Designing repeat proteins: well-expressed,soluble and stable proteins from combinatorial libraries of consensus ankyrin repeat proteins

We describe an efficient way to generate combinatorial libraries of stable, soluble and well-expressed ankyrin repeat (AR) proteins. Using a combination of sequence and structure consensus analyses, we designed a 33 amino acid residue AR module with seven randomized positions having a theoretical diversity of 7.2x10(7). Different numbers of this module were cloned between N and C-terminal capping repeats, i.e. ARs designed to shield the hydrophobic core of stacked AR modules. In this manner, combinatorial libraries of designed AR proteins consisting of four to six repeats were generated, thereby potentiating the theoretical diversity. All randomly chosen library members were expressed in soluble form in the cytoplasm of Escherichia coli in amounts up to 200 mg per 1 l of shake-flask culture. Virtually pure proteins were obtained in a single purification step. The designed AR proteins are monomeric and display CD spectra identical with those of natural AR proteins. At the same time, our AR proteins are highly thermostable, with T(m) values ranging from 66 degrees C to well above 85 degrees C. Thus, our combinatorial library members possess the properties required for biotechnological applications. Moreover, the favorable biophysical properties and the modularity of the AR fold may account, partly, for the abundance of natural AR proteins.     

A novel strategy to design binding molecules harnessing the modular nature of repeat proteins

Repeat proteins, such as ankyrin or leucine-rich repeat proteins, are ubiquitous binding molecules, which occur, unlike antibodies, intra- and extracellularly. Their unique modular architecture features repeating structural units (repeats), which stack together to form elongated repeat domains displaying variable and modular target-binding surfaces. Based on this modularity, we developed a novel strategy to generate combinatorial libraries of polypeptides with highly diversified binding specificities. This strategy includes the consensus design of self-compatible repeats displaying variable surface residues and their random assembly into repeat domains. We envision that such repeat protein libraries will be highly valuable sources for novel binding molecules especially suitable for intracellular applications.     

Designing repeat proteins: modular leucine-rich repeat protein libraries based on the mammalian ribonuclease inhibitor family

We present a novel approach to design repeat proteins of the leucine-rich repeat (LRR) family for the generation of libraries of intracellular binding molecules. From an analysis of naturally occurring LRR proteins, we derived the concept to assemble repeat proteins with randomized surface positions from libraries of consensus repeat modules. As a guiding principle, we used the mammalian ribonuclease inhibitor (RI) family, which comprises cytosolic LRR proteins known for their extraordinary affinities to many RNases. By aligning the amino acid sequences of the internal repeats of human, pig, rat, and mouse RI, we derived a first consensus sequence for the characteristic alternating 28 and 29 amino acid residue A-type and B-type repeats. Structural considerations were used to replace all conserved cysteine residues, to define less conserved positions, and to decide where to introduce randomized amino acid residues. The so devised consensus RI repeat library was generated at the DNA level and assembled by stepwise ligation to give libraries of 2-12 repeats. Terminal capping repeats, known to shield the continuous hydrophobic core of the LRR domain from the surrounding solvent, were adapted from human RI. In this way, designed LRR protein libraries of 4-14 LRRs (equivalent to 130-415 amino acid residues) were obtained. The biophysical analysis of randomly chosen library members showed high levels of soluble expression in the Escherichia coli cytosol, monomeric behavior as characterized by gel-filtration, and alpha-helical CD spectra, confirming the success of our design approach.     

Folding of a designed simple ankyrin repeat protein

Ankyrin repeats (AR) are 33-residue motifs containing a beta-turn, followed by two alpha-helices connected by a loop. AR occur in tandem arrangements and stack side-by-side to form elongated domains involved in very different cellular tasks. Recently, consensus libraries of AR repeats were constructed. Protein E1_5 represents a member of the shortest library, and consists of only a single consensus repeat flanked by designed N- and C-terminal capping repeats. Here we present a biophysical characterization of this AR domain. The protein is compactly folded, as judged from the heat capacity of the native state and from the specific unfolding enthalpy and entropy. From spectroscopic data, thermal and urea-induced unfolding can be modeled by a two-state transition. However, scanning calorimetry experiments reveal a deviation from the two-state behavior at elevated temperatures. Folding and unfolding at 5 degrees C both follow monoexponential kinetics with k(folding) = 28 sec(-1) and k(unfolding) = 0.9 sec(-1). Kinetic and equilibrium unfolding parameters at 5 degrees C agree very well. We conclude that E1_5 folds in a simple two-state manner at low temperatures while equilibrium intermediates become populated at higher temperatures. A chevron-plot analysis indicates that the protein traverses a very compact transition state along the folding/unfolding pathway. This work demonstrates that a designed minimal ankyrin repeat protein has the thermodynamic and kinetic properties of a compactly folded protein, and explains the favorable properties of the consensus framework.     

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.     

Dynamic combinatorial libraries of artificial repeat proteins

Repeat proteins are found in almost all cellular systems, where they are involved in diverse molecular recognition processes. Recent studies have suggested that de novo designed repeat proteins may serve as universal binders, and might potentially be used as practical alternative to antibodies. We describe here a novel chemical methodology for producing small libraries of repeat proteins, and screening in parallel the ligand binding of library members. The first stage of this research involved the total synthesis of a consensus-based three-repeat tetratricopeptide (TPR) protein (～14 kDa), via sequential attachment of the respective peptides. Despite the effectiveness of the synthesis and ligation steps, this method was found to be too demanding for the production of proteins containing variable number of repeats. Additionally, the analysis of binding of the individual proteins was time consuming. Therefore, we designed and prepared novel dynamic combinatorial libraries (DCLs), and show that their equilibration can facilitate the formation of TPR proteins containing up to eight repeating units. Interestingly, equilibration of the library building blocks in the presence of the biologically relevant ligands, Hsp90 and Hsp70, induced their oligomerization into forming more of the proteins with large recognition surfaces. We suggest that this work presents a novel simple and rapid tool for the simultaneous screening of protein mixtures with variable binding surfaces, and for identifying new binders for ligands of interest.     

Characterization and further stabilization of designed ankyrin repeat proteins by combining molecular dynamics simulations and experiments

Multiple molecular dynamics simulations with explicit solvent at room temperature and at 400 K were carried out to characterize designed ankyrin repeat (AR) proteins with full-consensus repeats. Using proteins with one to five repeats, the stability of the native structure was found to increase with the number of repeats. The C-terminal capping repeat, originating from the natural guanine-adenine-binding protein, was observed to denature first in almost all high-temperature simulations. Notably, a stable intermediate is found in experimental equilibrium unfolding studies of one of the simulated consensus proteins. On the basis of simulation results, this intermediate is interpreted to represent a conformation with a denatured C-terminal repeat. To validate this interpretation, constructs without C-terminal capping repeat were prepared and did not show this intermediate in equilibrium unfolding experiments. Conversely, the capping repeats were found to be essential for efficient folding in the cell and for avoiding aggregation, presumably because of their highly charged surface. To design a capping repeat conferring similar solubility properties yet even higher stability, eight point mutations adapting the C-cap to the consensus AR and adding a three-residue extension at the C-terminus were predicted in silico and validated experimentally. The in vitro full-consensus proteins were also compared with a previously published designed AR protein, E3_5, whose internal repeats show 80% identity in primary sequence. A detailed analysis of the simulations suggests that networks of salt bridges between β-hairpins, as well as additional interrepeat hydrogen bonds, contribute to the extraordinary stability of the full consensus.     

Folding landscapes of ankyrin repeat proteins: experiments meet theory

Nearly 6% of eukaryotic protein sequences contain ankyrin repeat (AR) domains, which consist of several repeats and often function in binding. AR proteins show highly cooperative folding despite a lack of long-range contacts. Both theory and experiment converge to explain that formation of the interface between elements is more favorable than formation of any individual repeat unit. IkappaBalpha and Notch both undergo partial folding upon binding perhaps influencing the binding free energy. The simple architecture, combined with identification of consensus residues that are important for stability, has enabled systematic perturbation of the energy landscape by single point mutations that affect stability or by addition of consensus repeats. The folding energy landscapes appear highly plastic, with small perturbations re-routing folding pathways.     

The telobox, a Myb-related telomeric DNA binding motif found in proteins from yeast, plants and human.

The yeast TTAGGG binding factor 1 (Tbf1) was identified and cloned through its ability to interact with vertebrate telomeric repeats in vitro. We show here that a sequence of 60 amino acids located in its C-terminus is critical for DNA binding. This sequence exhibits homologies with Myb repeats and is conserved among five proteins from plants, two of which are known to bind telomeric-related sequences, and two proteins from human, including the telomeric repeat binding factor (TRF) and the predicted C-terminal polypeptide, called orf2, from a yet unknown protein. We demonstrate that the 111 C-terminal residues of TRF and the 64 orf2 residues are able to bind the human telomeric repeats specifically. We propose to call the particular Myb-related motif found in these proteins the 'telobox'. Antibodies directed against the Tbf1 telobox detect two proteins in nuclear and mitotic chromosome extracts from human cell lines. Moreover, both proteins bind specifically to telomeric repeats in vitro. TRF is likely to correspond to one of them. Based on their high affinity for the telomeric repeat, we predict that TRF and orf2 play an important role at human telomeres.     

Allosteric inhibition of aminoglycoside phosphotransferase by a designed ankyrin repeat protein

Aminoglycoside phosphotransferase (3')-IIIa (APH) is a bacterial kinase that confers antibiotic resistance to many pathogenic bacteria and shares structural homology with eukaryotic protein kinases. We report here the crystal structure of APH, trapped in an inactive conformation by a tailor-made inhibitory ankyrin repeat (AR) protein, at 2.15 A resolution. The inhibitor was selected from a combinatorial library of designed AR proteins. The AR protein binds the C-terminal lobe of APH and thereby stabilizes three alpha helices, which are necessary for substrate binding, in a significantly displaced conformation. BIAcore analysis and kinetic enzyme inhibition experiments are consistent with the proposed allosteric inhibition mechanism. In contrast to most small-molecule kinase inhibitors, the AR proteins are not restricted to active site binding, allowing for higher specificity. Inactive conformations of pharmaceutically relevant enzymes, as can be elucidated with the approach presented here, represent powerful starting points for rational drug design.     

A hot-spot motif characterizes the interface between a designed ankyrin-repeat protein and its target ligand

Nonantibody scaffolds such as designed ankyrin repeat proteins (DARPins) can be rapidly engineered to detect diverse target proteins with high specificity and offer an attractive alternative to antibodies. Using molecular simulations, we predicted that the binding interface between DARPin off7 and its ligand (maltose binding protein; MBP) is characterized by a hot-spot motif in which binding energy is largely concentrated on a few amino acids. To experimentally test this prediction, we fused MBP to a transmembrane domain to properly orient the protein into a polymer-cushioned lipid bilayer, and characterized its interaction with off7 using force spectroscopy. Using this, to our knowledge, novel technique along with surface plasmon resonance, we validated the simulation predictions and characterized the effects of select mutations on the kinetics of the off7-MBP interaction. Our integrated approach offers scientific insights on how the engineered protein interacts with the target molecule.     

Designed ankyrin repeat proteins as scaffolds for multivalent recognition

Ankyrin repeat (AR) proteins are composed of tandem repeats of a basic structural motif of ca. 33 amino acid residues that form a β-turn followed by two antiparallel α-helices. Multiple repeats stack together in a modular fashion to form a scaffold that is ideally suited for the presentation of multiple functional groups and/or recognition elements. Here we describe a biosynthetic strategy that takes advantage of the modular nature of these proteins to generate multivalent ligands that are both chemically homogeneous and structurally well-defined. Glycosylated AR proteins cluster the tetrameric lectin concanavalin A (Con A) at a rate that is comparable to the rate of Con A aggregation mediated by globular protein conjugates and variable density linear polymers. Thus, AR proteins define a new class of multivalent ligand scaffolds that have significant potential application in the study and control of a variety of multivalent interactions.     

Ligand binding to the androgen receptor induces conformational changes that regulate phosphatase interactions

We describe a mechanism for protein phosphatase 2A (PP2A) targeting to the androgen receptor (AR) and provide insight into the more general issue of kinase and phosphatase interactions with AR. Simian virus 40 (SV40) small t antigen (ST) binding to N-terminal HEAT repeats in the PP2A A subunit induces structural changes transduced to C-terminal HEAT repeats. This enables the C-terminal HEAT repeats in the PP2A A subunit, including HEAT repeat 13, to discriminate between androgen- and androgen antagonist-induced AR conformations. The PP2A-AR interaction was used to show that an AR mutant in prostate cancer cells (T877A) is activated by multiple ligands without acquiring the same conformation as that induced by androgen. The correlation between androgen binding to AR and increased phosphorylation of the activation function 1 (AF-1) region implies that changes in AR conformation or chaperone composition are causal to kinase access to phosphorylation sites. However, AF-1 phosphorylation sites are kinase accessible prior to androgen binding. This suggests that androgens can enhance the phosphorylation state of AR either by negatively regulating the ability of the ligand-binding domain to bind phosphatases or by inducing an AR conformation that is resistant to phosphatase action. SV40 ST subverts this mechanism by promoting the direct transfer of PP2A onto androgen-bound AR, resulting in multisite dephosphorylation.     

Stabilizing ionic interactions in a full-consensus ankyrin repeat protein

Full-consensus designed ankyrin repeat proteins (DARPins), in which randomized positions of the previously described DARPin library have been fixed, are characterized. They show exceptionally high thermodynamic stabilities, even when compared to members of consensus DARPin libraries and even more so when compared to naturally occurring ankyrin repeat proteins. We determined the crystal structure of a full-consensus DARPin, containing an N-capping repeat, three identical internal repeats and a C-capping repeat at 2.05 Å resolution, and compared its structure with that of the related DARPin library members E3_5 and E3_19. This structural comparison suggests that primarily salt bridges on the surface, which arrange in a network with almost crystal-like regularity, increase thermostability in the full-consensus NI₃C DARPin to make it resistant to boiling. In the crystal structure, three sulfate ions complement this network. Thermal denaturation experiments in guanidine hydrochloride directly indicate a contribution of sulfate binding to the stability, providing further evidence for the stabilizing effect of surface-exposed electrostatic interactions and regular charge networks. The charged residues at the place of randomized residues in the DARPin libraries were selected based on sequence statistics and suggested that the charge interaction network is a hidden design feature of this protein family. Ankyrins can therefore use design principles from proteins of thermophilic organisms and reach at least similar stabilities.     

Characterization of the heparin-binding site of the mycobacterial heparin-binding hemagglutinin adhesin

The mycobacterial adhesin heparin-binding hemagglutinin (HBHA) contains several lysine-rich repeats at its carboxyl-terminal end. Using truncated recombinant HBHA forms and hybrid proteins containing HBHA repeats grafted onto the Escherichia coli maltose-binding protein (MBP), we found that these repeats are responsible for heparin binding. Immunofluorescence microscopy studies revealed that their deletion abrogates binding of HBHA to human pneumocytes. Conversely, when fused to MBP, the HBHA repeats confer pneumocyte adherence properties to the hybrid protein. Treatment of pneumocytes with glycosaminoglycan-degrading enzymes showed that HBHA binding depends on the presence of heparan sulfate chains on the cell surface. The epitope of a monoclonal antibody that inhibits mycobacterial adherence to epithelial cells was mapped within the lysine-rich repeats, confirming their involvement in mycobacterial adherence to epithelial cells. Surface plasmon resonance analyses showed that recombinant HBHA binds to immobilized heparin with fast association kinetics (k(a) = 5.62 (+/- 0.10) x 10(5) m(-1) s(-1)), whereas the dissociation kinetics were slower (k(d) = 0.015 (+/- 0.002) s(-1)), yielding a K(D) value of 26 nm. Similar analyses with grafted MBP indicated similar kinetic constants, indicating that the carboxyl-terminal repeats contain the entire heparin-binding site of HBHA. The molecular characterization of the interactions of HBHA with epithelial glycosaminoglycans should help to better understand mycobacterial adherence within the lungs and may ultimately lead to new approaches for therapy or immunoprophylaxis.     

High affinity binding of receptor-associated protein to heparin and low density lipoprotein receptor-related protein requires similar basic amino acid sequence motifs

The 39-kDa receptor-associated protein (RAP) is a specialized chaperone for members of the low density lipoprotein receptor gene family, which also binds heparin. Previous studies have identified a triplicate repeat sequence within RAP that appears to exhibit differential functions. Here we generated a series of truncated and site-directed RAP mutants in order to define the sites within RAP that are important for interacting with heparin and low density lipoprotein receptor-related protein (LRP). We found that high affinity binding of RAP to heparin is mediated by the carboxyl-terminal repeat of RAP, whereas both the carboxyl-terminal repeat and a combination of amino and central repeats exhibit high affinity binding to LRP. Several motifs were found to mediate the binding of RAP to heparin, and each contained a cluster of basic amino acids; among them, an intact R(282)VSR(285)SR(287)EK(289) motif is required for high affinity binding of RAP to heparin, whereas two other motifs, R(203)LR(205)R(206) and R(314)ISR(317)AR(319), also contribute to this interaction. We also found that intact motifs of both R(203)LR(205)R(206) and R(282)VSR(285)SR(287)EK(289) are required for high affinity binding of RAP to LRP, with the third motif, R(314)ISR(317)AR(319), contributing little to RAP-LRP interaction. We conclude that electrostatic interactions likely contribute significantly in the binding of RAP to both heparin and LRP and that high affinity interaction with both heparin and LRP appears to require mostly overlapping sequence motifs within RAP.