Designed armadillo repeat proteins as general peptide-binding scaffolds: consensus design and computational optimization of the hydrophobic core

Armadillo repeat proteins are abundant eukaryotic proteins involved in several cellular processes, including signaling, transport, and cytoskeletal regulation. They are characterized by an armadillo domain, composed of tandem armadillo repeats of approximately 42 amino acids, which mediates interactions with peptides or parts of proteins in extended conformation. The conserved binding mode of the peptide in extended form, observed for different targets, makes armadillo repeat proteins attractive candidates for the generation of modular peptide-binding scaffolds. Taking advantage of the large number of repeat sequences available, a consensus-based approach combined with a force field-based optimization of the hydrophobic core was used to derive soluble, highly expressed, stable, monomeric designed proteins with improved characteristics compared to natural armadillo proteins. These sequences constitute the starting point for the generation of designed armadillo repeat protein libraries for the selection of peptide binders, exploiting their modular structure and their conserved binding mode.     

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.     

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.     

Structures of designed armadillo repeat proteins binding to peptides fused to globular domains

Designed armadillo repeat proteins (dArmRP) are α‐helical solenoid repeat proteins with an extended peptide binding groove that were engineered to develop a generic modular technology for peptide recognition. In this context, the term “peptide” not only denotes a short unstructured chain of amino acids, but also an unstructured region of a protein, as they occur in termini, loops, or linkers between folded domains. Here we report two crystal structures of dArmRPs, in complex with peptides fused either to the N‐terminus of Green Fluorescent Protein or to the C‐terminus of a phage lambda protein D. These structures demonstrate that dArmRPs bind unfolded peptides in the intended conformation also when they constitute unstructured parts of folded proteins, which greatly expands possible applications of the dArmRP technology. Nonetheless, the structures do not fully reflect the binding behavior in solution, that is, some binding sites remain unoccupied in the crystal and even unexpected peptide residues appear to be bound. We show how these differences can be explained by restrictions of the crystal lattice or the composition of the crystallization solution. This illustrates that crystal structures have to be interpreted with caution when protein–peptide interactions are characterized, and should always be correlated with measurements in solution.     

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.     

Unique peptide substrate binding properties of 110-kDa heat-shock protein (Hsp110) determine its distinct chaperone activity

The molecular chaperone 70-kDa heat-shock proteins (Hsp70s) play essential roles in maintaining protein homeostasis. Hsp110, an Hsp70 homolog, is highly efficient in preventing protein aggregation but lacks the hallmark folding activity seen in Hsp70s. To understand the mechanistic differences between these two chaperones, we first characterized the distinct peptide substrate binding properties of Hsp110s. In contrast to Hsp70s, Hsp110s prefer aromatic residues in their substrates, and the substrate binding and release exhibit remarkably fast kinetics. Sequence and structure comparison revealed significant differences in the two peptide-binding loops: the length and properties are switched. When we swapped these two loops in an Hsp70, the peptide binding properties of this mutant Hsp70 were converted to Hsp110-like, and more impressively, it functionally behaved like an Hsp110. Thus, the peptide substrate binding properties implemented in the peptide-binding loops may determine the chaperone activity differences between Hsp70s and Hsp110s.     

The lid subdomain of DnaK is required for the stabilization of the substrate-binding site

We examined the effect of deletion of different segments in the helical subdomain (the so-called "lid") of the DnaK peptide-binding domain on peptide binding and protein stability. At 25 degrees C, wt DnaK and the deletion mutant proteins are able to stably bind peptides with similar affinity. However, at physiological (37 degrees C) and stress (42 degrees C) temperatures, removal of the N-terminal half of alphaB and the rest of the lid drastically decreases the ability of the protein to bind substrates. Differential scanning calorimetry and infrared spectroscopy show that this behavior is accompanied by destabilization of the peptide-binding domain. Our data suggest that the reversible interaction between the lid and beta-sandwich subdomains of DnaK peptide-binding domain is required for the stabilization of the loops that form the peptide-binding site, which in turn modulates the protein affinity for peptide substrates. This interaction might have functional implications because it could prevent rebinding of the peptide substrate, which would be forced to fold.     

Engineering repeat proteins of the immune system

Limitations associated with immunoglobulins have motivated the search for novel binding scaffolds. Repeat proteins have emerged as one promising class of scaffolds, but often are limited to binding protein and peptide targets. An exception is the repeat proteins of the immune system, which have in recent years served as an inspiration for binding scaffolds which can bind glycans and other classes of biomolecule. Like other repeat proteins, these proteins can be very stable and have a monomeric mode of binding, with elongated and highly variable binding surfaces. The ability to target glycans and glycoproteins fill an important gap in current tools for research and biomedical applications.     

Structure of Pumilio reveals similarity between RNA and peptide binding motifs

Translation regulation plays an essential role in the differentiation and development of animal cells. One well-studied case is the control of hunchback mRNA during early Drosophila embryogenesis by the trans-acting factors Pumilio, Nanos, and Brain Tumor. We report here a crystal structure of the critical region of Pumilio, the Puf domain, that organizes a multivalent repression complex on the 3' untranslated region of hunchback mRNA. The structure reveals an extended, rainbow shaped molecule, with tandem helical repeats that bear unexpected resemblance to the armadillo repeats in beta-catenin and the HEAT repeats in protein phosphatase 2A. Based on the structure and genetic experiments, we identify putative interaction surfaces for hunchback mRNA and the cofactors Nanos and Brain Tumor. This analysis suggests that similar features in helical repeat proteins are used to bind extended peptides and RNA.     

Radicicol-sensitive peptide binding to the N-terminal portion of GRP94

GRP94 is a molecular chaperone that carries immunologically relevant peptides from cell to cell, transferring them to major histocompatibility proteins for presentation to T cells. Here we examine the binding of several peptides to recombinant GRP94 and study the regulation and site of peptide binding. We show that GRP94 contains a peptide-binding site in its N-terminal 355 amino acids. A number of peptides bind to this site with low on- and off-rates and with specificity that is distinct from that of another endoplasmic reticulum chaperone, BiP/GRP78. Binding to the N-terminal fragment is sufficient to account for the peptide binding activity of the entire molecule. Peptide binding is inhibited by radicicol, a known inhibitor of the chaperone activities of HSP90-family proteins. However, the peptide-binding site is distinct from the radicicol-binding pocket, because both can bind to the N-terminal fragment simultaneously. Furthermore, peptide binding does not cause the same conformational change as does binding of radicicol. When the latter binds to the N-terminal domain, it induces a conformational change in the downstream, acidic domain of GRP94, as measured by altered gel mobility and loss of an antibody epitope. These results relate the peptide-binding activity of GRP94 to its other function as a chaperone.     

The N-terminal domain of Homer/Vesl is a new class II EVH1 domain

Cellular activities controlled by signal transduction processes such as cell motility and cell growth depend on the tightly regulated assembly of multiprotein complexes. Adapter proteins that specifically interact with their target proteins are key components required for the formation of these assemblies. Ena/VASP-homology 1 (EVH1) domains are small constituents of large modular proteins involved in microfilament assembly that specifically recognize proline-rich regions. EVH1 domain-containing proteins are present in neuronal cells, like the Homer/Vesl protein family that is involved in memory-generating processes. Here, we describe the crystal structure of the murine EVH1 domain of Vesl 2 at 2.2 A resolution. The small globular protein consists of a seven-stranded antiparallel beta-barrel with a C-terminal alpha-helix packing alongside the barrel. A shallow groove running parallel with beta-strand VI forms an extended peptide-binding site. Using peptide library screenings, we present data that demonstrate the high affinity of the Vesl 2 EVH1 domain towards peptide sequences containing a proline-rich core sequence (PPSPF) that requires additional charged amino acid residues on either side for specific binding. Our functional data, substantiated by structural data, demonstrate that the ligand-binding of the Vesl EVH1 domain differs from the interaction characteristics of the previously examined EVH1 domains of the Evl/Mena proteins. Analogous to the Src homology 3 (SH3) domains that bind their cognate ligands in two distinct directions, we therefore propose the existence of two distinct classes of EVH1 domains.     

High-throughput analysis of peptide-binding modules

Modular protein interaction domains (PIDs) that recognize linear peptide motifs are found in hundreds of proteins within the human genome. Some PIDs such as SH2, 14-3-3, Chromo, and Bromo domains serve to recognize posttranslational modification (PTM) of amino acids (such as phosphorylation, acetylation, methylation, etc.) and translate these into discrete cellular responses. Other modules such as SH3 and PSD-95/Discs-large/ZO-1 (PDZ) domains recognize linear peptide epitopes and serve to organize protein complexes based on localization and regions of elevated concentration. In both cases, the ability to nucleate-specific signaling complexes is in large part dependent on the selectivity of a given protein module for its cognate peptide ligand. High-throughput (HTP) analysis of peptide-binding domains by peptide or protein arrays, phage display, mass spectrometry, or other HTP techniques provides new insight into the potential protein-protein interactions prescribed by individual or even whole families of modules. Systems level analyses have also promoted a deeper understanding of the underlying principles that govern selective protein-protein interactions and how selectivity evolves. Lastly, there is a growing appreciation for the limitations and potential pitfalls associated with HTP analysis of protein-peptide interactomes. This review will examine some of the common approaches utilized for large-scale studies of PIDs and suggest a set of standards for the analysis and validation of datasets from large-scale studies of peptide-binding modules. We will also highlight how data from large-scale studies of modular interaction domain families can provide insight into systems level properties such as the linguistics of selective interactions.     

Topological characteristics of helical repeat proteins.

The recent elucidation of protein structures based upon repeating amino acid motifs, including the armadillo motif, the HEAT motif and tetratricopeptide repeats, reveals that they belong to the class of helical repeat proteins. These proteins share the common property of being assembled from tandem repeats of an alpha-helical structural unit, creating extended superhelical structures that are ideally suited to create a protein recognition interface.     

Elucidation of the binding preferences of peptide recognition modules: SH3 and PDZ domains

Peptide-binding domains play a critical role in regulation of cellular processes by mediating protein interactions involved in signalling. In recent years, the development of large-scale technologies has enabled exhaustive studies on the peptide recognition preferences for a number of peptide-binding domain families. These efforts have provided significant insights into the binding specificities of these modular domains. Many research groups have taken advantage of this unprecedented volume of specificity data and have developed a variety of new algorithms for the prediction of binding specificities of peptide-binding domains and for the prediction of their natural binding targets. This knowledge has also been applied to the design of synthetic peptide-binding domains in order to rewire protein-protein interaction networks. Here, we describe how these experimental technologies have impacted on our understanding of peptide-binding domain specificities and on the elucidation of their natural ligands. We discuss SH3 and PDZ domains as well characterized examples, and we explore the feasibility of expanding high-throughput experiments to other peptide-binding domains.     

Protein repeats: structures, functions, and evolution

Internal repetition within proteins has been a successful strategem on multiple separate occasions throughout evolution. Such protein repeats possess regular secondary structures and form multirepeat assemblies in three dimensions of diverse sizes and functions. In general, however, internal repetition affords a protein enhanced evolutionary prospects due to an enlargement of its available binding surface area. Constraints on sequence conservation appear to be relatively lax, due to binding functions ensuing from multiple, rather than, single repeats. Considerable sequence divergence as well as the short lengths of sequence repeats mean that repeat detection can be a particularly arduous task. We also consider the conundrum of how multiple repeats, which show strong structural and functional interdependencies, ever evolved from a single repeat ancestor. In this review, we illustrate each of these points by referring to six prolific repeat types (repeats in beta-propellers and beta-trefoils and tetratricopeptide, ankyrin, armadillo/HEAT, and leucine-rich repeats) and in other less-prolific but nonetheless interesting repeats.     

A combinatorial amino Acid code for RNA recognition by pentatricopeptide repeat proteins

The pentatricopeptide repeat (PPR) is a helical repeat motif found in an exceptionally large family of RNA-binding proteins that functions in mitochondrial and chloroplast gene expression. PPR proteins harbor between 2 and 30 repeats and typically bind single-stranded RNA in a sequence-specific fashion. However, the basis for sequence-specific RNA recognition by PPR tracts has been unknown. We used computational methods to infer a code for nucleotide recognition involving two amino acids in each repeat, and we validated this model by recoding a PPR protein to bind novel RNA sequences in vitro. Our results show that PPR tracts bind RNA via a modular recognition mechanism that differs from previously described RNA-protein recognition modes and that underpins a natural library of specific protein/RNA partners of unprecedented size and diversity. These findings provide a significant step toward the prediction of native binding sites of the enormous number of PPR proteins found in nature. Furthermore, the extraordinary evolutionary plasticity of the PPR family suggests that the PPR scaffold will be particularly amenable to redesign for new sequence specificities and functions.     

Structure of the armadillo repeat domain of plakophilin 1

The p120ctn subfamily of armadillo domain proteins has roles in modulating intercellular adhesion by cadherin-containing junctions. We have determined the crystal structure of the arm repeat domain from plakophilin-1 (PKP1), a member of the p120ctn subfamily that is found in desmosomes. The structure reveals that the domain has nine instead of the expected ten arm repeats. A sequence predicted to be an arm repeat is instead a large insert which serves as a wedge that produces a significant bend in the overall domain structure. Structure-based sequence alignments indicate that the nine repeats and large insert are common to this subfamily of armadillo proteins. A prominent basic patch on the surface of the protein may serve as a binding site for partners of these proteins.