Molecular dynamics simulations reveal structural differences among wild-type NPC1 protein and its mutant forms

Abstract

NPC1 is a 25-exon gene located on the long arm of chromosome 18q11.2 and encodes NPC1, a transmembrane protein comprising 1278 amino acid residues. Mutations in the NPC1 gene can cause Niemann-Pick disease type C (NP-C), a rare autosomal-recessive neurovisceral disease. We assessed mutant protein folding using computer-based molecular dynamics (MD) simulations and molecular docking of the three most common NPC1 mutations, all of which result in changes in a cysteine-rich luminal loop region of the protein: a) I1061T is the most commonly detected variant in patients with NP-C worldwide; b) P1007A is the second most common variant, frequently detected in Portuguese, British and German patients; c) G992W occurs most often in patients of Acadian descent. Analyses of molecular structural information and related cellular physiological processes revealed that mutant NPC1 proteins exhibited altered function despite being far from the N-terminal domain cholesterol binding. MD simulations revealed that mutant I1061T protein shows remarkable instability in comparison the WT and also de other mutants, and interestingly this mutant has been identified as the most common variant. In the case of the mutant P1007A, it is presumed that this substitution promotes larger structural changes than proline due to their greater hydrophobic properties.

Structural changes related to the G992W mutation may affect the physicochemical space of G992W variant protein because tryptophan induces hydrophobic interactions. Cholesterol docking studies focused on binding recognition showed differences in the binding positions of variants versus the wild-type protein that go some way to explaining the molecular pathogenesis.

Communicated by Ramaswamy H. Sarma     

Disease-causing mutations in proteins: structural analysis of the CYP1B1 mutations causing primary congenital glaucoma in humans

In this communication, we report an in-depth structure-based analysis of the human CYP1b1 protein carrying disease-causing mutations that are discovered in patients suffering from primary congenital glaucoma (PCG). The "wild-type" and the PCG mutant structures of the human CYP1b1 protein obtained from comparative modeling were subjected to long molecular dynamics simulations with an intention of studying the possible impact of these mutations on the protein structure and hence its function. Analysis of time evolution as well as time averaged values of various structural properties--especially of those of the functionally important regions: the heme binding region, substrate binding region, and substrate access channel--gave some insights into the possible structural characteristics of the disease mutant and the wild-type forms of the protein. In a nutshell, compared to the wild-type the core regions in the mutant structures are associated with subtle but significant changes, and the functionally important regions seem to adopt such structures that are not conducive for the wild-type-like functionality.     

Molecular dynamics as a tool to detect protein foldability. A mutant of domain B1 of protein G with non-native secondary structure propensities

The usefulness of molecular dynamics to assess the structural integrity of mutants containing several mutations has been investigated. Our goal was to determine whether molecular dynamics would be able to discriminate mutants of a protein having a close-to-wild-type fold, from those that are not folded under the same conditions. We used as a model the B1 domain of protein G in which we replaced the unique central alpha-helix by the sequence of the second beta-hairpin, which has a strong intrinsic propensity to form this secondary structure in solution. In the resulting protein, one-third of the secondary structure has been replaced by a non-native one. Models of the mutants were built based on the three-dimensional structure of the wild-type GB1 domain. During 2 ns of molecular dynamics simulations on these models, mutants containing up to 10 mutations in the helix retained the native fold, while another mutant with an additional mutation unfolded. This result is in agreement with our circular dichroism and NMR experiments, which indicated that the former mutants fold into a structure similar to the wild-type, as opposed to the latter mutant which is partly unfolded. Additionally, a mutant containing six mutations scattered through the surface of the domain, and which is unfolded, was also detected by the simulation. This study suggests that molecular dynamics calculations could be performed on molecular models of mutants of a protein to evaluate their foldability, prior to a mutagenesis experiment.     

用分子模拟方法研究HIV-1整合酶突变体的耐药性机理

二酮酸类化合物(DKAs)是目前最有前景的HIV-1整合酶(integrase, IN)抑制剂．为了解DKAs引起的多种耐药株共有的耐药性机理,选择3种S-1360引起的IN耐药突变体,用分子对接和分子动力学模拟,研究了野生型和突变型IN与S-1360的结合模式,基于该结合模式探讨了3种耐药突变体所共有的耐药性机理．结果表明：在突变体中,S-1360结合到耐药突变IN核心区中的位置靠近功能loop 3区却远离与 DNA结合的关键残基,结合位置不同导致S-1360的抑制作用部分丧失;残基138到166区域的柔性对IN发挥生物学功能很重要,S-1360能与DNA结合的关键残基N155及K159形成氢键,这2个氢键作用降低了该区域的柔性,突变体中无类似氢键,因而该区域柔性增高;在突变体中,S-1360的苯环远离病毒DNA结合区,不能阻止病毒DNA末端暴露给宿主DNA;T66I突变导致残基Ⅰ的长侧链占据IN的活性口袋,阻止抑制剂以与野生型中相同的方式结合到活性中心,这均是产生抗药性的重要原因．这些模拟结果与实验结果吻合,可为抗IN的抑制剂设计和改造提供帮助．     

U1A protein-stem loop 2 RNA recognition: prediction of structural differences from protein mutations

Molecular dynamics (MD) simulations were carried out to compare the free and bound structures of wild type U1A protein with several Phe56 mutant U1A proteins that bind the target stem loop 2 (SL2) RNA with a range of affinities. The simulations indicate the free U1A protein is more flexible than the U1A-RNA complex for both wild type and Phe56 mutant systems. A complete analysis of the hydrogen-bonding (HB) and non-bonded (VDW) interactions over the course of the MD simulations suggested that changes in the interactions in the free U1A protein caused by the Phe56Ala and Phe56Leu mutations may stabilize the helical character in loop 3, and contribute to the weak binding of these proteins to SL2 RNA. Compared with wild type, changes in HB and VDW interactions in Phe56 mutants of the free U1A protein are global, and include differences in β-sheet, loop 1 and loop 3 interactions. In the U1A-RNA complex, the Phe56Ala mutation leads to a series of differences in interactions that resonate through the complex, while the Phe56Leu and Phe56Trp mutations cause local differences around the site of mutation. The long-range networks of interactions identified in the simulations suggest that direct interactions and dynamic processes in both the free and bound forms contribute to complex stability.     

Describing inhibitor specificity for the amino acid transporter LAT1 from metainference simulations

The human L-type amino acid transporter 1 (LAT1; SLC7A5) is a membrane transporter of amino acids, thyroid hormones, and drugs such as the Parkinson’s disease drug levodopa (L-Dopa). LAT1 is found in the blood-brain barrier, testis, bone marrow, and placenta, and its dysregulation has been associated with various neurological diseases, such as autism and epilepsy, as well as cancer. In this study, we combine metainference molecular dynamics simulations, molecular docking, and experimental testing, to characterize LAT1-inhibitor interactions. We first conducted a series of molecular docking experiments to identify the most relevant interactions between LAT1’s substrate-binding site and ligands, including both inhibitors and substrates. We then performed metainference molecular dynamics simulations using cryoelectron microscopy structures in different conformations of LAT1 with the electron density map as a spatial restraint, to explore the inherent heterogeneity in the structures. We analyzed the LAT1 substrate-binding site to map important LAT1-ligand interactions as well as newly described druggable pockets. Finally, this analysis guided the discovery of previously unknown LAT1 ligands using virtual screening and cellular uptake experiments. Our results improve our understanding of LAT1-inhibitor recognition, providing a framework for rational design of future lead compounds targeting this key drug target.     

Cancer-related mutations in BRCA1-BRCT cause long-range structural changes in protein-protein binding sites: a molecular dynamics study

Cancer-associated mutations in the BRCT domain of BRCA1 (BRCA1-BRCT) abolish its tumor suppressor function by disrupting interactions with other proteins such as BACH1. Many cancer-related mutations do not cause sufficient destabilization to lead to global unfolding under physiological conditions, and thus abrogation of function probably is due to localized structural changes. To explore the reasons for mutation-induced loss of function, the authors performed molecular dynamics simulations on three cancer-associated mutants, A1708E, M1775R, and Y1853ter, and on the wild type and benign M1652I mutant, and compared the structures and fluctuations. Only the cancer-associated mutants exhibited significant backbone structure differences from the wild-type crystal structure in BACH1-binding regions, some of which are far from the mutation sites. Backbone differences of the A1708E mutant from the liganded wild type structure in these regions are much larger than those of the unliganded wild type X-ray or molecular dynamics structures. These BACH1-binding regions of the cancer-associated mutants also exhibited increases in their fluctuation magnitudes compared with the same regions in the wild type and M1562I mutant, as quantified by quasiharmonic analysis. Several of the regions of increased fluctuation magnitude correspond to correlated motions of residues in contact that provide a continuous path of fluctuating amino acids in contact from the A1708E and Y1853ter mutation sites to the BACH1-binding sites with altered structure and dynamics. The increased fluctuations in the disease-related mutants suggest an increase in vibrational entropy in the unliganded state that could result in a larger entropy loss in the disease-related mutants upon binding BACH1 than in the wild type. To investigate this possibility, vibrational entropies of the A1708E and wild type in the free state and bound to a BACH1-derived phosphopeptide were calculated using quasiharmonic analysis, to determine the binding entropy difference DeltaDeltaS between the A1708E mutant and the wild type. DeltaDeltaS was determined to be -4.0 cal mol(-1) K(-1), with an uncertainty of 2 cal mol(-1) K(-1); that is, the entropy loss upon binding the peptide is 4.0 cal mol(-1) K(-1) greater for the A1708E mutant, corresponding to an entropic contribution to the DeltaDeltaG of binding (-TDeltaDeltaS) 1.1 kcal mol(-1) more positive for the mutant. The observed differences in structure, flexibility, and entropy of binding likely are responsible for abolition of BACH1 binding, and illustrate that many disease- related mutations could have very long-range effects. The methods described here have potential for identifying correlated motions responsible for other long-range effects of deleterious mutations.     

Towards the identification of the allosteric Phe-binding site in phenylalanine hydroxylase

The enzyme phenylalanine hydroxylase (PAH) is defective in the inherited disorder phenylketonuria. PAH, a tetrameric enzyme, is highly regulated and displays positive cooperativity for its substrate, Phe. Whether Phe binds to an allosteric site is a matter of debate, despite several studies worldwide. To address this issue, we generated a dimeric model for Phe–PAH interactions, by performing molecular docking combined with molecular dynamics simulations on human and rat wild-type sequences and also on a human G46S mutant. Our results suggest that the allosteric Phe-binding site lies at the dimeric interface between the regulatory and the catalytic domains of two adjacent subunits. The structural and dynamical features of the site were characterized in depth and described. Interestingly, our findings provide evidence for lower allosteric Phe-binding ability of the G46S mutant than the human wild-type enzyme. This also explains the disease-causing nature of this mutant.     

Critical effects on binding of epidermal growth factor produced by amino acid substitutions

Epidermal growth factor (EGF) plays important roles in multiple biological processes, such as the regulation of cell growth, proliferation, and differentiation. EGF exerts their pharmacologic effects via receptor-mediated mechanism associated with high affinity to epidermal growth factor receptor (EGFR) on the cell surface. Overexpression of EGFR has been reported and implicated in the pathogenesis of many human cancers. The current study addresses the effects of mutations on binding properties of EGF to EGFR. Two mutant structures with three point mutations of conserved residues, Ile23, Arg41 and Leu47, which have been found to be important for the receptor binding, were built using homology modeling. The “wild type” (WT) and the mutant structures, after structural validations, were subjected to molecular dynamics simulations (MDSs). The primary aim of MDS was to investigate the possible impact of mutations on the protein structure and function. Analysis of root mean square deviation (RMSD), other time dependent structural properties and their averages provided some insights into the possible structural characteristics of the mutant and the WT forms of the EGF. RMSD analysis showed that WT EGF was more stable than the mutant structures. The docking analysis revealed that the binding energy of mutant EGFs to EGFR is lower than WT. Combination of the used computational approaches provides a way in understanding the impact of deleterious mutations in altering the EGF and EGFR interactions.     

Common conformational effects of p53 mutations

The tumor suppressor gene p53 has been identified as the most frequent target of genetic alterations in human cancers. Most of these mutations occur in highly conserved regions in the DNA-binding core domain of the p53 protein, suggesting that the amino acid residues in these regions are critical for maintaining normal p53 structure and function. We previously used molecular dynamics calculations to demonstrate that several amino acid substitutions in these regions that are induced by environmental carcinogens and found in human tumors produce certain common conformational changes in the mutant proteins that differ substantially from the wild-type structure. In order to determine whether these conformational changes are consistent for other p53 mutants, we have now used molecular dynamics to determine the structure of the DNA-binding core domain of seven other environmentally induced, cancer-related p53 mutants, namely His 175, Asp 245, Asn 245, Trp 248, Met 249, Ser 278, and Lys 286. The results indicate that all of these mutants differ substantially from the wild-type structure in certain discrete regions and that some of these conformational changes are similar for these mutants as well as those determined previously. The changes are also consistent with experimental evidence for alterations in structure in p53 mutants determined by epitope detectability using monoclonal antibodies directed against these regions of predicted conformational change.     

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.     

Insights into regioselective metabolism of mefenamic acid by cytochrome P450 BM3 mutants through crystallography,docking, molecular dynamics,and free energy calculations

Cytochrome P450 BM3 (CYP102A1) mutant M11 is able to metabolize a wide range of drugs and drug‐like compounds. Among these, M11 was recently found to be able to catalyze formation of human metabolites of mefenamic acid and other nonsteroidal anti‐inflammatory drugs (NSAIDs). Interestingly, single active‐site mutations such as V87I were reported to invert regioselectivity in NSAID hydroxylation. In this work, we combine crystallography and molecular simulation to study the effect of single mutations on binding and regioselective metabolism of mefenamic acid by M11 mutants. The heme domain of the protein mutant M11 was expressed, purified, and crystallized, and its X‐ray structure was used as template for modeling. A multistep approach was used that combines molecular docking, molecular dynamics (MD) simulation, and binding free‐energy calculations to address protein flexibility. In this way, preferred binding modes that are consistent with oxidation at the experimentally observed sites of metabolism (SOMs) were identified. Whereas docking could not be used to retrospectively predict experimental trends in regioselectivity, we were able to rank binding modes in line with the preferred SOMs of mefenamic acid by M11 and its mutants by including protein flexibility and dynamics in free‐energy computation. In addition, we could obtain structural insights into the change in regioselectivity of mefenamic acid hydroxylation due to single active‐site mutations. Our findings confirm that use of MD and binding free‐energy calculation is useful for studying biocatalysis in those cases in which enzyme binding is a critical event in determining the selective metabolism of a substrate. Proteins 2016; 84:383–396. © 2016 Wiley Periodicals, Inc.     

In silico and in vitro effects of the I30T mutation on myelin protein zero instability in the cell membrane

Charcot‐Marie‐Tooth (CMT) diseases are a heterogeneous group of genetic peripheral neuropathies caused by mutations in a variety of genes, which are involved in the development and maintenance of peripheral nerves. Myelin protein zero (MPZ) is expressed by Schwann cells, and MPZ mutations can lead to primarily demyelinating polyneuropathies including CMT type 1B. Different mutations demonstrate various forms of disease pathomechanisms, which may be beneficial in understanding the disease cellular pathology. Our molecular dynamics simulation study on the possible impacts of I30T mutation on the MPZ protein structure suggested a higher hydrophobicity and thus lower stability in the membranous structures. A study was also conducted to predict native/mutant MPZ interactions. To validate the results of the simulation study, the native and mutant forms of the MPZ protein were separately expressed in a cellular model, and the protein trafficking was chased down in a time course pattern. In vitro studies provided more evidence on the instability of the MPZ protein due to the mutation. In this study, qualitative and quantitative approaches were adopted to confirm the instability of mutant MPZ in cellular membranes.     

Modulation of interaction of mutant TP53 and wild type BRCA1 by alkaloids: a computational approach towards targeting protein-protein interaction as a futuristic therapeutic intervention strategy for breast cancer impediment

Protein–protein interactions (PPI) are a new emerging class of novel therapeutic targets. In order to probe these interactions, computational tools provide a convenient and quick method towards the development of therapeutics. Keeping this in view the present study was initiated to analyse interaction of tumour suppressor protein p53 (TP53) and breast cancer associated protein (BRCA1) as promising target against breast cancer. Using computational approaches such as protein–protein docking, hot spot analyses, molecular docking and molecular dynamics simulation (MDS), stepwise analyses of the interactions of the wild type and mutant TP53 with that of wild type BRCA1 and their modulation by alkaloids were done. Protein–protein docking method was used to generate both wild type and mutant complexes of TP53-BRCA1. Subsequently, the complexes were docked using sixteen different alkaloids, fulfilling ADMET and Lipinski’s rule of five criteria, and were compared with that of a well-known inhibitor of PPI, namely nutlin. The alkaloid dicentrine was found to be the best docked alkaloid among all the docked alklaloids as well as that of nutlin. Furthermore, MDS analyses of both wild type and mutant complexes with the best docked alkaloid i.e. dicentrine, revealed higher stability of mutant complex than that of the wild one, in terms of average RMSD, RMSF and binding free energy, corroborating the results of docking. Results suggested more pronounced interaction of BRCA1 with mutant TP53 leading to increased expression of mutated TP53 thus showing a dominant negative gain of function and hampering wild type TP53 function leading to tumour progression.     

Parkinson's disease-associated DJ-1 mutations impair mitochondrial dynamics and cause mitochondrial dysfunction

Mitochondrial dysfunction represents a critical event during the pathogenesis of Parkinson's disease (PD) and expanding evidences demonstrate that an altered balance in mitochondrial fission/fusion is likely an important mechanism leading to mitochondrial and neuronal dysfunction/degeneration. In this study, we investigated whether DJ-1 is involved in the regulation of mitochondrial dynamics and function in neuronal cells. Confocal and electron microscopic analysis demonstrated that M17 human neuroblastoma cells over-expressing wild-type DJ-1 (WT DJ-1 cells) displayed elongated mitochondria while M17 cells over-expressing PD-associated DJ-1 mutants (R98Q, D149A and L166P) (mutant DJ-1 cells) showed significant increase of fragmented mitochondria. Similar mitochondrial fragmentation was also noted in primary hippocampal neurons over-expressing PD-associated mutant forms of DJ-1. Functional analysis revealed that over-expression of PD-associated DJ-1 mutants resulted in mitochondria dysfunction and increased neuronal vulnerability to oxidative stress (H(2) O(2)) or neurotoxin. Further immunoblot studies demonstrated that levels of dynamin-like protein (DLP1), also known as Drp1, a regulator of mitochondrial fission, was significantly decreased in WT DJ-1 cells but increased in mutant DJ-1 cells. Importantly, DLP1 knockdown in these mutant DJ-1 cells rescued the abnormal mitochondria morphology and all associated mitochondria/neuronal dysfunction. Taken together, these studies suggest that DJ-1 is involved in the regulation of mitochondrial dynamics through modulation of DLP1 expression and PD-associated DJ-1 mutations may cause PD by impairing mitochondrial dynamics and function.     

Relation between sequence and structure of HIV-1 protease inhibitor complexes: a model system for the analysis of protein flexibility.

The flexibility of different regions of HIV-1 protease was examined by using a database consisting of 73 X-ray structures that differ in terms of sequence, ligands or both. The root-mean-square differences of the backbone for the set of structures were shown to have the same variation with residue number as those obtained from molecular dynamics simulations, normal mode analyses and X-ray B-factors. This supports the idea that observed structural changes provide a measure of the inherent flexibility of the protein, although specific interactions between the protease and the ligand play a secondary role. The results suggest that the potential energy surface of the HIV-1 protease is characterized by many local minima with small energetic differences, some of which are sampled by the different X-ray structures of the HIV-1 protease complexes. Interdomain correlated motions were calculated from the structural fluctuations and the results were also in agreement with molecular dynamics simulations and normal mode analyses. Implications of the results for the drug-resistance engendered by mutations are discussed briefly.     

Long-range coupling between separate docking sites in interleukin-1beta

The human cytokine interleukin-1beta (IL-1beta) interacts with the interleukin type I receptor using two large docking surfaces designated A and B. Crystallographic studies reveal that a single histidine residue (His30) in IL-1beta makes critical electrostatic interactions at the receptor/ligand interface. To study the function of this residue at site A, four mutant forms of IL-1beta (H30A, H30D, H30F and H30R) were investigated. The mutation that introduces charge repulsion at His30 destabilizes the protein, but paradoxically causes the least effect on receptor binding (H30D). Mutations that enhance hydrophobic or electrostatic interactions have little effect on protein stability yet markedly impair receptor binding (H30F, H30R). All mutations can transmit effects from site A to site B, as evidenced by changes in the binding of a single-chain antibody highly specific for site B. Dihedral scalar coupling constants for the wild-type IL-1beta and the four His mutant proteins showed changes in backbone angles in residues located around site B, some approximately 30 angstroms away from His30 in site A. A comparison of native solvent exchange in wild-type and mutated IL-1beta shows transmission of local destabilization along the hydrogen bond network of the beta-sheet. Taken together, the data indicate that a single residue in site A of IL-1beta can impact stability and function through perturbations in both local and long-range contacts.     

Common Conformational Effects in the P53 Protein of Vinyl Chloride-Induced Mutations

The tumor suppressor gene p53 has been identified as the most frequent site of genetic alterations in human cancers. Vinyl chloride, a known human carcinogen, has been associated with specific A T transversions at codons 179, 249, and 255 of the p53 gene. The mutations result in amino acid substitutions of His Leu at residue 179, Arg Trp at residue 249, and Ile Phe at residue 255 in highly conserved regions of the DNA-binding core domain of the p53 protein. We previously used molecular dynamics calculations to demonstrate that the latter two mutants contain certain common regions that differ substantially in conformation from the wild-type structure. In order to determine whether these conformational changes are consistent for other p53 mutants, we have now used molecular dynamics to determine the structure of the DNA-binding core domain of the Leu 179 p53 mutant. The results indicate that the Leu 179 mutant differs substantially from the wild-type structure in certain discrete regions that are similar to those noted previously in the other p53 mutants. One of these regions (residues 204–217) contains the epitope for the monoclonal antibody PAb240, which is concealed in the wild-type structure, but accessible in the mutant structure, and another region (residues 94–110) contains the epitope for the monoclonal antibody PAb1620, which is accessible in the wild-type structure, but concealed in the mutant structure. Immunologic analyses of tumor tissue known to contain this mutation confirmed these predicted conformational shifts in the mutant p53 protein.