Structural basis of allele variation of human thiopurine-S-methyltransferase

Human thiopurine S-methyltransferase (TPMT) exhibits considerable person-to-person variation in activity to thiopurine drugs. We have produced an N-terminal truncation of human TPMT protein, crystallized the protein in complex with the methyl donor product S-adenosyl-L-homocysteine, and determined the atomic structure to the resolution of 1.58 and 1.89 A, respectively, for the seleno-methionine incorporated and wild type proteins. The structure of TPMT indicates that the naturally occurring amino acid polymorphisms scatter throughout the structure, and that the amino acids whose alteration have the most influence on function are those that form intra-molecular stabilizing interactions (mainly van der Waals contacts). Furthermore, we have produced four TPMT mutant proteins containing variant alleles of TPMT*2, *3A, *3B, and *3C and examined the structure-function relationship of the mutant proteins based on their expression and solubility in bacteria and their thermostability profile.     

Correlations of cationic charges with salt sensitivity and microbial specificity of cystine-stabilized beta -strand antimicrobial peptides

The electrostatic interaction of the charge cluster of an amphipathic peptide antibiotic with microbial membranes is a salt-sensitive step that often determines organism specificity. We have examined the correlation between charge clusters and salt insensitivity and microbial specificity in linear, cyclic, and retro-isomeric cystine-stabilized beta-strand (CSbeta) tachyplesin (TP) in a panel of 10 test organisms. Cyclic tachyplesins consisting of 14 and 18 amino acids are constrained by an end-to-end peptide backbone and two or three disulfide bonds to cross-brace the anti-parallel beta-strand that approximates a "beta-tile" structure. Circular dichroism measurements of beta-tile TPs showed that they displayed ordered structures. Control peptides containing the same number of basic amino acids as TP but lacking disulfide constraints were highly salt sensitive. Cyclic TP analogues with six cationic charges were more broadly active and salt-insensitive than those with fewer cationic charges. Reducing their proximity or number of cationic charges, particularly those with three or fewer basic amino acids, led to a significant decrease in potency and salt insensitivity, but an increased selectivity to certain Gram-positive bacteria. An end-group effect of the dibasic N-terminal Lys of TP in the open-chain TP and its retroisomer was observed in certain Gram-negative bacteria under high-salt conditions, an effect that was not found in the cyclic analogs. These results suggest that a stable folded structure together with three or more basic amino acids closely packed in a charged region in CSbeta peptides is important for salt insensitivity and organism specificity.     

ESBRI: A web server for evaluating salt bridges in proteins

Salt bridges can play important roles in protein structure and function and have stabilizing and destabilizing effects in protein folding. ESBRI is a software available as web tool which analyses the salt bridges in a protein structure, starting from the atomic coordinates. In the case of protein complexes, the salt bridges between protein chains can be evaluated, as well as those among specific charged amino acids and the different protein subunits, in order to obtain useful information regard the protein-protein interaction.     

Secondary structure perturbations in salt-induced protein precipitates

The secondary structure implications of precipitation induced by a chaotropic salt, KSCN, and a structure stabilizing salt, Na₂SO₄, were studied for twelve different proteins. α-helix and β-sheet content of precipitate and native structures were estimated from the analysis of amide I band Raman spectra. A statistical analysis of the estimated perturbations in the secondary structure contents indicated that the most significant event is the formation of β-sheet structures with a concomitant loss of α-helix on precipitation with KSCN. The conformational changes for each protein were also analyzed with respect to elements of primary, secondary and tertiary structure existing in the native protein; primary structure was quantified by the fractions of hydrophobic and charged amino acids, secondary structure by x-ray estimates of α-helix and β-sheet contents of native proteins and tertiary structure by the dipole moment and solvent-accessible surface area. For the KSCN precipitates, factors affecting β-sheet content included the fraction of charged amino acids in the primary sequence and the surface area. Changes in α-helix content were influenced by the initial helical content and the dipole moment. The enhanced β-sheet contents of precipitates observed in this work parallel protein structural changes occurring in other aggregative phenomena.     

Mechanisms of interaction of amino acids with phospholipid bilayers during freezing

In this study we compare the ability of various amino acids to protect small unilamellar vesicles against damage during freeze/thaw. Liposomes were composed of 75% palmitoyloleoyl phosphatidylcholine and 25% phosphatidylserine. Damage to liposomes frozen in liquid nitrogen and thawed at 20 degrees C was assessed by resonance energy transfer. Cryoprotection by numerous amino acids was compared in the presence and absence of 350 mM NaCl. The majority of amino acids with hydrocarbon side chains increased membrane damage during freeze/thaw regardless of the presence of salt. However, amino acids with hydrocarbon side chains of less than three carbons long, e.g. glycine, alanine, and 2-aminobutyric acid, were cryoprotective only in the presence of salt. We suggest that NaCl selectively increases the solubility of such amino acids, allowing them to act as cryoprotectants. In contrast, amino acids with side chains containing charged amine groups were cryoprotective regardless of the presence of salt. The degree of charge on the second amine group is shown to be important for cryoprotection by these molecules. We present evidence that suggests an interaction between the positively charged, second amine group of the amino acid, and the negatively charged phospholipid headgroup.     

Interactions of macromolecules with salt ions: an electrostatic theory for the Hofmeister effect

Salting-out of proteins was discovered in the nineteenth century and is widely used for protein separation and crystallization. It is generally believed that salting-out occurs because at high concentrations salts and the protein compete for solvation water. Debye and Kirkwood suggested ideas for explaining salting-out (Debeye and MacAulay, Physik Z; 1925;131:22-29; Kirkwood, In: Proteins, amino acids and peptides as ions and dipolar ions. New York: Reinhold; 1943. p 586-622). However, a quantitative theory has not been developed, and such a theory is presented here. It is built on Kirkwood's idea that a salt ion has a repulsive interaction with an image charge inside a low dielectric cavity. Explicit treatment is given for the effect of other salt ions on the interaction between a salt ion and its image charge. When combined with the Debye-Hückel effect of salts on the solvation energy of protein charges (i.e., salting-in), the characteristic curve of protein solubility versus salt concentration is obtained. The theory yields a direct link between the salting-out effect and surface tension and is able to provide rationalizations for the effects of salt on the folding stability of several proteins.     

Insights of interaction between small and large subunits of ADP-glucose pyrophosphorylase from bread wheat (Triticum aestivum L.)

Lack of knowledge of three dimensional structures of small and large subunits of ADP- glucose pyrophosphorylase (AGPase) in wheat has hindered efforts to understand the binding specifities of substrate and catalytic mechanism. Thus, to understand the structure activity relationship, 3D structures were built by homology modelling based on crystal structure of potato tuber ADP-glucose pyrophosphorylase. Selected models were refined by energy minimization and further validated by Procheck and Prosa-web analysis. Ramachandran plot showed that overall main chain and side chain parameters are favourable. Moreover, Z-score of the models from Prosa-web analysis gave the conformation that they are in the range of the template. Interaction analysis depicts the involvement of six amino acids in hydrogen bonding (AGP-SThr422-AGP-LMet138, AGP- SArg420-AGP-LGly47, AGP-SSer259-AGP-LSer306, AGP-SGlu241-AGP-LIle311, AGPSGln113- AGP-LGlu286 and AGP-SGln70-AGP-LLys291). Fifteen amino acids of small subunit were able to make hydrophobic contacts with seventeen amino acids of large subunit. Furthermore, decrease in the solvent accessible surface area in the amino acids involved in interaction were also reported. All the distances were formed in between 2.27 to 3.78Å. The present study focussed on heterodimeric structure of (AGPase). This predicted complex not only enhance our understanding of the interaction mechanism between these subunits (AGP-L and AGP-S) but also enable to further study to obtain better variants of this enzyme for the improvement of the plant yield.     

Characterization of discontinuous epitope of prion protein recognized by the monoclonal antibody T2

The anti-prion protein (PrP) monoclonal antibody T2 has previously been prepared using PrP-knockout mice immunized with mouse recombinant PrP residues 121-231, however its interaction mechanism to PrP antigen has not been cleared. Here we identified and characterized the epitope of T2 antibody. The competitive ELISA with 20-mer synthetic peptides derived from PrP121-231 showed that T2 antibody had no affinity for these peptides. The analysis with deletion mutants of PrP revealed that 10 amino acids in the N terminus and 66 amino acids in the C terminus of PrP121-231 were necessary for reactivity with T2. Two far regions are necessary for complete affinity of the T2 antibody for PrP; either region alone is not sufficient to retain the affinity. The epitope recognized by T2 antibody is discontinuous and conformational. We examined the effect of disulfide bond and salt bridges. Alkylation of cysteine residues in C terminus of PrP121-231, which breaks a disulfide bond and disrupts the structure, had diminished the reactivity. Mutations induced in the PrP121-231 to break the disulfide bond or salt bridges, markedly had reduced the reactivity with T2 antibody. It suggests that T2 antibody recognized the structure maintained by the disulfide bond and salt bridges.     

Dimer/monomer status and in vivo function of salt‐bridge mutants of the plant UV‐B photoreceptor UVR8

UV RESISTANCE LOCUS8 (UVR8) is a photoreceptor for ultraviolet‐B (UV‐B) light that initiates photomorphogenic responses in plants. UV‐B photoreception causes rapid dissociation of dimeric UVR8 into monomers that interact with CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1) to initiate signal transduction. Experiments with purified UVR8 show that the dimer is maintained by salt‐bridge interactions between specific charged amino acids across the dimer interface. However, little is known about the importance of these charged amino acids in determining dimer/monomer status and UVR8 function in plants. Here we evaluate the use of different methods to examine dimer/monomer status of UVR8 and show that mutations of several salt‐bridge amino acids affect dimer/monomer status, interaction with COP1 and photoreceptor function of UVR8 in vivo. In particular, the salt‐bridges formed between arginine 286 and aspartates 96 and 107 are key to dimer formation. Mutation of arginine 286 to alanine impairs dimer formation, interaction with COP1 and function in vivo, whereas mutation to lysine gives a weakened dimer that is functional in vivo, indicating the importance of the positive charge of the arginine/lysine residue for dimer formation. Notably, a UVR8 mutant in which aspartates 96 and 107 are conservatively mutated to asparagine is strongly impaired in dimer formation but mediates UV‐B responses in vivo with a similar dose–response relationship to wild‐type. The UV‐B responsiveness of this mutant does not correlate with dimer formation and monomerisation, indicating that monomeric UVR8 has the potential for UV‐B photoreception, initiating signal transduction and responses in plants.     

Relationship between the Molecular Structure of the Nitrogen Source and the Activity of the Extracellular Lectins of Lentinus edodes (Berk.) Sing [Lentinula edodes (Berk.) Pegler] upon Submerged Cultivation

The activity of the extracellular lectins of Lentinus edodes (Berk.) Sing [Lentinula edodes (Berk.) Pegler] and the formation of a pigmented mycelial film by this fungus upon submerged cultivation in a synthetic medium were found to depend on the presence of some amino acids (particularly, asparagine) and Ca²⁺ and Mn²⁺ ions in the medium. Quantum-chemical calculations suggest that the different character of the interaction of amino acids with the aforementioned ions is due to differences in the hydrophobicity of the amino acids rather than to differences in the electron structure of the amino acid zwitterions.     

Low frequency dynamics of chymotrypsin by neutron spectroscopy

The low frequency vibrational modes of enzymes have large amplitudes of vibration which should be related to conformational changes that occur during enzyme action. In the present paper we present inelastic neutron scattering measurements for -chymotrypsin that show a peak in the low frequency spectrum. This peak is well defined at 77 K. Gaussian fits yield values of 0.93±0.05, 0.86±0.04, 0.81±0.05, and 0.87±0.06 THz for the peak position at wave vector transfers (Q) of 1.00, 1.40, 1.85, and 3.00 Å^-1, respectively. The full widths at half maximum are all greater than the resolution (0.2 THz) by at least a factor of two. At 298 K a weak peak at about 0.6 THz was observed for Q values of 1.0, 1.4 and 1.85 Å^-1. The data are interpreted in terms of the allowed oscillations of a large globular protein treated as an elastic sphere. Assuming a Raman active oscillation at 0.9 THz it is shown that a peak in the neutron scattering response at 0.6 THz may arise from a rotational shear mode of the chymotrypsin molecule.     

The nature of protein domain evolution: shaping the interaction network

The proteomes that make up the collection of proteins in contemporary organisms evolved through recombination and duplication of a limited set of domains. These protein domains are essentially the main components of globular proteins and are the most principal level at which protein function and protein interactions can be understood. An important aspect of domain evolution is their atomic structure and biochemical function, which are both specified by the information in the amino acid sequence. Changes in this information may bring about new folds, functions and protein architectures. With the present and still increasing wealth of sequences and annotation data brought about by genomics, new evolutionary relationships are constantly being revealed, unknown structures modeled and phylogenies inferred. Such investigations not only help predict the function of newly discovered proteins, but also assist in mapping unforeseen pathways of evolution and reveal crucial, co-evolving inter- and intra-molecular interactions. In turn this will help us describe how protein domains shaped cellular interaction networks and the dynamics with which they are regulated in the cell. Additionally, these studies can be used for the design of new and optimized protein domains for therapy. In this review, we aim to describe the basic concepts of protein domain evolution and illustrate recent developments in molecular evolution that have provided valuable new insights in the field of comparative genomics and protein interaction networks.     

A heterodimerizing leucine zipper coiled coil system for examining the specificity of a position interactions: amino acids I,V, L,N, A,and K

We use a heterodimerizing leucine zipper system to examine the contribution of the interhelical a-a' interaction to dimer stability for six amino acids (A, V, L, I, K, and N). Circular dichroism (CD) spectroscopy monitored the thermal denaturation of 36 heterodimers that generate six homotypic and 30 heterotypic a-a' interactions. Isoleucine (I-I) is the most stable homotypic a-a' interaction, being 9.2 kcal/mol per dimer more stable than the A-A interaction and 4.0 kcal/mol per dimer more stable than either the L-L or V-V interaction, and 7.0 kcal/mol per dimer more stable than the N-N interaction. Only lysine was less stable than alanine. An alanine-based double-mutant thermodynamic cycle calculated coupling energies between the a and a' positions in the heterodimer. The aliphatic amino acids L, V, and I prefer to form homotypic interactions with coupling energies of -0.6 to -0.9 kcal/mol per dimer, but the heterotypic aliphatic interactions have positive coupling energies of <1.0 kcal/mol per dimer. The asparagine homotypic interaction has a coupling energy of -0.5 kcal/mol per dimer, while heterotypic interactions with the aliphatic amino acids produce coupling energies ranging from 2.6 to 4.9 kcal/mol per dimer. The homotypic K-K interaction is 2.9 kcal/mol per dimer less stable than the A-A interaction, but the coupling energy is only 0.3 kcal/mol per dimer. Heterotypic interactions with lysine and either asparagine or aliphatic amino acids produce similar coupling energies ranging from -0.2 to -0.7 kcal/mol per dimer. Thus, of the amino acids that were examined, asparagine contributes the most to dimerization specificity because of the large positive coupling energies in heterotypic interactions with the aliphatic amino acids which results in the N-N homotypic interaction.     

Extent and nature of contacts between protein molecules in crystal lattices and between subunits of protein oligomers

A survey was compiled of several characteristics of the intersubunit contacts in 58 oligomeric proteins, and of the intermolecular contacts in the lattice for 223 protein crystal structures. The total number of atoms in contact and the secondary structure elements involved are similar in the two types of interfaces. Crystal contact patches are frequently smaller than patches involved in oligomer interfaces. Crystal contacts result from more numerous interactions by polar residues, compared with a tendency toward nonpolar amino acids at oligomer interfaces. Arginine is the only amino acid prominent in both types of interfaces. Potentials of mean force for residue–residue contacts at both crystal and oligomer interfaces were derived from comparison of the number of observed residue–residue interactions with the number expected by mass action. They show that hydrophobic interactions at oligomer interfaces favor aromatic amino acids and methionine over aliphatic amino acids; and that crystal contacts form in such a way as to avoid inclusion of hydrophobic interactions. They also suggest that complex salt bridges with certain amino acid compositions might be important in oligomer formation. For a protein that is recalcitrant to crystallization, substitution of lysine residues with arginine or glutamine is a recommended strategy. Proteins 28:494–514, 1997. © 1997 Wiley-Liss, Inc.     

Insight into the mode of action of the LRRK2 Y1699C pathogenic mutant

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most prevalent known cause of autosomal dominant Parkinson's disease. The LRRK2 gene encodes a Roco protein featuring a Ras of complex proteins (ROC) GTPase and a kinase domain linked by the C-terminal of ROC (COR) domain. Here, we explored the effects of the Y1699C pathogenic LRRK2 mutation in the COR domain on GTPase activity and interactions within the catalytic core of LRRK2. We observed a decrease in GTPase activity for LRRK2 Y1699C comparable to the decrease observed for the R1441C pathogenic mutant and the T1348N dysfunctional mutant. To study the underlying mechanism, we explored the dimerization in the catalytic core of LRRK2. ROC-COR dimerization was significantly weakened by the Y1699C or R1441C/G mutation. Using a competition assay, we demonstrated that the intra-molecular ROC : COR interaction is favoured over ROC : ROC dimerization. Interestingly, the intra-molecular ROC : COR interaction was strengthened by the Y1699C mutation. This is supported by a 3D homology model of the ROC-COR tandem of LRRK2, showing that Y1699 is positioned at the intra-molecular ROC : COR interface. In conclusion, our data provides mechanistic insight into the mode of action of the Y1699C LRRK2 mutant: the Y1699C substitution, situated at the intra-molecular ROC : COR interface, strengthens the intra-molecular ROC : COR interaction, thereby locally weakening the dimerization of LRRK2 at the ROC-COR tandem domain resulting in decreased GTPase activity.     

Unraveling the molecular effects of mutation L270P on Wiskkot–Aldrich syndrome protein: insights from molecular dynamics approach

Missense mutation L270P disrupts the auto-inhibited state of “Wiskkot–Aldrich syndrome protein” (WASP), thereby constitutively activating the mutant structure, a key event for pathogenesis of X-linked neutropenia (XLN). In this study, we comprehensively deciphered the molecular feature of activated mutant structure by all atom molecular dynamics (MD) approach. MD analysis revealed that mutant structure exposed a wide variation in the spatial environment of atoms, resulting in enhanced residue flexibility. The increased flexibility of residues favored to decrease the number of intra-molecular hydrogen bonding interactions in mutant structure. The reduction of hydrogen bonds in the mutant structure resulted to disrupt the local folding of secondary structural elements that eventually affect the proper folding of mutants. The unfolded state of mutant structure established more number of inter-molecular hydrogen bonding interaction at interface level due to the conformational variability, thus mediated high binding affinity with its interacting partner, Cdc42.     

Molecular insight into amyloid oligomer destabilizing mechanism of flavonoid derivative 2-(4′ benzyloxyphenyl)-3-hydroxy-chromen-4-one through docking and molecular dynamics simulations

Aggregation of amyloid peptide (Aβ) has been shown to be directly related to progression of Alzheimer’s disease (AD). Aβ is neurotoxic and its deposition and aggregation ultimately lead to cell death. In our previous work, we reported flavonoid derivative (compound 1) showing promising result in transgenic AD model of Drosophila. Compound 1 showed prevention of Aβ-induced neurotoxicity and neuroprotective efficacy in Drosophila system. However, mechanism of action of compound 1 and its effect on the amyloid is not known. We therefore performed molecular docking and atomistic, explicit-solvent molecular dynamics simulations to investigate the process of Aβ interaction, inhibition, and destabilizing mechanism. Results showed different preferred binding sites of compound 1 and good affinity toward the target. Through the course of 35 ns molecular dynamics simulation, conformations_5 of compound 1 intercalates into the hydrophobic core near the salt bridge and showed major structural changes as compared to other conformations. Compound 1 showed interference with the salt bridge and thus reducing the inter strand hydrogen bound network. This minimizes the side chain interaction between the chains A–B leading to disorder in oligomer. Contact map analysis of amino acid residues between chains A and B also showed lesser interaction with adjacent amino acids in the presence of compound 1 (conformations_5). The study provides an insight into how compound 1 interferes and disorders the Aβ peptide. These findings will further help to design better inhibitors for aggregation of the amyloid oligomer.     

Molecular dynamics and principal components of potassium binding with human telomeric intra-molecular G-quadruplex

Telomere assumes intra-molecular G-quadruplex that is a significant drug target for inhibiting telomerase maintenance of telomeres in cancer. Metal cations have been recognized as playing important roles in stabilizing G-quadruplex, but their binding processes to human telomeric G-quadruplex remain uncharacterized. To investigate the detailed binding procedures, molecular dynamics simulations were conducted on the hybrid [3+ 1] form-one human telomeric intra-molecular G-quadruplex. We show here that the binding of a potassium ion to a G-tetrad core is mediated by two alternative pathways. Principal component analysis illustrated the dominant concerted motions of G-quadruplex occurred at the loop domains. MM-PBSA calculations revealed that binding was energetically favorable and driven by the electrostatic interactions. The lower binding site was found more constructive favorable for binding. Our data provide useful information on a potassium-mediated stable structure of human telomeric intra-molecular G-quadruplex, implicating in ion disorder associated conformational changes and targeted drug design.     

Boron nitride nanotube-based biosensor for acetone detection: molecular structural mechanics-based simulation

In this study, an ultra-sensitive biosensor based on a single-walled boron nitride nanotube (SWBNNT) structure is proposed for acetone detection. The molecular structural mechanics-based simulation approach has been used to model the atomic structure of SWBNNTs. The cantilevered and bridged configurations of SWBNNT-based biosensor have been considered for analysis. The resonant frequency shift due to attached mass has been analysed for the mass-based detection of acetone molecules. The present simulation approach is validated by comparing obtained simulated results with the continuum mechanics-based analytical results. Along with detection of the attached molecule, identification of its intermediate landing position along the length of the nanotube is equally important for the better performance of the biosensor systems. The frequency shift-based analysis has been reported for the mass-based detection of acetone molecules as well as its intermediate landing position along the length of the nanotube. The resonant frequency shift variations of the higher order modes of vibration for both the considered configurations of SWBNNTs have been assistive for the identification of intermediate landing position of the acetone molecule. The proposed molecular structural mechanics-based simulation approach is found to be very effectual in terms of simulation of the real atomic structures of the nanotube. The proposed biosensor can achieve extremely high sensitivity at molecular level and it can be potentially used for real-time sensing capability for the acetone concentration for future health monitoring.