Screening of cyclic peptide phage libraries identifies ligands that bind streptavidin with high affinities

Giebel, Lutz B.; Cass, Robert; Milligan, Daniel L.; Young, Dennis R.; Arze, Rafael; Johnson, Charles R.

doi:10.1021/bi00047a006

Cited by 196 publications

(178 citation statements)

References 29 publications

Supporting

Mentioning

172

Contrasting

Order By: Relevance

“…The Round 0 mRNA pool was generated by T7 runoff transcription and purified by urea-PAGE. The purified mRNA was ligated to F30P (5′-dA 21 [C 9 ] 3 dAdCdC-P; C 9 = tri-(ethylene glycol) phosphate (Glen Research), P = puromycin (Glen Research)), via an oligonucleotide splint (MX 10 Ksplint: 5′… TTTTTTTTTTTTTACCGCTGCCAGAC …3′). Following PAGE purification of the ligation reaction, the template was dissolved in water and quantitated by absorbance at 260 nm.…”

Section: Synthesis Of MX 10 K Librarymentioning

confidence: 99%

Design of Cyclic Peptides That Bind Protein Surfaces with Antibody-Like Affinity

et al. 2007

View full text Add to dashboard Cite

There is a pressing need for new molecular tools to target protein surfaces with high affinity and specificity. Here, we describe cyclic messenger RNA display with a trillion-member covalent peptide macrocycle library. Using this library, we have designed a number of high-affinity, redoxinsensitive, cyclic peptides that target the signaling protein Gαi1. In addition to cyclization, our library construction took advantage of an expanded genetic code, utilizing nonsense suppression to insert N-methylphenylalanine as a 21st amino acid. The designed macrocycles exhibit several intriguing features. First, the core motif seen in all of the selected variants is the same and shares an identical context with respect to the macrocyclic scaffold, consistent with the idea that selection simultaneously optimizes both the cyclization chemistry and the structural placement of the binding epitope. Second, detailed characterization of one molecule, cyclic Gαi binding peptide (cycGiBP), demonstrates substantially enhanced proteolytic stability relative to that of the parent linear molecule. Third and perhaps most important, the cycGiBP peptide binds the target with very high affinity (K i ≈ 2.1 nM), similar to those of many of the best monoclonal antibodies and higher than that of the βγ heterodimer, an endogenous Gαi1 ligand. Overall the work provides a general route to design novel, low-molecular-weight, high-affinity ligands that target protein surfaces.Although networks of protein-protein interactions control function inside cells, it is increasingly clear that many of these players cannot be targeted using traditional druglike molecules (1). High-affinity, high-specificity ligands targeting protein surfaces are thus of considerable interest as tools for chemical genetics, as potential lead/surrogate compounds, and as new drugs. Nanotechnology could also greatly benefit from such molecules, particularly robust, inexpensive, low-molecular-weight ligands that could replace monoclonal antibodies (2). Designing these ligands still remains a significant challenge despite the tremendous advances in structural biology and computational chemistry.Our laboratory has been working to design new peptide ligands that target heterotrimeric Gprotein signaling. We have previously used messenger RNA (mRNA) display (3) to isolate new linear peptides that target Gαi1 (4-6). Gαi1 is a member of the Gα subunit family and serves as a molecular router, connecting the cell-surface G-protein-coupled receptors (GPCR) to down-stream effector pathways (see ref 7 for a review of GPCR signaling). Gα subunits function by collaboration with the Gβγ heterodimer and a transmembrane receptor. The Gαβγ heterotrimer associates with the cytosolic portion of GPCRs with GDP bound in the nucleotide pocket of Gα. Extracellular ligand binding to the GPCR results in

show abstract

Section: Synthesis Of MX 10 K Librarymentioning

confidence: 99%

Design of Cyclic Peptides That Bind Protein Surfaces with Antibody-Like Affinity

et al. 2007

View full text Add to dashboard Cite

show abstract

“…Because of the constrained nature of cyclic peptides, receptor binding affinities often become more favorable (28). Cyclic peptides can be exceptional mimotopes because they have the entropic benefit of binding antibody with their structure already constrained (17,23,41). Cysteine residues were inserted in J1, in place of Phe6 and Asn16, such that the disulfide bond would not affect Trp11.…”

Section: Discussionmentioning

confidence: 99%

Mimotopes of Apical Membrane Antigen 1: Structures of Phage-Derived Peptides Recognized by the Inhibitory Monoclonal Antibody 4G2dc1 and Design of a More Active Analogue

et al. 2007

View full text Add to dashboard Cite

Apical membrane antigen 1 (AMA1) of the malaria parasite Plasmodium falciparum is an integral membrane protein that plays a key role in merozoite invasion of host erythrocytes. A monoclonal antibody, 4G2dc1, recognizes correctly folded AMA1 and blocks merozoite invasion. Phage display was used to identify peptides that bind to 4G2dc1 and mimic an important epitope of AMA1. Three of the highest-affinity binders-J1, J3, and J7-were chosen for antigenicity and immunogenicity studies. J1 and J7 were found to be true antigen mimics since both peptides generated inhibitory antibodies in rabbits (J. L. Casey et al., Infect. Immun. 72:1126-1134, 2004). In the present study, the solution structures of all three mimotopes were investigated by nuclear magnetic resonance spectroscopy. J1 adopted a well-defined region of structure, which can be attributed in part to the interactions of Trp11 with surrounding residues. In contrast, J3 and J7 did not adopt an ordered conformation over the majority of residues, although they share a region of local structure across their consensus sequence. Since J1 was the most structured of the peptides, it provided a template for the design of a constrained analogue, J1cc, which shares a structure similar to that of J1 and has a disulfide-stabilized conformation around the Trp11 region. J1cc binds with greater affinity to 4G2dc1 than does J1. These peptide structures provide the foundation for a better understanding of the complex conformational nature of inhibitory epitopes on AMA1. With its greater conformational stability and higher affinity for AMA1, J1cc may be a better in vitro correlate of immunity than the peptides identified by phage display.Malaria infects 300 to 500 million people per year worldwide and causes 2 to 3 million deaths, mainly in children under 5 years of age. A considerable effort is being devoted to the development of a vaccine against malaria, and one of the leading candidates for inclusion in such a vaccine is apical membrane antigen 1 (AMA1), a type I integral membrane protein conserved throughout all Plasmodium species. AMA1 is critical for the invasion of host erythrocytes (36) and is translocated from the micronemes onto the parasite surface around the time of invasion (20). Although its precise role in invasion remains undefined, it has been postulated that AMA1 is involved in realignment of the parasite after attachment to the erythrocyte, ensuring that the apical prominence of the merozoite is in close proximity to the erythrocyte surface (8, 33). Recombinant AMA1 induced protective immune responses in mouse and monkey models of malaria (2, 10, 11, 13), and both monoclonal and polyclonal antibodies to AMA1 inhibit merozoite invasion of erythrocytes (2,9,11,12,25,34,42). The observation that it was not possible to obtain targeted gene disruptions of the AMA1 gene that knocked out the function of the protein further supports an important role for AMA1 in the invasion of host erythrocytes (43). Recently, a conditional knockout of Toxoplasma gondii AMA1 was cre...

show abstract

“…The BIAcore 2000 system, sensor chip, and coupling reagents were from Pharmacia Biotech Inc. Affinities for each peptide ligand were determined under identical conditions in a competition assay involving a cyclic, disulfide-containing streptavidin binding peptide, cyclo-Ac-AE[CHPQGPPC]IEGRK-NH 2 , whose binding affinity has been previously determined (36) by BIAcore, immobilized on the sensor chip surface via the ⑀-amino group of the C-terminal lysine residue using standard amine immobilization chemistry (20,21).…”

Section: Methodsmentioning

confidence: 99%

Topochemical Catalysis Achieved by Structure-based Ligand Design

Katz

Cass

Liu

et al. 1995

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Dimerization is a trigger for many important biological processes (1-4). Recently we described a structure-based design strategy for the preparation of ligands that dimerize receptors by topochemistry (5), which involves a chemical reaction mediated by holding reactants in close proximity and in productive orientations on a protein or crystal scaffold.The strategy for preparation by topochemistry of receptor dimerizing molecules is as follows. First, a protein ligand for a receptor monomer is developed by structure-based design or discovered by screening. The three-dimensional structure of the protein-ligand complex is then determined by crystallography. Ligating groups of the appropriate length and orientation are then introduced at sites in the ligand in a region distinct from the binding motif. Dimerization of two such ligands, each bound on a receptor monomer of a native receptor dimer complex, is directed by the receptor monomers, which present the reactive ligating groups of the ligands next to one another. A ligand dimer capable of dimerizing the receptor to produce a native or native-like receptor dimer is thus prepared by topochemistry.This strategy for preparing a receptor-dimerizing ligand was demonstrated (5) with streptavidin using a high affinity cyclic peptide ligand, cyclo-Ac-[CHPQGPPC]-NH 2 , with a K d of 670 nM at pH 7.3, discovered by screening phage libraries against the streptavidin target (36). Streptavidin, a tetrameric protein with a molecular mass of 13,200 Da/subunit produced by Streptomyces avidinii, functions as an antibiotic by binding the vitamin biotin with the highest affinity known for a noncovalent protein-ligand interaction, K d ϳ10 Ϫ15 M (6). This remarkable affinity forms the basis for many bioanalytical applications (7-10), as well as a paradigm for studying the structural basis of high affinity protein-ligand interactions (11-13, 37), and for applying structure-based ligand design strategies (14, 15).In streptavidin, there are four binding sites, one per subunit. In I222 crystals of streptavidin, one crystallographically equivalent pair, site 1 and site 1Ј, is near a crystallographic 2-fold axis; the other pair, site 2 and site 2Ј, is not. When bound to I222 crystals of streptavidin, the intramolecular disulfide bonds of neighboring cyclo-Ac-[CHPQGPPC]-NH 2 monomers bound at site 1, presented next to one another, undergo disulfide interchange. Thus a C2-symmetric peptide dimer adopting the symmetry of the crystal is produced with two intermolecular disulfide bonds passing through the 2-fold axis. A doubleheaded streptavidin dimerizing agent is thereby prepared by topochemistry. Concomitant with peptide dimerization is the dimerization of the associated bound streptavidin tetramers. We determined the crystal structure of the protein-peptide dimer complex and demonstrated by HPLC 1 and mass spectrometry that streptavidin crystals of space group I222 mediate the dimerization (5).Although I222 streptavidin crystals mediate the disulfide interchange reaction between neighboring...

show abstract

Screening of cyclic peptide phage libraries identifies ligands that bind streptavidin with high affinities

Cited by 196 publications

References 29 publications

Design of Cyclic Peptides That Bind Protein Surfaces with Antibody-Like Affinity

Design of Cyclic Peptides That Bind Protein Surfaces with Antibody-Like Affinity

Mimotopes of Apical Membrane Antigen 1: Structures of Phage-Derived Peptides Recognized by the Inhibitory Monoclonal Antibody 4G2dc1 and Design of a More Active Analogue

Topochemical Catalysis Achieved by Structure-based Ligand Design

Contact Info

Product

Resources

About