Designed De Novo α-Sheet Peptides Destabilize Bacterial Biofilms and Increase the Susceptibility of E. coli and S. aureus to Antibiotics.

Biofilm-associated microbes are 10-1000 times less susceptible to antibiotics. An emerging treatment strategy is to target the structural components of biofilm to weaken the extracellular matrix without introducing selective pressure. Biofilm-associated bacteria, including Escherichia coli and Staphylococcus aureus, generate amyloid fibrils to reinforce their extracellular matrix. Previously, de novo synthetic α-sheet peptides designed in silico were shown to inhibit amyloid formation in multiple bacterial species, leading to the destabilization of their biofilms. Here, we investigated the impact of inhibiting amyloid formation on antibiotic susceptibility. We hypothesized that combined administration of antibiotics and α-sheet peptides would destabilize biofilm formation and increase antibiotic susceptibility. Two α-sheet peptides, AP90 and AP401, with the same sequence but inverse chirality at every amino acid were tested: AP90 is L-amino acid dominant while AP401 is D-amino acid dominant. For E. coli, both peptides increased antibiotic susceptibility and decreased the biofilm colony forming units when administered with five different antibiotics, and AP401 caused a greater increase in all cases. For S. aureus, increased biofilm antibiotic susceptibility was also observed for both peptides, but AP90 outperformed AP401. A comparison of the peptide effects demonstrates how chirality influences biofilm targeting of gram-negative E. coli and gram-positive S. aureus. The observed increase in antibiotic susceptibility highlights the role amyloid fibrils play in the reduced susceptibility of bacterial biofilms to specific antibiotics. Thus, the co-administration of α-sheet peptides and existing antibiotics represents a promising strategy for the treatment of biofilm infections.

Human islet amyloid polypeptide-induced ß-cell cytotoxicity is linked to formation of α-sheet structure.

Type 2 diabetes (T2D) results from insulin secretory dysfunction arising in part from the loss of pancreatic islet ß-cells. Several factors contribute to ß-cell loss, including islet amyloid formation, which is observed in over 90% of individuals with T2D. The amyloid is comprised of human islet amyloid polypeptide (hIAPP). Here we provide evidence that early in aggregation, hIAPP forms toxic oligomers prior to formation of amyloid fibrils. The toxic oligomers contain α-sheet secondary structure, a nonstandard secondary structure associated with toxic oligomers in other amyloid diseases. De novo, synthetic α-sheet compounds designed to be nontoxic and complementary to the α-sheet structure in the toxic oligomers inhibit hIAPP aggregation and neutralize oligomer-mediated cytotoxicity in cell-based assays. In vivo administration of an α-sheet design to mice for 4 weeks revealed no evidence of toxicity nor did it elicit an immune response. Furthermore, the α-sheet designs reduced endogenous islet amyloid formation and mitigation of amyloid-associated ß-cell loss in cultured islets isolated from an hIAPP transgenic mouse model of islet amyloidosis. Characterization of the involvement of α-sheet in early aggregation of hIAPP and oligomer toxicity contributes to elucidation of the molecular mechanisms underlying amyloid-associated ß-cell loss.

Designed α-sheet peptides disrupt uropathogenic E. coli biofilms rendering bacteria susceptible to antibiotics and immune cells.

Uropathogenic Escherichia coli account for the largest proportion of nosocomial infections in the United States. Nosocomial infections are a major source of increased costs and treatment complications. Many infections are biofilm associated, rendering antibiotic treatments ineffective or cause additional complications (e.g., microbiome depletion). This work presents a potentially complementary non-antibiotic strategy to fight nosocomial infections by inhibiting the formation of amyloid fibrils, a proteinaceous structural reinforcement known as curli in E. coli biofilms. Despite extensive characterization of the fibrils themselves and their associated secretion system, mechanistic details of curli assembly in vivo remain unclear. We hypothesized that, like other amyloid fibrils, curli polymerization involves a unique secondary structure termed "α-sheet". Biophysical studies herein confirmed the presence of α-sheet structure in prefibrillar species of CsgA, the major component of curli, as it aggregated. Binding of synthetic α-sheet peptides to the soluble α-sheet prefibrillar species inhibited CsgA aggregation in vitro and suppressed amyloid fibril formation in biofilms. Application of synthetic α-sheet peptides also enhanced antibiotic susceptibility and dispersed biofilm-resident bacteria for improved uptake by phagocytic cells. The ability of synthetic α-sheet peptides to reduce biofilm formation, improve antibiotic susceptibility, and enhance clearance by macrophages has broad implications for combating biofilm-associated infections.

SOBA: Development and testing of a soluble oligomer binding assay for detection of amyloidogenic toxic oligomers.

The formation of toxic Amyloid ß-peptide (Aß) oligomers is one of the earliest events in the molecular pathology of Alzheimer's Disease (AD). These oligomers lead to a variety of downstream effects, including impaired neuronal signaling, neuroinflammation, tau phosphorylation, and neurodegeneration, and it is estimated that these events begin 10 to 20 y before the presentation of symptoms. Toxic Aß oligomers contain a nonstandard protein structure, termed α-sheet, and designed α-sheet peptides target this main-chain structure in toxic oligomers independent of sequence. Here we show that a designed α-sheet peptide inhibits the deleterious effects on neuronal signaling and also serves as a capture agent in our soluble oligomer binding assay (SOBA). Pre-incubated synthetic α-sheet-containing Aß oligomers produce strong SOBA signals, while monomeric and ß-sheet protofibrillar Aß do not. α-sheet containing oligomers were also present in cerebrospinal fluid (CSF) from an AD patient versus a noncognitively impaired control. For the detection of toxic oligomers in plasma, we developed a plate coating to increase the density of the capture peptide. The proof of concept was achieved by testing 379 banked human plasma samples. SOBA detected Aß oligomers in patients on the AD continuum, including controls who later progressed to mild cognitive impairment. In addition, SOBA discriminated AD from other forms of dementia, yielding sensitivity and specificity of 99% relative to clinical and neuropathological diagnoses. To explore the broader potential of SOBA, we adapted the assay for a-synuclein oligomers and confirmed their presence in CSF from patients with Parkinson's disease and Lewy body dementia.

The role of α-sheet structure in amyloidogenesis: characterization and implications.

Amyloid diseases are linked to protein misfolding whereby the amyloidogenic protein undergoes a conformational change, aggregates and eventually forms amyloid fibrils. While the amyloid fibrils and plaques are hallmarks of these diseases, they typically form late in the disease process and do not correlate with disease. Instead, there is growing evidence that smaller, soluble toxic oligomers form prior and appear to be early triggers of the molecular pathology underlying these diseases. Nearly 20 years ago, we proposed the α-sheet hypothesis after discovering that the early conformational changes observed during atomistic molecular dynamics simulations involve the formation of a non-standard protein structure, α-sheet. Furthermore, we proposed that toxic oligomers contain α-sheet structure and that preferentially targeting this structure could neutralize the toxicity, prevent further aggregation and serve as the basis for early detection of disease. Here, we present the origin of the α-sheet hypothesis and describe α-sheet structure and the corresponding mechanisms of conversion. We discuss experimental studies demonstrating that both mammalian and bacterial amyloid systems form α-sheet oligomers before converting to conventional ß-sheet fibrils. Furthermore, we show that the process can be inhibited with de novo designed α-sheet peptides complementary to the structure in the toxic oligomers.

Modulation of post-powerstroke dynamics in myosin II by 2'-deoxy-ADP.

Muscle myosins are molecular motors that hydrolyze ATP and generate force through coordinated interactions with actin filaments, known as cross-bridge cycling. During the cross-bridge cycle, functional sites in myosin 'sense' changes in interactions with actin filaments and the nucleotide binding region, resulting in allosteric transmission of information throughout the structure. We investigated whether the dynamics of the post-powerstroke state of the cross-bridge cycle are modulated in a nucleotide-dependent fashion. We compared molecular dynamics simulations of the myosin II motor domain (M) from Dictyostelium discoideum in the presence of ADP (M.ADP) versus 2'-deoxy-ADP bound myosin (M.dADP). We found that dADP was more flexible than ADP and the two nucleotides interacted with myosin in different ways. Replacement of ADP with dADP in the post-powerstroke state also altered the conformation of the actin binding region in myosin heads. Our results provide atomic level insights into allosteric communication networks in myosin that provide insight into the nucleotide-dependent dynamics of the cross-bridge cycle.

Myosin dynamics during relaxation in mouse soleus muscle and modulation by 2'-deoxy-ATP.

KEY POINTS: Skeletal muscle relaxation has been primarily studied by assessing the kinetics of force decay. Little is known about the resultant dynamics of structural changes in myosin heads during relaxation. The naturally occurring nucleotide 2-deoxy-ATP (dATP) is a myosin activator that enhances cross-bridge binding and kinetics. X-ray diffraction data indicate that with elevated dATP, myosin heads were extended closer to actin in relaxed muscle and myosin heads return to an ordered, resting state after contraction more quickly. Molecular dynamics simulations of post-powerstroke myosin suggest that dATP induces structural changes in myosin heads that increase the surface area of the actin-binding regions promoting myosin interaction with actin, which could explain the observed delays in the onset of relaxation. This study of the dATP-induced changes in myosin may be instructive for determining the structural changes desired for other potential myosin-targeted molecular compounds to treat muscle diseases. ABSTRACT: Here we used time-resolved small-angle X-ray diffraction coupled with force measurements to study the structural changes in FVB mouse skeletal muscle sarcomeres during relaxation after tetanus contraction. To estimate the rate of myosin deactivation, we followed the rate of the intensity recovery of the first-order myosin layer line (MLL1) and restoration of the resting spacing of the third and sixth order of meridional reflection (SM3 and SM6 ) following tetanic contraction. A transgenic mouse model with elevated skeletal muscle 2-deoxy-ATP (dATP) was used to study how myosin activators may affect soleus muscle relaxation. X-ray diffraction evidence indicates that with elevated dATP, myosin heads were extended closer to actin in resting muscle. Following contraction, there is a slight but significant delay in the decay of force relative to WT muscle while the return of myosin heads to an ordered resting state was initially slower, then became more rapid than in WT muscle. Molecular dynamics simulations of post-powerstroke myosin suggest that dATP induces structural changes in myosin that increase the surface area of the actin-binding regions, promoting myosin interaction with actin. With dATP, myosin heads may remain in an activated state near the thin filaments following relaxation, accounting for the delay in force decay and the initial delay in recovery of resting head configuration, and this could facilitate subsequent contractions.

Tumorigenic p53 mutants undergo common structural disruptions including conversion to α-sheet structure.

The p53 protein is a commonly studied cancer target because of its role in tumor suppression. Unfortunately, it is susceptible to mutation-associated loss of function; approximately 50% of cancers are associated with mutations to p53, the majority of which are located in the central DNA-binding domain. Here, we report molecular dynamics simulations of wild-type (WT) p53 and 20 different mutants, including a stabilized pseudo-WT mutant. Our findings indicate that p53 mutants tend to exacerbate latent structural-disruption tendencies, or vulnerabilities, already present in the WT protein, suggesting that it may be possible to develop cancer therapies by targeting a relatively small set of structural-disruption motifs rather than a multitude of effects specific to each mutant. In addition, α-sheet secondary structure formed in almost all of the proteins. α-Sheet has been hypothesized and recently demonstrated to play a role in amyloidogenesis, and its presence in the reported p53 simulations coincides with the recent re-consideration of cancer as an amyloid disease.

Cardiac myosin activation with 2-deoxy-ATP via increased electrostatic interactions with actin.

The naturally occurring nucleotide 2-deoxy-adenosine 5'-triphosphate (dATP) can be used by cardiac muscle as an alternative energy substrate for myosin chemomechanical activity. We and others have previously shown that dATP increases contractile force in normal hearts and models of depressed systolic function, but the structural basis of these effects has remained unresolved. In this work, we combine multiple techniques to provide structural and functional information at the angstrom-nanometer and millisecond time scales, demonstrating the ability to make both structural measurements and quantitative kinetic estimates of weak actin-myosin interactions that underpin sarcomere dynamics. Exploiting dATP as a molecular probe, we assess how small changes in myosin structure translate to electrostatic-based changes in sarcomere function to augment contractility in cardiac muscle. Through Brownian dynamics simulation and computational structural analysis, we found that deoxy-hydrolysis products [2-deoxy-adenosine 5'-diphosphate (dADP) and inorganic phosphate (Pi)] bound to prepowerstroke myosin induce an allosteric restructuring of the actin-binding surface on myosin to increase the rate of cross-bridge formation. We then show experimentally that this predicted effect translates into increased electrostatic interactions between actin and cardiac myosin in vitro. Finally, using small-angle X-ray diffraction analysis of sarcomere structure, we demonstrate that the proposed increased electrostatic affinity of myosin for actin causes a disruption of the resting conformation of myosin motors, resulting in their repositioning toward the thin filament before activation. The dATP-mediated structural alterations in myosin reported here may provide insight into an improved criterion for the design or selection of small molecules to be developed as therapeutic agents to treat systolic dysfunction.

α-Sheet secondary structure in amyloid ß-peptide drives aggregation and toxicity in Alzheimer's disease.

Alzheimer's disease (AD) is characterized by the deposition of ß-sheet-rich, insoluble amyloid ß-peptide (Aß) plaques; however, plaque burden is not correlated with cognitive impairment in AD patients; instead, it is correlated with the presence of toxic soluble oligomers. Here, we show, by a variety of different techniques, that these Aß oligomers adopt a nonstandard secondary structure, termed "α-sheet." These oligomers form in the lag phase of aggregation, when Aß-associated cytotoxicity peaks, en route to forming nontoxic ß-sheet fibrils. De novo-designed α-sheet peptides specifically and tightly bind the toxic oligomers over monomeric and fibrillar forms of Aß, leading to inhibition of aggregation in vitro and neurotoxicity in neuroblastoma cells. Based on this specific binding, a soluble oligomer-binding assay (SOBA) was developed as an indirect probe of α-sheet content. Combined SOBA and toxicity experiments demonstrate a strong correlation between α-sheet content and toxicity. The designed α-sheet peptides are also active in vivo where they inhibit Aß-induced paralysis in a transgenic Aß Caenorhabditis elegans model and specifically target and clear soluble, toxic oligomers in a transgenic APPsw mouse model. The α-sheet hypothesis has profound implications for further understanding the mechanism behind AD pathogenesis.

Molecular dynamics-derived rotamer libraries for d-amino acids within homochiral and heterochiral polypeptides.

Computational resources have contributed to the design and engineering of novel proteins by integrating genomic, structural and dynamic aspects of proteins. Non-canonical amino acids, such as d-amino acids, expand the available sequence space for designing and engineering proteins; however, the rotamer libraries for d-amino acids are usually constructed as the mirror images of l-amino acid rotamer libraries, an assumption that has not been tested. To this end, we have performed molecular dynamics (MD) simulations of model host-guest peptide systems containing d-amino acids. Our simulations systematically address the applicability of the mirror image convention as well as the effects of neighboring residue chirality. Rotamer libraries derived from these systems provide realistic rotamer distributions suitable for use in both rational and computational design workflows. Our simulations also address the impact of chirality on the intrinsic conformational preferences of amino acids, providing fundamental insights into the relationship between chirality and biomolecular dynamics. While d-amino acids are rare in naturally occurring proteins, they are used in designed proteins to stabilize a desired conformation, increase bioavailability or confer favorable biochemical and physical attributes. Here, we present d-amino acid rotamer libraries derived from MD simulations of alanine-based host-guest pentapeptides and show how certain residues can deviate from mirror image symmetry. Our simulations directly model d-amino acids as guest residues within the chiral l-Ala and d-Ala pentapeptide series to explicitly incorporate any contributions resulting from the chiralities of neighboring residues.

De Novo Designed α-Sheet Peptides Inhibit Functional Amyloid Formation of Streptococcus mutans Biofilms.

Streptococcus mutans is a bacterial species that predominates in the oral microbiome. S. mutans binds to the tooth surface, metabolizes sugars and produces acid, leading to cavity formation. S. mutans can also cause infectious endocarditis. Recent evidence suggests that S. mutans biofilms contain amyloid fibrils. Amyloids are insoluble fibrillar protein aggregates, and bacteria use functional amyloids to improve robustness of their biofilms. While the functional amyloids in bacteria such as Escherichia coli and Staphylococcus aureus have been heavily investigated, little is known about the mechanism of S. mutans amyloid formation. Previous results from our laboratory with the amyloidogenic proteins and peptides from the aforementioned bacteria and other mammalian amyloid systems suggest that amyloid formation progresses via an intermediate that adopts a unique secondary structure-α-sheet. De novo designed peptides with alternating l- and d-amino acid also adopt an α-sheet secondary structure and inhibit amyloid formation by binding to soluble oligomeric species during amyloidogenesis. Inhibition of fibrillization by α-sheet peptides suggests the presence of α-sheet during amyloid formation. To investigate the mechanism of functional amyloid formation in S. mutans, α-sheet peptides were compared to epigallocatechin gallate for their ability to inhibit fibril formation in S. mutans. Inhibition was demonstrated in a biofilm plate assay and on hydroxyapatite surfaces both in S. mutans alone and in bacteria from human saliva. The observed inhibition suggests that an α-sheet mediated mechanism may be operative during functional amyloid formation.

Chemical and Physical Variability in Structural Isomers of an l/d α-Sheet Peptide Designed To Inhibit Amyloidogenesis.

There has been much interest in synthetic peptides as inhibitors of aggregation associated with amyloid diseases. Of particular interest are compounds that target the cytotoxic soluble oligomers preceding the formation of mature, nontoxic fibrils. This study explores physical and chemical differences between two de novo-designed peptides that share an identical primary structure but differ in backbone chirality at six key positions. We show that the presence of alternating l/d-amino acid motifs dramatically increases aqueous solubility, enforces α-sheet secondary structure, and inhibits aggregation of the ß-amyloid peptide implicated in Alzheimer's disease, in addition to neutralizing its cytotoxicity. In contrast, the all-l-amino acid isomer does not form α-sheet structure and is insoluble and inactive.

An in silico algorithm for identifying stabilizing pockets in proteins: test case, the Y220C mutant of the p53 tumor suppressor protein.

The p53 tumor suppressor protein performs a critical role in stimulating apoptosis and cell cycle arrest in response to oncogenic stress. The function of p53 can be compromised by mutation, leading to increased risk of cancer; approximately 50% of cancers are associated with mutations in the p53 gene, the majority of which are in the core DNA-binding domain. The Y220C mutation of p53, for example, destabilizes the core domain by 4 kcal/mol, leading to rapid denaturation and aggregation. The associated loss of tumor suppressor functionality is associated with approximately 75 000 new cancer cases every year. Destabilized p53 mutants can be 'rescued' and their function restored; binding of a small molecule into a pocket on the surface of mutant p53 can stabilize its wild-type structure and restore its function. Here, we describe an in silico algorithm for identifying potential rescue pockets, including the algorithm's integration with the Dynameomics molecular dynamics data warehouse and the DIVE visual analytics engine. We discuss the results of the application of the method to the Y220C p53 mutant, entailing finding a putative rescue pocket through MD simulations followed by an in silico search for stabilizing ligands that dock into the putative rescue pocket. The top three compounds from this search were tested experimentally and one of them bound in the pocket, as shown by nuclear magnetic resonance, and weakly stabilized the mutant.

Peptides Composed of Alternating L- and D-Amino Acids Inhibit Amyloidogenesis in Three Distinct Amyloid Systems Independent of Sequence.

There is now substantial evidence that soluble oligomers are primary toxic agents in amyloid diseases. The development of an antibody recognizing the toxic soluble oligomeric forms of different and unrelated amyloid species suggests a common conformational intermediate during amyloidogenesis. We previously observed a common occurrence of a novel secondary structure element, which we call α-sheet, in molecular dynamics (MD) simulations of various amyloidogenic proteins, and we hypothesized that the toxic conformer is composed of α-sheet structure. As such, α-sheet may represent a conformational signature of the misfolded intermediates of amyloidogenesis and a potential unique binding target for peptide inhibitors. Recently, we reported the design and characterization of a novel hairpin peptide (α1 or AP90) that adopts stable α-sheet structure and inhibits the aggregation of the ß-Amyloid Peptide Aß42 and transthyretin. AP90 is a 23-residue hairpin peptide featuring alternating D- and L-amino acids with favorable conformational propensities for α-sheet formation, and a designed turn. For this study, we reverse engineered AP90 to identify which of its design features is most responsible for conferring α-sheet stability and inhibitory activity. We present experimental characterization (CD and FTIR) of seven peptides designed to accomplish this. In addition, we measured their ability to inhibit aggregation in three unrelated amyloid species: Aß42, transthyretin, and human islet amylin polypeptide. We found that a hairpin peptide featuring alternating L- and D-amino acids, independent of sequence, is sufficient for conferring α-sheet structure and inhibition of aggregation. Additionally, we show a correlation between α-sheet structural stability and inhibitory activity.

Designed α-sheet peptides inhibit amyloid formation by targeting toxic oligomers.

Previous studies suggest that the toxic soluble-oligomeric form of different amyloid proteins share a common backbone conformation, but the amorphous nature of this oligomer prevents its structural characterization by experiment. Based on molecular dynamics simulations we proposed that toxic intermediates of different amyloid proteins adopt a common, nonstandard secondary structure, called α-sheet. Here we report the experimental characterization of peptides designed to be complementary to the α-sheet conformation observed in the simulations. We demonstrate inhibition of aggregation in two different amyloid systems, ß-amyloid peptide (Aß) and transthyretin, by these designed α-sheet peptides. When immobilized the α-sheet designs preferentially bind species from solutions enriched in the toxic conformer compared with non-aggregated, nontoxic species or mature fibrils. The designs display characteristic spectroscopic signatures distinguishing them from conventional secondary structures, supporting α-sheet as a structure involved in the toxic oligomer stage of amyloid formation and paving the way for novel therapeutics and diagnostics.DOI: http://dx.doi.org/10.7554/eLife.01681.001.

DIVE: a data intensive visualization engine.

SUMMARY: Modern scientific investigation is generating increasingly larger datasets, yet analyzing these data with current tools is challenging. DIVE is a software framework intended to facilitate big data analysis and reduce the time to scientific insight. Here, we present features of the framework and demonstrate DIVE's application to the Dynameomics project, looking specifically at two proteins. AVAILABILITY AND IMPLEMENTATION: Binaries and documentation are available at http://www.dynameomics.org/DIVE/DIVESetup.exe.

Analyzing disease-associated protein structures with visual analytics.

Protein function is related to protein structure, and understanding disease-associated structural changes is critical both to understanding protein-mediated illness and to developing clinical therapies. We have developed a visual analytics tool called ContactWalker that uses molecular dynamics simulations to investigate the structural and physical differences between wild type and disease-associated protein structures. This tool has been used successfully to characterize the effects of the disease ataxia with vitamin E deficiency (AVED). We are now beginning to investigate mutations to other disease-associated proteins including the tumor suppressor protein p53.

A comparison of multiscale methods for the analysis of molecular dynamics simulations.

Molecular dynamics (MD) is the only technique available for obtaining dynamic protein data at atomic spatial resolution and picosecond or finer temporal resolution. In recent years, the cost of computational resources has decreased exponentially while the number of known protein structures, many of which are not characterized biochemically, has increased rapidly. These events have led to an increase in the use of MD in biological research, both to examine phenomena that cannot be resolved experimentally and to generate hypotheses that direct further experimental research. In fact, several databases of MD simulations have arisen in recent years. MD simulations, and especially MD simulation databases, contain massive amounts of data, yet interesting phenomena often occur over very short time periods and on the scale of only a few atoms. Analysis of such data must balance these fine-detail events with the global picture they create. Here, we address the multiscale nature of the problem by comparing several MD analysis methods to show their strengths and weaknesses at various scales using the wild-type and R282W mutant forms of the DNA-binding domain of protein p53. By leveraging these techniques together, we are able to pinpoint fine-detail and big picture differences between the protein's variants. Our analyses indicate that the R282W mutation of p53 destabilizes the L1 loop and loosens the H2 helix conformation, but the loosened L1 loop can be rescued by residue H115, preventing the R282W mutation from completely destabilizing the protein or abolishing activity.

Structural effects of the L145Q, V157F, and R282W cancer-associated mutations in the p53 DNA-binding core domain.

The p53 tumor suppressor is a transcription factor involved in many important signaling pathways, such as apoptosis and cell-cycle arrest. In over half of human cancers, p53 function is compromised by a mutation in its gene. Mutations in the p53 DNA-binding core domain destabilize the structure and reduce DNA-binding activity. We performed molecular dynamics simulations at physiological temperature to study the structural and dynamic effects of the L145Q, V157F, and R282W cancer-associated mutations in comparison to the wild-type protein. While there were common regions of destabilization in the mutant simulations, structural changes particular to individual mutations were also observed. Significant backbone deviations of the H2 helix and S7-S8 loop were observed in all mutant simulations; the H2 helix binds to DNA. In addition, the L145Q and V157F mutations, which are located in the ß-sandwich core of the domain, disrupted the ß-sheet structure and the loop-sheet-helix motif. The R282W mutation caused distortion of the loop-sheet-helix motif, but otherwise this mutant was similar to the wild-type structure. The introduction of these mutations caused rearrangement of the DNA-binding surface, consistent with their reduced DNA-binding activity. The simulations reveal detailed effects of the mutations on the stability and dynamics of p53 that may provide insight for therapeutic approaches.