tRNA structure and binding sites for cations

Equilibrium dialysis and electronic and nuclear resonance spectroscopy show that tRNA cooperatively binds divalent metal ions at very low concentrations (free metal concentration 3 × 10 ^?6 M). The first two methods show that different purified tRNAs have a very similar behavior, including initiator tRNA_F^met. tRNAs with an extra arm in the clover-leaf model, however, appear to have a slightly different behavior. The binding can be described in terms of two classes of sites. The cooperative association of divalent ions binding first does not parallel a cooperative change in the hyperchromism of the tRNA, while the non-cooperative association of the second class of divalent ions corresponds to the concentrations needed to obtain a cooperative melting of the tRNA. The temperature dependence of the number of binding sites and of their binding constants is also presented. The nature of the divalent ion gives the following efficiency: for the cooperativity Co⁺⁺>Mg⁺⁺>Mn⁺⁺ for the weak binding sites Mn⁺⁺>Co⁺⁺>Mg⁺⁺     

Interactions of alginates with univalent cations

Interactions of alginate with univalent cations in solution have been investigated by circular dichroism (c.d.) and rheological measurements. Poly-l-guluronate chain-segments show substantial enhancement (～ 50%) of c.d. ellipticity in the presence of excess of K⁺, with smaller changes for other univalent cations: Li⁺ < Na⁺ < K⁺ > Rb⁺ > Cs⁺ > NH₄⁺. The maximum c.d. change is attained by 0.3m, with no further increase at higher concentrations of cation. No significant dependence on polymer concentration is observed. Spectral changes for poly-d-mannuronate and heterotypic chain-sequences are much smaller. For intact alginates, the magnitude of c.d. change varies almost linearly with poly-l-guluronate content. Difference spectra (c.d. with excess of univalent counterion minus c.d. in distilled water) can be fitted accurately to two Gaussian bands at 211 and 198 nm, assigned to carboxyl n → π^* and π → π^* transitions, respectively. The perturbations induced by Li⁺, K⁺, Rb⁺, Cs⁺, and NH₄⁺ show a clear family relationship, and are mainly in the π → π^* spectral region. With Na⁺, by contrast, c.d. change is largely confined to the n → π^* transition, and is similar to that previously reported for intermolecular (“egg-box”) binding of divalent cations, consistent with results of rheological studies which indicate Na⁺-induced association of poly-l-guluronate chain-sequences. These associations are further enhanced on freezing and thawing. This combined evidence is interpreted in terms of three modes of interaction between univalent cations and alginate chains in solution: (a) ion-pair formation with carboxyl groups of mannuronate and isolated guluronate residues; (b) specific site-binding to contiguous guluronate residues; and (c) co-operative “egg-box” binding, particularly of Na⁺, between poly-l-guluronate chain-sequences.     

Cation binding sites on actin: a structural relationship between antigenic epitopes and cation exchange

Divalent cations such as Mg2+ and Ca2+, which bind specifically to actin, induce conformational changes that affect its antigenic structure. The distribution of antigenic epitopes on the sequence shows that these structural modifications involve epitopes related to monomer-monomer interfaces. In the N-terminal part, the 1-7 acidic extremity is not affected, in contrast with sequence 18-28. The ability of polycations such as diamine to modify the actin structure at concentrations below 0.1 microM strengthens the hypothesis that in vivo these compounds act locally and specifically on actin polymerization.     

The relationship between the chromophore moiety and the cation binding sites in bacteriorhodopsin

Bleaching of the purple membrane strongly reduces the number of divalent cation binding sites as well as their affinities. Conversely, deionization of the bleached membrane drastically inhibits the chromophore regeneration. Proteolysis experiments using bromelain show that the bleached membrane has an additional cleavage site probably located at the fifth loop, whereas in the blue membrane, the C-terminal tail is no longer susceptible to proteolysis. It is suggested that there exists a close relationship between the retinal environment and one or more of the cation binding sites.     

Specific binding sites for cations in bacteriorhodopsin.

The Asp-85 residue, located in the vicinity of the retinal chromophore, plays a key role in the function of bacteriorhodopsin (bR) as a light-driven proton pump. In the unphotolyzed pigment the protonation of Asp-85 is responsible for the transition from the purple form (lambda(max) = 570 nm) to the blue form (lambda(max) = 605 nm) of bR. This transition can also be induced by deionization (cation removal). It was previously proposed that the cations bind to the bR surface and raise the surface pH, or bind to a specific site in the protein, probably in the retinal vicinity. We have reexamined these possibilities by evaluating the interaction between Mn(2+) and a nitroxyl radical probe covalently bound to several mutants in which protein residues were substituted by cystein. We have found that Mn(2+), which binds to the highest-affinity binding site, significantly affects the EPR spectrum of a spin label attached to residue 74C. Therefore, it is concluded that the highest-affinity binding site is located in the extracellular side of the protein and its distance from the spin label at 74C is estimated to be approximately 9.8 +/- 0.7 A. At least part of the three to four low-affinity cation binding sites are located in the cytoplasmic side, because Mn(2+) bound to these binding sites affects spin labels attached to residues 103C and 163C located in the cytoplasmic side of the protein. The results indicate specific binding sites for the color-controlling cations, and suggest that the binding sites involve negatively charged lipids located on the exterior of the bR trimer structure.     

Interaction of lanthanide ions with Panulirus interruptus hemocyanin: Evidence for vicinity of some of the cation binding sites

The interaction of a number of lanthanide ions (namely terbium, praseodymium, erbium, lanthanum, gadolinium and europium) with Panulirus interruptus hemocyanin has been studied.Results from O₂-binding experiments indicate that all these ions may substitute for calcium as allosteric effectors of hemocyanin. Addition of the lanthanides to deoxygenated Panulirus hemocyanin saturated with Tb³⁺ results in a quenching of the terbium luminescence. The highly efficient quenching observed in the case of Eu³⁺ may indicate energy-transfer between Tb³⁺ and Eu³⁺. Since energy-transfer between lanthanides is only effective over very short distances, the data suggest that some of the cation binding sites of Panulirus hemocyanin are clustered.     

Amino acid sequence homology between ligands and their receptors: potential identification of binding sites

Mice were immunized with alpha-bungarotoxin (BGT), a nearly irreversible antagonist of the acetylcholine receptor (AChR), to produce monoclonal antibodies (Mabs). One of the Mabs (JMC2.7) bound not only to BGT, but to the AChR as well. To understand the molecular basis for this novel cross-reaction, the amino acid sequences of these proteins were searched for areas of similarity which might constitute the shared epitope. A number of short segments of sequence homology were found, one of them representing the BGT-binding site of the AChR. Because a portion of BGT resembles that part of the AChR that binds toxin, the self-binding of BGT was evaluated. As shown here, BGT binds specifically to itself to form dimers. In order to extend these observations, other ligand-receptor pairs were examined for sequence homology. The sodium channel and alpha-scorpion toxins were found to have distinct areas of similarity, as do interleukin 2 (IL-2) and the IL-2 receptor. As a general principle, we propose that peptide ligands and their receptors may often share amino acid sequence homology. In fact, the sites of interaction between two proteins may largely be determined by these regions of similarity.     

Microfibril-associated glycoprotein-1 binding to tropoelastin: multiple binding sites and the role of divalent cations.

Microfibrils and elastin are major constituents of elastic fibers, the assembly of which is dictated by multimolecular interactions. Microfibril-associated glycoprotein-1 (MAGP-1) is a microfibrillar component that interacts with the soluble elastin precursor, tropoelastin. We describe here the adaptation of a solid-phase binding assay that defines the effect of divalent cations on the interactions between MAGP-1 and tropoelastin. Using this assay, a strong calcium-dependent interaction was demonstrated, with a dissociation constant of 2.8 +/- 0.3 nm, which fits a single-site binding model. Manganese and magnesium bestowed a weaker association, and copper did not facilitate the protein interactions. Three constructs spanning tropoelastin were used to quantify their relative contributions to calcium-dependent MAGP-1 binding. Binding to a construct spanning a region from the N-terminus to domain 18 followed a single-site binding model with a dissociation constant of 12.0 +/- 2.2 nm, which contrasted with the complex binding behavior observed for fragments spanning domains 17-27 and domain 27 to the C-terminus. To further elucidate binding sites around the kallikrein cleavage site of domains 25/26, MAGP-1 was presented with constructs containing C-terminal deletions within the region. Construct M1659, which spans a region from the N-terminus of tropoelastin to domain 26, inclusive, bound MAGP-1 with a dissociation constant of 9.7 +/- 2.0 nm, which decreased to 4.9 +/- 1.0 nm following the removal of domain 26 (M155n), thus displaying only half the total capacity to bind MAGP-1. These results demonstrate that MAGP-1 is capable of cumulative binding to distinct regions on tropoelastin, with different apparent dissociation constants and different amounts of bound protein.     

Interaction of serum amyloid A with human cystatin C—identification of binding sites

Serum amyloid A (SAA) is a multifunctional acute‐phase protein whose natural role seems to be participation in many physiologic and pathological processes. Prolonged increased SAA level in a number of chronic inflammatory and neoplastic diseases gives rise to reactive systemic amyloid A amyloidosis, where the N‐terminal 76‐amino acid residue‐long segment of SAA is deposited as amyloid fibrils. Recently, a specific interaction between SAA and the ubiquitous inhibitor of cysteine proteases—human cystatin C (hCC)—has been described. Here, we report further evidence corroborating this interaction, and the identification of the SAA and hCC binding sites in the SAA–hCC complex, using a combination of selective proteolytic excision and high‐resolution mass spectrometry. The shortest binding site in the SAA sequence was determined as SAA(86–104), whereas the binding site in hCC sequence was identified as hCC(96–102). Binding specificities of both interacting sequences were ascertained by affinity experiments (ELISA) and by registration of mass spectrum of SAA–hCC complex. Copyright © 2012 John Wiley & Sons, Ltd.     

Tubulin binding sites on gamma-tubulin: identification and molecular characterization

gamma-Tubulin is essential to microtubule organization in eukaryotic cells. It is believed that gamma-tubulin interacts with tubulin to accomplish its cellular functions. However, such an interaction has been difficult to demonstrate and to characterize at the molecular level. gamma-Tubulin is a poorly soluble protein, not amenable to biochemical studies in a purified form as yet. Therefore basic questions concerning the existence and properties of tubulin binding sites on gamma-tubulin have been difficult to address. Here we have performed a systematic search for tubulin binding sites on gamma-tubulin using the SPOT peptide technique. We find a specific interaction of tubulin with six distinct domains on gamma-tubulin. These domains are clustered in the central part of the gamma-tubulin primary amino acid sequence. Synthetic peptides corresponding to the tubulin binding domains of gamma-tubulin bind with nanomolar K(d)s to tubulin dimers. These peptides do not interfere measurably with microtubule assembly in vitro and associate with microtubules along the polymer length. On the tertiary structure, the gamma-tubulin peptides cluster to surface regions on both sides of the molecule. Using SPOT analysis, we also find peptides interacting with gamma-tubulin in both the alpha- and beta-tubulin subunits. The tubulin peptides cluster to surface regions on both sides of the alpha- and beta- subunits. These data establish gamma-tubulin as a tubulin ligand with unique tubulin-binding properties and suggests that gamma-tubulin and tubulin dimers associate through lateral interactions.     

Electronmicroscopic detection of cation binding sites on the erythrocyte membrane

Numerous Ca2+-binding sites were localized ultrahistochemically at the inner surface of the erythrocyte membrane and small amounts at the outer surface. La3+-binding sites were demonstrated at the outer surface only. The results were discussed in relationship to the binding capacity of the filamentous matrix and the glycocalyx of the human erythrocyte membrane.     

Identification of preferred actinomycin-DNA binding sites by the combinatorial method REPSA.

An important question in the study of ligand-DNA interactions is the determination of binding specificity. Here, we used the combinatorial method restriction endonuclease protection, selection, and amplification (REPSA) to identify the preferred duplex DNA-binding sites of the antineoplastic agent actinomycin D. After 10 rounds of REPSA, over 95% of the cloned DNAs exhibited significantly reduced FokI restriction endonuclease cleavage in the presence of 1 microM actinomycin. A chi(2) statistical analysis of their sequences found that 39 of the 45 clones contained one or more copies of the sequence 5'-(T/A)GC(A/T)-3', giving a p<0.001 for this consensus. A DNase I footprinting analysis of the cloned DNAs found that all possessed relatively high affinity actinomycin-binding sites with apparent dissociation constants between 12 and 258nM (average 98nM). The average footprint encompassed 7.6 bases and in most cases (90%) included one or more consensus sequences. Interestingly, several of the selected clones contained overlapping consensus sequences (e.g., 5'-TGCTGCT-3'), suggesting that such close proximity DNA-binding sites may actually be preferred by actinomycin under physiological conditions.     

High-throughput identification of interacting protein-protein binding sites

Background  

With the advent of increasing sequence and structural data, a number of methods have been proposed to locate putative protein binding sites from protein surfaces. Therefore, methods that are able to identify whether these binding sites interact are needed.     

Divalent cation binding to the high- and low-affinity sites on G-actin

Metal binding to skeletal muscle G-actin has been assessed by equilibrium dialysis using 45Ca2+ and by kinetic measurements of the increase in the fluorescence of N-acetyl-N'-(5-sulfo-1-naphthyl)-ethylenediamine-labeled actin. Two classes of cation binding sites were found on G-actin which could be separated on the basis of their Ca2+ affinity: a single high-affinity site with a Kd considerably less than 1 microM and three identical moderate-affinity binding sites with a Kd of 18 microM. The data for the Mg2+-induced fluorescence enhancement of actin labeled with N-acetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine support a previously suggested mechanism [Frieden, C. (1982) J. Biol. Chem. 257, 2882-2886] in which Ca2+ is replaced by Mg2+ at the moderate affinity site(s), followed by a slow actin isomerization. This isomerization occurs independently of Ca2+ release from the high-affinity site. The fluorescence data do not support a mechanism in which this isomerization is directly related to Ca2+ release from the high-affinity site. Fluorescence changes of labeled actin associated with adding metal chelators are complex and do not reflect the same change induced by Mg2+ addition. Fluorescence changes in the labeled actin have also been observed for the addition of Cd2+ or Mn2+ instead of Mg2+. It is proposed actin may undergo a host of subtle conformational changes dependent on the divalent cation bound. We have also developed a method by which progress curves of a given reaction can be analyzed by nonlinear regression fitting of kinetic simulations to experimental reaction time courses.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction between the erythromycin and chloramphenicol binding sites on the Escherichica coli ribosome.

The effects of chloramphenical on the binding kinetics of a fluorescein isothiocyanate derivative of 9(S)-erythromycylamine with 70S and 50S ribosomes have been studied by direct fluorimetric measurements. While chloramphenicol had little effect on the second-order 70S binding rate of the erythromycin analogue, it substantially reduced the dissociation rate of the fluorescent antibiotic-70S ribosome complex. This could be explained by simultaneous binding of both antibiotics to the 70S ribosome. The kinetic results suggest that chloramphenicol-saturated 70S particles bind the erythromycin analogue four times stronger and this was confirmed by direct binding studies. In additon, chloramphenicol causes a twofold increase in the intrinsic fluorescence of the 70S-bound analogue. This increase in fluorescence was used to study the kinetics of chloramphenicol binding to 70S ribosomes containing the fluorescent derivative. The fluorescence change followed first-order kinetics, suggesting that chloramphenicol induces a conformational change in the 70S particle. This could explain both its effect on erythromycin binding and on the fluorescence of bound analogue. Less detailed results with the 50S particle indicate a qualitively similar picture of erythromycin-chloramphenicol interactions.     

Phosphate binding sites identification in protein structures

Nearly half of known protein structures interact with phosphate-containing ligands, such as nucleotides and other cofactors. Many methods have been developed for the identification of metal ions-binding sites and some for bigger ligands such as carbohydrates, but none is yet available for the prediction of phosphate-binding sites. Here we describe Pfinder, a method that predicts binding sites for phosphate groups, both in the form of ions or as parts of other non-peptide ligands, in proteins of known structure. Pfinder uses the Query3D local structural comparison algorithm to scan a protein structure for the presence of a number of structural motifs identified for their ability to bind the phosphate chemical group. Pfinder has been tested on a data set of 52 proteins for which both the apo and holo forms were available. We obtained at least one correct prediction in 63% of the holo structures and in 62% of the apo. The ability of Pfinder to recognize a phosphate-binding site in unbound protein structures makes it an ideal tool for functional annotation and for complementing docking and drug design methods. The Pfinder program is available at http://pdbfun.uniroma2.it/pfinder.     

Role of structural water for prediction of cation binding sites in apoproteins

Structures of many metal-binding proteins are often obtained without structural cations in their apoprotein forms. Missing cation coordinates are usually updated from structural templates constructed from many holoprotein structures. Such templates usually do not include structural water, the important contributor to the ion binding energy. Structural templates are also inconvenient for taking into account structural modifications around the binding site at apo-/holo- transitions. An approach based upon statistical potentials readily takes into account structural modifications associated with binding as well as contribution of structural water molecules. Here, we construct a set of statistical potentials for Mg²⁺, Ca²⁺, and Zn²⁺ contacting with protein atoms of a different type or structural water oxygens. Each type of the cations tends to form tight contacts with protein atoms of specific types. Structural water contributes relatively more into the binding pseudo-energy of Mg²⁺ and Ca²⁺ than of Zn²⁺. We have developed PIONCA (Protein-Ion Calculator), a fast CUDA GPGPU-based algorithm that predicts ion-binding sites in apoproteins. Comparative tests demonstrate that PIONCA outperforms most of the tools based on structural templates or docking. Our software can be also used for locating bound cations in holoprotein structures with missing cation heteroatoms. PIONCA is equipped with an interactive web interface based upon JSmol.     

The environment of the high-affinity cation binding site on actin and the separation between cation and ATP sites as revealed by proton NMR and fluorescence spectroscopy

The lanthanide ions Lu3+ (diamagnetic) and Gd3+ (paramagnetic broadening probe) were used to displace Ca2+ from the high-affinity cation binding site on G-actin. The effects of these higher-affinity ions on the proton nuclear magnetic resonance spectrum of actin were recorded. The aliphatic proton envelope in the Gd-actin sample exhibited a complex array of changes due to the proximity of Gd to several aliphatic residues. No such changes were observed in the diamagnetic Lu-actin control spectrum. By contrast, the aromatic proton envelope remained largely unaffected in both Gd-actin and Lu-actin samples. However, the adenosine moiety on the actin-bound ATP became increasingly mobilized without the triphosphate chain being released from the ATP binding site. Maximum adenosine mobilization occurred with approximately 1 mol of lanthanide ion bound per mol of actin. The absence of changes in the aromatic proton envelope suggests that the high-affinity cation binding site is in a region well removed from the adenosine moiety of bound ATP as well as any aromatic side-chains. The separation of the ATP and cation sites was further explored using the fluorescent ATP analogues FTP and epsilon-ATP. Tb3+ bound to the high-affinity cation site was found to be separated by 16 A from the FTP chromophore bound to the nucleotide binding site on actin. Since this distance is greater than can be accommodated on a model of the Tb-ATP complex, we conclude that the sites are physically separate. This conclusion was further reinforced by experiments involving the quenching of epsilon-ATP fluorescence by Mn2+.(ABSTRACT TRUNCATED AT 250 WORDS)