Chaperone-Assisted Crystallography with DARPins
2008; Elsevier BV; Volume: 16; Issue: 10 Linguagem: Inglês
10.1016/j.str.2008.08.010
ISSN1878-4186
AutoresGaby Sennhauser, Markus G. Grütter,
Tópico(s)RNA and protein synthesis mechanisms
ResumoThe structure of proteins that are difficult to crystallize can often be solved by forming a noncovalent complex with a helper protein—a crystallization "chaperone." Although several such applications have been described to date, their handling usually is still very laborious. A valuable addition to the present repertoire of binding proteins is the recently developed designed ankyrin repeat protein (DARPin) technology. DARPins are built based on the natural ankyrin repeat protein fold with randomized surface residue positions allowing specific binding to virtually any target protein. The broad potential of these binding proteins for X-ray crystallography is illustrated by five cocrystal structures that have been determined recently comprising target proteins from distinct families, namely a sugar binding protein, two kinases, a caspase, and a membrane protein. This article reviews the opportunities of this technology for structural biology and the structural aspects of the DARPin-protein complexes. The structure of proteins that are difficult to crystallize can often be solved by forming a noncovalent complex with a helper protein—a crystallization "chaperone." Although several such applications have been described to date, their handling usually is still very laborious. A valuable addition to the present repertoire of binding proteins is the recently developed designed ankyrin repeat protein (DARPin) technology. DARPins are built based on the natural ankyrin repeat protein fold with randomized surface residue positions allowing specific binding to virtually any target protein. The broad potential of these binding proteins for X-ray crystallography is illustrated by five cocrystal structures that have been determined recently comprising target proteins from distinct families, namely a sugar binding protein, two kinases, a caspase, and a membrane protein. This article reviews the opportunities of this technology for structural biology and the structural aspects of the DARPin-protein complexes. Despite the continuous technical advances in protein crystallography due to the worldwide effort in structural genomics programs that promote the application of automated procedures in cloning, expression, purification, crystallization, data collection using synchrotron radiation, and computational crystallography, the determination of some protein structures remains a difficult task. Today, the main limitation is the growth of well-diffracting crystals—a pure trial-and-error process—because the formation of protein crystals depends on several unpredictable variables. In particular, advances in microscaling methodology involving pipetting robots and automated imaging of individual experiments have allowed high throughput approaches boosting the field. However, even with these advances, many proteins resist forming suitable crystals on their own because of inherent structural flexibility and instability. Consequently, conformation stabilizing methods that help to crystallize proteins seem attractive. Approaches in this direction are removal or mutation of surface residues known to be flexible (Lawson et al., 1991Lawson D.M. Artymiuk P.J. Yewdall S.J. Smith J.M. Livingstone J.C. Treffry A. Luzzago A. Levi S. Arosio P. 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This increases the chances to obtain crystals of proteins that otherwise adopt several conformations and therefore resist forming crystals. In addition, novel surfaces can potentially provide better crystal contacts. Especially for membrane proteins, protein ligands also increase the hydrophilic surface area of an otherwise small non-transmembrane region. This strategy for the crystallization of membrane proteins has repeatedly led to successful structure determinations, as seen for many antibody fragment-membrane protein complexes (Dutzler et al., 2003Dutzler R. Campbell E.B. MacKinnon R. Gating the selectivity filter in ClC chloride channels.Science. 2003; 300: 108-112Crossref PubMed Scopus (630) Google Scholar, Hunte et al., 2000Hunte C. Koepke J. Lange C. Rossmanith T. Michel H. 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Cocrystallization of a target protein in complex with a proteinaceous ligand of known structure potentially provides another advantage, namely the structure solution by molecular replacement allowing to avoid the often time-consuming determination of phases via heavy atoms as in single/multiple anomalous dispersion or multiple isomorphous replacement techniques. DNA and RNA binding proteins are often cocrystallized with their natural binding partner, fragments of DNA or RNA stabilizing the conformation of the protein (Anderson et al., 1984Anderson J. Ptashne M. Harrison S.C. Cocrystals of the DNA-binding domain of phage 434 repressor and a synthetic phage 434 operator.Proc. Natl. Acad. Sci. USA. 1984; 81: 1307-1311Crossref PubMed Scopus (75) Google Scholar). In many cases, however, the protein of interest lacks a natural binding partner, the natural binding partner might not be known, or it might not be available for other reasons. Therefore, proteinaceous ligands, either natural or synthetic, have received a great deal of attention in the field of structural biology. Antibody and fragments thereof are the most successful molecules to be used as high-affinity and specific binding proteins in biomedical research and have not only been used as a binding protein but also assist in crystallization (Amit et al., 1986Amit A.G. Mariuzza R.A. Phillips S.E. Poljak R.J. Three-dimensional structure of an antigen-antibody complex at 2.8 A resolution.Science. 1986; 233: 747-753Crossref PubMed Scopus (961) Google Scholar). In 1995, Ostermeier et al. selected monoclonal antibodies for the first time against a membrane protein, namely cytochrome c oxidase of Paracoccus denitrificans, finally allowing the successful structure determination of the oxidase in complex with a FV fragment (Iwata et al., 1995Iwata S. Ostermeier C. Ludwig B. Michel H. 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High-affinity single-domain binding proteins with a binary-code interface.Proc. Natl. Acad. Sci. USA. 2007; 104: 6632-6637Crossref PubMed Scopus (138) Google Scholar), and repeat proteins (Binz et al., 2004Binz H.K. Amstutz P. Kohl A. Stumpp M.T. Briand C. Forrer P. Grütter M.G. Plückthun A. High-affinity binders selected from designed ankyrin repeat protein libraries.Nat. Biotechnol. 2004; 22: 575-582Crossref PubMed Scopus (495) Google Scholar) have shown that these molecules are potential alternatives to antibody fragments in structural biology. These binding molecules were developed as tools for basic research, but their potential use in commercial applications (as specific reagents in biomedical research and as potential therapeutic molecules) was immediately apparent and quickly recognized (Gill and Damle, 2006Gill D.S. Damle N.K. Biopharmaceutical drug discovery using novel protein scaffolds.Curr. Opin. Biotechnol. 2006; 17: 653-658Crossref PubMed Scopus (57) Google Scholar). Many novel binding proteins have improved on the limitations of antibodies, in particular their rather low production yield and intrinsic stability. Simultaneously, they retain the beneficial properties of antibodies, namely specific and tight binding. Structural biologists likewise have a great interest in using these molecules as chaperones in crystallization. In this review, we focus on one scaffold, the DARPins. The potential of DARPins as promising compounds in drug discovery and drug development has been reviewed recently (Stumpp and Amstutz, 2007Stumpp M.T. Amstutz P. DARPins: a true alternative to antibodies.Curr. Opin. Drug Discov. Dev. 2007; 10: 153-159PubMed Google Scholar). We examine the five recently determined X-ray structures of DARPin complexes and address several questions concerning DARPin-target interactions. The diversity of the successfully chosen antigens is paralleled by the diversity of the interaction partners of natural ankyrin repeat proteins, which indicates that the technology can be generally applied to a very broad range of target molecules. The main advantage of antibodies is their ability to recognize a wide range of target molecules with high affinity and specificity. Besides proteins, antibodies can bind compounds such as peptides, sugars and small molecules. However, natural antibodies are complex multidomain molecules not well suited for cocrystallization due to their flexible linker regions between domains and their bivalent character. Therefore, only antibody fragments have been applied for cocrystallization purposes. Fab or Fv fragments contain the complete antigen recognition sites and are therefore sufficient to retain the specific antibody-antigen interaction. Nevertheless, these fragments often derive from monoclonal antibodies, and their classical production by hybridoma technology is time-consuming, expensive, and based on the use of living organisms. The selection using recombinant and synthetic libraries have facilitated their generation (Rothe et al., 2008Rothe C. Urlinger S. Lohning C. Prassler J. Stark Y. Jager U. Hubner B. Bardroff M. Pradel I. Boss M. et al.The human combinatorial antibody library HuCAL GOLD combines diversification of all six CDRs according to the natural immune system with a novel display method for efficient selection of high-affinity antibodies.J. Mol. Biol. 2008; 376: 1182-1200Crossref PubMed Scopus (192) Google Scholar) and led to successful structure determinations (Fellouse et al., 2007Fellouse F.A. Esaki K. Birtalan S. Raptis D. Cancasci V.J. Koide A. Jhurani P. Vasser M. Wiesmann C. 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Biol. 2002; 12: 503-508Crossref PubMed Scopus (152) Google Scholar) but the problems associated with all antibody-derived crystallization chaperones inspire continuously to search for alternative binding proteins with similar binding capabilities but superior properties regarding structure, stability and throughput. DARPins are derived from the ankyrin repeat motif present in numerous naturally occurring proteins. In nature, ankyrin repeat containing proteins are involved in a wide variety of biological activities and are present in all three superkingdoms (Bork, 1993Bork P. Hundreds of ankyrin-like repeats in functionally diverse proteins: mobile modules that cross phyla horizontally?.Proteins. 1993; 17: 363-374Crossref PubMed Scopus (422) Google Scholar). Best documented is their involvement in specific protein-protein interactions. The diversity of their roles in a cellular context is further reflected in their localization, which can be the nucleus, cytoplasm, and the extracellular space, where these proteins interact with a large diversity of partners. The number of repeats presented in a single ankyrin repeat protein and thus involved in binding is highly variable so that ankyrin domains can bind to host target molecules that vary considerably in size and shape. The 33-residue sequence motif of an ankyrin repeat forms a well-defined architecture, consisting of a β turn, followed by a pair of antiparallel α helices and a loop that builds the connection to the next repeat (Figure 1A). The characteristic secondary structure components fold into an L-shaped conformation where the helices form the stem and the loop projects outward at an angle of about 90°. Not uncommon are insertions between or within repeats, mainly in the β turn/loop region (Sedgwick and Smerdon, 1999Sedgwick S.G. Smerdon S.J. The ankyrin repeat: a diversity of interactions on a common structural framework.Trends Biochem. Sci. 1999; 24: 311-316Abstract Full Text Full Text PDF PubMed Scopus (626) Google Scholar). These insertions can be either a short helical segment or more complex motifs. The fully assembled ankyrin domain is elongated and slightly curved, manifested particularly in high repeat number ankyrin repeat proteins (Figure 1B). In naturally occurring complexes involving ankyrin repeat proteins, the concave surface, formed by the β turn and the first α helix, is commonly involved in binding the target molecules as shown by the X-ray structures determined to date: p16INK4a-CDK6 (Russo et al., 1998Russo A.A. Tong L. Lee J.O. Jeffrey P.D. Pavletich N.P. Structural basis for inhibition of the cyclin-dependent kinase Cdk6 by the tumour suppressor p16INK4a.Nature. 1998; 395: 237-243Crossref PubMed Scopus (394) Google Scholar), p19INK4d-CDK6 (Brotherton et al., 1998Brotherton D.H. Dhanaraj V. Wick S. 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Plückthun A. Designing repeat proteins: well-expressed, soluble and stable proteins from combinatorial libraries of consensus ankyrin repeat proteins.J. Mol. Biol. 2003; 332: 489-503Crossref PubMed Scopus (415) Google Scholar). Heeding the lessons of nature, they created DNA libraries that encode DARPins consisting of different numbers of repeat modules and special capping repeats attached to the N and C termini of the protein (Figure 1B). Therefore, the molecules are termed NxC, where x indicates the number of ankyrin repeat modules. The consensus-designed idealized repeat module consists of conserved residues essential for the formation and stabilization of the repeat module itself and the interrepeat stacking interactions. These positions are invariant and build the framework of the individual repeats. In addition, positions not contributing to the structural integrity of the basic fold were defined to be adaptive surface residues (Figure 1A). These residues are positioned at the typical interface region of the natural ankyrin repeat, namely the tip of the β turn and along the exposed surface of the first α-helix (Figure 1C). At these positions, the introduced diversification allows any amino acid except proline and glycine (structurally unfavorable) and cysteine (could form unwanted disulfide bonds). This design resulted in a virtually unlimited repertoire of molecular surfaces. Rapid and easy production is a prerequisite for alternative scaffolds suitable for cocrystallization. Repeat protein libraries (Binz et al., 2004Binz H.K. Amstutz P. Kohl A. Stumpp M.T. Briand C. Forrer P. Grütter M.G. Plückthun A. High-affinity binders selected from designed ankyrin repeat protein libraries.Nat. Biotechnol. 2004; 22: 575-582Crossref PubMed Scopus (495) Google Scholar, Binz et al., 2003Binz H.K. Stumpp M.T. Forrer P. Amstutz P. Plückthun A. Designing repeat proteins: well-expressed, soluble and stable proteins from combinatorial libraries of consensus ankyrin repeat proteins.J. Mol. Biol. 2003; 332: 489-503Crossref PubMed Scopus (415) Google Scholar) can be selected using powerful library selection technologies such as ribosome display (RD) (Hanes and Plückthun, 1997Hanes J. Plückthun A. In vitro selection and evolution of functional proteins by using ribosome display.Proc. Natl. Acad. Sci. USA. 1997; 94: 4937-4942Crossref PubMed Scopus (848) Google Scholar) or phage display (Smith, 1985Smith G.P. Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface.Science. 1985; 228: 1315-1317Crossref PubMed Scopus (2842) Google Scholar), finally yielding specific binders to the target protein. RD is a complete in vitro technology based on in vitro translation in which noncovalent ternary complexes consisting of mRNA, ribosome, and nascent protein chain are formed. These ternary complexes can then be tested for binding to a particular target protein. This system thereby guarantees the coupling of the genotype (mRNA) to the phenotype (protein) and allows immediate access to the genetic information of the binders. To achieve this, the coding sequence of the protein is genetically fused to a C-terminal spacer, allowing the correct folding of the nascent polypeptide chain, and the construct lacks a Stop codon, thus preventing release of the mRNA and the polypeptide from the ribosome. Low temperature and high concentrations of magnesium further stabilize the ternary complexes. The disassembly of the complexes results from depletion of magnesium, and the DNA is recovered via reverse transcription. RD is particularly well suited for large libraries, because no transformation steps limit the applicable library size. Furthermore, as a cell-free system, it enables the selection of cytotoxic proteins or of proteins with limited in vivo stability. All the DARPin binders presented here were isolated from an N3C library and were selected by RD. DARPin binders could also be successfully selected from an N2C library, but these did not yield crystals. Ankyrin repeat proteins consisting of four to five repeats in total are very abundant in nature (Bork, 1993Bork P. Hundreds of ankyrin-like repeats in functionally diverse proteins: mobile modules that cross phyla horizontally?.Proteins. 1993; 17: 363-374Crossref PubMed Scopus (422) Google Scholar) and also many of the solved natural ankyrin repeat structures in complex with their target display such repeat numbers. The target proteins chosen here were all recombinantly expressed, purified, and either in vivo-biotinylated at a specific lysine in the AviTag sequence (MBP, APH, and Caspase-2), in vitro-biotinylated at diverse lysines on the surface (AcrB), or GST-tagged (Plk-1) for immobilization. Within a few RD cycles (usually 3 to 4), it was possible to enrich the resulting residual library with proteins that specifically and with high affinity bind to the target. A subset of the DARPins obtained by RD was then tested individually for binding using approaches suitable for the particular target protein or the intended use of the binder. For MBP (Binz et al., 2004Binz H.K. Amstutz P. Kohl A. Stumpp M.T. Briand C. Forrer P. Grütter M.G. Plückthun A. High-affinity binders selected from designed ankyrin repeat protein libraries.Nat. Biotechnol. 2004; 22: 575-582Crossref PubMed Scopus (495) Google Scholar) and Plk-1 (Bandeiras et al., 2008Bandeiras T.M. Hillig R.C. Matias P.M. Eberspaecher U. Fanghanel J. Thomaz M. Miranda S. Crusius K. Putter V. Amstutz P. et al.Structure of wild-type Plk-1 kinase domain in complex with a selective DARPin.Acta Crystallogr. D Biol. Crystallogr. 2008; 64: 339-353Crossref PubMed Scopus (34) Google Scholar), a standard ELISA was applied to identify a high-affinity binder. In the case of caspase-2, an in vitro enzymatic activity test combined with a standard ELISA yielded an inhibitor (Schweizer et al., 2007Schweizer A. Roschitzki-Voser H. Amstutz P. Briand C. Gulotti-Georgieva M. Prenosil E. Binz H.K. Capitani G. Baici A. Plückthun A. Grütter M.G. Inhibition of caspase-2 by a designed ankyrin repeat protein: specificity, structure, and inhibition mechanism.Structure. 2007; 15: 625-636Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). For APH (Amstutz et al., 2005Amstutz P. Binz H.K. Parizek P. Stumpp M.T. Kohl A. Grütter M.G. Forrer P. Plückthun A. Intracellular kinase inhibitors selected from combinatorial libraries of designed ankyrin repeat proteins.J. Biol. Chem. 2005; 280: 24715-24722Crossref PubMed Scopus (100) Google Scholar, Kohl et al., 2005Kohl A. Amstutz P. Parizek P. Binz H.K. Briand C. Capitani G. Forrer P. Plückthun A. Grütter M.G. Allosteric inhibition of aminoglycoside phosphotransferase by a designed ankyrin repeat protein.Structure. 2005; 13: 1131-1141Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar) and AcrB (Sennhauser et al., 2007Sennhauser G. Amstutz P. Briand C. Storchenegger O. Grütter M.G. Drug export pathway of multidrug exporter AcrB revealed by DARPin inhibitors.PLoS Biol. 2007; 5: e7Crossref PubMed Scopus (247) Google Scholar), an in vivo assay was applied in which the inhibition of the target protein through the DARPins resulted in hypersusceptibility of the bacteria. Normally, the DNA of around 20 DARPins obtained from these assays was sequenced per target. The pools often seemed already quite enriched, revealing groups with similar sequences, although identical sequences were rarely observed. Depending on the sequence variability, five to ten complexes were then purified (yields between 50 and 200 mg/l), characterized, and subjected to crystallization. As a model protein for the approach of selecting a specific DARPin protein by RD, the maltose binding protein from Escherichia coli was chosen and represents the first successful example for the selection and crystallization of a particular target protein in complex with a DARPin. The structure of the complex was determined at 2.3 Å resolution and the phases for this structure could be determined using a known structure of an unselected DARPin previously solved (Kohl et al., 2003Kohl A. Binz H.K. Forrer P. Stumpp M.T. Plückthun A. Grütter M.G. Designed to be stable: crystal structure of a consensus ankyrin repeat protein.Proc. Natl. Acad. Sci. USA. 2003; 100: 1700-1705Crossref PubMed Scopus (218) Google Scholar). This was possible because the size of the DARPin (∼18 kDa) relative to the size of maltose-binding protein (MBP) (∼43 kDa) was appropriate, and there was only one complex in the asymmetric unit. Off7 binds the open form of MBP not involving the sugar-binding cleft but binding three helices at one side of the elongated MBP (Figure 2A). Four lysines forming a positive surface patch which account for 60% of the buried surface area of MBP upon complexation interact closely with off7 (Figure 3A). (A–D) Center: ribbon diagrams of the X-ray structures of the individual complexes. Target proteins and DARPins are colored blue and purple, respectively. Interfaces are highlighted in yellow. Left: the targets have been rotated clockwise. Right: the DARPins have been rotated counterclockwise. Residues that approach within 4 Å of the interaction partner are colored by element (C, N, and O atoms are colored yellow, blue, and red, respectively). (A) DARPin off7 in complex with MBP (PDB entry code 1svx). (B) DARPin 3a in complex with APH (PDB
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