Formation Of Hydrophobic Core Research Articles

Investigating natural plant products as potential inhibitors to disrupt NS1 β-roll domain polymerisation in DENV2: a detailed computational chemistry approach

ABSTRACT The aim of this investigation is to design a novel drug, screened through various computational methods, that harnesses the historical evidence of plant products inhibiting viral infections. The beta-roll domain of Flavivirus NS1, crucial for its stability, membrane interaction and viral RNA replication, underscores its significance in disease pathogenesis, being the sole non-structural protein detected in the sera of DENV-infected patients. Our goal is to develop optimised compounds that disrupt NS1 β-roll domain polymerisation, mitigating hydrophobic core formation, to effectively inhibit DENV2 cytopathic infection by leveraging natural interference with viral replication. We created 10 plant-based ligand libraries and applied scaffold hopping and bioisosteric replacement to improve them. Computational methodologies, including virtual screening, molecular dynamics simulation, contact matrix and dynamic cross correlation matrix, were employed for analysing the dynamics of the protein with the association of ligand. ADMET analysis of ligand 126 revealed a favourable ADME profile with no observed toxicity properties. Calculations of pKd, pKi and pIC50 values indicated a heightened affinity of the ligand for the receptor protein, suggesting enhanced inhibitory action and a reduced dissociation rate. These encouraging findings suggest a greater potency of the drug, warranting further in vitro testing for validation.

Amino acids and glycine derivatives differently affect refolding of mesophilic and thermophilic like α-amylases: implications in protein refolding and aggregation

α-amylases are industrially important enzymes which are used in different starch-based industries. They are adapted to different environmental conditions like extremes of temperature, pH and salinity. Herein, α-amylases from Bacillus amyloliquifaciens (BAA) and Bacillus licheniformis (BLA), representing mesophilic and thermophilic-like proteins, respectively, have been used to investigate the effect of naturally occurring osmolytes like arginine, proline, glycine and its methyl derivatives, sarcosine and betaine on their refolding. In this study, we have shown that among amino acids and glycine derivatives, betaine is the most promising osmolyte, while arginine and glycine exhibit moderately positive effect at their lower concentrations on the refolding of BAA only. Except betaine, all other osmolytes above 0.25 M showed inhibitory effect on the native enzyme activity of BLA and BAA. However, aggregation kinetics monitored by static light scattering indicates suppression of aggregation by all of these osmolytes. Further investigation by tryptophan and ANS fluorescence spectroscopy indicates the formation of compact hydrophobic core in the presence of the osmolytes. The morphology of protein aggregates having different sizes was visualized by atomic force microscopy ,and it was observed that amorphous aggregates of variable heights were formed. Our study highlights the importance of differential effects of arginine, proline, glycine, sarcosine and betaine on the native state as well as on refolding of BLA and BAA which may be helpful in devising strategies for developing effective protein formulation and prevention of aggregation of industrially and therapeutically important proteins. Communicated by Ramaswamy H. Sarma

Hydrophobic core formation and secondary structure elements in uranyl(VI)-binding peptides.

Cyclic peptides as well as a modified EF-hand motif of calmodulin have been newly designed to achieve high affinity towards uranyl(VI). Cyclic peptides may be engineered to bind uranyl(VI) to its backbone under acidic conditions, which may enhance its selectivity. For the modified EF-hand motif of calmodulin, strong electrostatic interactions between uranyl(VI) and negatively charged side chains play an important role in achieving high affinity; however, it is also essential to have a secondary structure element and formation of hydrophobic cores in the metal-bound state of the peptide.

Structural characterization of the thermal unfolding pathway of human VEGFR1 D2 domain.

Folding stability is a crucial feature of protein evolution and is essential for protein functions. Thus, the comprehension of protein folding mechanisms represents an important complement to protein structure and function, crucial to determine the structural basis of protein misfolding. In this context, thermal unfolding studies represent a useful tool to get a molecular description of the conformational transitions governing the folding/unfolding equilibrium of a given protein. Here, we report the thermal folding/unfolding pathway of VEGFR1D2, a member of the immunoglobulin superfamily by means of a high‐resolution thermodynamic approach that combines differential scanning calorimetry with atomic‐level unfolding monitored by NMR. We show how VEGFR1D2 folding is driven by an oxidatively induced disulfide pairing: the key event in the achievement of its functional structure is the formation of a small hydrophobic core that surrounds a disulfide bridge. Such a ‘folding nucleus’ induces the cooperative transition to the properly folded conformation supporting the hypothesis that a disulfide bond can act as a folding nucleus that eases the folding process.

Thermodynamics of hydrophobic-polar model proteins on the face-centered cubic lattice.

The HP model, a coarse-grained protein representation with only hydrophobic (H) and polar (P) amino acids, has already been extensively studied on the simple cubic (SC) lattice. However, this geometry severely restricts possible bond angles, and a simple improvement is to instead use the face-centered cubic (fcc) lattice. In this paper, the density of states and ground state energies are calculated for several benchmark HP sequences on the fcc lattice using the replica-exchange Wang-Landau algorithm and a powerful set of Monte Carlo trial moves. Results from the fcc lattice proteins are directly compared with those obtained from a previous lattice protein folding study with a similar methodology on the SC lattice. A thermodynamic analysis shows comparable folding behavior between the two lattice geometries, but with a greater rate of hydrophobic-core formation persisting into lower temperatures on the fcc lattice.

Synthesis and Fabrication of Gelatin-Based Elastomeric Hydrogels Through Cosolvent-Induced Polymer Restructuring

Hydrogels have a wide range of applications in tissue engineering, drug delivery, device fabrication for biological studies and stretchable electronics. For biomedical applications, natural polymeric hydrogels have general advantages such as biodegradability and non-toxic by products as well as biocompatibility. However, applications of nature derived hydrogels have been severely limited by their poor mechanical properties. For example, most of the protein derived hydrogels do not exhibit high stretchability like methacrylated gelatin hydrogel have ⁓11% failure strain when stretched [1]. Moreover, protein derived elastomeric hydrogels that are fabricated from low molecular weight synthetics peptides require laborious process of synthesis and purification. Biopolymers like gelatin, produced in bulk for pharma and food industry can provide an alternative for development of elastomeric hydrogels. Here, we report the synthesis of ureidopyrimidinone (Upy) functionalized gelatin and its fabrication into soft elastomeric hydrogels through supramolecular interactions that could exhibit high failure strain (318.73 ± 44.35%). The hydrogels were fabricated through a novel method involving co-solvent optimization and structural transformation with 70% water content. It is anticipated that the hydrogel fabrication method involved the formation of hydrophobic cores of ureidopyrimidinone groups inside hydrogel which introduced elastomeric properties to the resulting hydrogel.

Mechanism of Vitamin D Receptor Ligand-Binding Domain Regulation Studied by gREST Simulations.

The vitamin D receptor ligand-binding domain (VDR-LBD) undergoes conformational changes upon ligand binding. In this nuclear receptor family, agonistic or antagonistic activities are controlled by the conformation of the helix (H)12. However, all crystal structures of VDR-LBD reported to date correspond to the active H12 conformation, regardless of agonist/antagonist binding. To understand the mechanism of VDR-LBD regulation structurally, conformational samplings of agonist- and antagonist-bound rat VDR-LBD were performed using the generalized replica exchange with solute tempering (gREST) method. The gREST simulations demonstrated different structural responses of rat VDR-LBD to agonist or antagonist binding, whereas in conventional molecular dynamics simulations, the conformation was the same as that of the crystal structures, regardless of agonist/antagonist binding. In the gREST simulations, a spontaneous conformational change of H12 was observed only for the antagonist complex. The different responses to agonist/antagonist binding were attributed to hydrophobic core formation at the ligand-binding pocket and cooperative rearrangements of H11. The gREST method can be applied to the examination of structure-activity relationships for multiple VDR-LBD ligands.

Site-Specific Backbone and Side-Chain Contributions to Thermodynamic Stabilizing Forces of the WW Domain.

The native structure of a protein is stabilized by a number of interactions such as main-chain hydrogen bonds and side-chain hydrophobic contacts. However, it has been challenging to determine how these interactions contribute to protein stability at single amino acid resolution. Here, we quantified site-specific thermodynamic stability at the molecular level to extend our understanding of the stabilizing forces in protein folding. We derived the free energy components of individual amino acid residues separately for the folding of the human Pin WW domain based on simulated structures. A further decomposition of the thermodynamic properties into contributions from backbone and side-chain groups enabled us to identify the critical residues in the secondary structure and hydrophobic core formation, without introducing physical modifications to the system as in site-directed mutagenesis methods. By relating the structural and thermodynamic changes upon folding for each residue, we find that the simultaneous formation of the backbone hydrogen bonds and side-chain contacts cooperatively stabilizes the folded structure. The identification of stabilizing interactions in a folding protein at atomic resolution will provide molecular insights into understanding the origin of the protein structure and into engineering a more stable protein.

Genotype-Structure-Phenotype Correlations of Disease-Associated IGF1R Variants and Similarities to Those of INSR Variants.

We previously reported genotype-phenotype correlations in 12 missense variants causing severe insulin resistance, located in the second and third fibronectin type III (FnIII) domains of the insulin receptor (INSR), containing the α-β cleavage and part of insulin-binding sites. This study aimed to identify genotype-phenotype correlations in FnIII domain variants of IGF1R, a structurally related homolog of INSR, which may be associated with growth retardation, using the recently reported crystal structures of IGF1R. A structural bioinformatics analysis of five previously reported disease-associated heterozygous missense variants and a likely benign variant in the FnIII domains of IGF1R predicted that the disease-associated variants would severely impair the hydrophobic core formation and stability of the FnIII domains or affect the α-β cleavage site, while the likely benign variant would not affect the folding of the domains. A functional analysis of these variants in CHO cells showed impaired receptor processing and autophosphorylation in cells expressing the disease-associated variants but not in those expressing the wild-type form or the likely benign variant. These results demonstrated genotype-phenotype correlations in the FnIII domain variants of IGF1R, which are presumably consistent with those of INSR and would help in the early diagnosis of patients with disease-associated IGF1R variants.

Enzyme catalysis prior to aromatic residues: Reverse engineering of a dephospho-CoA kinase.

The wide variety of protein structures and functions results from the diverse properties of the 20 canonical amino acids. The generally accepted hypothesis is that early protein evolution was associated with enrichment of a primordial alphabet, thereby enabling increased protein catalytic efficiencies and functional diversification. Aromatic amino acids were likely among the last additions to genetic code. The main objective of this study was to test whether enzyme catalysis can occur without the aromatic residues (aromatics) by studying the structure and function of dephospho-CoA kinase (DPCK) following aromatic residue depletion. We designed two variants of a putative DPCK from Aquifex aeolicus by substituting (a) Tyr, Phe and Trp or (b) all aromatics (including His). Their structural characterization indicates that substituting the aromatics does not markedly alter their secondary structures but does significantly loosen their side chain packing and increase their sizes. Both variants still possess ATPase activity, although with 150-300 times lower efficiency in comparison with the wild-type phosphotransferase activity. The transfer of the phosphate group to the dephospho-CoA substrate becomes heavily uncoupled and only the His-containing variant is still able to perform the phosphotransferase reaction. These data support the hypothesis that proteins in the early stages of life could support catalytic activities, albeit with low efficiencies. An observed significant contraction upon ligand binding is likely important for appropriate organization of the active site. Formation of firm hydrophobic cores, which enable the assembly of stably structured active sites, is suggested to provide a selective advantage for adding the aromatic residues.

Dispersing Effect of Core Cross-linked Star Polycarboxylate Superplasticizers on the Cement Paste

Researches on topological modification of polycarboxylate superplasticizers to improve the water-reducing or slump-retainning properties have been a hot topic in rencent years. Herein, a core crosslinked star polycarboxylate superplasticizers (SPCE) were synthesized by crosslinking the comb polycarboxylate superplasticizers (CPCE) in aqueous solution. Crosslinkers and spacing monomers were added into the polymerization to promote the formation of hydrophobic core. A larger molecular weight with 75% of yields of the SPCE were observed by gel permeation chromatography (GPC). The dispersing effect of SPCE on the cement paste was then investigated by varying the dosage of crosslinkers and spacing monomers. It was found that the dispersing ability of SPCE became weakened compared to the CPCE. More of the crosslinkers led to smaller flow spread, as revealed by cement paste experiments. Introduction of the spacing monomers contributed to the formation of SPCE. However, the dispersing ability was also weakened. Best conversion efficiency and water-reducing ability was obtained when the molar ratio of crosslinker and spacing monomer to CPCE was set as 3:5:1.

Oligomers, fibrils and aggregates formed by alpha-synuclein: role of solution conditions

The classical Hofmeister series orders ions into kosmotropes and chaotropes, based on their interaction with the solvent, water. The role of protein is mostly ignored probably because most of the proteins studied are natively folded and broadly follow this classification pattern. Recent reports suggest that the interaction of ions is different with solvent molecules of proximal layer and bulk. Intrinsically disordered proteins (IDPs) differ from globular proteins in the fraction of polar vis-à-vis hydrophobic amino acids and the absence of distinct secondary and tertiary structures. The kosmotrope, ammonium sulphate, increases the compactness of the polypeptide conformation, with differing effects for globular proteins and IDPs. For globular proteins, lowered flexibility corresponds to a more stable native structure. Using oligomer-specific and aggregation-specific antibodies and comparing with fibrillation results, we show for alpha-synuclein, an IDP, ammonium sulphate-induced compaction results in the formation of the aggregation-prone hydrophobic core, which combines with other similar moieties to form the fibrillar ‘seed’. SEC-HPLC and SAXS analysis show the presence of the threshold oligomers. In the presence of the aggregation suppressor, arginine too, an oligomer is formed. This oligomer, however, is ‘dead’, and does not move further along the aggregation pathway. Thus, alpha-synuclein undergoes compaction in the presence of protein stabilisers, with differing consequences. In case of the chaotropes, KSCN and urea, aggregation of alpha-synuclein is partially inhibited. However, the amounts and types of aggregates formed are different in the two cases. Thus, the classical catalogue of molecules into protein stabilisers and destabilisers requires a relook for IDPs. Communicated by Ramaswamy H. Sarma

New diagnostic method for Alzheimer's disease based on the toxic conformation theory of amyloid β.

Recent investigations suggest that soluble oligomeric amyloid β (Aβ) species may be involved in early onset of Alzheimer's disease (AD). Using systematic proline replacement, solid-state NMR, and ESR, we identified a toxic turn at position 22 and 23 of Aβ42, the most potent neurotoxic Aβ species. Through radicalization, the toxic turn can induce formation of the C-terminal hydrophobic core to obtain putative Aβ42 dimers and trimers. Synthesized dimer and trimer models showed that the C-terminal hydrophobic core plays a critical role in the formation of high molecular weight oligomers with neurotoxicity. Accordingly, an anti-toxic turn antibody (24B3) that selectively recognizes a toxic dimer model of E22P-Aβ42 was developed. Sandwich enzyme-linked immunosorbent assay with 24B3 and 82E1 detected a significantly higher ratio of Aβ42 with a toxic turn to total Aβ42 in cerebrospinal fluid of AD patients compared with controls, suggesting that 24B3 could be useful for early onset of AD diagnosis.

Model of Early Stage Intermediate in Respect to Its Final Structure.

The model, describing a method of determining the structure of an early intermediate in the process of protein folding to analyze nonredundant PDB protein bases, allows determining the relationship between the sequence of tetrapeptides and their structural forms expressed by structural codes. The contingency table expressing such a relationship can be used to predict the structure of polypeptides by proposing a structural form with a precision limited to the structural code. However, by analyzing structural forms in native forms of proteins based on the fuzzy oil drop model, one can also determine the status of polypeptide chain fragments with respect to the assumptions of this model. Whether the probability distributions for both compliant and noncompliant forms were similar or whether the tetrapeptide sequences showed some differences at a level of a set of structural codes was investigated. The analysis presented here indicated that some sequences in both forms revealed differences in probability distributions expressed as a negative statistically significant correlation coefficient. This meant that the identified sections (tetrapeptides) took different forms against the fuzzy oil drop model. It may suggest that the information of the final status with respect to hydrophobic core formation is already carried by the structure of the early-stage intermediate.

Structure and dynamics of G protein-coupled receptor–bound ghrelin reveal the critical role of the octanoyl chain

Ghrelin plays a central role in controlling major biological processes. As for other G protein-coupled receptor (GPCR) peptide agonists, the structure and dynamics of ghrelin bound to its receptor remain obscure. Using a combination of solution-state NMR and molecular modeling, we demonstrate that binding to the growth hormone secretagogue receptor is accompanied by a conformational change in ghrelin that structures its central region, involving the formation of a well-defined hydrophobic core. By comparing its acylated and nonacylated forms, we conclude that the ghrelin octanoyl chain is essential to form the hydrophobic core and promote access of ghrelin to the receptor ligand-binding pocket. The combination of coarse-grained molecular dynamics studies and NMR should prove useful in improving our mechanistic understanding of the complex conformational space explored by a natural peptide agonist when binding to its GPCR. Such information should also facilitate the design of new ghrelin receptor-selective drugs.

Autonomous Sequences in Myoglobin Emerging from X-ray Structure of Holomyoglobin.

The proposed continuous folding structure units are fundamental to analyze protein structure. Here, we could elucidate for the first time two types of hydrophobic core networks in apomyoglobin using continuous folding structure units. In myoglobin, two autonomous sequences emerged clearly. We could thus characterize the autonomous sequences using well-defined hydrophobic core networks within respective semifolds. A hydrophobic core is defined as a pair of topology-local hydrophobic amino acids in different folding structures. Hydrophobic core formation is indispensable to stabilize the different folding structures via an efficient hydrophobic interaction. Autonomous sequences in myoglobin encode tertiary structure information for semifolds. These sequences fold autonomously into small sets of continuous folding structure units to grow separate semifolds on each separate framework. The autonomous sequence can be defined as the local sequence assigned to the small set of continuous folding structure units. They create the discrete hydrophobic region in a semifold by assembly of their hydrophobic regions. Semifolds were characterized by discrete hydrophobic regions stabilized by respective type I hydrophobic core networks, which were present within each semifold. The discrete hydrophobic region of a semifold propagated itself with that of a different semifold by hydrophobic interactions in type II hydrophobic core network, which was present between different semifolds, as observed by the X-ray structures of semifolds. The most significant feature of semifolds in apomyoglobin was that they could be verified by the X-ray structure of holomyoglobin regardless of the instability of folds characteristic to autonomous sequence fragments. This work presents the first description of autonomous sequences.

The Formation of Hydrophobic Core Regulates the Protein Folding of Villin Elucidated with Parallel Cascade Selection Molecular Dynamics

Villin is a fast-folding protein and has a unique hydrophobic core at the center in its folded form. In the present study, the folding process of Villin was investigated with parallel cascade selec...

Exploring the flap dynamics of the South African HIV subtype C protease in presence of FDA-approved inhibitors: MD study.

HIV-1 protease (HIV PR) is considered as one of the most attractive targets for the treatment of HIV and the impact of flap dynamics of HIV PR on the binding affinities of protease inhibitors (PIs) is a crucial ongoing research field. Recently, our research group evaluated the binding affinities of different FDA approved PIs against the South African HIV-1 subtype C (C-SA) protease (PR). The CSA-HIV PR displayed weaker binding affinity for most of the clinical PIs compared to HIV-1 B subtype for West and Central Europe, the Americas. In the current work, the flap dynamics of four different systems of HIV-1 C-SA PR complexed to FDA approved second generation PIs and its impact on binding was explored over the molecular dynamic trajectories. It was observed that the interactions of the selected drugs with the binding site residues of the protease may not be the major contributor for affinity towards PIs. Various post-MD analyses were performed, also entropic contributions, solvation free energies and hydrophobic core formation interactions were studied to assess how the flap dynamics of C-SA PR which is affected by such factors. From these contributions, large van der Waals interactions and low solvation free energies were found to be major factors for the higher activity of ATV against C-SA HIV PR. Furthermore, a comparatively stable hydrophobic core may be responsible for higher stability of the PR flaps of the ATV complex. The outcome of this study provides significant guidance to how the flap dynamics of C-SA PR is affected by various factors as a result of the binding affinity of various protease inhibitors. It will also assist with the design of potent inhibitors against C-SA HIV PR that apart from binding in the active site of PR can interacts with the flaps to prevent opening of the flaps resulting in inactivation of the protease.

Genotype–Phenotype Correlations and Structural Basis of INSR and IGF1R Mutations Causing Severe Insulin/IGF-1 Resistance

Mutations of the insulin receptor (INSR) gene cause severe insulin resistance, resulting in several syndromes with various abnormal phenotypes associated with variable severity of INSR dysfunction. As a member of the insulin receptor family, the IGF-I receptor (IGF1R) shows the same domain structure as INSR containing the fibronectin type III (FnIII) domains where there are an α-β cleavage site and a part of the ligand binding site. Mutations of IGF1R can cause intrauterine and postnatal growth retardation. We compiled 3 mutations of the FnIII-2 and FnIII-3 domains in IGF1R registered in HGMD, ClinVar and UniProt databases as causing growth retardation as well as the SNP with >1% allele frequency in IGF1R from the 1000 Genomes Project data, ESP6500, HGVD and 3.5KJPN, a reference panel of Japanese individuals. We conducted in silico structural analysis of the mutations of the FnIII domains in IGF1R which revealed that the pathogenic mutations were predicted to severely impair stability of the FnIII domains and hydrophobic core formation or affect processing site, while the SNP was predicted not to affect folding of the domains. In addition, IGF-1 proreceptor processing was impaired in cells expressing mutations of FnIII compared to cells expressing WT form. Previously, we performed structural analysis of mutations of the FnIII domains in INSR, which revealed that missense mutations causing the most severe Donohue syndrome were predicted to severely impair hydrophobic core formation and stability of the FnIII domains, while those causing less severe Rabson-Mendenhall syndrome were predicted to lead to localized destabilization and not to affect folding of the FnIII domains. Genotype-structure-phenotype correlations of variants of the FnIII domains in INSR and IGF1R identified in these analyses will enable early diagnosis of pathogenic mutations in INSR and IGF1R and provide a valuable insight into the molecular mechanism of severe insulin or IGF-1 resistance. Disclosure J. Hosoe: None. H. Kadowaki: None. F. Miya: None. M. Takakura: None. T. Tsunoda: None. N. Shojima: None. T. Yamauchi: Research Support; Self; Novo Nordisk Inc., Daiichi Sankyo Company, Limited, Sanofi, Takeda, Tanabe-Mitsubishi. T. Kadowaki: Consultant; Self; Novo Nordisk A/S, AstraZeneca, Merck Sharp & Dohme Corp.. Research Support; Self; Kissei Pharmaceutical Co., Ltd., Taisho Pharmaceutical Co., Ltd., Sanofi, Kyowa Hakko Kirin Co., Ltd., Novo Nordisk A/S, Astellas Pharma, Daiichi Sankyo Company, Limited, Takeda, Mitsubishi Tanabe Pharma Corporation, Ono Pharmaceutical Co., Ltd., Merck Sharp & Dohme Corp., Nippon Boehringer Ingelheim Co. Ltd.. Speaker's Bureau; Self; Astellas Pharma, AstraZeneca, Merck Sharp & Dohme Corp., Ono Pharmaceutical Co., Ltd., Takeda, Eli Lilly and Company, Nippon Boehringer Ingelheim Co. Ltd., Novo Nordisk A/S.

Efficient UV repair requires disengagement of the CSB winged helix domain from the CSB ATPase domain

The ATP-dependent chromatin remodeler CSB is implicated in a variety of different DNA repair mechanisms, including transcription-coupled nucleotide excision repair (TC-NER), base excision repair and DNA double strand break (DSB) repair. However, how CSB is regulated in these various repair processes is not well understood. Here we report that the first 30 amino acids of CSB along with two phosphorylation events on S10 and S158, previously reported to be required for CSB function in homologous recombination (HR)-mediated repair, are dispensable for repairing UV-induced DNA damage, suggesting that the regulation of CSB in these two types of repair are carried out by distinct mechanisms. In addition, we show that although the central ATPase domain of CSB is engaged in interactions with both the N- and C-terminal regions, these interactions are disrupted following UV-induced DNA damage. The UV-induced disengagement of the C-terminal region of CSB from the ATPase domain requires two conserved amino acids W1486 and L1488, which are thought to contribute to the hydrophobic core formation of the winged helix domain (WHD) at its C-terminus. Failure to undergo UV-induced dissociation of the C-terminal region of CSB from the ATPase domain is associated with impairment in its UV-induced chromatin association, its UV-induced post-translational modification as well as cell survival. Collectively, these findings suggest that UV-induced dissociation of CSB domain interactions is a necessary step in repairing UV-induced DNA damage and that the WHD of CSB plays a key role in this dissociation.