intercalation into the outer membrane, whereas PhoPQ-mediated LPS modification protects against this process by having innately increased crystallinity. Since induced ordering has been observed with other AMPs and drugs, LPS modification may represent a general mechanism by which Gram-negative bacteria protect against host innate immunity.

Empirical and Computational Comparison of Alternative Therapeutic Exon Skip Repairs for Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a common and devastating genetic disease primarily caused by exon deletions that create a genetic frameshift in dystrophin. Exon skipping therapy seeks to correct this by masking an exon during the mRNA maturation process, restoring dystrophin expression, but creating an edited protein missing both the original defect and the therapeutically skipped region. Crucially, it is possible to correct many defects in alternative ways, by skipping an exon either before or after the patient's defect. This results in alternatively edited, hybrid proteins that might have different properties and therapeutic consequences. We examined three such dystrophin exon-skipped edits, Δe45-53, Δe46-54, and Δe47-55, comprising two pairs of alternative repairs of Δe46-53 and Δe47-54 DMD defects. We found that in both cases, Δe46-54 was the more stable repair as determined by a variety of thermodynamic and biochemical measurements. We also examined the origin of these differences with molecular dynamics simulations, which showed that these stability differences were the result of different types of structural perturbations. For example, in one edit there was partial unfolding at the edit site that caused domain-localized perturbations while in another there was unfolding at the protein domain junctions distal to the edit site that increased molecular flexibility. These results demonstrate that alternative exon skip repairs of the same underlying defect can have very different consequences at the level of protein structure and stability and furthermore that these can arise by different mechanisms, either locally or by more subtle long-range perturbations.