Insights on Rigidity and Flexibility at the Global and Local Levels of Protein Structures and Their Roles in Homologous Psychrophilic, Mesophilic, and Thermophilic Proteins: A Computational Study.

The rigidity and flexibility of homologous psychrophilic (P), mesophilic (M), and thermophilic (T) proteins have been investigated at the global and local levels in terms of "packing factors" and "atomic fluctuations" obtained from B-factors. For comparison of atomic fluctuations, correction of errors by considering errors in B-factors from all sources in a consolidated manner and conversion of the fluctuations to the same temperature have been suggested and validated. The results indicate no differences in the global values like the average packing factor among the three classes of protein homologues, but at local levels there are differences. A comparison of homologous protein triplets show that the average atomic fluctuations at a given temperature mainly obey the order P > M > T. Packing factors and the atomic fluctuations are anti-correlated, suggesting that altering the rigidity of the active site might be a potential strategy to make tailor-made psychrophilic or thermophilic proteins from their mesophilic homologues. The computer codes developed and used in this work are available at the link https://github.com/Munna-Sarkar/proteins-rigidity-flexibility.git.

Population genetic and biophysical evidences reveal that purifying selection shapes the genetic landscape of Plasmodium falciparum RH ligands in Chhattisgarh and West Bengal, India.

BACKGROUND: Reticulocyte binding protein-like homologs (RHs) are currently being evaluated as anti-erythrocytic stage vaccine targets against Plasmodium falciparum malaria. Present study explores the possible evolutionary drivers shaping the genetic organization of Pfrhs in Indian parasite population. It simultaneously evaluates a putative gain-of-function variant of PfRH5, a keystone member of PfRH family. METHODS: Receptor binding regions of Pfrh1, Pfrh2a/b, Pfrh4 and whole Pfrh5 were amplified using blood samples of P. falciparum malaria patients from Chhattisgarh and West Bengal and sequenced. Assembled sequences were analysed using MEGA7 and DnaSPv6. Binding affinities of recombinant PfRH5 proteins with basigin (BSG) were compared using in silico (CHARMM and AUTODOCK) and in vitro (Circular dichroism, fluorescence spectroscopy and isothermal titration calorimetry) methods. RESULTS: Pfrh1 (0.5), Pfrh2a/b (0.875), Pfrh4 (0.667) and Pfrh5 (0.778) sequence changes corresponded to low frequency (< 0.05) variants which resulted in an overall negative Tajima's D. Since mismatch distribution of none of the Pfrh loci corroborated with the model of demographic expansion, a possible role of natural selection formulating Pfrh sequence diversity was investigated. Among the 5 members, Pfrh5 displayed very high dN/dS (5.7) ratio. Nevertheless, the model of selective sweep due to presence of any advantageous substitutions could not be invoked as polymorphic nonsynonymous sites (17/18) for Pfrh5 exceeded significantly over the divergent (62/86) ones (p = 0.0436). The majority of extant PfRH5 sequences (52/83) differed from the reference Pf3D7 allele by a single amino acid mismatch (C203Y). This non-conservative alteration was predicted to lower the total interaction energy of that PfRH5variant with BSG, compared to PfRH53D7. Biophysical evidences validated the proposition that PfRH5variant formed a more stable complex with BSG. Thermodynamic association constant for interaction of BSG with PfRH5variant was also found to be higher (Kavariant = 3.63E6 ± 2.02E6 M-1 and Ka3D7 = 1.31E6 ± 1.21E6 M-1). CONCLUSIONS: Together, the study indicates that the genetic architecture of Pfrhs is principally shaped by purifying selection. The most abundant and ubiquitous PfRH5 variant harbouring 203Y, exhibits a greater affinity for BSG compared to PfRH53D7 possessing 203C allele. The study underscores the importance of selecting the functional allele that best represents circulating strains in natural parasite populations as vaccine targets.

Non-synonymous amino acid alterations in PfEBA-175 modulate the merozoite ligand's ability to interact with host's Glycophorin A receptor.

The pathological outcome of malaria due to Plasmodium falciparum infection depends largely on erythrocyte invasion by blood-stage merozoites which employ a cascade of interactions occurring between parasite ligands and RBC receptors. In a previous study exploring the genetic diversity of region-II of PfEBA-175, a ligand that plays a crucial part in parasite's RBC entry through Glycophorin A (GPA) receptor, we demonstrated that F2 domain of region-II underwent positive selection in Indian P. falciparum population through the accumulation of non-synonymous polymorphisms. Here, we examine the functional impact of two highly prevalent non-synonymous alterations in F2, namely Q584E & E592A, using a battery of molecular, biophysical and in-silico techniques. Application of circular dichroism, FTIR, fluorescence spectroscopy reveals that secondary and three-dimensional folding of recombinant-F2 protein carrying 584E and 592A residues (F2-Mut) differs significantly from that carrying 584Q and 592E (F2-3D7). A comparison of spectroscopic and thermodynamic parameters shows that F2-Mut is capable of forming a complex with GPA with higher efficiency compared to F2-3D7. In silico docking predicts both artemisinin and artesunate possess the capacity of slipping into the GPA binding crevices of PfEBA-175 and disrupt PfEBA-GPA association. However, the estimated affinity of artesunate towards PfEBA-175 with 584E and 592A residues is higher than that of artemisinin. Thermodynamic parameters computed using isotherms are concordant with this in-silico prediction. Together, our data suggest that the presence of amino acid alterations in F2 provide structural and functional stability favoring PfEBA-GPA interaction and artesunate can efficiently disrupt the interaction between GPA and PfEBA-175 even carrying altered amino acid residues. The present study alerts the malaria research community by presenting evidence that the parasite is gaining evolutionary fitness by cultivating genetic alterations in many of its proteins.

Porphyrins to restrict progression of pancreatic cancer by stabilizing KRAS G-quadruplex: In silico, in vitro and in vivo validation of anticancer strategy.

KRAS, a frequently mutated G-quadruplex forming proto-oncogene is responsible for almost every type of cancer which can form a parallel G-quadruplex structure in the promoter region. G-quadruplex structure is one of the most important drug targets for modern cancer therapy for their unique structure and specificity. Here, we have screened several synthetic porphyrin-based compounds as potential KRAS G-quadruplex stabilizing ligands, using molecular modeling and docking studies. Two novel porphyrins: Porphyrin-1(Cobalt containing) and Porphyrin-2 (Palladium containing) evidenced high affinity towards KRAS-promoter/G-quadruplex. As KRAS mutation is prevalent in pancreatic cancer, the efficacy of these ligands against human pancreatic ductal carcinoma cell line PANC-1 and MiaPaCa2 were examined. Both the Porphyrins exhibited significant cytotoxicity and block metastasis by inhibiting Epithelial to messenchymal transition. In vivo studies confirmed both porphyrin compounds to be effective against EAC tumors along with significantly low toxicity against normal Swiss albino mice. The expression of KRAS gene in porphyrin-treated PANC-1, MiaPaCa2 and tumor-derived EAC cells were drastically reduced at both protein and RNA levels. Thus interaction of porphyrin-based ligands with G-quadruplex DNA at the promoter region of KRAS, might be utilized as a target for anticancer therapeutic strategy.

Genetic structure of two erythrocyte binding antigens of Plasmodium falciparum reveals a contrasting pattern of selection.

Erythrocyte binding antigens 175 (EBA-175) and 140 (EBA-140) play key roles in erythrocyte invasion by binding to glycophorin A (GPA) and C (GPC) respectively in human malaria. Since antigenic variation in malaria endemic region is a major barrier to development of effective vaccine, we explore the nature and pattern of sequence diversity of these two vaccine candidates in Kolkata, India. Population genetic parameters based on parasite sequences representing region II of Pfeba-175 and Pfeba-140 genes were estimated using DnaSP V.5.10 and MEGA version 6.0. A novel molecular docking approach was implemented to assess the binding affinities of Kolkata Pfeba-175 variants with GPA. P. falciparum Kolkata isolates experienced a recent population expansion as documented by negative Tajima's D, Fu & Li's statistics, unimodal mismatch distribution and star-like median-joining network for both loci. Positive selection seemed to play a major role in shaping the diversity of Pfeba-175 (dN/dS=2.45, and McDonald-Kreitman P-value=0.04) with successive accumulation of Q584K/E, E592A and R664S deriving high frequency haplotypes designated here as F2KH3 and F2KH1. In silico molecular docking demonstrated that polypeptides encoded by F2KH1 and F2KH3 were capable of engaging the parasite ligand into energetically favorable interaction with GPA. Our data demonstrated emergence of Pfeba-175 sequences harboring selectively advantageous nonsynonymous substitutions on Pf3D7 sequence background in the Kolkata parasite isolates. A contrasting pattern of Pf3D7-centric expansion of parasite sequences was noted for Pfeba-140. Together, this study provides a firm genetic and biological support favoring a dominant role of EBA-175 in erythrocyte invasion.

An insight to the binding of ellagic acid with human serum albumin using spectroscopic and isothermal calorimetry studies.

Ellagic acid (EA), a natural polyphenol evidence several pharmacological benefits. The binding profile of EA with human serum albumin (HSA) has been explored and investigated by Isothermal titration calorimetry (ITC), circular dichroism (CD) spectroscopy, time-correlated single-photon counting (TCSPC), absorbance spectroscopy, steady-state fluorescence spectroscopy, and modelling studies. The ITC data analysis revealed the binding Constant (Ka), ΔH, ΔS and ΔG values to be 15.5×104M-1, -116.2±18.1 Kcal mol-1, -366 cal mol-1K-1 and -7.13 Kcal mol-1 respectively with a unique binding site at HSA. EA effectively quenched the intrinsic fluorescence of HSA by static quenching, whereas TCSPC data also revealed association of dynamic quenching also. Thermodynamic analysis confirmed that hydrophobic and mainly hydrogen bonding interaction played important role in stabilizing the HSA-EA complex. It further dictates the binding reaction to be enthalpy driven. The secondary structure of HSA was altered upon binding with EA. CD spectroscopic data indicated the fraction of alpha helicity to be decreased from 52% to 40% upon binding to EA. This study will provide an insight on evaluation of this bioactive interaction during transport and releasing efficiency at the target site in human physiological system since HSA is the most important carrier protein in blood serum.

Interaction of KRAS G-quadruplex with natural polyphenols: A spectroscopic analysis with molecular modeling.

Researchers are endeavoring to find out new therapeutics for curing cancer and G-quadruplex DNA has already been identified as a prospective one in this venture. Stabilizing G-quadruplex structures of telomere has emerged to be an important strategy in this context. Mutation in KRAS is mostly responsible for pancreatic, lung and colon cancer. In this present study we explored binding and conformational behaviour of G-quadruplex with different ligands by utilizing several biophysical techniques. Natural polyphenols like Curcumin and Ellagic acid were observed to bind with the G-quadruplex and enhance the melting temperature significantly indicating higher stability. UV-vis spectroscopy confirms formation of G quadruplex-ligand complex for both the compounds with specific binding affinity. Fluorimetric studies revealed that Ellagic acid had stronger binding affinity, 1.10×10(5)M(-1) compared to Curcumin, 1.6×10(4)M(-1) towards G-quadruplex. Interestingly, Curcumin provides greater stability by stacking on the top of the quadruplex structure with the help of the loops compared to Ellagic acid as is evident by docking studies. The keto form of curcumin showed stronger affinity than the enol form. We have developed a general model to estimate the influence of the ligands towards stabilizing the G-quadruplex subsequently characterizing the binding profile to enlighten prospective therapeutics.

An in silico method for designing thermostable variant of a dimeric mesophilic protein based on its 3D structure.

Designing proteins with enhanced thermostability has been a major interest of protein engineering because of its potential industrial applications. Here, we have presented a computational method for designing dimeric thermostable protein based on rational mutations on a mesophilic protein. Experimental and structural data indicate that the surface stability of a protein is a major factor controlling denaturation of a protein and ion-pairs are most efficient in enhancing the stability of the surfaces of the monomers and the interface between them. Our mutation based strategy is to first identify several polar or charged residues on the protein surface, interacting weakly with the rest of the protein and then replacing the side-chains of suitable neighboring residues to increase the interaction between these two residues. In stabilizing the interface, mutation is done in the interface for forming an ion pairs between the monomers. Application of this design strategy to a homo-dimeric protein and a hetero-dimeric protein as examples has produced excellent results. In both the cases the designed mutated proteins including the individual monomers and the interfaces were found to be considerably more stable than the respective mesophilic proteins as judged by self-energies and residue-wise interaction patterns. This method is easily applicable to any multi-meric proteins.

Do homologous thermophilic-mesophilic proteins exhibit similar structures and dynamics at optimal growth temperatures? A molecular dynamics simulation study.

Structure and dynamics both are known to be important for the activity of a protein. A fundamental question is whether a thermophilic protein and its mesophilic homologue exhibit similar dynamics at their respective optimal growth temperatures. We have addressed this question by performing molecular dynamics (MD) simulations of a natural mesophilic-thermophilic homologue pair at their respective optimal growth temperatures to compare their structural, dynamical, and solvent properties. The MD simulations were done in explicit aqueous solvent under periodic boundary and constant pressure and temperature (CPT) conditions and continued for 10.0 ns using the same protocol for the two proteins, excepting the temperatures. The trajectories were analyzed to compare the properties of the two proteins. Results indicated that the dynamical behaviors of the two proteins at the respective optimal growth temperatures were remarkably similar. For the common residues in the thermophilic protein, the rms fluctuations have a general trend to be slightly higher compared to that in the mesophilic counterpart. Lindemann parameter values indicated that only a few residues exhibited solid-like dynamics while the protein as a whole appeared as a molten globule in each case. Interestingly, the water-water interaction was found to be strikingly similar in spite of the difference in temperatures while, the protein-water interaction was significantly different in the two simulations.

Predicting hERG activities of compounds from their 3D structures: development and evaluation of a global descriptors based QSAR model.

A QSAR based predictive model of hERG activity in terms of 'global descriptors' has been developed and evaluated. The QSAR was developed by training 77 compounds covering a wide range of activities and was validated based on an external 'test set' of 80 compounds using neural network method. Statistical parameters and examination of enrichment factor indicated the effectiveness of the present model. Randomization test demonstrated the robustness of the model and cross-validation test further validated the QSAR. Domain of applicability test indicated to the high degree of reliability of the predicted results. Satisfactory performance in classifying compounds into 'active' and 'inactive' groups was also obtained. The cases where the QSAR failed, the possible sources of errors have been discussed.

Turning a mesophilic protein into a thermophilic one: a computational approach based on 3D structural features.

In spite of considerable improvement of our understanding of factors responsible for protein thermostability, rational designing of thermostable variants of mesophilic proteins is not yet fully established. The present paper describes an effective computational strategy that we have developed to identify the most suitable mutations converting a chosen mesophilic protein into a thermophilic one starting from its 3D structure. The approach is based on the concept that stabilization of several surface residues should enhance the global stability of the protein. The method relies on the estimation of electrostatic and van der Waals interactions in computing the interaction among the side chains of individual residues and the rest of the protein. The polar or charged residues whose side chains interact weakly with the rest of the original protein are identified first. Then, for each such identified residue (A), another residue (B) in its spatial vicinity is identified. The side chain of the residue (B) is then replaced by a suitable conformer of a residue that is electrostatically complementary to the residue (A) to enhance local interactions and hence the stability of the protein. The steric effect is taken care of through van der Waals interactions. We reject the mutations that improve interactions only locally along the sequence as it is unlikely to enhance the global stability of the 3D architecture. We use the difference in self-energies (DeltaEself) as a measure of the stability difference between the original and its mutant variant. This paper presents two test cases with demonstration of the enhanced stability of such mutated proteins and validates the strategy by considering five experimentally known thermophilic-mesophilic protein pairs.

A simple approach for indexing the oral druglikeness of a compound: discriminating druglike compounds from nondruglike ones.

A knowledge-based simple score has been developed for indexing the oral druglikeness of compounds based on the concept that oral druglikeness should be independent of the drug targets and, thus, are closely related to the global absorption, distribution, metabolism, and excretion related properties. We have considered several simple molecular descriptors as the key determinants of druglikeness. The patterns of the distributions of these molecular descriptors for a set of drug molecules have been extracted using a nonlinear neural network method. We assumed direct correlations of these patterns to the expectation values that a given compound may behave like a drug. On the basis of this assumption, we have defined a simple druglike index or score (DLS) combining the contributions coming from the descriptors considered. This index scales the druglikeness of a compound in the range 0.0-1.0, 1.0 being the highest druglikeness. The index applied for a drug data set, a mixed data set, and three different bioactive databases produced expected features and indicated that even the marketed drugs have druglike scores varying over a considerable range. A total of 73.3% of the drugs considered showed DLS > 0.5, while it is only 44.7% for the HIC-Up compounds (unbiased ligand database). For the ChemBank, Asinex-Gold collection, and NCI databases 61.2%, 76.0%, and 79.1% of the compounds have DLS > 0.5.

Exploring the potential of complex formation between a mutant DNA and the wild type protein counterpart: a MM and MD simulation approach.

We have demonstrated that the methods of molecular modeling and molecular dynamics simulation might be used to assess whether a specific mutation in the DNA would destabilize a known DNA-protein complex. The approach is based on probing the changes in the interaction that would be induced into the complex if within the already formed wild type complex the mutation could be introduced. We have used Hoxc8-DNA complex as a test system where it is known that the Hoxc8 binding affinity of the DNA is completely lost upon mutation of the DNA by replacing TAAT stretch to GCCG. Mutation was obtained by changing the relevant base pairs into the DNA of the model of the corresponding wild type complex developed by homology modeling and MD simulation in water for 2.0 ns. Comparison of the structure, dynamics and interactions between the hypothetical mutant model with those of the similarly refined wild type model shows that the loss of affinity of the mutant DNA to Hoxc8 has two different origins: (i) loss of several strong H-bonds as the direct consequences of mutation and (ii) reduced H-bonds in the common parts due to a net loss or inferior H-bonding geometry induced by the mutation as indirect effects. The net change in the interaction energy between the DNA and the protein in the best possible configuration indicated the experimentally observed destabilization effects. No significant change in the groove width was observed and no correlation was found between the water-bridges and the loss of affinity.

Homology modeling based solution structure of Hoxc8-DNA complex: role of context bases outside TAAT stretch.

The 3D structure of neither Hoxc8 nor Hoxc8-DNA complex is known. The repressor protein Hoxc8 binds to the TAAT stretch of the promoter of the osteopontin gene and modulates its expression. Over expression of the osteopontin gene is related to diseases like osteoporosis, multiple sclerosis, cancer et cetera. In this paper we have proposed a 3D structure of Hoxc8-DNA complex obtained by Homology modeling and molecular dynamics (MD) simulation in explicit water. The crystal structure (9ant.pdb) of Antennapedia homeodomain in complex with its DNA sequence was chosen as the template based on (i) high sequence identity (85% for the protein and 60% for the DNA) and (ii) the presence of the TAAT stretch in interaction with the protein. The resulting model was refined by MD simulation for 2.0ns in explicit water. This refined model was then characterized in terms of the structural and the interactional features to improve our understanding of the mechanism of Hoxc8-DNA recognition. The interaction pattern shows that the residues Ile(195), Gln(198), and Asn(199), and the bases S2-(4)TAATG(8) are most important for recognition suggesting the stretch TAATG as the "true recognition element" in the present case. A strong and long-lived water bridge connecting Gln(198) and the base of S1-C(7) complementary to S2-G(8) was observed. Our predicted model of Hoxc8-DNA complex provides us with features that are consistent with the available experimental data on Hoxc8 and the general features of other homeodomain-DNA complexes. The predictions based on the model are also amenable to experimental verification.

Statistical analysis of pair-wise compatibility of spatially nearest neighbor and adjacent residues in alpha-helix and beta-strands: application to a minimal model for secondary structure prediction.

Secondary structural elements like alpha-helix and beta-strands possess distinctly different structural features and thus the relative positioning of the nearest neighbor residues, and also the sequence-wise adjacent residues is important in determining the structural preference. In the present work we have statistically examined the pair-wise compatibility pattern of physically nearest neighbors and separately the adjacent residue pairs along the sequence in between the nearest neighbor partners in alpha-helices and beta-strands. It has been demonstrated that the patterns and hence, the physical basis of the compatibility of adjacent residue pairs and the spatially nearest neighbors are significantly different in most cases. The influence of tertiary contacts on the pair-wise compatibility is shown to be significant for beta-strands while it is small for alpha-helices. Based on the compatibility of physically nearest neighbors and the sequence-wise adjacent residue pairs, a minimal model has been constructed to predict the alpha-helices, beta-strands and coils of a protein from its sequence. Application of this method to 100 sequences shows that it has a predictive capability comparable to that of other more sophisticated statistical methods.

Some practical aspects of free energy calculations from molecular dynamics simulation.

In this work, we address two critical aspects of calculation of the free energy differences in molecular systems from molecular simulations. The first aspect involves checking whether the calculated free energy difference depends significantly on the extent of perturbation used for accomplishment of a given transformation. The second aspect of interest is to verify if the sampling errors in calculating the free energy differences between the wild-type molecule and a mutated one in its free state and in a complex are similar, or not, for a finite-length dynamic simulation. The reliability of the free energy estimates obtained from molecular simulations using thermodynamic cycles depends in part on this fact. For investigating these aspects, we use a self-transformation scheme in which a transformation of a part of a molecular system into itself is considered. We perform MD simulations of DNA fragments in which a part of a specific base is subjected to such a self-transformation. Results indicate that the estimated free energy differences do not depend significantly on the extent of perturbation used to achieve the transformation. Interestingly, the variation in the cumulative free energy difference, ΔA, with the coupling parameter, λ, depends significantly on the extent of perturbation. We examine the physical basis of the observed nature of the variation of the accumulated free energy difference, ΔA, against the λ value in the case of a self-transformation. In a thermodynamic cycle, the sampling errors due to the finite-length simulation for the molecular system are found to be similar to each other for the two perturbations (free and in a complex) justifying the use of such approach in calculating ΔΔA in molecular complexes. ©1999 John Wiley & Sons, Inc. J Comput Chem 20: 877-885, 1999.