Metal-ion affinity and specificity in EF-hand proteins: coordination geometry and domain plasticity in parvalbumin.

BACKGROUND: The EF-hand family is a large set of Ca(2+)-binding proteins that contain characteristic helix-loop-helix binding motifs that are highly conserved in sequence. Members of this family include parvalbumin and many prominent regulatory proteins such as calmodulin and troponin C. EF-hand proteins are involved in a variety of physiological processes including cell-cycle regulation, second messenger production, muscle contraction, microtubule organization and vision. RESULTS: We have determined the structures of parvalbumin mutants designed to explore the role of the last coordinating residue of the Ca(2+)-binding loop. An E101D substitution has been made in the parvalbumin EF site. The substitution decreases the Ca(2+)-binding affinity 100-fold and increases the Mg(2+)-binding affinity 10-fold. Both the Ca(2+)- and Mg(2+)-bound structures have been determined, and a structural basis has been proposed for the metal-ion-binding properties. CONCLUSIONS: The E101D mutation does not affect the Mg(2+) coordination geometry of the binding loop, but it does pull the F helix 1.1 A towards the loop. The E101D-Ca(2+) structure reveals that this mutant cannot obtain the sevenfold coordination preferred by Ca(2+), presumably because of strain limits imposed by tertiary structure. Analysis of these results relative to previously reported structural information supports a model wherein the characteristics of the last coordinating residue and the plasticity of the Ca(2+)-binding loop delimit the allowable geometries for the coordinating sphere.     

Evaluation of cooperative interactions between substructures of iso-1-cytochrome c using double mutant cycles

A double mutant cycle has been used to evaluate interaction energies between the global stabilizer mutation asparagine 52 --> isoleucine (N52I) in iso-1-cytochrome c and mutations producing single surface histidines at positions 26, 33, 39, 54, 73, 89, and 100. These histidine mutation sites are distributed through the four cooperative folding units of cytochrome c. The double mutant cycle starts with the iso-1-cytochrome c variant AcTM, a variant with no surface histidines and with asparagine at position 52. Isoleucine is added singly at position 52, AcTMI52 variant, as are the surface histidines, AcHX variants, where X indicates the histidine sequence position. The double mutant variants, AcHXI52, provide the remaining corner of the double mutant cycle. The stabilities of all variants were determined by guanidine hydrochloride denaturation and interaction energies were calculated between position 52 and each histidine site. Six of the seven double mutants show additive (AcH33I52, AcH39I52, AcH54I52, AcH89I52, and AcH100I52) stability effects or weak interaction energies (AcH73I52) of the histidine mutations and the N52I mutation, consistent with cooperative effects on protein folding and stability being sparsely distributed through the protein structure. The AcH26I52 variant shows a strong favorable interaction energy, 2.0 +/- 0.5 kcal/mol, between the N52I mutation in one substructure and the addition of His 26 to an adjacent substructure. The data are consistent with an entropic stabilization of the intersubstructure hydrogen bond between His 26 and Glu 44 by the Ile 52 mutation.     

Engineering new metal specificity in EF-hand peptides

Peptides (33-34 amino acids long) corresponding to the helix-turn-helix (EF-hand) motif of the calcium binding site I of Paramecium tetraurelia calmodulin have been synthesized. The linear sequence was unable to acquire a native-like conformation and calcium binding. However, incorporation of a well-positioned disulfide bond bridging the two putative helical regions greatly improved the ordered structure and binding properties. Analyzed by electrospray mass spectrometry, circular dichroism and time-resolved laser-induced fluorescence, such a disulfide-stabilized peptide is shown to acquire a calcium-dependent helical conformation and exhibits native-like affinity for calcium, terbium and europium ions with 30+/-1, 3.5+/-0.6 and 0.6+/-0.1 microM dissociation constants, respectively. Comparable affinities were calculated within the biological construct comprising the entire domain I of Arabidopsis taliana calmodulin. Single sequence mutation (Glu25Asp) in the binding loop of the peptide abolishes calcium affinity, but preserves lanthanide affinity, showing that metal selectivity can be modulated by specific mutations. Such disulfide-stabilized peptides represent useful models to engineer metal specificity in new calmodulin proteins, facilitating the development of new systems to monitor metal pollution in biosensors and to increase metal binding capability of bacterial and plant cells in bioremediation techniques.     

Engineering strontium binding affinity in an EF-hand motif: a quantum chemical and molecular dynamics study

Proteins with the ability to specifically bind strontium would potentially be of great use in the field of nuclear waste management. Unfortunately, no such peptides or proteins are known -- indeed, it is uncertain whether they exist under natural conditions due to low environmental concentrations of strontium. To investigate the possibility of devising such molecules, one of us (CV), in a previous experimental study, proposed starting from an EF-hand motif of the protein calmodulin and mutating some residues to change the motif's specificity for calcium into one for strontium. In this paper, which represents a theoretical complement to the experimental work, we analyzed small-molecule crystallographic structures and performed quantum chemical calculations to identify possible mutations. We then constructed seven mutant sequences of the EF-hand motif and analyzed their dynamical and binding behaviors using molecular dynamics simulations and free-energy calculations (using the MM/PBSA method). As a result of these analyzes we were able to isolate some characteristics that could lead to mutant peptides with enhanced strontium affinity.     

Sugar-binding and crystallographic studies of an arabinose-binding protein mutant (Met108Leu) that exhibits enhanced affinity and altered specificity

In addition to hydrogen bonds, van der Waals forces contribute to the affinity of protein-carbohydrate interactions. Nonpolar van der Waals contacts in the complexes of the L-arabinose-binding protein (ABP) with monosaccharides have been studied by means of site-directed mutagenesis, equilibrium and rapid kinetic binding techniques, and X-ray crystallography. ABP, a periplasmic transport receptor of Escherichia coli, binds L-arabinose, D-galactose, and D-fucose with preferential affinity in the order of Ara greater than Gal much greater than Fuc. Well-refined, high-resolution structures of ABP complexed with the three sugars revealed that the structural differences in the ABP-sugar complexes are localized around C5 of the sugars, where the equatorial H of Ara has been substituted for CH3 (Fuc) or CH2OH (Gal). The side chain of Met108 undergoes a sterically dictated, ligand-specific, conformational change to optimize nonpolar interactions between its methyl group and the sugar. We found that the Met108Leu ABP binds Gal tighter than wild-type ABP binds Ara and exhibits a preference for ligand in the order of Gal much greater than Fuc greater than Ara. The differences in affinity can be attributed to differences in the dissociation rates of the ABP-sugar complexes. We have refined at better than 1.7-A resolution the crystal structures of the Met108Leu ABP complexed with each of the sugars and offer a molecular explanation for the altered binding properties.     

Energetics of side chain packing in staphylococcal nuclease assessed by systematic double mutant cycles.

All 44 possible double mutant permutations of isoleucine, leucine, and valine were constructed in 11 pairings of six sites in the core of staphylococcal nuclease. The stabilities of these mutants were determined by guanidine hydrochloride denaturation. Comparison of the stabilities of all double mutants with those expected from addition of the corresponding single mutants showed that the effects of the two single mutations are energetically independent of each other in 30 of the double mutants. However, a substantial minority, 14, of the double mutants have stability effects that are not additive. In these cases, it appears that direct van der Waals contacts between the two side chains are present. The requirement of direct van der Waals contact for the interdependence of mutational stability effects is somewhat surprising in light of results previously reported by others. In addition, it was found that double mutants that did not alter or lower the overall number of atoms in the core and that showed nonadditive behavior were more stable than expected from addition of the effects of the corresponding single mutants. A net increase in the number of atoms in the core usually, but not always, resulted in a mutant that was less stable than expected. In contrast to previous staphylococcal nuclease double mutants, energetically significant changes to the denatured state do not appear to be occurring in these packing mutants. These conclusions imply that attempts to engineer protein stability based on single mutant data will be generally successful if overall core size is preserved and if residues are not in van der Waals contact.     

Engineering aluminum binding affinity in an isolated EF-hand from troponin C: a computational site-directed mutagenesis study

Peptides with the ability to specifically bind aluminum would potentially be of great use in the fields of biochemistry and environmental chemistry. Unfortunately no such peptides are known. An aluminum-specific peptide may be used as an in vivo chelator, for metalloprotein design, for understanding metal-ion induced folding and metal-ion trafficking, and as an environmental sensor to monitor metal pollution in the environment. Plants genetically engineered to produce an aluminum binding peptide might be useful in environmental remediation in areas of high free aluminum ion concentration. In this paper, which is the theoretical complement to the experimental work, we analyzed crystallographic structures of EF-hands bound to various metals in order to determine the ligand distances and identities to compare to metal-ion size, charge, electronegativity, and coordination number and performed energy minimization calculations to identify possible mutations. We then constructed various mutant sequences in silico in an isolated EF-hand from troponin C and analyzed their binding behavior using molecular mechanics for binding to Tb(3+) as compared to Al(3+). As a result of these analyses we were able to isolate some characteristics that could lead to mutant peptides with enhanced aluminum activity that we plan to test experimentally in the future. We also performed metal-ion binding studies with the isolated EF-hand used in the computational work to examine the ability of Al(3+) and comparative metals to bind the peptide. In competition studies, the peptide demonstrated preference for Tb(3+) over Al(3+).     

In vitro and in vivo complementation of the Helicobacter pylori arginase mutant using an intergenic chromosomal site

BACKGROUND: Gene complementation strategies are important in validating the roles of genes in specific phenotypes. Complementation systems in Helicobacter pylori include shuttle vectors, which transform H. pylori at relatively low frequencies, and chromosomally based approaches. Chromosomal complementation strategies are susceptible to polar effects and disruption of other H. pylori genes, leading to unwanted pleiotropic effects. MATERIALS AND METHODS: A new complementation strategy was developed for H. pylori by utilizing a suicide plasmid vector that contains fragments of an H. pylori intergenic region (hp0203-hp0204), a chloramphenicol acetyltransferase cassette (cat), and a multiple-cloning site. Genes of interest could be cloned into the intergenic plasmid and the genes integrated into H. pylori by homologous recombination into the intergenic chromosomal region without disrupting any annotated H. pylori gene. The complementation system was validated using the gene encoding arginase (rocF). RESULTS: A rocF mutant unable to hydrolyze or consume l-arginine regained these functions by complementation with the wild-type rocF gene. Complemented strains also had restored arginase protein as determined by Western blot analysis. The complementation system could be successfully applied to multiple H. pylori strains. The intergenic region varied in length and sequence across 17 H. pylori strains, but the flanking-3' ends of the hp0203 and hp0204 coding regions were highly conserved. Inserting a cat cassette and wild-type rocF into the intergenic region did not alter the ability of strain SS1 to colonize mice. CONCLUSIONS: This complementation strategy should greatly facilitate genetic experiments in H. pylori.     

Purification of capping protein using the capping protein binding site of CARMIL as an affinity matrix

Capping protein (CP) is a ubiquitously expressed, heterodimeric actin binding protein that is essential for normal actin dynamics in cells. The existing methods for purifying native CP from tissues and recombinant CP from bacteria are time-consuming processes that involve numerous conventional chromatographic steps and functional assays to achieve a homogeneous preparation of the protein. Here, we report the rapid purification of Acanthamoeba CP from amoeba extracts and recombinant mouse CP from E. coli extracts using as an affinity matrix GST-fusion proteins containing the CP binding site from Acanthamoeba CARMIL and mouse CARMIL-1, respectively. This improved method for CP purification should facilitate the in vitro analysis of CP structure, function, and regulation.     

Analysis of type I signal peptidase affinity and specificity for preprotein substrates

Type I signal peptidases (SPases) are membrane-bound endopeptidases responsible for the catalytic cleavage of signal peptides from secretory proteins. Here, we analysed the interaction between a bacterial type I SPase and preprotein substrates using surface plasmon resonance. The use of a home-made biosensor surface based on a mixed self-assembled monolayer of thiols on gold allowed qualitative and kinetic analysis. In vitro binding of purified preproteins to a covalently immobilised bacterial SPase was found to be rather efficient (apparent K(D)=10(-7)-10(-8)M). The signal peptide was shown to be a prerequisite for SPase binding and the nature of the mature part of the preprotein significantly affected SPase binding affinity. The developed biosensor containing immobilised SPase is of great importance for analysis of specificity at substrate binding level and for drug screening. In fact, this is the first report of a membrane protein that was covalently attached to a biosensor surface and that retained binding capacity.     

Optimization of affinity, specificity and function of designed influenza inhibitors using deep sequencing

We show that comprehensive sequence-function maps obtained by deep sequencing can be used to reprogram interaction specificity and to leapfrog over bottlenecks in affinity maturation by combining many individually small contributions not detectable in conventional approaches. We use this approach to optimize two computationally designed inhibitors against H1N1 influenza hemagglutinin and, in both cases, obtain variants with subnanomolar binding affinity. The most potent of these, a 51-residue protein, is broadly cross-reactive against all influenza group 1 hemagglutinins, including human H2, and neutralizes H1N1 viruses with a potency that rivals that of several human monoclonal antibodies, demonstrating that computational design followed by comprehensive energy landscape mapping can generate proteins with potential therapeutic utility.     

Energetic analysis of an antigen/antibody interface: alanine scanning mutagenesis and double mutant cycles on the HyHEL-10/lysozyme interaction.

Alanine scanning mutagenesis of the HyHEL-10 paratope of the HyHEL-10/HEWL complex demonstrates that the energetically important side chains (hot spots) of both partners are in contact. A plot of deltadeltaG(HyHEL-10_mutant) vs. deltadeltaG(HEWL_mutant) for the five of six interacting side-chain hydrogen bonds is linear (Slope = 1). Only 3 of the 13 residues in the HEWL epitope contribute >4 kcal/mol to the free energy of formation of the complex when replaced by alanine, but 6 of the 12 HyHEL-10 paratope amino acids do. Double mutant cycle analysis of the single crystallographically identified salt bridge, D32H/K97, shows that there is a significant energetic penalty when either partner is replaced with a neutral side-chain amino acid, but the D32(H)N/K97M complex is as stable as the WT. The role of the disproportionately high number of Tyr residues in the CDR was evaluated by comparing the deltadeltaG values of the Tyr --> Phe vs. the corresponding Tyr --> Ala mutations. The nonpolar contacts in the light chain contribute only about one-half of the total deltadeltaG observed for the Tyr --> Ala mutation, while they are significantly more important in the heavy chain. Replacement of the N31L/K96 hydrogen bond with a salt bridge, N31D(L)/K96, destabilizes the complex by 1.4 kcal/mol. The free energy of interaction, deltadeltaG(int), obtained from double mutant cycle analysis showed that deltadeltaG(int) for any complex for which the HEWL residue probed is a major immunodeterminant is very close to the loss of free energy observed for the HyHEL-10 single mutant. Error propagation analysis of double mutant cycles shows that data of atypically high precision are required to use this method meaningfully, except where large deltadeltaG values are analyzed.     

Understanding the relative affinity and specificity of the substrate binding site of protein kinase B for substrate-mimetic inhibitors

Designing selective inhibitor of protein kinase B (PKB/Akt) is an area of intense research to develop potential anticancer drugs. As a general point of strategy, the peptide substrate-binding site only responds to a highly specific sequence of amino acids. Targeting the substrate-mimetic inhibitors to the peptide substrate-binding site has the potential for better selectivity. It is therefore of great interest to understand the peptide substrate binding mode of PKB, as well as its specificity and affinity for different substrate-mimetic inhibitors. In the present study, we used molecular dynamic simulations to better understand the interactions of the PKB substrate-binding site with the substrate-mimetic inhibitors. Our computational models successfully mirrored PKB’s selectivity for the substrate-mimetic inhibitors. Furthermore, the key residues interacting with the substrate-mimetic inhibitor were discussed by analysing the different interaction modes of these inhibitors, with different inhibitory potencies, binding to PKB and by comparing the different binding free energy contributions of corresponding residues around the binding pocket. The pharmacophoric requirements were then also summarised for the substrate-mimetic inhibitor binding to PKB. It is expected that this work will provide useful chemical or biochemical informatics for the design of novel and potent substrate-mimetic inhibitors of PKB.     

Excavating an active site: the nucleobase specificity of ribonuclease A

Ribonuclease A (RNase A) catalyzes the cleavage of RNA after pyrimidine nucleotides. When bound in the active site, the base of a pyrimidine nucleotide forms hydrogen bonds with the side chain of Thr45. Here, the role of Thr45 was probed by using the wild-type enzyme, its T45G variant, X-ray diffraction analysis, and synthetic oligonucleotides as ligands and substrates. Catalytic specificity was determined with the fluorogenic substrate: 6-carboxyfluorescein approximately dArXdAdA approximately 6-carboxytetramethylrhodamine (6-FAM approximately dArXdAdA approximately 6-TAMRA), where X = C, U, A, or G. Wild-type RNase A cleaves 10(6)-fold faster when X = C than when X = A. Likewise, its affinity for the non-hydrolyzable oligonucleotide 6-FAM approximately d(CAA) is 50-fold greater than for 6-FAM approximately d(AAA). T45G RNase A cleaves 6-FAM approximately dArAdAdA approximately 6-TAMRA 10(2)-fold faster than does the wild-type enzyme. The structure of crystalline T45G RNase A, determined at 1.8-A resolution by X-ray diffraction analysis, does not reveal new potential interactions with a nucleobase. Indeed, the two enzymes have a similar affinity for 6-FAM approximately d(AAA). The importance of pentofuranosyl ring conformation to nucleotide specificity was probed with 6-FAM approximately d(AU(F)AA), where U(F) is 2'-deoxy-2'-fluorouridine. The conformation of the pentofuranosyl ring in dU(F) is known to be more similar to that in rU than dU. The affinity of wild-type RNase A for 6-FAM approximately d(AU(F)AA) is 50-fold lower than for 6-FAM approximately d(AUAA). This discrimination is lost in the T45G enzyme. Together, these data indicate that the side chain of Thr45 plays multiple roles-interacting favorably with pyrimidine nucleobases but unfavorably with purine nucleobases. Moreover, a ribose-like ring disfavors the interaction of Thr45 with a pyrimidine nucleobase, suggesting that Thr45 enhances catalysis by ground-state destabilization.     

The role of disorder in RNA binding affinity and specificity

Most RNA-binding modules are small and bind few nucleotides. RNA-binding proteins typically attain the physiological specificity and affinity for their RNA targets by combining several RNA-binding modules. Here, we review how disordered linkers connecting RNA-binding modules govern the specificity and affinity of RNA–protein interactions by regulating the effective concentration of these modules and their relative orientation. RNA-binding proteins also often contain extended intrinsically disordered regions that mediate protein–protein and RNA–protein interactions with multiple partners. We discuss how these regions can connect proteins and RNA resulting in heterogeneous higher-order assemblies such as membrane-less compartments and amyloid-like structures that have the characteristics of multi-modular entities. The assembled state generates additional RNA-binding specificity and affinity properties that contribute to further the function of RNA-binding proteins within the cellular environment.