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The molecular interactions of the sarcomeric proteins are essential in the regulation of various cardiac functions. Mutations in the gene MYBPC3 coding for cardiac myosin-binding protein-C (cMyBP-C), a multi-domain protein, are the most common cause of hypertrophic cardiomyopathy (HCM). The N-terminal complex, C1-motif-C2 is a central region in cMyBP-C for the regulation of cardiac muscle contraction. However, the mechanism of binding/unbinding of this complex during health and disease is unknown. Here, we study possible mechanisms of unbinding using steered molecular dynamics simulations for the complex in the wild type, in single mutations (E258K in C1, E441K in C2), as well as in a double mutation (E258K in C1 + E441K in C2), which are associated with severe HCM. The observed molecular events and the calculation of force utilized for the unbinding suggest the following: (i) double mutation can encourage the formation of rigid complex that required large amount of force and long-time to unbind, (ii) C1 appears to start to unbind ahead of C2 regardless of the mutation, and (iii) unbinding of C2 requires larger amount of force than C1. This molecular insight suggests that key HCM-causing mutations might significantly modify the native affinity required for the assembly of the domains in cMyBP-C, which is essential for normal cardiac function.

Hereditary hearing loss (HHL) is a common disorder characterized by a huge genetic heterogeneity. The definition of a correct molecular diagnosis is essential for proper genetic counseling, recurrence risk estimation, and therapeutic options. From 20 to 40% of patients carry mutations in GJB2 gene, thus, in more than half of cases it is necessary to look for causative variants in the other genes so far identified (~100). In this light, the use of next-generation sequencing technologies has proved to be the best solution for mutational screening, even though it is not always conclusive. Here we describe a combined approach, based on targeted re-sequencing (TRS) of 96 HHL genes followed by high-density SNP arrays, aimed at the identification of the molecular causes of non-syndromic HHL (NSHL). This strategy has been applied to study 103 Italian unrelated cases, negative for mutations in GJB2, and led to the characterization of 31% of them (i.e., 37% of familial and 26.3% of sporadic cases). In particular, TRS revealed TECTA and ACTG1 genes as major players in the Italian population. Furthermore, two de novo missense variants in ACTG1 have been identified and investigated through protein modeling and molecular dynamics simulations, confirming their likely pathogenic effect. Among the selected patients analyzed by SNP arrays (negative to TRS, or with a single variant in a recessive gene) a molecular diagnosis was reached in ~36% of cases, highlighting the importance to look for large insertions/deletions. Moreover, copy number variants analysis led to the identification of the first case of uniparental disomy involving LOXHD1 gene. Overall, taking into account the contribution of GJB2, plus the results from TRS and SNP arrays, it was possible to reach a molecular diagnosis in ~51% of NSHL cases. These data proved the usefulness of a combined approach for the analysis of NSHL and for the definition of the epidemiological picture of HHL in the Italian population.

Mutations in the gene coding for cardiac myosin binding protein-C (cMyBP-C), a multi-domain (C0-C10) protein, are a major causative factor for inherited hypertrophic cardiomyopathy. Patients carrying mutations in this gene have an extremely heterogeneous clinical course, with some progressing to end-stage heart failure. The cause of this variability is unknown. We here describe molecular modeling of a double mutation in domains C1 (E258K) and C2 (E441K) in a patient with severe HCM phenotype. The three-dimensional structure for the C1-motif-C2 complex was constructed with double and single mutations being introduced. Molecular dynamic simulations were performed for 10 ns under physiological conditions. The results showed that both E258K and E441K in isolation can predominantly affect the native domain as well as the nearby motif via conformational changes and result in an additive effect when they coexist. These changes involve important regions of the motif such as phosphorylation and potential actin-binding sites. Moreover, the charge reversal mutations altered the surface electrostatic properties of the complex. In addition, we studied protein expression, which showed that the mutant proteins were expressed and we can suppose that the severe phenotype was not due to haploinsufficiency. However, additional studies on human gene expression will need to confirm this hypothesis. The double mutation affecting the regulatory N-terminal of cMyBP-C have the potential of synergistically interfering with the binding to neighbouring domains and other sarcomeric proteins. These effects may account for the severe phenotype and clinical course observed in the complex cMyBP-C genotypes.

The ability of cells to secrete extracellular matrix proteins is an important property in the repair, replacement, and regeneration of living tissue. Cells that populate tissue-engineered constructs need to be able to emulate these functions. The motifs, KTTKS or palmitoyl-KTTKS (peptide amphiphile), have been shown to stimulate production of collagen and fibronectin in differentiated cells. Molecular modeling was used to design different forms of active peptide motifs to enhance the efficacy of peptides to increase collagen and fibronectin production using terminals KTTKS/SKTTK/SKTTKS connected by various hydrophobic linkers, V4A3/V4A2/A4G3. Molecular dynamic simulations showed SKTTKS-V4A3-SKTTKS (P3), with palindromic (SKTTKS) motifs and SKTTK-V4A2-KTTKS (P5), maintained structural integrity and favorable surface electrostatic distributions that are required for functionality. In vitro studies showed that peptides, P3 and P5, showed low toxicity to human adipose-derived stem cells (hADSCs) and significantly increased the production of collagen and fibronectin in a concentration-dependent manner compared with the original active peptide motif. The 4-day treatment showed that stem cell markers of hADSCs remained stable with P3. The molecular design of novel peptides is a promising strategy for the development of intelligent biomaterials to guide stem cell function for tissue engineering applications.

Human cytochrome P450 (CYP) 3A4 extensively contributes to metabolize 50% of the marketed drugs. Recently, a CYP3A4 structure with two molecules of ketoconazole (2KT) was identified. However, channels for egresses of these inhibitors are unexplored. Thus, we applied molecular dynamics simulations followed by channel analyses. Two simulations of empty and 2KT-bound CYP3A4 results revealed the multiple ligand-induced conformational changes in channel forming regions, which appear to be important for the regulation of channels. In addition, we observed that the channel-3 entrance is closed due to the large structural deviation of the key residues from Phe-cluster. F215 and F220 are known as entrance blockers of channel-2 in metyrapone-bound CYP3A4. Currently, F220 blocks the channel-3 along with F213 and F241. Therefore, it suggested that channel-1 and 2 could potentially serve as egress routes for 2KT. It is also supported by the results from MOLAxis analyses, in which the frequency of channel occurrence and bottleneck radius during simulation favor channel-1 and 2. Several bottleneck residues of these channels may have critical roles in 2KT egresses, especially S119. Our modeling study for multiple ligand-channeling of CYP3A4 could be very helpful to gain new insights into channel selectivity of CYP3A4.

Silent information regulator 2 (Sir2) or sirtuins are NAD(+)-dependent deacetylases, which hydrolyze the acetyl-lysine residues. In mammals, sirtuins are classified into seven different classes (SIRT1-7). SIRT1 was reported to be involved in age related disorders like obesity, metabolic syndrome, type II diabetes mellitus and Parkinson’s disease. Activation of SIRT1 is one of the promising approaches to treat these age related diseases. In this study, we have used HipHop module of CATALYST to identify a series of pharmacophore models to screen SIRT1 enhancing molecules. Three molecules from Sirtris Pharmaceuticals were selected as training set and 607 sirtuin activator molecules were used as test set. Five different hypotheses were developed and then validated using the training set and the test set. The results showed that the best pharmacophore model has four features, ring aromatic, positive ionization and two hydrogen-bond acceptors. The best hypothesis from our study, Hypo2, screened high number of active molecules from the test set. Thus, we suggest that this four feature pharmacophore model could be helpful to screen novel SIRT1 activator molecules. Hypo2-virtual screening against Maybridge database reveals seven molecules, which contains all the critical features. Moreover, two new scaffolds were identified from this study. These scaffolds may be a potent lead for the SIRT1 activation.

Osteoporosis is influenced by genetic factors. The interindividual variability in the activity of CYP3A, the metabolic enzyme of sex hormones, may result from genetic polymorphisms. In a study of 2,178 women of ages 40-79 years, the presence of the CYP3A4*18 variant was found to be significantly associated with low bone mass. In vitro functional analyses indicate that CYP3A4*18 is a gain-of-function mutation in sex steroid metabolism, resulting in rapid oxidation of estrogens and testosterone; in vivo pharmacokinetics using midazolam (MDZ) verify the altered activity of the CYP3A4*18, showing lower metabolic turnover in the mutant than in the wild type. Molecular modeling reveals the structural changes in the substrate recognition sites of CYP3A4*18 that can cause changes in enzymatic activity and that potentially account for the difference between the catalytic activities of estrogen and MDZ, depending on the genotype. The results indicate that a genetic variation in the CYP3A4 gene--as a gain-of-function mutation in the metabolism of certain CYP3A substrates, including sex steroids--may predispose individuals to osteoporosis.

Cardiac myosin binding protein-C (cMyBP-C) is a multi-domain (C0-C10) protein that regulates heart muscle contraction through interaction with myosin, actin and other sarcomeric proteins. Several mutations of this protein cause familial hypertrophic cardiomyopathy (HCM). Domain C1 of cMyBP-C plays a central role in protein interactions with actin and myosin. Here, we studied structure-function relationship of three disease causing mutations, Arg177His, Ala216Thr and Glu258Lys of the domain C1 using computational biology techniques with its available X-ray crystal structure. The results suggest that each mutation could affect structural properties of the domain C1, and hence it's structural integrity through modifying intra-molecular arrangements in a distinct mode. The mutations also change surface charge distributions, which could impact the binding of C1 with other sarcomeric proteins thereby affecting contractile function. These structural consequences of the C1 mutants could be valuable to understand the molecular mechanisms for the disease.

Hypertrophic cardiomyopathy is the most common inherited heart diseases associated with a large number of mutations in several sarcomere proteins that regulate striated muscle contraction. The 3-dimensional structural and biophysical consequences of these mutations and their impact on the genotype-phenotype relationship remains poorly understood. Mutations in cardiac TroponinT (cTnT) have been reported to purport an increased risk of sudden cardiac deaths. Mutational hotspots such as codon 102 (Arg92 Moolman et al., 1997) of cTnT have been observed in several populations, including recently by our group in Egypt. In an attempt to explain this observation, we have performed molecular modelling of specific key mutations at codon 102, Arg102. The X-ray structure of the core domain of human cardiac troponin complex (cTnC, cTnI and cTnT) has been used for this study after constructing the missing regions. The resultant structure and the mutants were subjected to structural rigidity analysis and mutational modelling. In addition, molecular dynamics simulations were performed to understand the binding of functionally important calcium ions with the troponin complex. Our results suggest that the location of Arg102 in cTnT is instrumental to create a molecular bridge through hydrogen bonds (Figure) between secondary structural elements, in order to hold the core structure together. The loss of this molecular bridge by the key mutations (such as Arg102Leu) could produce conformational changes and affect natural binding of calcium ions, which are essential for regulation of muscle functions. This mechanism can explain the important structural consequences of mutations in this codon, associated with frequent progression of LV dysfunction, as well as the occurrence of ventricular arrhythmias secondary to the intracellular consequences of profound energetic and Ca++ handling derangement.

The cardiac muscle contraction is regulated by a set of proteins known as sarcomeric proteins, which are components of thick and thin filaments. Mutation in these proteins especially cardiac myosin binding protein-C, a multi-domain (C0-C10) protein, is one of the major causes of hypertrophic cardiomyopathy (HCM). However, structure-function relationship of this protein is unclear. Mutation E258K, which is located in the C-terminal of domain C1, has been shown to be associated with HCM in Egypt. Hence, the purpose of the study was to understand the molecular basis of this missense mutation. Molecular modelling study was performed using the available crystal structure of the domain C1. Here, we have carried out molecular dynamic simulations for both WT and E258K for 10 ns, and the structures that showed major changes were considered for further analyses. Our results suggest that the mutation can change the local structural stability through altering a series of intramolecular interactions. Moreover, as the mutation results in replacement of a negative amino acid by a positive one, thus, it affects the surface electrostatic properties of the domain (Figure). Hence, it might interfere with the binding to neighbouring domains and with the other sarcomeric proteins such as actin and myosin. E258K appears to affect the structural integrity of the domain C1 both directly and indirectly. It is hoped that these findings can help to understand the molecular mechanism of the disease.

A molecular structure is an essential source to identify ligand binding sites in orphan human cytochrome P450 4A22 (CYP4A22) that belongs to family 4, which is known to be involved in the regulation of blood pressure. Thus, a homology model has been constructed for CYP4A22 and refined by molecular dynamics simulation (MDS). Subsequently, molecular docking was performed with possible substrates, arachidonic acid (essential fatty acid, AA) and erythromycin (therapeutic drug, ERY). These complexes were also subjected to MDS, which helped in predicting the energetically favorable binding sites for these ligands. Putative substrate recognition sites (SRSs) of this protein provide highly hydrophobic binding pockets for the target ligands. A few key ligand binding residues identified in this study indicates that they could also play a major role in ligand-channeling (F122, L132 and C230). Furthermore, it appears that they might serve critical support for the catalytic reaction center (E321, F450, P449 and R455). Structural analysis of channels proposed that the conformational changes might have originated from the active site upon ligand binding and transferred to the rest of the protein via SRSs, which could thereby regulate the channels in CYP4A22. Most of our prediction results are supported by other research groups. In summary, the first molecular modeling study of CYP4A22 yields structural knowledge, which would be helpful to design structure-based-drugs and functional experiments for the target protein.

In Bacillus subtilis, CodW peptidase and CodX ATPase function together as a distinctive ATP-dependent protease called CodWX, which participates in protein degradation and regulates cell division. The molecular structure of CodX and the assembly structure of CodW-CodX have not yet been resolved. Here we present the first three-dimensional structure of CodX N-terminal (N) and C-terminal (C) domain including possible structure of intermediate (I) domain based on the crystal structure of homologous Escherichia coli HslU ATPase. Moreover, the biologically relevant CodWX (W(6)W(6)X(6)) octadecamer complex structure was constructed using the recently identified CodW-HslU hybrid crystal structure. Molecular dynamics (MD) simulation shows a reasonably stable structure of modeled CodWX and explicit behavior of key segments in CodX N and C domain: nucleotide binding residues, GYVG pore motif and CodW-CodX interface. Predicted structure of the possible I domain is flexible in nature with highly coiled hydrophobic region (M153-M206) that could favor substrate binding and entry. Electrostatic surface potential observation unveiled charge complementarity based CodW-CodX interaction pattern could be a possible native interaction pattern in the interface of CodWX. CodX GYVG pore motif structural features, flexible nature of glycine (G92 and G95) residues and aromatic ring conformation preserved Y93 indicated that it may follow the similar mode during the proteolysis mechanism as in the HslU closed state. This molecular modeling study uncovers the significance of CodX N and C domain in CodWX complex and provides possible explanations which would be helpful to understand the CodWX-dependent proteolysis mechanism of B. subtilis.