Molecular Dynamic Simulations Reveal the Structural Determinants of Fatty Acid Binding to Oxy-Myoglobin

The mechanism(s) by which fatty acids are sequestered and transported in muscle have not been fully elucidated. A potential key player in this process is the protein myoglobin (Mb). Indeed, there is a catalogue of empirical evidence supporting direct interaction of globins with fatty acid metabolites; however, the binding pocket and regulation of the interaction remains to be established. In this study, we employed a computational strategy to elucidate the structural determinants of fatty acids (palmitic & oleic acid) binding to Mb. Sequence analysis and docking simulations with a horse (Equus caballus) structural Mb reference reveals a fatty acid-binding site in the hydrophobic cleft near the heme region in Mb. Both palmitic acid and oleic acid attain a “U” shaped structure similar to their conformation in pockets of other fatty acid-binding proteins. Specifically, we found that the carboxyl head group of palmitic acid coordinates with the amino group of Lys45, whereas the carboxyl group of oleic acid coordinates with both the amino groups of Lys45 and Lys63. The alkyl tails of both fatty acids are supported by surrounding hydrophobic residues Leu29, Leu32, Phe33, Phe43, Phe46, Val67, Val68 and Ile107. In the saturated palmitic acid, the hydrophobic tail moves freely and occasionally penetrates deeper inside the hydrophobic cleft, making additional contacts with Val28, Leu69, Leu72 and Ile111. Our simulations reveal a dynamic and stable binding pocket in which the oxygen molecule and heme group in Mb are required for additional hydrophobic interactions. Taken together, these findings support a mechanism in which Mb acts as a muscle transporter for fatty acid when it is in the oxygenated state and releases fatty acid when Mb converts to deoxygenated state.     

Assessing Structural Determinants of Zn2+ Binding to Human HV1 via Multiple MD Simulations

Voltage-gated proton channels (H_V1) are essential for various physiological tasks but are strongly inhibited by Zn²⁺ cations. Some determinants of Zn²⁺ binding have been elucidated experimentally and in computational studies. However, the results have always been interpreted under the assumption that Zn²⁺ binds to monomeric H_V1 despite evidence that H_V1 expresses as a dimer and that the dimer has a higher affinity for zinc than the monomer and experimental data that suggest coordination in the dimer interface. The results of former studies are also controversial, e.g., supporting either one single or two binding sites. Some structural determinants of the binding are still elusive. We performed a series of molecular dynamics simulations to address different structures of the human proton channel, the monomer and two plausible dimer conformations, to compare their respective potential to interact with and bind Zn²⁺ via the essential histidines. The series consisted of several copies of the system to generate independent trajectories and increase the significance compared to a single simulation. The amount of time simulated totals 29.9 μs for 126 simulations of systems comprising ∼59,000 to ∼187,000 atoms. Our approach confirms the existence of two binding sites in monomeric and dimeric human H_V1. The dimer interface is more efficient for attracting and binding Zn²⁺ via the essential histidines than the monomer or a dimer with the histidines in the periphery. The higher affinity is due to the residues in the dimer interface that create an attractive electrostatic potential funneling the zinc cations toward the binding sites.     

Atomistic Simulations of Pore Formation and Closure in Lipid Bilayers

Cellular membranes separate distinct aqueous compartments, but can be breached by transient hydrophilic pores. A large energetic cost prevents pore formation, which is largely dependent on the composition and structure of the lipid bilayer. The softness of bilayers and the disordered structure of pores make their characterization difficult. We use molecular-dynamics simulations with atomistic detail to study the thermodynamics, kinetics, and mechanism of pore formation and closure in DLPC, DMPC, and DPPC bilayers, with pore formation free energies of 17, 45, and 78 kJ/mol, respectively. By using atomistic computer simulations, we are able to determine not only the free energy for pore formation, but also the enthalpy and entropy, which yields what is believed to be significant new insights in the molecular driving forces behind membrane defects. The free energy cost for pore formation is due to a large unfavorable entropic contribution and a favorable change in enthalpy. Changes in hydrogen bonding patterns occur, with increased lipid-water interactions, and fewer water-water hydrogen bonds, but the total number of overall hydrogen bonds is constant. Equilibrium pore formation is directly observed in the thin DLPC lipid bilayer. Multiple long timescale simulations of pore closure are used to predict pore lifetimes. Our results are important for biological applications, including the activity of antimicrobial peptides and a better understanding of membrane protein folding, and improve our understanding of the fundamental physicochemical nature of membranes.     

Atomistic Simulations of Pore Formation and Closure in Lipid Bilayers

Cellular membranes separate distinct aqueous compartments, but can be breached by transient hydrophilic pores. A large energetic cost prevents pore formation, which is largely dependent on the composition and structure of the lipid bilayer. The softness of bilayers and the disordered structure of pores make their characterization difficult. We use molecular-dynamics simulations with atomistic detail to study the thermodynamics, kinetics, and mechanism of pore formation and closure in DLPC, DMPC, and DPPC bilayers, with pore formation free energies of 17, 45, and 78 kJ/mol, respectively. By using atomistic computer simulations, we are able to determine not only the free energy for pore formation, but also the enthalpy and entropy, which yields what is believed to be significant new insights in the molecular driving forces behind membrane defects. The free energy cost for pore formation is due to a large unfavorable entropic contribution and a favorable change in enthalpy. Changes in hydrogen bonding patterns occur, with increased lipid-water interactions, and fewer water-water hydrogen bonds, but the total number of overall hydrogen bonds is constant. Equilibrium pore formation is directly observed in the thin DLPC lipid bilayer. Multiple long timescale simulations of pore closure are used to predict pore lifetimes. Our results are important for biological applications, including the activity of antimicrobial peptides and a better understanding of membrane protein folding, and improve our understanding of the fundamental physicochemical nature of membranes.     

Understanding Nucleotide-Regulated FtsZ Filament Dynamics and the Monomer Assembly Switch with Large-Scale Atomistic Simulations

Bacterial cytoskeletal protein FtsZ assembles in a head-to-tail manner, forming dynamic filaments that are essential for cell division. Here, we study their dynamics using unbiased atomistic molecular simulations from representative filament crystal structures. In agreement with experimental data, we find different filament curvatures that are supported by a nucleotide-regulated hinge motion between consecutive FtsZ monomers. Whereas GTP-FtsZ filaments bend and twist in a preferred orientation, thereby burying the nucleotide, the differently curved GDP-FtsZ filaments exhibit a heterogeneous distribution of open and closed interfaces between monomers. We identify a coordinated Mg²⁺ ion as the key structural element in closing the nucleotide site and stabilizing GTP filaments, whereas the loss of the contacts with loop T7 from the next monomer in GDP filaments leads to open interfaces that are more prone to depolymerization. We monitored the FtsZ monomer assembly switch, which involves opening/closing of the cleft between the C-terminal domain and the H7 helix, and observed the relaxation of isolated and filament minus-end monomers into the closed-cleft inactive conformation. This result validates the proposed switch between the low-affinity monomeric closed-cleft conformation and the active open-cleft FtsZ conformation within filaments. Finally, we observed how the antibiotic PC190723 suppresses the disassembly switch and allosterically induces closure of the intermonomer interfaces, thus stabilizing the filament. Our studies provide detailed structural and dynamic insights into modulation of both the intrinsic curvature of the FtsZ filaments and the molecular switch coupled to the high-affinity end-wise association of FtsZ monomers.     

Understanding Nucleotide-Regulated FtsZ Filament Dynamics and the Monomer Assembly Switch with Large-Scale Atomistic Simulations

Bacterial cytoskeletal protein FtsZ assembles in a head-to-tail manner, forming dynamic filaments that are essential for cell division. Here, we study their dynamics using unbiased atomistic molecular simulations from representative filament crystal structures. In agreement with experimental data, we find different filament curvatures that are supported by a nucleotide-regulated hinge motion between consecutive FtsZ monomers. Whereas GTP-FtsZ filaments bend and twist in a preferred orientation, thereby burying the nucleotide, the differently curved GDP-FtsZ filaments exhibit a heterogeneous distribution of open and closed interfaces between monomers. We identify a coordinated Mg²⁺ ion as the key structural element in closing the nucleotide site and stabilizing GTP filaments, whereas the loss of the contacts with loop T7 from the next monomer in GDP filaments leads to open interfaces that are more prone to depolymerization. We monitored the FtsZ monomer assembly switch, which involves opening/closing of the cleft between the C-terminal domain and the H7 helix, and observed the relaxation of isolated and filament minus-end monomers into the closed-cleft inactive conformation. This result validates the proposed switch between the low-affinity monomeric closed-cleft conformation and the active open-cleft FtsZ conformation within filaments. Finally, we observed how the antibiotic PC190723 suppresses the disassembly switch and allosterically induces closure of the intermonomer interfaces, thus stabilizing the filament. Our studies provide detailed structural and dynamic insights into modulation of both the intrinsic curvature of the FtsZ filaments and the molecular switch coupled to the high-affinity end-wise association of FtsZ monomers.     

Molecular Dynamics Simulations and Classical Multidimensional Scaling Unveil New Metastable States in the Conformational Landscape of CDK2

Protein kinases are key regulatory nodes in cellular networks and their function has been shown to be intimately coupled with their structural flexibility. However, understanding the key structural mechanisms of large conformational transitions remains a difficult task. CDK2 is a crucial regulator of cell cycle. Its activity is finely tuned by Cyclin E/A and the catalytic segment phosphorylation, whereas its deregulation occurs in many types of cancer. ATP competitive inhibitors have failed to be approved for clinical use due to toxicity issues raised by a lack of selectivity. However, in the last few years type III allosteric inhibitors have emerged as an alternative strategy to selectively modulate CDK2 activity. In this study we have investigated the conformational variability of CDK2. A low dimensional conformational landscape of CDK2 was modeled using classical multidimensional scaling on a set of 255 crystal structures. Microsecond-scale plain and accelerated MD simulations were used to populate this landscape by using an out-of-sample extension of multidimensional scaling. CDK2 was simulated in the apo-form and in complex with the allosteric inhibitor 8-anilino-1-napthalenesulfonic acid (ANS). The apo-CDK2 landscape analysis showed a conformational equilibrium between an Src-like inactive conformation and an active-like form. These two states are separated by different metastable states that share hybrid structural features with both forms of the kinase. In contrast, the CDK2/ANS complex landscape is compatible with a conformational selection picture where the binding of ANS in proximity of the αC helix causes a population shift toward the inactive conformation. Interestingly, the new metastable states could enlarge the pool of candidate structures for the development of selective allosteric CDK2 inhibitors. The method here presented should not be limited to the CDK2 case but could be used to systematically unmask similar mechanisms throughout the human kinome.     

Temperature-Sensitive RB Mutations Linked to Incomplete Penetrance of Familial Retinoblastoma in 12 Families

The tumor-suppressor activity of the retinoblastoma protein (RB) is encoded within a protein-binding ("pocket") domain that is targeted for mutations in all cases of familial retinoblastoma and in many common adult cancers. Although familial retinoblastoma is a paradigm for a highly penetrant, recessive model of tumorigenesis, the molecular basis for the phenotype of incomplete penetrance of familial retinoblastoma is undefined. We studied the RB pocket-binding properties of three independent, mutant RB alleles that are present in the germline of 12 kindreds with the phenotype of incomplete penetrance of familial retinoblastoma. Each arises from alterations of single codons within the RB pocket domain (designated "delta 480," "661W," or "712R"). Under the same conditions, we studied the properties of wild-type (WT) RB, an RB point mutant isolated from a lung carcinoma sample (706F) and an adjacent, in vitro-generated point mutant (707W). The delta 480, 661W, and 712R mutants lack pocket protein-binding activity in vitro but retain the WT ability to undergo cyclin-mediated phosphorylation in vivo. Each of the low-penetrant RB mutants exhibits marked enhancement of pocket protein binding when the cells are grown at reduced temperature. In contrast, in this temperature range, no change in binding activity is seen with WT RB, the 706F mutant, or the 707W mutant. We have demonstrated that many families with incomplete penetrance of familial retinoblastoma carry unstable, mutant RB alleles with temperature-sensitive pocket protein-binding activity. The variable frequency for tumor development in these families may result from reversible fluctuations in a threshold level of RB pocket-binding activity.     

OM Mutations in DROSOPHILA ANANASSAE Are Linked to Insertions of a Transposable Element

It has been hypothesized that Om mutability in Drosophila ananassae (involving spontaneous mutation at 20 loci, resulting in semidominant, nonpleiotropic eye morphology defects) was due to insertion of a transposable element, tom. One particularly unstable X-linked Om allele produced several derivatives, one of which has a more extreme Om phenotype and was accompanied by a singed bristle mutant, sn ^9g. DNA probes from the sn locus of D. melanogaster were used to clone the homologous region of D. ananassae. Analysis of sn^9g DNA detected a 6.5-kb insert. Genomic Southern blotting and in situ hybridization techniques showed that this insert is repetitive and dispersed. The existence of the tom element is supported by genetic mapping that established homology between the 6.5-kb sn^9g insert and Om mutants at the four X-linked loci tested.     

Dual RNA-Seq of Human Leprosy Lesions Identifies Bacterial Determinants Linked to Host Immune Response

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Quantitative Fundus Autofluorescence for the Evaluation of Retinal Diseases

The retinal pigment epithelium (RPE) is juxtaposed to the overlying sensory retina, and supports the function of the visual system. Among the tasks performed by the RPE are phagocytosis and processing of outer photoreceptor segments through lysosome-derived organelles. These degradation products, stored and referred to as lipofuscin granules, are composed partially of bisretinoids, which have broad fluorescence absorption and emission spectra that can be detected clinically as fundus autofluorescence with confocal scanning laser ophthalmoscopy (cSLO). Lipofuscin accumulation is associated with increasing age, but is also found in various patterns in both acquired and inherited degenerative diseases of the retina. Thus, studying its pattern of accumulation and correlating such patterns with changes in the overlying sensory retina are essential to understanding the pathophysiology and progression of retinal disease. Here, we describe a technique employed by our lab and others that uses cSLO in order to quantify the level of RPE lipofuscin in both healthy and diseased eyes.     

Integrating NMR,SAXS, and Atomistic Simulations: Structure and Dynamics of a Two-Domain Protein

Multidomain proteins with two or more independently folded functional domains are prevalent in nature. Whereas most multidomain proteins are linked linearly in sequence, roughly one-tenth possess domain insertions where a guest domain is implanted into a loop of a host domain, such that the two domains are connected by a pair of interdomain linkers. Here, we characterized the influence of the interdomain linkers on the structure and dynamics of a domain-insertion protein in which the guest LysM domain is inserted into a central loop of the host CVNH domain. Expanding upon our previous crystallographic and NMR studies, we applied SAXS in combination with NMR paramagnetic relaxation enhancement to construct a structural model of the overall two-domain system. Although the two domains have no fixed relative orientation, certain orientations were found to be preferred over others. We also assessed the accuracies of molecular mechanics force fields in modeling the structure and dynamics of tethered multidomain proteins by integrating our experimental results with microsecond-scale atomistic molecular dynamics simulations. In particular, our evaluation of two different combinations of the latest force fields and water models revealed that both combinations accurately reproduce certain structural and dynamical properties, but are inaccurate for others. Overall, our study illustrates the value of integrating experimental NMR and SAXS studies with long timescale atomistic simulations for characterizing structural ensembles of flexibly linked multidomain systems.     

Probing the Mechanism of pH-Induced Large-Scale Conformational Changes in Dengue Virus Envelope Protein Using Atomistic Simulations

One of the key steps in the infection of the cell by dengue virus is a pH-induced conformational change of the viral envelope proteins. These envelope proteins undergo a rearrangement from a dimer to a trimer, with large conformational changes in the monomeric unit. In this article, metadynamics simulations were used to enable us to understand the mechanism of these large-scale changes in the monomer. By using all-atom, explicit solvent simulations of the monomers, the stability of the protein structure is studied under low and high pH conditions. Free energy profiles obtained along appropriate collective coordinates demonstrate that pH affects the domain interface in both the conformations of E monomer, stabilizing one and destabilizing the other. These simulations suggest a mechanism with an intermediate detached state between the two monomeric structures. Using further analysis, we comment on the key residue interactions responsible for the instability and the pH-sensing role of a histidine that could not otherwise be studied experimentally. The insights gained from this study and methodology can be extended for studying similar mechanisms in the E proteins of the other members of class II flavivirus family.     

Structural Determinants of RGS-RhoGEF Signaling Critical to Entamoeba histolytica Pathogenesis

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Highlights? E. histolytica expresses a convergently evolved RGS-RhoGEF protein ? EhRGS-RhoGEF possesses a conventional nine-helix RGS domain ? Activated EhRGS-RhoGEF induces S2 cell spreading through Rac1/2 ? EhRGS-RhoGEF modulates amoebic migration and host cell killing     

Atomistic Simulations of Phosphatidylcholines and Cholesteryl Esters in High-Density Lipoprotein-Sized Lipid Droplet and Trilayer: Clues to Cholesteryl Ester Transport and Storage

Cholesteryl esters (CEs) are the water-insoluble transport and storage form of cholesterol. For both transport and storage, phospholipids and proteins embrace the CEs to form an amphipathic monolayer that surrounds the CEs. CEs are transported extracellularly in lipoproteins and are stored intracellularly as cytoplasmic lipid droplets. To clarify the molecular phenomena related to the above structures, we conducted atomistic molecular-dynamics simulations for a spherical, approximately high density lipoprotein sized lipid droplet comprised of palmitoyl-oleoyl-phosphatidylcholine (POPC) and cholesteryl oleate (CO) molecules. An additional simulation was conducted for a lamellar lipid trilayer consisting of the same lipid constituents. The density profiles showed that COs were located in the core of the spherical droplet. In trilayer simulations, CO molecules were also in the core and formed two denser strata. This is remarkable because the intra- and intermolecular behaviors of the COs were similar to previous findings from bulk COs in the fluid phase. In accordance with previous experimental studies, the solubility of COs in the POPC monolayers was found to be low. The orientation distribution of the sterol moiety with respect to the normal of the system was found to be broad, with mainly isotropic or slightly parallel orientations observed deep in the core of the lipid droplet or the trilayer, respectively. In both systems, the orientation of the sterol moiety changed to perpendicular with respect to the normal close to the phopsholipid monolayers. Of interest, within the POPC monolayers, the intramolecular conformation of the COs varied from the previously proposed horseshoe-like conformation to a more extended one. From a metabolic point of view, the observed solubilization of CEs into the phospholipid monolayers, and the conformation of CEs in the phospholipid monolayers are likely to be important regulatory factors of CE transport and hydrolysis.     

Human Papillomavirus Genotype Distribution in Czech Women and Men with Diseases Etiologically Linked to HPV

Background

The HPV prevalence and genotype distribution are important for the estimation of the impact of HPV-based cervical cancer screening and HPV vaccination on the incidence of diseases etiologically linked to HPVs. The HPV genotype distribution varies across different geographical regions. Therefore, we investigated the type-specific HPV prevalence in Czech women and men with anogenital diseases.

Methods

We analyzed 157 squamous cell carcinoma samples, 695 precancerous lesion samples and 64 cervical, vulvar and anal condylomata acuminate samples. HPV detection and typing were performed by PCR with GP5+/6+ primers, reverse line blot assay and sequencing.

Results

Thirty different HPV genotypes were detected in our study, HPV 16 being the most prevalent type both in precancerous lesions (45%) and squamous cell carcinomas (59%). In benign lesions, HPV 6 (72%) was the most common type. Altogether, 61% of carcinoma samples and 43% of precancerous lesion samples contained HPV 16 and/or 18. The presence of HPV types related to the vaccinal ones (HPV 31, 45, 33, 52, 58) were detected in 16% of carcinoma samples and 18% of precancerous lesion samples. HPV 16 and/or 18 were present in 76% of cervical cancer samples, 33% of CIN1, 43% CIN2 and 71% of CIN3 samples. HPV types 6 and/or 11 were detected in 84% samples of condylomata acuminate samples.

Conclusions

The prevalence of vaccinal and related HPV types in patients with HPV-associated diseases in the Czech Republic is very high. We may assume that the implementation of routine vaccination against HPV would greatly reduce the burden of HPV-associated diseases in the Czech Republic.     

HITS-CLIP and Integrative Modeling Define the Rbfox Splicing-Regulatory Network Linked to Brain Development and Autism

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