Structural behavior of RNA-binding proteins in free and complex states: ribosomal protein L25 and 5S RNA from Escherichia coli

A novel approach has been proposed to evaluate the steadiness of polar clusters containing RNA-binding sites on the protein surface. The degree of clusterization of RNA-binding polar residues can be a measure of the steadiness of corresponding polar clusters. Ribosomal protein L25 from E. coli forms a complex with a fragment of 5S rRNA by means of two binding sites S1 and S2. We have examined cluster distribution of RNA-contacting polar residues on the protein surface by using the data of two states: complex state (in crystal and solution) and free state (in solution). For the crystal, the extent of clusterization of binding sites S1 and S2 are estimated to be 74.1 and 100%, respectively. For the free state in solution, the degrees of clusterization of these two sites are 22.8 and 68.6%, respectively. Thus, we have obtained a steadiness quantitative measure of two different types of protein sites for binding to RNA: one for the already existing protein binding site, and the other for the RNA-induced protein binding site. It was shown that definite variations of the protein structure in crystal and in solution can be of significant functional meaning. The result could be applied to the structural behavior of numerous protein complexes with double-stranded RNA and DNA.     

Structural behavior of RNA-binding proteins in the free state and in complex with RNA: <Emphasis Type="Italic">Escherichia coli</Emphasis> ribosomal protein L25 and 5S rRNA

A novel approach was proposed to evaluate the steadiness of polar clusters containing the RNA-binding sites on the protein surface. The degree of clustering of RNA-binding polar residues was used as a measure of the steadiness of the corresponding polar clusters. Escherichia coli ribosomal protein L25 utilizes two binding sites, S1 and S2, to complexate with a 5S rRNA fragment. The cluster distribution of RNA-contacting polar residues on the protein surface was studied using the structural data on the complex (in crystal and in solution) and the free state (in solution). The degree of polar residue clustering in S1 and S2 in crystal was estimated at 71.4 and 100%, respectively. For the free state in solution, the degree of clustering of the two sites was 22.8 and 68.6%, respectively. Thus, the steadiness was quantitatively estimated for the RNA-binding sites of two different types, one preexisting in the protein and the other induced by the RNA structure upon complexation. The difference between the protein structures in crystal and in solution was found to be functionally significant. The results can be extrapolated to numerous complexes of proteins with double-stranded RNA and DNA.     

Formation of metal nanoclusters on specific surface sites of protein molecules

During vacuum condensation of metals on frozen proteins, nanoclusters are preferentially formed at specific surface sites (decoration). Understanding the nature of metal/protein interaction is of interest for structure analysis and is also important in the fields of biocompatibility and sensor development. Studies on the interaction between metal and distinct areas on the protein which enhance or impede the probability for cluster formation require information on the structural details of the protein's surface underlying the metal clusters. On three enzyme complexes, lumazine synthase from Bacillus subtilis, proteasome from Thermoplasma acidophilum and GTP cyclohydrolase I from Escherichia coli, the decoration sites as determined by electron microscopy (EM) were correlated with their atomic surface structures as obtained by X-ray crystallography. In all three cases, decoration of the same protein results in different cluster distributions for gold and silver. Gold decorates surface areas consisting of polar but uncharged residues and with rough relief whereas silver clusters are preferentially formed on top of protein pores outlined by charged and hydrophilic residues and filled with frozen buffer under the experimental conditions. A common quality of both metals is that they strictly avoid condensation on hydrophobic sites lacking polar and charged residues. The results open ways to analyse the binding mechanism of nanoclusters to small specific sites on the surface of hydrated biomacromolecules by non-microscopic, physical-chemical methods. Understanding the mechanism may lead to advanced decoration techniques resulting in fewer background clusters. This would improve the analysis of single molecules with regard to their symmetries and their orientation in the adsorbed state and in precrystalline assemblies as well as facilitate the detection of point defects in crystals caused by misorientation or by impurities.     

Molecular dynamics simulations of a calmodulin-peptide complex in solution

We have performed an 4-ns MD simulation of calmodulin complexed with a target peptide in explicit water, under realistic conditions of constant temperature and pressure, in the presence of a physiological concentration of counterions and using Ewald summation to avoid truncation of long-range electrostatic forces. During the simulation the system tended to perform small fluctuations around a structure similar to, but somewhat looser than the starting crystal structure. The calmodulin-peptide complex was quite rigid and did not exhibit any large amplitude domain motions such as previously seen in apo- and calcium-bound calmodulin. We analyzed the calmodulin-peptide interactions by calculating buried surface areas, CHARMM interaction energies and continuum model interaction free energies. In the trajectory, the protein surface area buried by contact with the peptide is 1373 A(2) approximately evenly divided between the calmodulin N-terminal, C-terminal and central linker regions. A majority of this buried surface, 803 A(2), comes from nonpolar residues, in contrast to the protein as a whole, for which the surface is made up of mostly polar and charged groups. Our continuum calculations indicate that the largest favorable contribution to peptide binding comes from burial of molecular surface upon complex formation. Electrostatic contributions are favorable but smaller in the trajectory structures, and actually unfavorable for binding in the crystal structure. Since nonpolar groups make up most of buried surface of the protein, our calculations suggest that the hydrophobic effect is the main driving force for binding the helical peptide to calmodulin, consistent with thermodynamic analysis of experimental data. Besides the burial of nonpolar surface area, secondary contributions to peptide binding come from burial of polar surface and electrostatic interactions. In the nonpolar interactions a crucial role is played by the nine methionines of calmodulin. In the electrostatic interactions the negatively charged protein residues and positively charged peptide residues play a dominant role.     

Molecular Dynamics Simulations of a Calmodulin-peptide Complex in Solution

Abstract

We have performed an 4-ns MD simulation of calmodulin complexed with a target peptide in explicit water, under realistic conditions of constant temperature and pressure, in the presence of a physiological concentration of counterions and using Ewald summation to avoid truncation of long-range electrostatic forces. During the simulation the system tended to perform small fluctuations around a structure similar to, but somewhat looser than the starting crystal structure. The calmodulin-peptide complex was quite rigid and did not exhibit any large amplitude domain motions such as previously seen in apo- and calcium-bound calmodulin. We analyzed the calmodulin-peptide interactions by calculating buried surface areas, CHARMM interaction energies and continuum model interaction free energies. In the trajectory, the protein surface area buried by contact with the peptide is 1373 Ų, approximately evenly divided between the calmodulin N-terminal, C-terminal and central linker regions. A majority of this buried surface, 803 ·A², comes from nonpolar residues, in contrast to the protein as a whole, for which the surface is made up of mostly polar and charged groups. Our continuum calculations indicate that the largest favorable contribution to pep- tide binding comes from burial of molecular surface upon complex formation. Electrostatic contributions are favorable but smaller in the trajectory structures, and actually unfavorable for binding in the crystal structure. Since nonpolar groups make up most of buried surface of the protein, our calculations suggest that the hydrophobic effect is the main driving force for binding the helical peptide to calmodulin, consistent with thermodynamic analysis of experimental data. Besides the burial of nonpolar surface area, secondary contributions to peptide binding come from burial of polar surface and electrostatic interactions. In the nonpolar interactions a crucial role is played by the nine methionines of calmodulin. In the electrostatic interactions the negatively charged protein residues and positively charged peptide residues play a dominant role.     

On the Importance of Polar Interactions for Complexes Containing Intrinsically Disordered Proteins

There is a growing recognition for the importance of proteins with large intrinsically disordered (ID) segments in cell signaling and regulation. ID segments in these proteins often harbor regions that mediate molecular recognition. Coupled folding and binding of the recognition regions has been proposed to confer high specificity to interactions involving ID segments. However, researchers recently questioned the origin of the interaction specificity of ID proteins because of the overrepresentation of hydrophobic residues in their interaction interfaces. Here, we focused on the role of polar and charged residues in interactions mediated by ID segments. Making use of the extended nature of most ID segments when in complex with globular proteins, we first identified large numbers of complexes between globular proteins and ID segments by using radius-of-gyration-based selection criteria. Consistent with previous studies, we found the interfaces of these complexes to be enriched in hydrophobic residues, and that these residues contribute significantly to the stability of the interaction interface. However, our analyses also show that polar interactions play a larger role in these complexes than in structured protein complexes. Computational alanine scanning and salt-bridge analysis indicate that interfaces in ID complexes are highly complementary with respect to electrostatics, more so than interfaces of globular proteins. Follow-up calculations of the electrostatic contributions to the free energy of binding uncovered significantly stronger Coulombic interactions in complexes harbouring ID segments than in structured protein complexes. However, they are counter-balanced by even higher polar-desolvation penalties. We propose that polar interactions are a key contributing factor to the observed high specificity of ID segment-mediated interactions.     

Non‐interacting surface solvation and dynamics in protein–protein interactions

Protein–protein interactions control a plethora of cellular processes, including cell proliferation, differentiation, apoptosis, and signal transduction. Understanding how and why proteins interact will inevitably lead to novel structure‐based drug design methods, as well as design of de novo binders with preferred interaction properties. At a structural and molecular level, interface and rim regions are not enough to fully account for the energetics of protein–protein binding, even for simple lock‐and‐key rigid binders. As we have recently shown, properties of the global surface might also play a role in protein–protein interactions. Here, we report on molecular dynamics simulations performed to understand solvent effects on protein–protein surfaces. We compare properties of the interface, rim, and non‐interacting surface regions for five different complexes and their free components. Interface and rim residues become, as expected, less mobile upon complexation. However, non‐interacting surface appears more flexible in the complex. Fluctuations of polar residues are always lower compared with charged ones, independent of the protein state. Further, stable water molecules are often observed around polar residues, in contrast to charged ones. Our analysis reveals that (a) upon complexation, the non‐interacting surface can have a direct entropic compensation for the lower interface and rim entropy and (b) the mobility of the first hydration layer, which is linked to the stability of the protein–protein complex, is influenced by the local chemical properties of the surface. These findings corroborate previous hypotheses on the role of the hydration layer in shielding protein–protein complexes from unintended protein–protein interactions. Proteins 2015; 83:445–458. © 2014 Wiley Periodicals, Inc.     

A phospholipid substrate molecule residing in the membrane surface mediates opening of the lid region in group IVA cytosolic phospholipase A2

The Group IVA (GIVA) phospholipase A(2) associates with natural membranes in response to an increase in intracellular Ca(2+) along with increases in certain lipid mediators. This enzyme associates with the membrane surface as well as binding a single phospholipid molecule in the active site for catalysis. Employing deuterium exchange mass spectrometry, we have identified the regions of the protein binding the lipid surface and conformational changes upon a single phospholipid binding in the absence of a lipid surface. Experiments were carried out using natural palmitoyl arachidonyl phosphatidylcholine vesicles with the intact GIVA enzyme as well as the isolated C2 and catalytic domains. Lipid binding produced changes in deuterium exchange in eight different regions of the protein. The regions with decreased exchange included Ca(2+) binding loop one, which has been proposed to penetrate the membrane surface, and a charged patch of residues, which may be important in interacting with the polar head groups of phospholipids. The regions with an increase in exchange are all located either in the hydrophobic core underneath the lid region or near the lid and hinge regions from 403 to 457. Using the GIVA phospholipase A(2) irreversible inhibitor methyl-arachidonyl fluorophosphonate, we were able to isolate structural changes caused only by pseudo-substrate binding. This produced results that were very similar to natural lipid binding in the presence of a lipid interface with the exception of the C2 domain and region 466-470. This implies that most of the changes seen in the catalytic domain are due to a substrate-mediated, not interface-mediated, lid opening, which exposes the active site to water. Finally experiments carried out with inhibitor plus phospholipid vesicles showed decreases at the C2 domain as well as charged residues on the putative membrane binding surface of the catalytic domain revealing the binding sites of the enzyme to the lipid surface.     

Exploring the Composition of Protein-Ligand Binding Sites on a Large Scale

The residue composition of a ligand binding site determines the interactions available for diffusion-mediated ligand binding, and understanding general composition of these sites is of great importance if we are to gain insight into the functional diversity of the proteome. Many structure-based drug design methods utilize such heuristic information for improving prediction or characterization of ligand-binding sites in proteins of unknown function. The Binding MOAD database if one of the largest curated sets of protein-ligand complexes, and provides a source of diverse, high-quality data for establishing general trends of residue composition from currently available protein structures. We present an analysis of 3,295 non-redundant proteins with 9,114 non-redundant binding sites to identify residues over-represented in binding regions versus the rest of the protein surface. The Binding MOAD database delineates biologically-relevant “valid” ligands from “invalid” small-molecule ligands bound to the protein. Invalids are present in the crystallization medium and serve no known biological function. Contacts are found to differ between these classes of ligands, indicating that residue composition of biologically relevant binding sites is distinct not only from the rest of the protein surface, but also from surface regions capable of opportunistic binding of non-functional small molecules. To confirm these trends, we perform a rigorous analysis of the variation of residue propensity with respect to the size of the dataset and the content bias inherent in structure sets obtained from a large protein structure database. The optimal size of the dataset for establishing general trends of residue propensities, as well as strategies for assessing the significance of such trends, are suggested for future studies of binding-site composition.     

An analysis of the relationship between hydration and protein-DNA interactions.

Eleven protein-DNA crystal structures were analyzed to test the hypothesis that hydration sites predicted in the first hydration shell of DNA mark the positions where protein residues hydrogen-bond to DNA. For nine of those structures, protein atoms, which form hydrogen bonds to DNA bases, were found within 1.5 A of the predicted hydration positions in 86% of the interactions. The correspondence of the predicted hydration sites with the hydrogen-bonded protein side chains was significantly higher for bases inside the conserved DNA recognition sequences than outside those regions. In two CAP-DNA complexes, predicted base hydration sites correctly marked 71% (within 1.5 A) of protein atoms, which form hydrogen bonds to DNA bases. Phosphate hydration was compared to actual protein binding sites in one CAP-DNA complex with 78% marked contacts within 2.0 A. These data suggest that hydration sites mark the binding sites at protein-DNA interfaces.     

Identification of receptor binding sites by competitive peptide mapping: phages T1, T5, and phi 80 and colicin M bind to the gating loop of FhuA.

Previously we proposed a transmembrane model of the FhuA receptor protein in the outer membrane of Escherichia coli. Removal of the largest loop at the cell surface converted the FhuA transport protein into an open channel and rendered cells resistant to the FhuA-specific phages T1, T5, and phi 80 and to colicin M. In the present study we employed acetylated hexapeptide amides covering the entire surface loop to investigate binding of the phages and of colicin M. Competitive peptide mapping proved to be a powerful technique to uncover three ligand binding sites within a region of 34 amino acid residues. Hexapeptides derived from three specific regions of the surface loop inhibited infection of cells by the phages and killing by colicin M. Two of these regions were common among all four FhuA ligands. Electron microscopy of phage T5 revealed that one inhibitory peptide triggered a strong conformational change leading to the release of DNA from the phage head. These results suggest that the FhuA gating loop is the target for specific binding of phages T1, T5, and phi 80 and colicin M.     

Analysis of Heparin-Binding Sites in Human Lipoprotein Lipase Using Synthetic Peptides

Synthetic nonbasic peptides based on the type I repeats of thrombospondin (TSP) and four peptides corresponding to the predicted basic clusters in lipoprotein lipase (LPL) have been analyzed for heparin binding. In the present report we examine the structural requirement for the binding of these peptides to heparin-Sepharose column. The peptide containing the sequence Phe-Ser-Trp-Ser-Asp-Trp-Trp-Ser (residues 388–395 in lipoprotein lipase, which include the consensus TSP type I sequence) showed strong binding to heparin. Both the first and second Trp residues in this sequence were essential for tight heparin binding. Substitution of either of the Trp residues by an Ala resulted in the complete loss of heparin binding. The peptides representing the four basic cluster regions of lipoprotein lipase showed variable heparin binding. Strong retention was observed for peptides representing cluster 1 (residues 261–287) and cluster 3 (residues 147–151) peptides followed by cluster 2 (residues 290–302) peptide. A peptide corresponding to LPL cluster 4 (residues 405–414) did not show binding to heparin column. The present study confirms the presence of specific heparin-binding sites in LPL. Furthermore, this study also demonstrates the potential use of synthetic peptides to investigate the interaction between peptides and heparin as an alternative approach to site-directed mutagenesis in selected regions of large protein molecules. The affinity of these peptides toward heparin can be explored to block molecular interactions at these specific sites or to carry and deliver other coupled molecules at the site(s) of attachment of these peptides for therapeutic applications.     

Role of extracellular charged amino acids in the yeast alpha-factor receptor

The yeast pheromone receptor, Ste2p, is a G protein coupled receptor that initiates cellular responses to alpha-mating pheromone, a 13 residue peptide that carries a net positive charge at physiological pH. We have examined the role of extracellular charged groups on the receptor in response to the pheromone. Substitutions of Asn or Ala for one extracellular residue, Asp275, affected both pheromone binding and signaling, suggesting that this position interacts directly with ligand. The other seven extracellular acidic residues could be individually replaced by polar residues with no detectable effects on receptor function. However, substitution of Ala for each of these seven residues resulted in impairment of signaling without affecting pheromone binding, implying that the polar nature of these residues promotes receptor activation. In contrast, substitution of Ala for each of the six positively charged residues at the extracellular surface of Ste2p did not affect signaling.