STIM1 gates TRPC channels, but not Orai1, by electrostatic interaction

The receptor-evoked Ca(2+) signal includes activation of the store-operated channels (SOCs) TRPCs and the Orais. Although both are gated by STIM1, it is not known how STIM1 gates the channels and whether STIM1 gates the TRPCs and Orais by the same mechanism. Here, we report the molecular mechanism by which STIM1 gates TRPC1, which involves interaction between two conserved, negatively charged aspartates in TRPC1((639)DD(640)) with the positively charged STIM1((684)KK(685)) in STIM1 polybasic domain. Charge swapping and functional analysis revealed that exact orientation of the charges on TRPC1 and STIM1 are required, but all positive-negative charge combinations on TRPC1 and STIM1, except STIM1((684)EE(685))+TRPC1((639)RR(640)), are functional as long as they are reciprocal, indicating that STIM1 gates TRPC1 by intermolecular electrostatic interaction. Similar gating was observed with TRPC3((697)DD(698)). STIM1 gates Orai1 by a different mechanism since the polybasic and S/P domains of STIM1 are not required for activation of Orai1 by STIM1.     

Prediction of interaction sites from apo 3D structures when the holo conformation is different

The predictability of catalytic and binding sites from apo structures is addressed for proteins that undergo significant conformational change upon binding. Theoretical microscopic titration curves (THEMATICS), an electrostatics-based method for the prediction of functional sites, is performed on a test set of 24 proteins with both apo and holo structures available. For 23 of these 24 proteins (96%), THEMATICS predicts the correct catalytic or binding site for both the apo and holo forms. For only one of the 24 proteins, THEMATICS makes the correct prediction for the holo structure but fails for the apo structure. The metrics used by THEMATICS to identify functional residues generally are larger in absolute value for the functional residues in the holo forms compared to the corresponding residues in the apo forms. However, even in the apo forms, these identifying metrics are still statistically significantly larger for functional residues than for residues not involved in catalysis or binding. This indicates that some of the unusual electrostatic properties of functional residues are preserved in the apo conformation. Evidence is presented that certain residues immediately surrounding the active catalytic and binding residues impart functionally important chemical and electrostatic properties to the active residues. At least parts of these microenvironments exist in the unbound conformations, such that THEMATICS is able to distinguish the functional residues even in the apo structures.     

Similar folds with different stabilization mechanisms: the cases of prion and doppel proteins

Background  

Protein misfolding is the main cause of a group of fatal neurodegenerative diseases in humans and animals. In particular, in Prion-related diseases the normal cellular form of the Prion Protein PrP (PrP ^C ) is converted into the infectious PrP ^Sc through a conformational process during which it acquires a high β-sheet content. Doppel is a protein that shares a similar native fold, but lacks the scrapie isoform. Understanding the molecular determinants of these different behaviours is important both for biomedical and biophysical research.     

Similar but different: ligand-induced activation of the insulin and epidermal growth factor receptor families

The insulin and epidermal growth factor receptor families are among the most intensively studied proteins in biology. They are closely related members of the receptor tyrosine kinase superfamily and deregulated signaling by members of either receptor family has been implicated in the progression of a variety of cancers. These receptors have thus emerged as validated therapeutic targets for the development of anti-tumour agents. Recent studies have revealed detail of the ligand-binding sites in the insulin receptor family, as well as detail of conformational change upon ligand binding in the epidermal growth factor receptor family. Taken together, these findings and further data relating to kinase activation highlight the fact that while the receptor families share common structural elements, the structural detail of their functioning is remarkably different.     

Protein farnesylation in plants--conserved mechanisms but different targets

Protein farnesylation has an important role in the regulation of plant development and signal transduction, but the exact function of this modification is not well understood. The identification of protein farnesyltransferase substrates, together with the genetic analysis of mutants that are deficient in protein farnesylation, should significantly increase our knowledge of this form of protein modification in plants.     

Similar chemistry, but different bond preferences in inter versus intra-protein interactions

Proteins fold into a well-defined structure as a result of the collapse of the polypeptide chain, while transient protein-complex formation mainly is a result of binding of two folded individual monomers. Therefore, a protein-protein interface does not resemble the core of monomeric proteins, but has a more polar nature. Here, we address the question of whether the physico-chemical characteristics of intraprotein versus interprotein bonds differ, or whether interfaces are different from folded monomers only in the preference for certain types of interactions. To address this question we assembled a high resolution, nonredundant, protein-protein interaction database consisting of 1374 homodimer and 572 heterodimer complexes, and compared the physico-chemical properties of these interactions between protein interfaces and monomers. We performed extensive statistical analysis of geometrical properties of interatomic interactions of different types: hydrogen bonds, electrostatic interactions, and aromatic interactions. Our study clearly shows that there is no significant difference in the chemistry, geometry, or packing density of individual interactions between interfaces and monomeric structures. However, the distribution of different bonds differs. For example, side-chain-side-chain interactions constitute over 62% of all interprotein interactions, while they make up only 36% of the bonds stabilizing a protein structure. As on average, properties of backbone interactions are different from those of side chains, a quantitative difference is observed. Our findings clearly show that the same knowledge-based potential can be used for protein-binding sites as for protein structures. However, one has to keep in mind the different architecture of the interfaces and their unique bond preference.     

Similar numbers but different repertoires of olfactory receptor genes in humans and chimpanzees

Animals recognize their external world through the detection of tens of thousands of chemical odorants. Olfactory receptor (OR) genes encode proteins for detecting odorant molecules and form the largest multigene family in mammals. It is known that humans have fewer OR genes and a higher fraction of OR pseudogenes than mice or dogs. To investigate whether these features are human specific or common to all higher primates, we identified nearly complete sets of OR genes from the chimpanzee and macaque genomes and compared them with the human OR genes. In contrast to previous studies, here we show that the number of OR genes ( approximately 810) and the fraction of pseudogenes (51%) in chimpanzees are very similar to those in humans, though macaques have considerably fewer OR genes. The pseudogenization rates and the numbers of genes affected by positive selection are also similar between humans and chimpanzees. Moreover, the most recent common ancestor between humans and chimpanzees had a larger number of functional OR genes (>500) and a lower fraction of pseudogenes (41%) than its descendents, suggesting that the OR gene repertoires are in a phase of deterioration in both lineages. Interestingly, despite the close evolutionary relationship between the 2 species, approximately 25% of their functional gene repertoires are species specific due to massive gene losses. These findings suggest that the tempo of evolution of OR genes is similar between humans and chimpanzees, but the OR gene repertoires are quite different between them. This difference might be responsible for the species-specific ability of odor perception.     

Prediction of the most favorable configuration in the ACBP-membrane interaction based on electrostatic calculations

Acyl-CoA binding proteins (ACBPs) are highly conserved 10 kDa cytosolic proteins that bind medium- and long-chain acyl-CoA esters. They act as intracellular carriers of acyl-CoA and play a role in acyl-CoA metabolism, gene regulation, acyl-CoA-mediated cell signaling, transport-mediated lipid synthesis, membrane trafficking and also, ACBPs were indicated as a possible inhibitor of diazepam binding to the GABA-A receptor. To estimate the importance of the non-specific electrostatic energy in the ACBP-membrane interaction, we computationally modeled the interaction of HgACBP with both anionic and neutral membranes. To compute the Free Electrostatic Energy of Binding (dE), we used the Finite Difference Poisson Boltzmann Equation (FDPB) method as implemented in APBS. In the most energetically favorable orientation, ACBP brings charged residues Lys18 and Lys50 and hydrophobic residues Met46 and Leu47 into membrane surface proximity. This conformation suggests that these four ACBP amino acids are most likely to play a leading role in the ACBP-membrane interaction and ligand intake. Thus, we propose that long range electrostatic forces are the first step in the interaction mechanism between ACBP and membranes.     

Similar integration but different stability of Alus and LINEs in the human genome

Alus and LINEs (LINE1) are widespread classes of repeats that are very unevenly distributed in the human genome. The majority of GC-poor LINEs reside in the GC-poor isochores whereas GC-rich Alus are mostly present in GC-rich isochores. The discovery that LINES and Alus share similar target site duplication and a common AT-rich insertion site specificity raised the question as to why these two families of repeats show such a different distribution in the genome. This problem was investigated here by studying the isochore distributions of subfamilies of LINES and Alus characterized by different degrees of divergence from the consensus sequences, and of Alus, LINEs and pseudogenes located on chromosomes 21 and 22. Young Alus are more frequent in the GC-poor part of the genome than old Alus. This suggests that the gradual accumulation of Alus in GC-rich isochores has occurred because of their higher stability in compositionally matching chromosomal regions. Densities of Alus and LINEs increase and decrease, respectively, with increasing GC levels, except for the telomeric regions of the analyzed chromosomes. In addition to LINEs, processed pseudogenes are also more frequent in GC-poor isochores. Finally, the present results on Alu and LINE stability/exclusion predict significant losses of Alu DNA from the GC-poor isochores during evolution, a phenomenon apparently due to negative selection against sequences that differ from the isochore composition.     

Cultured skin cells interaction with polylactide surface coated by different collagen structures

The purpose of this work was an optimization of polylactide film surfaces designed for human keratinocytes cultivation. The polylactide films were coated by collagen 1. The experiments showed that uniform covering of polymer surface by collagen, and formation of different collagen structures depend on the mode of the protein application. The differences in collagen distribution on the polymer surface influened the keratinocytes growth in culture. Analysis of keratinocytes alignment, as well as cytoskeleton organization demonstrated that fibrillar collagen promoted more even keratinocytes distribution in comparison with the distribution on molecular collagen.     

Elongation factor Tu-targeted antibiotics: four different structures, two mechanisms of action

Elongation factor Tu (EF-Tu), the carrier of aa-tRNA to the mRNA-programmed ribosome, is the target of four families of antibiotics of unrelated structure, of which the action is supported by two basic mechanisms. Kirromycin and enacyloxin block EF-Tu.GDP on the ribosome; pulvomycin and GE2270 A inhibit the interaction of EF-Tu.GTP with aa-tRNA. The crystallographic analysis has unveiled the structural background of their actions, explaining how antibiotics of unrelated structures and binding modes and sites can employ similar mechanism of action. The selective similarities and differences of their binding sites and the induced EF-Tu conformations make understand how nature can affect the activities of a complex regulatory enzyme by means of low-molecular compounds, and have proposed a suitable approach for drug design.     

Similar taxonomic richness but different communities of ectomycorrhizas in native forests and non-native plantation forests

This investigation sought to examine if there was a difference between the ectomycorrhizal (ECM) communities in plots of native oak and introduced Scots pine and Sitka spruce forest. The ECM communities in four plots of each forest type were described, from five soil cores collected in each plot, by morphotyping, internal transcribed spacer (ITS)-restriction fragment length polymorphism matching of mycorrhizas and sporocarps and ITS sequencing. Fifty-one distinct taxa were distinguished; 25 were identified to species level, 11 to genus and 15 remained unidentified. Seventy-one ECM species were recorded as sporocarps from the forest plots; most (43 species) were found in the Sitka spruce plots. The below-ground ECM communities of the different forest types did not differ significantly with respect to species richness of taxa on roots, but differed in species composition. Multivariate analysis produced a clear separation of the communities of the different forest types using below-ground data, but the above-ground sporocarp data did not separate the forest types. Moreover, results of a Mantel test found no relationship between the above- and below-ground similarity matrices. The oak plots had the most distinctive ECM community, with Laccaria amethystina and Elaphomyces granulatus being frequent. The Sitka spruce plots showed the lowest intra-forest type similarity and were often dominated by "nursery type" ectomycorrhizas. There was only 10% similarity between the above- and below-ground ECM species in these plots, different colonisation methods of ectomycorrhizal taxa and insufficient below-ground sampling being possible reasons for this disparity. Our results indicate that plantations of non-native Sitka spruce can support similar levels of ECM diversity as native forests.     

Vesicle fusion with bilayer lipid membrane controlled by electrostatic interaction

The fusion of proteoliposomes is a promising approach for incorporating membrane proteins in artificial lipid membranes. In this study, we employed an electrostatic interaction between vesicles and supported bilayer lipid membranes (s-BLMs) to control the fusion process. We combined large unilamellar vesicles (LUVs) containing anionic lipids, which we used instead of proteoliposomes, and s-BLMs containing cationic lipids to control electrostatic interaction. Anionic LUVs were never adsorbed or ruptured on the SiO₂ substrate with a slight negative charge, and selectively fused with cationic s-BLMs. The LUVs can be fused effectively to the target position. Furthermore, as the vesicle fusion proceeds and some of the positive charges are neutralized, the attractive interaction weakens and finally the vesicle fusion saturates. In other words, we can control the number of LUVs fused with s-BLMs by controlling the concentration of the cationic lipids in the s-BLMs. The fluidity of the s-BLMs after vesicle fusion was confirmed to be sufficiently high. This indicates that the LUVs attached to the s-BLMs were almost completely fused, and there were few intermediate state vesicles in the fusion process. We could control the position and amount of vesicle fusion with the s-BLMs by employing an electrostatic interaction.     

Folding mechanisms of proteins with high sequence identity but different folds

The problem of how a protein folds from a linear chain of amino acids to the three-dimensional structure necessary for function is often investigated using proteins with a low degree of sequence identity that adopt different folds. The design of pairs of proteins with a high degree of sequence identity but different folds offers the opportunity for a complementary study; in two highly similar sequences, which residues are the most important in directing folding to a particular structure? Here we use molecular dynamics simulations to characterize the folding-unfolding pathways of a pair of proteins designed by Bryan and co-workers [Alexander, P. A., et al. (2005) Biochemistry 44, 14045-14054; He, Y. N., et al. (2005) Biochemistry 44, 14055-14061]. Despite being 59% identical, the two protein sequences fold to two different structures. The first sequence folds to the alpha+beta protein G structure and the second to the all-alpha-helical protein A structure. We show that the final protein structure is determined early along the folding pathway. In folding to the protein G structure, the single alpha-helix (alpha1) and the beta3-beta4 turn fold early. Formation of the hairpin turn essentially prevents folding to helical structure in this region of the protein. This early structure is then consolidated by formation of long-range hydrophobic interactions between alpha1 and the beta3-beta4 turn. The protein A sequence differs both in the residues that form the beta3-beta4 turn and also in many of the residues that form the early hydrophobic interactions in the protein G structure. Instead, in the protein A sequence, a more hierarchical mechanism is observed, with helices folding before many of the tertiary interactions are formed. We find that small, but critical, sequence differences determine the topology of the protein early along the folding pathway, which help to explain the process by which one fold can evolve into another.     

Annexin V membrane interaction: an electrostatic potential study

The possible role of electrostatic interactions for membrane binding and pore formation of annexin V has been analysed on the basis of a simple dielectric model. It is suggested that the binding of phospholipids to annexin V is regulated, at least initially, by the protein's electrostatic potential. The calculations show that a strong local gradient of the electrostatic potential exists at the membrane-protein interface and a membrane pore may be generated by electroporation. The observed specificity and regulation of ion conduction is suggested to reside in the protein part of the pore. On the basis of the three-dimensional structures of the protein and its hypothetical membrane complex, and electrophysiological measurements, a mechanical model of the transmembrane voltage regulation of the annexin's ion conduction properties is proposed.     

On the calculation of electrostatic interactions in proteins

In this paper we present a classical treatment of electrostatic interactions in proteins. The protein is treated as a region of low dielectric constant with spherical charges embedded within it, surrounded by an aqueous solvent of high dielectric constant, which may contain a simple electrolyte. The complete analysis includes the effects of solvent screening, polarization forces, and self energies, which are related to solvation energies. Formulae, and sample calculations of forces and energies, are given for the special case of a spherical protein. Our analysis and model calculations point out that any consistent treatment of electrostatic interactions in proteins should account for the following. Solvent polarization is an important factor in the calculation of pairwise electrostatic interactions. Solvent polarization substantially affects both electrostatic energies and forces acting upon charges. No simple expression for the effective dielectric constant, Deff, can generally be valid, since Deff is a sensitive function of position. Solvent screening of pairwise interactions involving dipolar groups is less effective than the screening of charges. In fact for many interactions involving dipoles, solvent screening can be essentially ignored. The self energy of charges makes a large contribution to the total electrostatic energy of a protein. This must be compensated by specific interactions with other groups in the protein. Strategies for applying our analysis to proteins whose structures are known are discussed.     

HPr proteins of different microorganisms studied by hydrogen-1 high-resolution nuclear magnetic resonance: similarities of structures and mechanisms

The HPr proteins of Streptococcus lactis, Streptococcus faecalis, Bacillus subtilis, and Escherichia coli were studied by 1H NMR at 360 MHz. The "active-center" histidines of all HPr proteins are characterized by a low pK value between 5.6 and 6.1 and similar spectral parameters. Phosphorylation of the histidyl residues leads to an increase of the pK value of 2-3 units and spectral changes characteristic for N-1 phosphorylation of the histidyl ring. The spectra of the HPr proteins of S. lactis, S. Faecalis, B. subtilis, and Staphylococcus aureus reveal many similarities, whereas the spectrum of the E. coli protein is different with exception of the active-center histidine. The HPr protein of S. lactis is formylated at its terminal amino group.     

Neuronal mechanisms of interaction between the red nucleus and other brain stem structures

Axon collaterals of rubrospinal neurons running to many brain stem structures were identified in acute experiments on cats by a technique of intracellular recording of antidromic action potentials in conjunction with collision testing. A systemic principle of organization of rubrospinal influences and also a tendency toward synchronous arrival of rubrospinal impulses at various brain stem centers were demonstrated. Most of these centers are relay nuclei, sending direct afferent projections to regions of the cerebellum which, in turn, control activity of the red nucleus. Besides such a loop, effecting dynamic cerebellar control over motor function, transmission of somatosensory information from nuclei of the dorsal columns of the spinal cord directly to the red nucleus was demonstrated. Special features of mono- and polysynaptic EPSPs evoked by stimulation of nuclei of the dorsal columns indicate that such PSPs arise in different regions of the soma-dendritic membrane of red nucleus neurons. The mechanisms of integration of descending motor volleys by the red nucleus are discussed.L. A. Orbeli Institute of Physiology, Academy of Sciences of the Armenian SSR, Erevan. Translated from Neirofiziologiya, Vol. 16, No. 5, pp. 665–678, September–October, 1984.     

Amazing stability of the arginine-phosphate electrostatic interaction

Electrostatic interactions between a basic epitope containing adjacent arginine residues and an acidic epitope containing a phosphorylated serine are involved in receptor heteromerization. In the present study, we demonstrate that this arginine-phosphate electrostatic interaction possesses a "covalent-like" stability. Hence, these bonds can withstand fragmentation by mass spectrometric collision-induced dissociation at energies similar to those that fragment covalent bonds and they demonstrate an extremely low dissociation constant by plasmon resonance. The present work also highlights the importance of phosphorylation-dephosphorylation events in the modulation of this electrostatic attraction. Phosphorylation of the acidic epitope, a casein kinase one consensus site, makes it available to interact with the basic epitope. On the other hand, phosphorylation of serine and/or threonine residues adjacent to the basic epitope, a protein kinase A consensus site, slows down the attraction between the epitopes. Although analyzed here in the frame of receptor heteromerization, the arginine-phosphate electrostatic interaction most likely represents a general mechanism in protein-protein interactions.