Prevalent Mutations of Human Prion Protein: A Molecular Modeling and Molecular Dynamics Study

Abstract

Point mutations in the human prion protein gene, leading to amino acid substitutions in the human prion protein contribute to conversion of PrP^C to PrP^Sc and amyloid formation, resulting in prion diseases such as familial Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Scheinker disease (GSS), and fatal familial insomnia. We have investigated impressions of prevalent mutations including Q217R, D202N, F198S, on the human prion protein and compared the mutant models with wild types. Structural analyses of models were performed with molecular modeling and molecular dynamics simulation methods. According to our results, frequently occurred mutations are observed in conserved and fully conserved sequences of human prion protein and the most fluctuation values occur in the Helix 1 around residues 144–152 and C-terminal end of the Helix 2. Our analysis of results obtained from MD simulation clearly shows that this long-range effect plays an important role in the conformational fluctuations in mutant structures of human prion protein. Results obtained from molecular modeling such as creation or elimination of some hydrogen bonds, increase or decrease of the accessible surface area and molecular surface, loss or accumulation of negative or positive charges on specific positions, and altering the polarity and pKa values, show that amino acid point mutations, though not urgently change the stability of PrP, might have some local impacts on the protein interactions which are required for oligomerization into fibrillar species.     

Prevalent mutations of human prion protein: a molecular modeling and molecular dynamics study

Point mutations in the human prion protein gene, leading to amino acid substitutions in the human prion protein contribute to conversion of PrPC to PrPSc and amyloid formation, resulting in prion diseases such as familial Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Scheinker disease (GSS), and fatal familial insomnia. We have investigated impressions of prevalent mutations including Q217R, D202N, F198S, on the human prion protein and compared the mutant models with wild types. Structural analyses of models were performed with molecular modeling and molecular dynamics simulation methods. According to our results, frequently occurred mutations are observed in conserved and fully conserved sequences of human prion protein and the most fluctuation values occur in the Helix 1 around residues 144-152 and C-terminal end of the Helix 2. Our analysis of results obtained from MD simulation clearly shows that this long-range effect plays an important role in the conformational fluctuations in mutant structures of human prion protein. Results obtained from molecular modeling such as creation or elimination of some hydrogen bonds, increase or decrease of the accessible surface area and molecular surface, loss or accumulation of negative or positive charges on specific positions, and altering the polarity and pKa values, show that amino acid point mutations, though not urgently change the stability of PrP, might have some local impacts on the protein interactions which are required for oligomerization into fibrillar species.     

Fragment molecular orbital calculations reveal that the E200K mutation markedly alters local structural stability in the human prion protein

The E200K mutation of the human prion protein (PrP) is known to cause familial Creutzfeldt–Jakob disease. In order to elucidate the effects of the mutation on the local structural stability of PrP, we performed ab initio fragment molecular orbital calculations for the wild-type human PrP and the E200K variant modeled under neutral and mild acidic conditions. The calculations revealed that this substitution markedly altered the intramolecular interactions in the PrP, suggesting that the local structural instabilities induced by the E200K mutation might cause initial denaturation of the PrP and its subsequent conversion to a pathogenic form. This work presents a new approach for quantitatively elucidating structural instabilities in proteins that cause misfolding diseases.     

High hydrophobic amino acid exposure is responsible of the neurotoxic effects induced by E200K or D202N disease-related mutations of the human prion protein

Mutations in prion protein are thought to be causative of inherited prion diseases favoring the spontaneous conversion of the normal prion protein into the scrapie-like pathological prion protein. We previously reported that, by controlled thermal denaturation, human prion protein fragment 90-231 acquires neurotoxic properties when transformed in a β-rich conformation, resembling the scrapie-like conformation. In this study we generated prion protein fragment 90-231 bearing mutations identified in familial prion diseases (D202N and E200K), to analyze their role in the induction of a neurotoxic conformation. Prion protein fragment 90-231(wild type) and the D202N mutant were not toxic in native conformation but induced cell death only after thermal denaturation. Conversely, prion protein fragment 90-231(E200K) was highly toxic in its native structure, suggesting that E200K mutation per se favors the acquisition of a peptide neurotoxic conformation. To identify the structural determinants of prion protein fragment 90-231 toxicity, we show that while the wild type peptide is structured in α-helix, hPrP90-231 E200K is spontaneously refolded in a β-structured conformer characterized by increased proteinase K resistance and propensity to generate fibrils. However, the most significant difference induced by E200K mutation in prion protein fragment 90-231 structure in native conformation we observed, was an increase in the exposure of hydrophobic amino-acids on protein surface that was detected in wild type and D202N proteins only after thermal denaturation. In conclusion, we propose that increased hydrophobicity is one of the main determinants of toxicity induced by different mutations in prion protein-derived peptides.     

Solution structure of the E200K variant of human prion protein. Implications for the mechanism of pathogenesis in familial prion diseases

Prion propagation in transmissible spongiform encephalopathies involves the conversion of cellular prion protein, PrP(C), into a pathogenic conformer, PrP(Sc). Hereditary forms of the disease are linked to specific mutations in the gene coding for the prion protein. To gain insight into the molecular basis of these disorders, the solution structure of the familial Creutzfeldt-Jakob disease-related E200K variant of human prion protein was determined by multi-dimensional nuclear magnetic resonance spectroscopy. Remarkably, apart from minor differences in flexible regions, the backbone tertiary structure of the E200K variant is nearly identical to that reported for the wild-type human prion protein. The only major consequence of the mutation is the perturbation of surface electrostatic potential. The present structural data strongly suggest that protein surface defects leading to abnormalities in the interaction of prion protein with auxiliary proteins/chaperones or cellular membranes should be considered key determinants of a spontaneous PrP(C) --> PrP(Sc) conversion in the E200K form of hereditary prion disease.     

Comparison studies of the structural stability of rabbit prion protein with human and mouse prion proteins

Background: Prion diseases are fatal and infectious neurodegenerative diseases affecting humans and animals. Rabbits are one of the few mammalian species reported to be resistant to infection from prion diseases isolated from other species (I. Vorberg et al., Journal of Virology 77 (3) (2003) 2003-2009). Thus the study of rabbit prion protein structure to obtain insight into the immunity of rabbits to prion diseases is very important.Findings: The paper is a straight forward molecular dynamics simulation study of wild-type rabbit prion protein (monomer cellular form) which apparently resists the formation of the scrapie form. The comparison analyses with human and mouse prion proteins done so far show that the rabbit prion protein has a stable structure. The main point is that the enhanced stability of the C-terminal ordered region especially helix 2 through the D177-R163 salt-bridge formation renders the rabbit prion protein stable. The salt bridge D201-R155 linking helixes 3 and 1 also contributes to the structural stability of rabbit prion protein. The hydrogen bond H186-R155 partially contributes to the structural stability of rabbit prion protein.Conclusions: Rabbit prion protein was found to own the structural stability, the salt bridges D177-R163, D201-R155 greatly contribute and the hydrogen bond H186-R155 partially contributes to this structural stability. The comparison of the structural stability of prion proteins from the three species rabbit, human and mouse showed that the human and mouse prion protein structures were not affected by the removing these two salt bridges. Dima et al. (Biophysical Journal 83 (2002) 1268-1280 and Proceedings of the National Academy of Sciences of the United States of America 101 (2004) 15335-15340) also confirmed this point and pointed out that “correlated mutations that reduce the frustration in the second half of helix 2 in mammalian prion proteins could inhibit the formation of PrP^Sc”.     

Structural and functional analysis of aldolase B mutants related to hereditary fructose intolerance

Hereditary fructose intolerance (HFI) is a recessively inherited disorder of carbohydrate metabolism caused by impaired function of human liver aldolase (B isoform). 25 enzyme-impairing mutations have been identified in the aldolase B gene. We have studied the HFI-related mutant recombinant proteins W147R, A149P, A174D, L256P, N334K and delta6ex6 in relation to aldolase B function and structure using kinetic assays and molecular graphics analysis. We found that these mutations affect aldolase B function by decreasing substrate affinity, maximal velocity and/or enzyme stability. Finally, the functional and structural analyses of the non-natural mutant Q354E provide insight into the catalytic role of Arg(303), whose natural mutants are associated to HFI.     

Exploring structural and thermodynamic stabilities of human prion protein pathogenic mutants D202N, E211Q and Q217R

The central event in the pathogenesis of prion protein (PrP) is a profound conformational change from its α-helical (PrP(C)) to its β-sheet-rich isoform (PrP(Sc)). Many single amino acid mutations of PrP are associated with familial prion diseases, such as D202N, E211Q, and Q217R mutations located at the third native α-helix of human PrP. In order to explore the underlying structural and dynamic effects of these mutations, we performed all-atom molecular dynamics (MD) simulations for the wild-type (WT) PrP and its mutants. The obtained results indicate that these amino acid substitutions have subtle effects on the protein structures, but show large changes of the overall electrostatic potential distributions. We can infer that the changes of PrP electrostatic surface due to the studied mutations may influence the intermolecular interactions during the aggregation process. In addition, the mutations also affect the thermodynamic stabilities of PrP.     

The Unfolded State of the Murine Prion Protein and Properties of Single-point Mutants Related to Human Prion Diseases

The prion protein can exist both in a normal cellular isoform and in a pathogenic conformational isoform. The latter is responsible for the development of different neurodegenerative diseases, for example Creutzfeldt-Jakob disease or fatal familial insomnia. To convert the native benign state of the protein into a highly ordered fibrillar aggregate, large-scale rearrangements of the tertiary structure are necessary during the conversion process and intermediates that are at least partially unfolded are present during fibril formation. In addition to the sporadic conversion into the pathogenic isoform, more than 20 familial diseases are known that are caused by single point mutations increasing the probability of aggregation and neurodegeneration. Here, we demonstrate that the chemically denatured states of the mouse and human prion proteins have very similar structural and dynamic characteristics. Initial studies on the single point mutants E196K, F198S, V203I and R208H of the oxidized mouse construct, which are related to human prion diseases, reveal significant differences in the rate of aggregation. Aggregation for mutants V203I and R208H is slower than it is for the wild type, and the constructs E196K and F198S show accelerated aggregation. These differences in aggregation behaviour are not correlated with the thermal stability of the mutants, indicating different mechanisms promoting the conformational conversion process.     

Molecular dynamics study of the dominant-negative E219K polymorphism in human prion protein

Human prion diseases are associated with misfolding or aggregation of the Human Prion Protein (HuPrP). Missense mutations in the HuPrP gene, contribute to conversion of HuPrP^C to HuPrP^Sc and amyloid formation. Based on our previous comprehensive study, three missense mutations, from two different functional groups, i.e. disease-related mutations, and protective mutations, were selected and extensive molecular dynamics simulations were performed on these three mutants to compare their dynamics and conformations with those of the wildtype HuPrP. In addition to simulations of monomeric forms of mutants, in order to study the dominant-negative effect of protective mutation (E219K), 30-ns simulations were performed on E219K-wildtype and wildtype-wildtype dimeric forms. Our results indicate that, although after 30-ns simulations the global three-dimensional structure of models remain fairly intact, the disease-related mutations (V210I and Q212P) introduce local structural changes, i.e. close contact changes and secondary structure changes, in addition to global flexibility changes. Furthermore, our results support the loss of hydrophobic interaction due to the mutations in hydrophobic core that has been reported by previous NMR and computational studies. On the other hand, this protective mutation (E219K) results in helix elongation, and significant increases of overall flexibility of E219K mutant during 30-ns simulation. In conclusion, the simulations of dimeric forms suggest that the dominant-negative effect of this protective mutation (E219K) is due to the incompatible structures and dynamics of allelic variants during conversion process.     

Comparative analysis of essential collective dynamics and NMR-derived flexibility profiles in evolutionarily diverse prion proteins

Collective motions on ns-µs time scales are known to have a major impact on protein folding, stability, binding and enzymatic efficiency. It is also believed that these motions may have an important role in the early stages of prion protein misfolding and prion disease. In an effort to accurately characterize these motions and their potential influence on the misfolding and prion disease transmissibility we have conducted a combined analysis of molecular dynamic simulations and NMR-derived flexibility measurements over a diverse range of prion proteins. Using a recently developed numerical formalism, we have analyzed the essential collective dynamics (ECD) for prion proteins from eight different species including human, cow, elk, cat, hamster, chicken, turtle and frog. We also compared the numerical results with flexibility profiles generated by the random coil index (RCI) from NMR chemical shifts. Prion protein backbone flexibility derived from experimental NMR data and from theoretical computations show strong agreement with each other, demonstrating that it is possible to predict the observed RCI profiles employing the numerical ECD formalism. Interestingly, flexibility differences in the loop between second b strand (S2) and the second a helix (HB) appear to distinguish prion proteins from species that are susceptible to prion disease and those that are resistant. Our results show that the different levels of flexibility in the S2-HB loop in various species are predictable via the ECD method, indicating that ECD may be used to identify disease resistant variants of prion proteins, as well as the influence of prion proteins mutations on disease susceptibility or misfolding propensity.Key words: prion proteins structural stability, molecular dynamics simulation, essential collective dynamics, protein dynamic domains, biomolecular NMR, rigid loop     

Distinct hydration properties of wild-type and familial point mutant A53T of α-synuclein associated with Parkinson's disease

The propensity of α-synuclein to form amyloid plays an important role in Parkinson's disease. Three familial mutations, A30P, E46K, and A53T, correlate with Parkinson's disease. Therefore, unraveling the structural effects of these mutations has basic implications in understanding the molecular basis of the disease. Here, we address this issue through comparing details of the hydration of wild-type α-synuclein and its A53T mutant by a combination of wide-line NMR, differential scanning calorimetry, and molecular dynamics simulations. All three approaches suggest a hydrate shell compatible with a largely disordered state of both proteins. Its fine details, however, are different, with the mutant displaying a somewhat higher level of hydration, suggesting a bias to more open structures, favorable for protein-protein interactions leading to amyloid formation. These differences disappear in the amyloid state, suggesting basically the same surface topology, irrespective of the initial monomeric state.     

Different misfolding mechanisms converge on common conformational changes

Prion diseases are caused by misfolding and aggregation of the prion protein (PrP). Pathogenic mutations such as Y218N and E196K are known to cause Gerstmann-Sträussler-Scheinker syndrome and Creutzfeldt-Jakob disease, respectively. Here we describe molecular dynamics simulations of these mutant proteins to better characterize the detailed conformational effects of these sequence substitutions. Our results indicate that the mutations disrupt the wild-type native PrP^C structure and cause misfolding. Y218N reduced hydrophobic packing around the X-loop (residues 165–171), and E196K abolished an important wild-type salt bridge. While differences in the mutation site led PrP mutants to misfold along different pathways, we observed multiple traits of misfolding that were common to both mutants. Common traits of misfolding included: 1) detachment of the short helix (HA) from the PrP core; 2) exposure of side chain F198; and 3) formation of a nonnative strand at the N-terminus. The effect of the E196K mutation directly abolished the wild-type salt bridge E196-R156, which further destabilized the F198 hydrophobic pocket and HA. The Y218N mutation propagated its effect by increasing the HB-HC interhelical angle, which in turn disrupted the packing around F198. Furthermore, a nonnative contact formed between E221 and S132 on the S1-HA loop, which offered a direct mechanism for disrupting the hydrophobic packing between the S1-HA loop and HC. While there were common misfolding features shared between Y218N and E196K, the differences in the orientation of HB and HC and the X-loop conformation might provide a structural basis for identifying different prion strains.     

Conformational polymorphism of wild-type and mutant prion proteins: Energy landscape analysis

Conformational transitions are thought to be the prime mechanism of prion diseases. In this study, the energy landscapes of a wild-type prion protein (PrP) and the D178N and E200K mutant proteins were mapped, enabling the characterization of the normal isoforms (PrP(C)) and partially unfolded isoforms (PrP(PU)) of the three prion protein analogs. It was found that the three energy landscapes differ in three respects: (i) the relative stability of the PrP(C) and the PrP(PU) states, (ii) the transition pathways from PrP(C) to PrP(PU), and (iii) the relative stability of the three helices in the PrP(C) state. In particular, it was found that although helix 1 (residues 144-156) is the most stable helix in wild-type PrP, its stability is dramatically reduced by both mutations. This destabilization is due to changes in the charge distribution that affects the internal salt bridges responsible for the greater stability of this helix in wild-type PrP. Although both mutations result in similar destabilization of helix 1, they a have different effect on the overall stability of PrP(C) and of PrP(PU) isoforms and on structural properties. The destabilization of helix 1 by mutations provides additional evidences to the role of this helix in the pathogenic transition from the PrP(C) to the pathogenic isoform PrP(SC).     

Atomic insight into prion disorder: An intricate detail gained by 0.5 μs molecular dynamics simulation of preventive G127V and deleterious D178V mutation in prion protein

In this study we are looking into two contradicting mutations found in prion protein (PrP) viz G127V and D178V, that are reportedly protective and pathogenic, respectively. Despite significant advances in comprehension of the role of pathogenic mutations, the role of protective mutation in amyloid fold inhibition still lacks a substantial basis. To understand the structural basis of protective mutation, molecular dynamics simulation coupled with protein-protein docking and molecular mechanics/Poisson-Boltzmann surface area analysis was used to understand the instant structural variability brought about by these mutations alone and in combination on PrP and prion-prion complex. Atomic-scale investigations successfully revealed that the binding pattern of prion-prion varies differentially in protective and pathogenic mutations with secondary structure showing distinct contrasting patterns, which could supposedly be a critical factor for differential prion behavior in protective and pathogenic mutations. Considering the reported role of an amyloid fold in prion-prion binding, the contrasting pattern has given us a lead in comprehending the role of these mutations and has been used in this study to look for small molecules that can inhibit amyloid fold for prion-prion interaction in pathogenic mutant carrying PrP.     

Dynamic diagnosis of familial prion diseases supports the β2-α2 loop as a universal interference target

Background

Mutations in the cellular prion protein associated to familial prion disorders severely increase the likelihood of its misfolding into pathogenic conformers. Despite their postulation as incompatible elements with the native fold, these mutations rarely modify the native state structure. However they variably have impact on the thermodynamic stability and metabolism of PrP^C and on the properties of PrP^Sc aggregates. To investigate whether the pathogenic mutations affect the dynamic properties of the HuPrP(125-229) α-fold and find possible common patterns of effects that could help in prophylaxis we performed a dynamic diagnosis of ten point substitutions.

Methodology/Principal Findings

Using all-atom molecular dynamics simulations and novel analytical tools we have explored the effect of D178N, V180I, T183A, T188K, E196K, F198S, E200K, R208H, V210I and E211Q mutations on the dynamics of HuPrP(125-228) α-fold. We have found that while preserving the native state, all mutations produce dynamic changes which perturb the coordination of the α2-α3 hairpin to the rest of the molecule and cause the reorganization of the patches for intermolecular recognition, as the disappearance of those for conversion inhibitors and the emergence of an interaction site at the β2-α2 loop region.

Conclusions/Significance

Our results suggest that pathogenic mutations share a common pattern of dynamical alterations that converge to the conversion of the β2-α2 loop into an interacting region that can be used as target for interference treatments in genetic diseases.     

Correlation of Local Changes in the Temperature-Dependent Conformational Flexibility of Thioredoxins with Their Thermostability

For the development of a method capable of predicting single point mutations substantially affecting protein thermostability, we studied the effect of the E85R and R82E mutations on the thermostability of thioredoxins from Escherichia coli (Trx) andBacillus acidocaldarius (BacTrx), respectively. The basic method of investigation was the molecular dynamics simulation of 3D protein models in an explicit solvent at different temperatures (300 and 373 K). Some thermolabile regions in Trx, BacTrx, and their mutants were revealed by analyzing the temperature effect on the molecular dynamics of the protein molecule. The effect of single point mutations on the temperature changes of the protein conformation flexibility in several thermolabile regions was found. The results of the simulations are in accord with experimental data indicating that the mutation E85R increases Trx thermostability, whereas the mutation R82E decreases BacTrx thermostability. The thermostability of these proteins was revealed to depend on ionic interactions between the thermolabile regions. The single point mutations change the parameters of these interactions and make them more favorable in the E85R-Trx mutant and less favorable in the R82E-BacTrx mutant.     

Structural characteristics of the nonallosteric human cytosolic malic enzyme

Human cytosolic NADP⁺-dependent malic enzyme (c-NADP-ME) is neither a cooperative nor an allosteric enzyme, whereas mitochondrial NAD(P)⁺-dependent malic enzyme (m-NAD(P)-ME) is allosterically activated by fumarate. This study examines the molecular basis for the different allosteric properties and quaternary structural stability of m-NAD(P)-ME and c-NADP-ME. Multiple residues corresponding to the fumarate-binding site were mutated in human c-NADP-ME to correspond to those found in human m-NAD(P)-ME. Additionally, the crystal structure of the apo (ligand-free) human c-NADP-ME conformation was determined. Kinetic studies indicated no significant difference between the wild-type and mutant enzymes in K_m,NADP, K_m,malate, and k_cat. A chimeric enzyme, [51-105]_c-NADP-ME, was designed to include the putative fumarate-binding site of m-NAD(P)-ME at the dimer interface of c-NADP-ME; however, this chimera remained nonallosteric. In addition to fumarate activation, the quaternary structural stability of c-NADP-ME and m-NAD(P)-ME is quite different; c-NADP-ME is a stable tetramer, whereas m-NAD(P)-ME exists in equilibrium between a dimer and a tetramer. The quaternary structures for the S57K/N59E/E73K/S102D and S57K/N59E/E73K/S102D/H74K/D78P/D80E/D87G mutants of c-NADP-ME are tetrameric, whereas the K57S/E59N/K73E/D102S m-NAD(P)-ME quadruple mutant is primarily monomeric with some dimer formation. These results strongly suggest that the structural features near the fumarate-binding site and the dimer interface are highly related to the quaternary structural stability of c-NADP-ME and m-NAD(P)-ME. In this study, we attempt to delineate the structural features governing the fumarate-induced allosteric activation of malic enzyme.     

Molecular dynamics simulations on human fibulin-4 mutants D203A and E126K reveal conformational changes in EGF domains potentially responsible for enhanced protease lability and impaired extracellular matrix assembly

Fibulin-4 is a 50 kDa glycoprotein of elastic fibers and plays an important role in development and function of elastic tissues. Fibulin-4 consists of a tandem array of five calcium-binding epidermal growth factor-like modules flanked by N- and C-terminal domains. Mutations in the human fibulin-4 gene EFEMP2 have been identified in patients affected with various arteriopathies including aneurysm, arterial tortuosity, or stenosis, but the molecular basis of most genotype-phenotype correlations is unknown. Here we present biochemical and computer modelling approaches designed to gain further insight into changes in structure and function of two fibulin-4 mutations (E126K and D203A), which are potentially involved in Ca²⁺ binding in the EGF2 and EGF4 domain, respectively. Using recombinantly produced fibulin-4 mutant and wild type proteins we show that both mutations introduced additional protease cleavage sites, impaired extracellular assembly into fibers, and affected binding to to fibrillin-1, latent TGF-β-binding proteins, and the lysyl oxidase LOXL2. Molecular dynamics studies indicated that the E126K and D203A mutations do not necessarily result in a direct loss of the complexed Ca²⁺ ion after 500 ns simulation time, but in significantly enhanced fluctuations within the connecting loop between EGF3 and EGF4 domains and other conformational changes. In contrast, intentionally removing Ca²⁺ from EGF4 (D203A ΔCa) predicted dramatic changes in the protein structure. These results may explain the changes in protease cleavage sites, reduced secretion and impaired extracellular assembly of the E126K and D203A fibulin-4 mutants and provide further insight into understanding the molecular basis of the associated clinical phenotypes.     

Impact of Gln94Glu mutation on the structure and function of protection of telomere 1, a cause of cutaneous familial melanoma

Abstract

Protection of telomere 1 (POT1) is a key component of shelterin complex, essential for maintaining telomere length and its regulation. It consists of N-terminal domain (residues 1–299), which interacts with telomeric ssDNA, and the C-terminal domain (residues 320–634) that binds to the tripeptidyl-peptidase I (TPP1). A large number of naturally occurring mutations in the POT1 gene are associated with glioma, cardiac angiosarcoma and cutaneous familial melanoma (FM). In particular, Q94E mutation disrupts the interaction of POT1 with telomeric DNA which subsequently enhances telomere uncapping and elongation and promotes the development of cutaneous familial melanoma. To understand the underlying mechanism of familial melanoma developed by Q94E-mutation, we have performed extensive structure analysis of WT and mutant protein followed by molecular dynamics simulations. Q94E mutation causes a dramatic change in the structure and stability of POT1 protein. A considerable decrease in the flexibility, fluctuation and solvent accessibility of Q94E was observed in comparison to the WT, indicating overall destabilization of protein. Essential dynamics and Anisotropic Network Mode analysis have quantified a significant change in direction and magnitude of conformational motion in Q94E mutant compared to WT. A significant loss of frustration due to Q94E mutation was also observed. Our findings indicate the loss of protein stability and dynamics of POT1 protein by Q94E mutation may be associated with the familial melanoma. Abbreviations ANM anisotropic network mode

ED essential dynamics

FM familial melanoma

MD molecular dynamics

POT1 protection of telomere 1

Rg radius of gyration

RMSD root-mean-square deviation

RMSF root-mean-square fluctuations

SASA solvent accessible surface area

SIFT sorting Intolerant from Tolerant

TPP1 tripeptidyl-peptidase I

WT wild type

Communicated by Ramaswamy H. Sarma