Curvature of designed armadillo repeat proteins allows modular peptide binding

Designed armadillo repeat proteins (dArmRPs) were developed to create a modular peptide binding technology where each of the structural repeats binds two residues of the target peptide. An essential prerequisite for such a technology is a dArmRP geometry that matches the peptide bond length. To this end, we determined a large set (n = 27) of dArmRP X-ray structures, of which 12 were previously unpublished, to calculate curvature parameters that define their geometry. Our analysis shows that consensus dArmRPs exhibit curvatures close to the optimal range for modular peptide recognition. Binding of peptide ligands can induce a curvature within the desired range, as confirmed by single-molecule FRET experiments in solution. On the other hand, computationally designed ArmRPs, where side chains have been chosen with the intention to optimally fit into a geometrically optimized backbone, turned out to be more divergent in reality, and thus not suitable for continuous peptide binding. Furthermore, we show that the formation of a crystal lattice can induce small but significant deviations from the curvature adopted in solution, which can interfere with the evaluation of repeat protein scaffolds when high accuracy is required. This study corroborates the suitability of consensus dArmRPs as a scaffold for the development of modular peptide binders.     

Crystal structures of designed armadillo repeat proteins: Implications of construct design and crystallization conditions on overall structure

Designed armadillo repeat proteins (dArmRP) are promising modular proteins for the engineering of binding molecules that recognize extended polypeptide chains. We determined the structure of a dArmRP containing five internal repeats and 3rd generation capping repeats in three different states by X‐ray crystallography: without N‐terminal His₆‐tag and in the presence of calcium (YM₅A/Ca²⁺), without N‐terminal His₆‐tag and in the absence of calcium (YM₅A), and with N‐terminal His₆‐tag and in the presence of calcium (His‐YM₅A/Ca²⁺). All structures show different quaternary structures and superhelical parameters. His‐YM₅A/Ca²⁺ forms a crystallographic dimer, which is bridged by the His₆‐tag, YM₅A/Ca²⁺ forms a domain‐swapped tetramer, and only in the absence of calcium and the His₆‐tag, YM₅A forms a monomer. The changes of superhelical parameters are a consequence of calcium binding, because calcium ions interact with negatively charged residues, which can also participate in the modulation of helix dipole moments between adjacent repeats. These observations are important for further optimizations of dArmRPs and provide a general illustration of how construct design and crystallization conditions can influence the exact structure of the investigated protein.     

Modular peptide binding: From a comparison of natural binders to designed armadillo repeat proteins

Several binding scaffolds that are not based on immunoglobulins have been designed as alternatives to traditional monoclonal antibodies. Many of them have been developed to bind to folded proteins, yet cellular networks for signaling and protein trafficking often depend on binding to unfolded regions of proteins. This type of binding can thus be well described as a peptide–protein interaction. In this review, we compare different peptide-binding scaffolds, highlighting that armadillo repeat proteins (ArmRP) offer an attractive modular system, as they bind a stretch of extended peptide in a repeat-wise manner. Instead of generating each new binding molecule by an independent selection, preselected repeats – each complementary to a piece of the target peptide – could be designed and assembled on demand into a new protein, which then binds the prescribed complete peptide. Stacked armadillo repeats (ArmR), each typically consisting of 42 amino acids arranged in three α-helices, build an elongated superhelical structure which enables binding of peptides in extended conformation. A consensus-based design approach, complemented with molecular dynamics simulations and rational engineering, resulted in well-expressed monomeric proteins with high stability. Peptide binders were selected and several structures were determined, forming the basis for the future development of modular peptide-binding scaffolds.     

Structure-based optimization of designed Armadillo-repeat proteins

The armadillo domain is a right‐handed super‐helix of repeating units composed of three α‐helices each. Armadillo repeat proteins (ArmRPs) are frequently involved in protein–protein interactions, and because of their modular recognition of extended peptide regions they can serve as templates for the design of artificial peptide binding scaffolds. On the basis of sequential and structural analyses, different consensus‐designed ArmRPs were synthesized and show high thermodynamic stabilities, compared to naturally occurring ArmRPs. We determined the crystal structures of four full‐consensus ArmRPs with three or four identical internal repeats and two different designs for the N‐ and C‐caps. The crystal structures were refined at resolutions ranging from 1.80 to 2.50 Å for the above mentioned designs. A redesign of our initial caps was required to obtain well diffracting crystals. However, the structures with the redesigned caps caused domain swapping events between the N‐caps. To prevent this domain swap, 9 and 6 point mutations were introduced in the N‐ and C‐caps, respectively. Structural and biophysical analysis showed that this subsequent redesign of the N‐cap prevented domain swapping and improved the thermodynamic stability of the proteins. We systematically investigated the best cap combinations. We conclude that designed ArmRPs with optimized caps are intrinsically stable and well‐expressed monomeric proteins and that the high‐resolution structures provide excellent structural templates for the continuation of the design of sequence‐specific modular peptide recognition units based on armadillo repeats.     

Exploring new strategies for grafting binding peptides onto protein loops using a consensus‐designed tetratricopeptide repeat scaffold

Peptide display approaches, in which peptide epitopes of known binding activities are grafted onto stable protein scaffolds, have been developed to constrain the peptide in its bioactive conformation and to enhance its stability. However, peptide grafting can be a lengthy process requiring extensive computational modeling and/or optimisation by directed evolution techniques. In this study, we show that ultra‐stable consensus‐designed tetratricopeptide repeat (CTPR) proteins are amenable to the grafting of peptides that bind the Kelch‐like ECH‐associated protein 1 (Keap1) onto the loop between adjacent repeats. We explore simple strategies to optimize the grafting process and show that modest improvements in Keap1‐binding affinity can be obtained by changing the composition of the linker sequence flanking either side of the binding peptide.     

Mapping discontinuous protein‐binding sites via structure‐based peptide libraries: combining in silico and in vitro approaches

To perform their various functions, protein surfaces often have to interact with each other in a specific way. Usually, only parts of a protein are accessible and can act as binding sites. Because proteins consist of polypeptide chains that fold into complex three‐dimensional shapes, binding sites can be divided into two different types: linear sites that follow the primary amino acid sequence and discontinuous binding sites, which are made up of short peptide fragments that are adjacent in spatial proximity. Such discontinuous binding sites dominate protein–protein interactions, but are difficult to identify. To meet this challenge, we combined a computational, structure‐based approach and an experimental, high‐throughput method. SUPERFICIAL is a program that uses protein structures as input and generates peptide libraries to represent the protein's surface. A large number of the predicted peptides can be simultaneously synthesised applying the SPOT technology. The results of a binding assay subsequently help to elucidate protein–protein interactions; the approach is applicable to any kind of protein. The crystal structure of the complex of hen egg lysozyme with the well‐characterised murine IgG1 antibody HyHEL‐5 is available, and the complex is known to have a discontinuous binding site. Using SUPERFICIAL, the entire surface of lysozyme was translated into a peptide library that was synthesised on a cellulose membrane using the SPOT technology and tested against the HyHEL‐5 antibody. In this way, it was possible to identify two peptides (longest common sequence and peptide 19) that represented the discontinuous epitope of lysozyme. Copyright © 2012 John Wiley & Sons, Ltd.     

A novel approach to segregate and identify functional loop regions in protein structures using their Ramachandran maps

The loops which connect or flank helices/sheets in protein structures are known to be functionally important. However, ironically they also belong to the part of protein whose structure is least accurately predicted. Here, a new method to isolate and analyze loop regions in protein structure is proposed using the spatial coordinates of the solved three‐dimensional structure. The extent of dispersion among points of successive amino acid residues in the Ramachandran map of protein region is utilized to calculate the Mean Separation between these points in the Ramachandran Plot (MSRP). Based on analysis of 2935 protein secondary structure regions obtained using DSSP software, spanning a range from 2 to 64 residues, taken from a set of 170 proteins, it is shown that helices (MSRP < 17) and strands (MSRP < 64) stand effectively demarcated from the loop regions (MSRP > 130). Analysis of 43 DNA binding and 98 ligand binding proteins revealed several loop regions with clear change in MSRP subsequent to binding. The population of such loops correlated with the magnitude of backbone displacement in the protein subsequent to binding. Can changes in MSRP quantify the temporal oscillations in dihedral angles among structured/unstructured regions in proteins? Molecular dynamics simulations (10 ns) revealed that deviations in MSRP among different snapshots in the trajectory were at least twofold higher for unstructured proteins in comparison with ordered proteins. The above results validate the use of MSRP parameter as a tool to identify and investigate functionally active loops and unstructured regions in protein structures. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Conformational analysis of peptides corresponding to all the secondary structure elements of protein L B1 domain: secondary structure propensities are not conserved in proteins with the same fold.

The solution conformation of three peptides corresponding to the two beta-hairpins and the alpha-helix of the protein L B1 domain have been analyzed by circular dichroism (CD) and nuclear magnetic resonance spectroscopy (NMR). In aqueous solution, the three peptides show low populations of native and non-native locally folded structures, but no well-defined hairpin or helix structures are formed. In 30% aqueous trifluoroethanol (TFE), the peptide corresponding to the alpha-helix adopts a high populated helical conformation three residues longer than in the protein. The hairpin peptides aggregate in TFE, and no significant conformational change occurs in the NMR observable fraction of molecules. These results indicate that the helical peptide has a significant intrinsic tendency to adopt its native structure and that the hairpin sequences seem to be selected as non-helical. This suggests that these sequences favor the structure finally attained in the protein, but the contribution of the local interactions alone is not enough to drive the formation of a detectable population of native secondary structures. This pattern of secondary structure tendencies is different to those observed in two structurally related proteins: ubiquitin and the protein G B1 domain. The only common feature is a certain propensity of the helical segments to form the native structure. These results indicate that for a protein to fold, there is no need for large native-like secondary structure propensities, although a minimum tendency to avoid non-native structures and to favor native ones could be required.     

Flexibility and inhibitor binding in cdc25 phosphatases

Cdc25 phosphatases involved in cell cycle checkpoints are now active targets for the development of anti‐cancer therapies. Rational drug design would certainly benefit from detailed structural information for Cdc25s. However, only apo‐ or sulfate‐bound crystal structures of the Cdc25 catalytic domain have been described so far. Together with previously available crystalographic data, results from molecular dynamics simulations, bioinformatic analysis, and computer‐generated conformational ensembles shown here indicate that the last 30–40 residues in the C‐terminus of Cdc25B are partially unfolded or disordered in solution. The effect of C‐terminal flexibility upon binding of two potent small molecule inhibitors to Cdc25B is then analyzed by using three structural models with variable levels of flexibility, including an equilibrium distributed ensemble of Cdc25B backbone conformations. The three Cdc25B structural models are used in combination with flexible docking, clustering, and calculation of binding free energies by the linear interaction energy approximation to construct and validate Cdc25B‐inhibitor complexes. Two binding sites are identified on top and beside the Cdc25B active site. The diversity of interaction modes found increases with receptor flexibility. Backbone flexibility allows the formation of transient cavities or compact hydrophobic units on the surface of the stable, folded protein core that are unexposed or unavailable for ligand binding in rigid and densely packed crystal structures. The present results may help to speculate on the mechanisms of small molecule complexation to partially unfolded or locally disordered proteins. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Efficient identification of tubby‐binding proteins by an improved system of T7 phage display

Mutation in the tubby gene causes adult‐onset obesity, progressive retinal, and cochlear degeneration with unknown mechanism. In contrast, mutations in tubby‐like protein 1 (Tulp1), whose C‐terminus is highly homologous to tubby, only lead to retinal degeneration. We speculate that their diverse N‐terminus may define their distinct disease profile. To elucidate the binding partners of tubby, we used tubby N‐terminus (tubby‐N) as bait to identify unknown binding proteins with open‐reading‐frame (ORF) phage display. T7 phage display was engineered with three improvements: high‐quality ORF phage display cDNA library, specific phage elution by protease cleavage, and dual phage display for sensitive high throughput screening. The new system is capable of identifying unknown bait‐binding proteins in as fast as ～4–7 days. While phage display with conventional cDNA libraries identifies high percentage of out‐of‐frame unnatural short peptides, all 28 tubby‐N‐binding clones identified by ORF phage display were ORFs. They encode 16 proteins, including 8 nuclear proteins. Fourteen proteins were analyzed by yeast two‐hybrid assay and protein pull‐down assay with ten of them independently verified. Comparative binding analyses revealed several proteins binding to both tubby and Tulp1 as well as one tubby‐specific binding protein. These data suggest that tubby‐N is capable of interacting with multiple nuclear and cytoplasmic protein binding partners. These results demonstrated that the newly‐engineered ORF phage display is a powerful technology to identify unknown protein–protein interactions. Copyright © 2009 John Wiley & Sons, Ltd.     

Two distinct classes of cochaperones compete for the EEVD motif in heat shock protein 70 to tune its chaperone activities

Chaperones of the heat shock protein 70 (Hsp70) family engage in protein–protein interactions with many cochaperones. One “hotspot” for cochaperone binding is the EEVD motif, found at the extreme C terminus of cytoplasmic Hsp70s. This motif is known to bind tetratricopeptide repeat domain cochaperones, such as the E3 ubiquitin ligase CHIP. In addition, the EEVD motif also interacts with a structurally distinct domain that is present in class B J-domain proteins, such as DnaJB4. These observations suggest that CHIP and DnaJB4 might compete for binding to Hsp70’s EEVD motif; however, the molecular determinants of such competition are not clear. Using a collection of EEVD-derived peptides, including mutations and truncations, we explored which residues are critical for binding to both CHIP and DnaJB4. These results revealed that some features, such as the C-terminal carboxylate, are important for both interactions. However, CHIP and DnaJB4 also had unique preferences, especially at the isoleucine position immediately adjacent to the EEVD. Finally, we show that competition between these cochaperones is important in vitro, as DnaJB4 limits the ubiquitination activity of the Hsp70–CHIP complex, whereas CHIP suppresses the client refolding activity of the Hsp70–DnaJB4 complex. Together, these data suggest that the EEVD motif has evolved to support diverse protein–protein interactions, such that competition between cochaperones may help guide whether Hsp70-bound proteins are folded or degraded.     

Structural properties of carnation mottle virus p7 movement protein and its RNA-binding domain

Plant viral movement proteins (MPs) participate actively in the intra- and intercellular movement of RNA plant viruses to such an extent that MP dysfunction impairs viral infection. However, the molecular mechanism(s) of their interaction with cognate nucleic acids are not well understood, partly due to the lack of structural information. In this work, a protein dissection approach was used to gain information on the structural and RNA-binding properties of this class of proteins, as exemplified by the 61-amino acid residue p7 MP from carnation mottle virus (CarMV). Circular dichroism spectroscopy showed that CarMV p7 is an alpha/beta RNA-binding soluble protein. Using synthetic peptides derived from the p7 sequence, we have identified three distinct putative domains within the protein. EMSA showed that the central region, from residue 17 to 35 (represented by peptide p7(17-35)), is responsible for the RNA binding properties of CarMV p7. This binding peptide populates a nascent alpha-helix in water solution that is further stabilized in the presence of either secondary structure inducers, such as trifluoroethanol and monomeric SDS, or RNA (which also changes its conformation upon binding to the peptide). Thus, the RNA recognition appears to occur via an "adaptive binding" mechanism. Interestingly, the amino acid sequence and structural properties of the RNA-binding domain of p7 seem to be conserved among carmoviruses and some other RNA-binding proteins and peptides. The low conserved N terminus of p7 (peptide p7(1-16)) is unstructured in solution. In contrast, the highly conserved C terminus motif (peptide p7(40-61)) adopts a beta-sheet conformation in aqueous solution. Alanine scanning mutagenesis of the RNA-binding motif showed how selected positive charged amino acids are more relevant than others in the RNA binding process and how hydrophobic amino acid side chains would participate in the stabilization of the protein-RNA complex.     

Dual display of proteins on the yeast cell surface simplifies quantification of binding interactions and enzymatic bioconjugation reactions

Yeast surface display, a well‐established technology for protein analysis and engineering, involves expressing a protein of interest as a genetic fusion to either the N‐ or C‐terminus of the yeast Aga2p mating protein. Historically, yeast‐displayed protein variants are flanked by peptide epitope tags that enable flow cytometric measurement of construct expression using fluorescent primary or secondary antibodies. Here, we built upon this technology to develop a new yeast display strategy that comprises fusion of two different proteins to Aga2p, one to the N‐terminus and one to the C‐terminus. This approach allows an antibody fragment, ligand, or receptor to be directly coupled to expression of a fluorescent protein readout, eliminating the need for antibody‐staining of epitope tags to quantify yeast protein expression levels. We show that this system simplifies quantification of protein‐protein binding interactions measured on the yeast cell surface. Moreover, we show that this system facilitates co‐expression of a bioconjugation enzyme and its corresponding peptide substrate on the same Aga2p construct, enabling enzyme expression and catalytic activity to be measured on the surface of yeast.     

Is Asp‐His‐Ser/Thr‐Trp tetrad hydrogen‐bond network important to WD40‐repeat proteins: A statistical and theoretical study

WD40‐repeat proteins are abundant and play important roles in forming protein complexes. The domain usually has seven WD40 repeats, which folds into a seven β‐sheet propeller with each β‐sheet in a four‐strand structure. An analysis of 20 available WD40‐repeat proteins in Protein Data Bank reveals that each protein has at least one Asp‐His‐Ser/Thr‐Trp (D‐H‐S/T‐W) hydrogen‐bonded tetrad, and some proteins have up to six or seven such tetrads. The relative positions of the four residues in the tetrads are also found to be conserved. A sequence alignment analysis of 560 WD40‐repeat protein sequences in human reveals very similar features, indicating that such tetrad may be a general feature of WD40‐repeat proteins. We carried out density functional theory and found that these tetrads can lead to significant stabilization including hydrogen‐bonding cooperativity. The hydrogen bond involving Trp is significant. These results lead us to propose that the tetrads may be critical to the stability and the mechanism of folding of these proteins. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Coiled coils at the edge of configurational heterogeneity. Structural analyses of parallel and antiparallel homotetrameric coiled coils reveal configurational sensitivity to a single solvent-exposed amino acid substitution

A detailed understanding of the mechanisms by which particular amino acid sequences can give rise to more than one folded structure, such as for proteins that undergo large conformational changes or misfolding, is a long-standing objective of protein chemistry. Here, we describe the crystal structures of a single coiled-coil peptide in distinct parallel and antiparallel tetrameric configurations and further describe the parallel or antiparallel crystal structures of several related peptide sequences; the antiparallel tetrameric assemblies represent the first crystal structures of GCN4-derived peptides exhibiting such a configuration. Intriguingly, substitution of a single solvent-exposed residue enabled the parallel coiled-coil tetramer GCN4-pLI to populate the antiparallel configuration, suggesting that the two configurations are close enough in energy for subtle sequence changes to have important structural consequences. We present a structural analysis of the small changes to the helix register and side-chain conformations that accommodate the two configurations and have supplemented these results using solution studies and a molecular dynamics energetic analysis using a replica exchange methodology. Considering the previous examples of structural nonspecificity in coiled-coil peptides, the findings reported here not only emphasize the predisposition of the coiled-coil motif to adopt multiple configurations but also call attention to the associated risk that observed crytstal structures may not represent the only (or even the major) species present in solution.     

Comparison of NMR and crystal structures of membrane proteins and computational refinement to improve model quality

Membrane proteins are challenging to study and restraints for structure determination are typically sparse or of low resolution because the membrane environment that surrounds them leads to a variety of experimental challenges. When membrane protein structures are determined by different techniques in different environments, a natural question is “which structure is most biologically relevant?” Towards answering this question, we compiled a dataset of membrane proteins with known structures determined by both solution NMR and X‐ray crystallography. By investigating differences between the structures, we found that RMSDs between crystal and NMR structures are below 5 Å in the membrane region, NMR ensembles have a higher convergence in the membrane region, crystal structures typically have a straighter transmembrane region, have higher stereo‐chemical correctness, and are more tightly packed. After quantifying these differences, we used high‐resolution refinement of the NMR structures to mitigate them, which paves the way for identifying and improving the structural quality of membrane proteins.     

Quantitative analysis and prediction of curvature in leucine‐rich repeat proteins

Leucine‐rich repeat (LRR) proteins form a large and diverse family. They have a wide range of functions most of which involve the formation of protein–protein interactions. All known LRR structures form curved solenoids, although there is large variation in their curvature. It is this curvature that determines the shape and dimensions of the inner space available for ligand binding. Unfortunately, large‐scale parameters such as the overall curvature of a protein domain are extremely difficult to predict. Here, we present a quantitative analysis of determinants of curvature of this family. Individual repeats typically range in length between 20 and 30 residues and have a variety of secondary structures on their convex side. The observed curvature of the LRR domains correlates poorly with the lengths of their individual repeats. We have, therefore, developed a scoring function based on the secondary structure of the convex side of the protein that allows prediction of the overall curvature with a high degree of accuracy. We also demonstrate the effectiveness of this method in selecting a suitable template for comparative modeling. We have developed an automated, quantitative protocol that can be used to predict accurately the curvature of leucine‐rich repeat proteins of unknown structure from sequence alone. This protocol is available as an online resource at http://www.bioinf.manchester.ac.uk/curlrr/ . Proteins 2009. © 2009 Wiley‐Liss, Inc.