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Directed discovery of bivalent peptide ligands to an SH3 domain

¹ Department of Human Biological Chemistry and Genetics, and Sealy Center for Structural Biology, and
² Department of Pathology, Center for Biodefense and Emerging Infectious Diseases, and Sealy Center for Vaccine Development, The University of Texas Medical Branch at Galveston, Galveston, Texas 77555, USA

Reprint requests to: Robert O. Fox, Department of Human Biological Chemistry and Genetics, 301 University Blvd., Mail Route 0647, The University of Texas Medical Branch at Galveston, Galveston, TX 77555-0647, USA; e-mail: fox{at}bloch.utmb.edu; fax: (409) 747-4745.

(RECEIVED October 2, 2003; FINAL REVISION December 5, 2003; ACCEPTED December 5, 2003)

	Abstract

TOP Abstract Introduction Results and Discussion Materials and methods References

 
The Caenorhabditis elegans SEM-5 SH3 domains recognize proline-rich peptide segments with modest affinity. We developed a bivalent peptide ligand that contains a naturally occurring proline-rich binding sequence, tethered by a glycine linker to a disulfide-closed loop segment containing six variable residues. The glycine linker allows the loop segment to explore regions of greatest diversity in sequence and structure of the SH3 domain: the RT and n-Src loops. The bivalent ligand was optimized using phage display, leading to a peptide (PP-G₄-L) with 1000-fold increased affinity for the SEM-5 C-terminal SH3 domain over that of a natural ligand. NMR analysis of the complex confirms that the peptide loop segment is targeted to the RT and n-Src loops and parts of the -sheet scaffold of this SH3 domain. This binding region is comparable to that targeted by a natural non-PXXP peptide to the p67^phox SH3 domain, a region not known to be targeted in the Grb2 SH3 domain family. PP-G₄-L may aid in the discovery of additional binding partners of Grb2 family SH3 domains.

Keywords: SH3 domain; signal transduction; phage display; combinatorial library; peptide ligand; bivalent ligand; NMR spectroscopy; non-PXXP binding site

³ These authors contributed equally to this work.

⁴ Present addresses: The Department of Internal Medicine, The Division of Infectious Diseases, The University of Texas Medical Branch, Galveston, TX 77555-0435, USA;

⁵ Centocor, Inc., Johnson and Johnson, 200 Great Valley Parkway, M/S R-3-1, Malvern, PA 19355, USA;

⁶ Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843, USA.

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.03470504.

	Introduction

TOP Abstract Introduction Results and Discussion Materials and methods References

 
Src homology 3 domains (SH3 domains) participate in diverse signaling pathways by binding to unique proline-rich peptide motifs, and thus serve as important targets for drug design (Scott and Smith 1990; Lim et al. 1994a; Kuriyan and Cowburn 1997). However, the binding affinity for this interaction is relatively low (K_d = 1–200 µM) (Nguyen et al. 1998). The interactions between proline-rich peptides and the family of SH3 domains involve a common binding surface recognizing a proline-rich region, and unique RT (named for conserved arginine and threonine residues) and n-Src loops, which recognize a single specific amino acid at the N or C terminus of the proline-rich sequence (Scott and Smith 1990; Feng et al. 1994; Lim et al. 1994b; Kuriyan and Cowburn 1997).

SH3 domain binding to these proline-rich peptides involves a relatively small interaction surface (~400 Ų) and several conserved hydrogen-bonding interactions. The reliance of binding affinity on hydrophobic interactions results in relatively promiscuous SH3-peptide recognition. For many SH3 domains, biological specificity may be achieved only by exploiting multiple binding sites, or by tertiary interactions with the parent protein and its target (Kuriyan and Cowburn 1997).

The binding affinity of the SEM-5 C-terminal SH3 domain to mSos-derived proline-rich sequences have been reported for Ac-PPPVPPRRR-amide (40 µM; Lim et al. 1994a) and for Ac-PPPVPPR-amide (190 µM; Nguyen et al. 1998). A physiologically relevant affinity and specificity for SEM-5 protein is obtained by interactions involving multiple PXXP sequences (P is proline and X is any amino acid) in the Sos protein target with two SH3 domains within the SEM-5 multidomain protein. Previously, efforts to increase SH3 ligand affinity and specificity to single SH3 domains have focused primarily on replacing sequences immediately flanking the PXXP motif with natural or nonnatural amino acids (Rickles et al. 1995; Braisted and Wells 1996; Pisabarro and Serrano 1996; Cunningham and Wells 1997; Posern et al. 1998; Lewitzky et al. 2001; Douangamath et al. 2002; Fazi et al. 2002; Kami et al. 2002; Tong et al. 2002).

Recently, non-PXXP sequences have been identified that bind tightly to a variety of SH3 domains (Nguyen et al. 1998, 2000; Barnett et al. 2000; Kang et al. 2000; Lewitzky et al. 2001; Douangamath et al. 2002; Kami et al. 2002). These sequences bind SH3 domains in an -helical conformation to one of two sites each distinct from the prolinerich peptide binding site. The role of these sites appears to represent additional regulatory regions on this small domain. Targeted ligands to non-PXXP sites would provide a valuable tool to understand the biological role of these regions. Further, specific tight-binding ligands to PXXP and non-PXXP sites would provide useful reagents for a chemical biology approach to signal transduction.

Bivalent ligands, wherein two independent molecules targeting a single protein are coupled by a linker, result in molecules with submicromolar affinities when the component moieties bind in the millimolar range (Jencks 1981; Cowburn et al. 1995; Shuker et al. 1996; Cussac et al. 1999; Erlanson et al. 2000; Parang et al. 2001; Tamiz et al. 2001). The free energy of binding of the bivalent compound is given by the sum of the intrinsic binding free energy of each fragment plus a term that accounts for the loss in translational degrees of freedom due to joining the two moieties (Jencks 1981). The length and geometry of the linker joining the two independent ligands are critical to establish a favorable bivalent binding to the individual recognition sites on the receptor (Cowburn et al. 1995; Cussac et al. 1999).

Here, we used a bivalent ligand optimized by phage display to develop peptides with high affinity and specificity to the C-terminal SEM-5 SH3 domain as potential inhibitors for the investigation of signal transduction pathways or as possible anti-oncogenic drug leads. We have designed a phage display library with a disulfide-closed variable hexapeptide loop tethered via a glycine linker to a natural proline-rich binding sequence from the mSos nucleotide exchange protein. The tethering of this diverse loop library to the natural ligand sequence permits us to explore additional binding sites on the SH3 domain. We expected that binding of the mSos sequence would place the loop peptide over regions on the SH3 domain that have higher sequence diversity (i.e., the RT and n-Src loops), allowing us to select a peptide specific to unique sequence elements of this SEM-5 SH3 domain. A subset of the non-PXXP binding sites can be reached by the variable loop. Chemical shift perturbation studies using NMR spectroscopy have been employed to map the binding regions for various peptide sequences on the SEM-5 SH3 domain, and to determine the structural binding features responsible for their higher affinity.

	Results and Discussion

TOP Abstract Introduction Results and Discussion Materials and methods References

 
Design and identification of tight binding bivalent ligands to SEM-5 SH3 domain
A Fuse-5 phage-displayed random peptide library (PPPVP PRGGGGCXXXXXXC, where X represents the 20 naturally occurring amino acids) was screened to select for a peptide sequence showing high-affinity binding interactions with the SEM-5 C terminal SH3 domain. The peptide library links a naturally occurring proline-rich region via a tetra-glycine linker to a disulfide-constrained, randomized loop. Members of the library allow us to explore regions of greatest sequence and structural diversity among SH3 domains: the n-Src and RT loops. The length of the glycine linker was selected to allow the loop residues to completely interact with the regions adjacent to the polyproline pocket on the SH3 domain and to reach one of the non-PXXP binding sites. The cysteine residues allow the formation of a disulfide bond to restrict the conformational space sampled by each loop sequence. This minimizes the loss in conformational entropy of the ligand upon binding to the receptor domain.

The phage selection converged to a single sequence, PPPVPPRGGGGCLYTRYWC (PP-G₄-L), after multiple rounds of biopanning. Phage eluted at pH 2 converged to a single amino acid sequence after round 3, while phage eluted at pH 3 converged to that same sequence after round 4. The resulting peptide was chemically synthesized and its binding to fluorescein-labeled SEM-5 SH3 domain was determined by fluorescence anisotropy. The PP-G₄-L peptide bound to its target protein SEM-5 SH3 with a K_d of 48 nM (Table 1). This represents ~1000-fold enhancement in affinity over the mSos polyproline peptide segment alone: Ac-PPPVPPR-amide (190 µM; Nguyen et al. 1998), and is the highest affinity of a selected peptide to any SH3 domain yet reported.

The sequence Ac-PPPVPPRGGGCLYTRYWCGRK-amide (PP-G₃-L) was chemically synthesized to investigate the effect of linker length on the bivalent ligand affinity for the SH3 domain. The peptide PP-G₃-L, in which the glycine linker was shortened by one residue, bound more tightly to the SH3 domain (K_d = 25 nM) that the longer PP-G₄-L ligand (K_d = 48 nM). The increased binding affinity can be attributed to the reduced conformational space sampled by the PP-G₃-L conformers, due to the reduced conformational ensemble associated with the shorter glycine segment.

NMR spectroscopy maps peptide interactions to the surface of the SH3 domain
The binding interactions of the mSos proline-rich peptide, PP-G₄-L, and L peptides were mapped to surface residues of ¹⁵N-labeled SEM-5 C-terminal SH3 domain in 2D ¹⁵N-¹H HSQC NMR-based chemical shift and line width perturbation studies. The relatively weak interaction between the SH3 domain and the mSos peptide results in a protein–peptide complex that is in fast exchange on the NMR time scale, as evidenced by chemical shift perturbations. However, the selective broadening of the SH3 domain amide (NH) cross-peaks upon titration with PP-G₄-L and L peptides demonstrate relatively stronger binding of these peptides to the SH3 domain, producing a protein–peptide complex that is in intermediate exchange on the NMR timescale (Fig. 1).

View larger version (22K): [in this window] [in a new window]   Figure 1. 1H-15N HSQC spectra of SH3 domain alone (black contours) and with PP-G4-L (red contours; molar ratio of ligand to protein, ~1.5 : 1). Black peaks alone indicate that the resonances from the complex (red) have been significantly broadened.

View larger version (20K): [in this window] [in a new window]   Figure 2. Graphical representation of chemical shift and line width perturbations of SEM-5 SH3 domain residues by m-Sos (A), PPG4L (B), and L (C) peptides. The perturbations observed in the side chain atoms are depicted in blue. Regions of higher perturbation on the SEM-5 SH3 domain are shown with horizontal red bars for all three peptides. The non-PXXP binding sites for Grb2 and p67phox are indicated by green and magenta bars, respectively, in (C). The weighted chemical shift differences for SEM-5 SH3 domain titration with mSos are calculated as the square root of [(H)2 + 0.5(N)2], where H and N are changes in proton and nitrogen chemical shifts in ppm units.

View larger version (22K): [in this window] [in a new window]   Figure 3. Ribbon diagrams of SEM-5 SH3 domain color coded to specify the regions of interactions with mSos (A), LGT-E-D3 (B), and PPG4L (C) peptides. Color coding from gold to orange to red indicates increasing perturbation or binding interactions of the domain residues with the respective peptides.

View larger version (38K): [in this window] [in a new window]   Figure 4. Comparison of the non-PXXP peptide binding sites on SH3 domains. (A) Sequence alignment of the SEM-5 C-terminal SH3 domain with SH3 domains sequences that bind non-PXXP peptides. The secondary structure elements above refer to the SEM-5 C-terminal SH3 domain. The residues that are broadened substantially by the L peptide (>30% reduction in signal intensity on peptide addition) for the SEM-5 domain (red) are compared with cross-saturation results reported for the other domains (red; Kami et al. 2002). (B) Mapping of non-PXXP binding regions (based on the sequence alignment) for SEM-5 and p67phox SH3 domains on the structure of SEM-5 C terminal SH3 domain. (C) Mapping of non-PXXP binding regions for SEM-5 and Grb2 SH3 domains on the structure of SEM-5 C terminal SH3 domain. The regions colored red and green are non-PXXP binding regions unique to SEM-5, p67phox and Grb2 C terminal SH3 domains, respectively; regions in gold are common non-PXXP binding regions for both the SH3 domains.

	Materials and methods

TOP Abstract Introduction Results and Discussion Materials and methods References

 
Construction of phage display library
The DNA oligo: 5'-B-C TAT TCT CAC TCG GCC GAC GGG GCT GGT ACC ATG CCT CCC CCT GTT CCT CCT CGT GGT GGT GGT GGT TGC NNK NNK NNK NNK NNK NNK TGC GGT GGA TCC GCC GCT GGG GCC GAA ACT GTT GAA-3' was used to prepare a Fuse-5 phage display library encoding -PPPVPPRGGGGCXXXXXXC-peptide sequences following an established protocol (Scott and Smith 1990). (5'-B: biotinylated 5' end; "N" represents an equal mixture of the deoxynucleotides G, A, T, and C; "K" represents an equal mixture of G and T; and X represents a mixture of all 20 naturally occurring amino acids). The oligo was double stranded, with a single primer oligo: 5'-B-TTTCAACAGTTTCGGCCCCAG-3' using a thermal cycler and, digested with BglI, and the oligo ends and undigested oligos were removed by incubating with streptavidin-agarose beads (Gibco-BRL, Cat.#15942-014) for 30 min at 25°C, followed by microcentrifugation at 2500 rpm for 2 min. The library of DNA fragments was cloned into the SfiI sites of the Fuse-5 vector and transfected into Escherichia coli strain DH5. The resulting library contained 1.2 x 10⁸ unique clones. (Oligo Synthesis: Keck Foundation Biotechnology Resource Laboratory, Yale University School of Medicine).

SEM-5 protein expression, purification, and phage display library selection
The C-terminal SEM-5 SH3 domain (residues 155–214) was expressed and purified as described (Lim et al. 1994a). We biotinylated SEM-5 SH3 using N-(hydroxysuccinamide)-NHS-LC-Biotin (Pierce, Cat. #21335) as described (Smith 1985). ESI mass spectrometry confirmed incorporation of 1~3 biotins per Sem 5 molecule. For library selection, streptavidin magnesphere paramagnetic particles (SA-PMPs; Promega Cat. #Z5481) were rinsed three times with Tris-buffered saline (TBS; 50 mM Tris-HCl, pH 7.5, 0.15 M NaCl). Biotinylated SH3 (100 µg) was added to 0.6 mg SA-PMPs in 600 µL TBS and incubated at 4°C for 18 h. The SA-PMPs were then incubated with 10 µmole of biotin at 4°C for 4 h to block unbound sites. The library phage virions (input = 10¹² cfu) were added and incubated with the beads in 700 µL TBS at 4°C for 18 h. The SA-PMPs were then washed with TBS-Tween (0.5%) 700 µL 6X at 4°C to remove unbound phage. The bound phage were eluted with 400 µL glycine-HCl (100 mM Glycine-HCl, pH adjusted with HCl 1 mg/mL BSA, 0.1 mg/mL phenol red) at pH 5, 4, 3, and 2 for 20 min at 4°C. Tris buffer (1 M Tris, pH 9.1) was used for neutralization. The pH eluates were transfected into ER2537 F'(NEB) E. coli cells for library expansion. RF DNA from a single clone was sequenced at each enrichment cycle.

Peptide synthesis and purification
The following sequences were synthesized:

mSos peptide: Ac-PPPVPPR-amide,

PP-G₄-L: Ac-PPPVPPRGGGGCLYTRYWCGRK -amide

PP-G₃-L: Ac-PPPVPPRGGGCLYTRYWCGRK -amide

G₄-L: Ac-GGGGCLYTRYWCGRK-amide

L: Ac-CLYTRYWCGRK -amide.

PP-G₄-L peptide was obtained from the original library selection, and PP-G₃-L and L peptides were modified peptides from the original library selection. All peptides were synthesized with Fmoc/tBu solid-phase peptide synthesis (ABI 431A). The N termini of the peptides were acetylated, and an amide group was added to the C terminus of each peptide. Peptides (200 µg/mL) were oxidized in 10–100 mM Tris (pH 8.0) with 10–20 µM CuCl₂. Peptides were purified by RP-HPLC on a C18 column and their identities confirmed to be monomers by ESI mass spectrometry. The mass results are given in Table 1. The peptides were oxidized to form disulfide bridges in 100 mM Tris (pH 8.0) and 10 µM Cu²⁺ at 25°C for 4 h.

Binding affinity
The SEM-5 SH3 domain was labeled at Cys209 using a thiol-reactive reagent (fluorescein-5-maleimide) as described by the manufacturer (Molecular Probes, cat. no. F-150). This site occurs on the opposite face of the SH3 domain from the proline-rich binding surface. Fluorescence measurements were performed at 25°C in HEPES buffer at pH 7.5 (20 mM HEPES, 50 mM NaCl) using a Beacon fluorescence polarization spectrometer (PANVERA). The excitation and emission wavelength were 490 and 525 nm, respectively.

NMR analysis
All NMR spectra were collected at 25°C on a Varian UnityPlus 750 MHz or 600 MHz instruments using a triple resonance probe equipped with a pulsed-field gradient. Sequential assignments are reported elsewhere (Ferreon et al. 2003). Titrations of ¹⁵N-labeled SEM-5 C-terminal SH3 domain with the mSos, PP-G₄-L, and L peptides were monitored using ¹H-¹⁵N HSQC spectra. The final ligand : peptide molar ratio was ~2 : 1 for the mSos, ~1.5 : 1 for PP-G₄-L, and ~2 : 1 for L peptide. All spectra were recorded as 64 x 2048 complex matrices with 16 scans per t₁ point. Spectral widths of 2400 and 7500 Hz were employed in D₁ and D₂, respectively. The NMR spectra were processed using NMRPipe software (Delaglio et al. 1995). Peak heights measured with NMRView were used for the titrations.

	Acknowledgments

 
This research was supported by grants from The Defense Advanced Research Projects Agency (9624-107 FP to R.E.S.), Center for Disease Control (U90/CCU618754-02 to R.E.S.), the NIH (GM51332 and AI056326 [GenBank] to R.O.F.), and The Robert A. Welch Foundation (H-1345 to R.O.F.) and The Sealy and Smith Foundation for Medical Research. A Jeane B. Kempner Postdoctoral Fellowship to M.R.F. and a McLaughlin Postdoctoral Fellowship to R.K. are gratefully acknowledged. M.M. was supported by a training fellowship from the W.M. Keck Foundation of the Gulf Coast Consortia through the Keck Center for Computational and Structural Biology. We thank Drs. David Gorenstein and David Volk for providing the SH3 domain chemical shift assignments prior to publication; Amy Huinker for mass spectrometry analyses; Drs. Alex Kurosky and Stefan Serabyn of the UTMB Protein Center Core Laboratory for peptide synthesis; and Dr. Tom Wood of the molecular Biology Core for DNA sequencing. Both Cores are supported in part by NIEHS Center Grant ES 06676. We thank Dr. David Konkel for editing the manuscript. We thank Dr. Kaushik Dutta, New York Structural Biology Center, for his valuable comments and suggestions.

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.

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