Design of N-substituted Peptomer Ligands for EVH1 Domains
2003; Elsevier BV; Volume: 278; Issue: 38 Linguagem: Inglês
10.1074/jbc.m305934200
ISSN1083-351X
AutoresJürgen Zimmermann, Ronald Kühne, Rudolf Volkmer, Thomas Jarchau, Ulrich Walter, Hartmut Oschkinat, Linda Ball,
Tópico(s)Biochemical and Structural Characterization
ResumoEna/VASP proteins are implicated in cytoskeletal reorganization during actin-dependent motility processes. Recruitment to subcellular sites of actin polymerization is mediated by the highly conserved N-terminal EVH1 domain, which interacts with target proteins containing proline-rich motifs. The VASP EVH1 domain specifically binds peptides with the consensus motif FPPPP present in all its binding partners, including the Listerial ActA protein. Previous studies have shown that the Phe and first and final Pro residues are highly conserved and cannot be substituted with any other natural amino acid without significant loss of binding affinity. We have incorporated peptoid building blocks (sarcosine derived, non-natural amino acids) into the peptide SFEFPPPPTEDEL from the Listerial ActA protein and were able to substitute the most highly conserved residues of this motif while maintaining binding to the VASP EVH1 domain with affinities in the range of 45-180 μm. We then used NMR chemical shift perturbations to locate specific domain residues involved in particular interactions. These studies may open up the way for designing selective modulators of VASP function for biological studies and for the development of novel therapeutics for diseases involving pathologically altered cell adhesion or cell motility. Ena/VASP proteins are implicated in cytoskeletal reorganization during actin-dependent motility processes. Recruitment to subcellular sites of actin polymerization is mediated by the highly conserved N-terminal EVH1 domain, which interacts with target proteins containing proline-rich motifs. The VASP EVH1 domain specifically binds peptides with the consensus motif FPPPP present in all its binding partners, including the Listerial ActA protein. Previous studies have shown that the Phe and first and final Pro residues are highly conserved and cannot be substituted with any other natural amino acid without significant loss of binding affinity. We have incorporated peptoid building blocks (sarcosine derived, non-natural amino acids) into the peptide SFEFPPPPTEDEL from the Listerial ActA protein and were able to substitute the most highly conserved residues of this motif while maintaining binding to the VASP EVH1 domain with affinities in the range of 45-180 μm. We then used NMR chemical shift perturbations to locate specific domain residues involved in particular interactions. These studies may open up the way for designing selective modulators of VASP function for biological studies and for the development of novel therapeutics for diseases involving pathologically altered cell adhesion or cell motility. Low affinity, protein-protein interactions mediated by proline-rich motifs (PRMs), 1The abbreviations used are: PRM, proline-rich motif; DIC, diisopropylcarbodiimide; Fmoc, 9-fluorenylmethoxycarbonyl; GST, glutathione S-transferase; HOBT, hydroxybenzotriazole; HSQC, heteronuclear single quantum correlation (spectroscopy); MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; MOPS, 4-morpholinepropanesulfonic acid; CSP, chemical shift perturbation; TBS, Tris-buffered saline. and PRM-binding domains are becoming increasingly recognized as important players in signal transduction pathways (1Williamson M.P. Biochem. J. 1994; 297: 249-260Crossref PubMed Scopus (838) Google Scholar, 2Kay B.K.P. Williamson M. Sudol M. FASEB J. 2000; 14: 231-241Crossref PubMed Scopus (1043) Google Scholar). Well known examples include the interactions of Src homology 3 (SH3)-, WW-, and Ena-VASP homology 1 (EVH1) domains with targets containing PxxP, PPx(Y/P)LPP, and FPxφP motifs, respectively, which typically bind with affinities in the range of 5-100 μm. Finely tuned interactions of this type are vital for the accurate regulation of important signal transduction processes involved in proliferation, migration, or differentiation of cells in the adult organism or during its development. The high degree of conservation found in the target PRMs can be rationalized on the basis of high resolution structural data now available for many of these complexes. Proline is the only natural N-substituted amino acid, and its unusual backbone structure gives rise to a unique mechanism for highly specific recognition (3Nguyen J.T. Turck C.W. Cohen F.E. Zuckermann R.N. Lim W.A. Science. 1998; 282: 2088-2092Crossref PubMed Scopus (277) Google Scholar). The low affinities often involved make protein-protein interaction modules difficult targets for drug design. However, the importance of these interactions has already triggered several attempts to overcome the principal problems involved in designing ligands for protein surfaces (4Kuruvilla F.G. Shamji A.F. Sternson S.M. Hergenrother P.J. Schreiber S.L. Nature. 2002; 416: 653-657Crossref PubMed Scopus (337) Google Scholar). Ena/VASP proteins are important components of signaling cascades that modulate actin cytoskeletal dynamics in response to extracellular stimuli (5Harbeck B. Huttelmaier S. Schluter K. Jockusch B.M. Illenberger S. J. Biol. Chem. 2000; 275: 30817-30825Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). They are involved in various cellular and subcellular actin-based motility processes that are dependent on highly organized, transient, microfilament networks that lead to the formation of plasma membrane protrusions such as lamellipodia or filopodia and have also been found to be crucial for supporting intracellular motility of the bacterial pathogen Listeria monocytogenes (6Renfranz P.J. Beckerle M.C. Curr. Opin. Cell Biol. 2002; 14: 88-103Crossref PubMed Scopus (86) Google Scholar). Mechanistically, Ena/VASP proteins are thought to promote local actin filament growth either by recruitment of polymerization-competent profilactin to Ena/VASP-binding proteins or by steric exclusion of polymerization-inhibiting actin-binding proteins from growing actin filament ends (for review see Refs. 7Cameron L.A. Giardini P.A. Soo F.S. Theriot J.A. Nat. Rev. Mol. Cell. Biol. 2000; 1: 110-119Crossref PubMed Scopus (136) Google Scholar and 8Krause M. Bear J.E. Loureiro J.J. Gertler F.B. J. Cell Sci. 2002; 115: 4721-4726Crossref PubMed Scopus (87) Google Scholar). Ena/VASP proteins are known to be involved in a diverse range of activities, including formation of epithelial sheets, spreading and aggregation of thrombocytes, migration of fibroblasts or neurons, and polarization of lymphocytes or macrophages, as well as being essential for the virulence and cell to cell spreading of Listeria. Recruitment of Ena/VASP proteins to subcellular sites of actin polymerization is mediated by their conserved, N-terminally located EVH1 domain via interactions with target proteins containing PRMs (9Reinhard M. Jarchau T. Walter U. Trends Biochem. Sci. 2001; 26: 243-249Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). The EVH1 domain of Ena/VASP proteins (residues 1-115) interacts directly with several target peptide sequences containing the FPxφP consensus core motif (where φ is a hydrophobic residue). Binding partners to date include the focal adhesion proteins, vinculin (10Brindle N.P. Holt M.R. Davies J.E. Price C.J. Critchley D.R. Biochem. J. 1996; 318: 753-757Crossref PubMed Scopus (162) Google Scholar, 11Reinhard M. Rüdiger M. Jokusch B.M. Walter U. FEBS Lett. 1996; 399: 103-107Crossref PubMed Scopus (131) Google Scholar) and zyxin (12Reinhard M. Jouvenal K. Triquier D. Walter U. Proc. Nat. Acad. Sci. U. S. A. 1995; 92: 7956-7960Crossref PubMed Scopus (161) Google Scholar, 13Drees B. Friederich E. Fradelizi J. Louvard D. Beckerle M.C. Golsteyn R.M. J. Biol. Chem. 2000; 275: 22503-22511Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar), the axon guidance proteins, Roundabout (Robo) (14Bashaw G.J. Kidd T. Murray D. Pawson T. Goodman C.S. Cell. 2000; 101: 703-715Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar) and Semaphorin 6A-1 (15Klostermann A. Lutz B. Gertler F. Behl C. J. Biol. Chem. 2000; 275: 39647-39653Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar), the Fyn-binding/SLP-76-associated protein (Fyb/SLAP) (16Krause M. Sechi A.S. Konradt M. Monner D. Gertler F.B. Wehland J. J. Cell Biol. 2000; 149: 181-194Crossref PubMed Scopus (256) Google Scholar), the lipoma preferred partner protein (17Hoffman L.M. Nix D.A. Benson B. Boot-Hanford R. Gustafsson E. Jamora C. Menzies A.S. Goh K.L. Jensen C.C. Gertler F.B. Fuchs E. Fassler R. Beckerle M.C. Mol. Cell. Biol. 2003; 23: 70-79Crossref PubMed Scopus (79) Google Scholar), and SAX-3 (18Yu T.W. Hao J.C. Lim W. Tessier-Lavigne M. Bargmann C.I. Nat. Neurosci. 2002; 5: 1147-1154Crossref PubMed Scopus (124) Google Scholar) proteins, as well as the Listeria surface protein, ActA (19Niebuhr K. Ebel F. Frank R. Reinhard M. Domann E. Carl U.D. Walter U. Gertler F.B. Wehland J. Chakraborty T. EMBO J. 1997; 16: 5433-5444Crossref PubMed Scopus (332) Google Scholar). Because the three-dimensional structure of the EVH1 domains of several Ena/VASP proteins are now known (20Prehoda K.E. Lee D.J. Lim W.A. Cell. 1999; 97: 471-480Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 21Fedorov A.A. Fedorov E. Gertler F. Almo S.C. Nat. Struct. Biol. 1999; 6: 661-665Crossref PubMed Scopus (99) Google Scholar, 22Ball L.J. Kühne R. Hoffmann B. Häffner A. Schmieder P. Volkmer-Engert R. Hof M. Wahl M. Schneider-Mergener J. Walter U. Oschkinat H. Jarchau T. EMBO J. 2000; 19: 4903-4914Crossref PubMed Scopus (99) Google Scholar), this opens the way for the rational design of modulators which would act by binding specifically to the EVH1 domain. Small-molecule modulators of Ena/VASP proteins, delivered to cells in a dose-dependent manner, could then be used to obtain valuable information complementary to genetic knockout data (4Kuruvilla F.G. Shamji A.F. Sternson S.M. Hergenrother P.J. Schreiber S.L. Nature. 2002; 416: 653-657Crossref PubMed Scopus (337) Google Scholar, 23Nguyen J.T. Porter M. Amoui M. Miller W.T. Zuckermann R.N. Lim W.A. Chem. Biol. 2000; 7: 463-473Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar) by providing means to perturb protein interactions in a specific, rapid, and tunable manner (for a recent review see Ref. 24Shogren-Knaak M.A. Alaimo P.J. Shokat K.M. Annu. Rev. Cell Dev. Biol. 2001; 17: 405-433Crossref PubMed Scopus (93) Google Scholar). Such inhibitors would be useful in a number of different ways: (i) to selectively inhibit the interaction of EVH1 domains with their natural ligands, allowing controlled and detailed studies of EVH1-mediated signaling events; (ii) in appropriately modified forms, as molecular tags to monitor the localization, distribution, and rates of formation/dissociation of EVH1-mediated interactions within the cell; and (iii) as potential precursors/lead molecules in the development of future generations of novel therapeutics for treatment of diseases where partial inhibition of EVH1-mediated events would be desirable (for example, in modulating pathologically altered cell adhesion or cell motility in inflammatory and metastatic diseased states and in combating virulence of intracellular pathogens). Here we make use of the available high resolution structural data on complexes of several EVH1 domains with peptides containing FPxφP motifs to guide the design of novel EVH1 inhibitors. Specifically, we decided to take advantage of ActA peptides to derive VASP EVH1 inhibitors. The Listerial surface protein, ActA, was found to be singularly necessary for VASP recruitment even in the presence of native VASP binding partners (13Drees B. Friederich E. Fradelizi J. Louvard D. Beckerle M.C. Golsteyn R.M. J. Biol. Chem. 2000; 275: 22503-22511Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 19Niebuhr K. Ebel F. Frank R. Reinhard M. Domann E. Carl U.D. Walter U. Gertler F.B. Wehland J. Chakraborty T. EMBO J. 1997; 16: 5433-5444Crossref PubMed Scopus (332) Google Scholar, 25Domann E. Wehland J. Rohde M. Pistor S. Hartl M. Goebel W. Leimeister-Wachter M. Wuenscher M. Chakraborty T. EMBO J. 1992; 11: 1981-1990Crossref PubMed Scopus (325) Google Scholar, 26Kocks C. Gouin E. Tabouret M. Berche P. Ohayon H. Cossart P. Cell. 1992; 68: 521-531Abstract Full Text PDF PubMed Scopus (654) Google Scholar, 27Machner M.P. Urbanke C. Barzik M. Otten S. Sechi A.S. Wehland J. Heinz D.W. J. Biol. Chem. 2001; 276: 40096-40103Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar) by efficiently and competitively binding to the EVH1 domain of VASP via FPPPP motifs in the ActA sequence thus enabling the bacterial pathogen to successfully hijack the host cell actin polymerization machinery to support its own motility (19Niebuhr K. Ebel F. Frank R. Reinhard M. Domann E. Carl U.D. Walter U. Gertler F.B. Wehland J. Chakraborty T. EMBO J. 1997; 16: 5433-5444Crossref PubMed Scopus (332) Google Scholar, 22Ball L.J. Kühne R. Hoffmann B. Häffner A. Schmieder P. Volkmer-Engert R. Hof M. Wahl M. Schneider-Mergener J. Walter U. Oschkinat H. Jarchau T. EMBO J. 2000; 19: 4903-4914Crossref PubMed Scopus (99) Google Scholar, 28Kang F. Laine R.O. Bubb M.R. Soutwick F.S. Purich D.L. Biochemistry. 1997; 36: 8384-8392Crossref PubMed Scopus (105) Google Scholar). Although ActA contains four tandem repeats of short sequences containing the FPxφP motif, each capable individually of binding to one EVH1 domain (27Machner M.P. Urbanke C. Barzik M. Otten S. Sechi A.S. Wehland J. Heinz D.W. J. Biol. Chem. 2001; 276: 40096-40103Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar), it was found that the third of these repeats, corresponding to residues 332-344 of the ActA sequence (numbered in this work as 1SFEFPPPPTEDEL13), bound the EVH1 domain most strongly (19Niebuhr K. Ebel F. Frank R. Reinhard M. Domann E. Carl U.D. Walter U. Gertler F.B. Wehland J. Chakraborty T. EMBO J. 1997; 16: 5433-5444Crossref PubMed Scopus (332) Google Scholar). We therefore used derivatives of this peptide for our studies. Our previous work in vitro showed that the proline residues at positions Pro5 and Pro8 were absolutely necessary for EVH1 domain binding (22Ball L.J. Kühne R. Hoffmann B. Häffner A. Schmieder P. Volkmer-Engert R. Hof M. Wahl M. Schneider-Mergener J. Walter U. Oschkinat H. Jarchau T. EMBO J. 2000; 19: 4903-4914Crossref PubMed Scopus (99) Google Scholar). When either of these prolines was replaced by any other natural amino acid, complete loss of VASP EVH1-binding affinity was observed in almost every case. The high resolution structures of the EVH1 domains from Mena, Evl, and VASP proteins, in complex with FPXφP-containing peptides (20Prehoda K.E. Lee D.J. Lim W.A. Cell. 1999; 97: 471-480Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 21Fedorov A.A. Fedorov E. Gertler F. Almo S.C. Nat. Struct. Biol. 1999; 6: 661-665Crossref PubMed Scopus (99) Google Scholar, 22Ball L.J. Kühne R. Hoffmann B. Häffner A. Schmieder P. Volkmer-Engert R. Hof M. Wahl M. Schneider-Mergener J. Walter U. Oschkinat H. Jarchau T. EMBO J. 2000; 19: 4903-4914Crossref PubMed Scopus (99) Google Scholar), showed that this conservation was due to the highly efficient packing of the N-substituted Pro5 and Pro8 pyrrolidine rings into hydrophobic grooves between a small cluster of closely spaced, aromatic side chains on the EVH1 domain surface (22Ball L.J. Kühne R. Hoffmann B. Häffner A. Schmieder P. Volkmer-Engert R. Hof M. Wahl M. Schneider-Mergener J. Walter U. Oschkinat H. Jarchau T. EMBO J. 2000; 19: 4903-4914Crossref PubMed Scopus (99) Google Scholar, 29Ball L.J. Jarchau T. Oschkinat H. Walter U. FEBS Lett. 2002; 513: 45-52Crossref PubMed Scopus (111) Google Scholar). Our first aim was therefore to study the effects of replacing these prolines with alternative N-substituted amino acid peptoid building blocks that would maintain the required N-substituted backbone structure. Previous workers used alternative N-substituted amino acids in place of prolines to create a number of peptomers for both SH3 and WW domains with affinities comparable to the natural peptides (3Nguyen J.T. Turck C.W. Cohen F.E. Zuckermann R.N. Lim W.A. Science. 1998; 282: 2088-2092Crossref PubMed Scopus (277) Google Scholar, 4Kuruvilla F.G. Shamji A.F. Sternson S.M. Hergenrother P.J. Schreiber S.L. Nature. 2002; 416: 653-657Crossref PubMed Scopus (337) Google Scholar, 23Nguyen J.T. Porter M. Amoui M. Miller W.T. Zuckermann R.N. Lim W.A. Chem. Biol. 2000; 7: 463-473Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). We applied a similar approach to the study of EVH1-domain:peptide interactions to help us to understand more about the specific roles of the conserved prolines in EVH1-recognition and to what extent variations in this N-substituted side chain can be tolerated at these sites. We also identified other conserved residues in positions flanking the FPxφP core motif using the SPOT method (30Frank R. Tetrahedron. 1992; 48: 9217-9232Crossref Scopus (925) Google Scholar). The effects of non-natural amino acid substitution at these positions was then also investigated. Preparation of the VASP EVH1 Domain; VASP-(1-115)—The DNA sequence of VASP-(1-115) was cloned into the plasmid pGEX-4T-1 (Amersham Biosciences). Protein for SPOT scans was expressed in 2× YT medium (16 g of bactotryptone (Difco), 10 g of bacto yeast extract (Difco), 5 g of NaCl in 1 liter, 2% glucose, 10 μg/ml ampicillin) and purified as described previously (22Ball L.J. Kühne R. Hoffmann B. Häffner A. Schmieder P. Volkmer-Engert R. Hof M. Wahl M. Schneider-Mergener J. Walter U. Oschkinat H. Jarchau T. EMBO J. 2000; 19: 4903-4914Crossref PubMed Scopus (99) Google Scholar). Protein molecular masses were verified by mass spectrometry. Uniformly 15N-labeled VASP (1-115) for NMR titrations, was expressed in an MOPS minimal medium (31Neidhardt F.C. Bloch P.L. Smith D.F. J. Bacteriol. 1974; 119: 736-747Crossref PubMed Google Scholar) containing 15NH4Cl as the sole nitrogen source. All samples for NMR spectroscopy were prepared at pH 6.0, in a buffer containing 20 mm KH2PO4, 50 mm KCl, 0.2 mm NaN3. Preparation of Peptomers on a Planar Cellulose Support—Cellulose membranes (Whatman 50) were prepared as described previously (32Volkmer-Engert R. Hoffmann B. Schneider-Mergener J. Tetrahedron Lett. 1997; 38: 1029-1032Crossref Scopus (45) Google Scholar). Cellulose-bound peptomers were semi-automatically prepared using an Abimed ASP222 robot. The peptide building blocks were synthesized according to the standard SPOT synthesis protocols (30Frank R. Tetrahedron. 1992; 48: 9217-9232Crossref Scopus (925) Google Scholar) as described in detail previously (33Kramer A. Schneider-Mergener J. Methods Mol. Biol. 1998; 87: 25fPubMed Google Scholar, 34Wenschuh H. Volkmer-Engert R. Schmidt M. Schulz M. Schneider-Mergener J. Reineke U. Biopolymers. 2000; 55: 188-206Crossref PubMed Scopus (174) Google Scholar, 35Frank R. Overwin H. Methods Mol. Biol. 1996; 66: 149fPubMed Google Scholar). Peptoid building block 1, sarcosine, was used in the Fmoc-protected form and treated in the chemical synthesis like proline, not as a peptoid building block. Reactions were monitored by bromphenol blue staining of membranes after each Fmoc deprotection step. Screening of the Peptomer Array—The membrane was soaked in methanol and washed twice with 50 ml of methanol, three times with 50 ml of TBS (prepared from a 10× solution comprising 80 g of NaCl, 2 g of KCl, and 61 g of Tris in 1 liter of water and adjusted to pH 8.0), and blocked with blocking reagent overnight (5 ml of blocking buffer from Roche Diagnostics, Mannheim, Germany, 5 ml of TBS, 250 μl of Tween 20 (10% in water), and 2.5 g of saccharose in 50 ml of MilliQ water). The membrane was then washed once with 50 ml of T-TBS (TBS plus 0.05% (v/v) Tween 20 (added from a 10% (v/v) solution in water)) and incubated with a solution of GST-VASP EVH1 (50 μm in TBS) for 3 h. Following incubation the membrane was washed three times with 50 ml of TBS and incubated for a further 2 h with a monoclonal rabbit GST antibody (Sigma). After further washing with 50 ml of TBS, the membrane was then incubated for 0.5 h with the second anti-rabbit IgG peroxidase-labeled antibody (Sigma, Deisenhofen, Germany). Analysis and quantification of binding was carried out using a chemiluminescence substrate (SuperSignal West Pico, Pierce) and LumiImagerTM (Roche Diagnostics). Images were processed using the program CorelDraw. Larger Scale Peptomer Synthesis—For the larger scale preparation (several milligrams) of each peptomer, syntheses were carried out in parallel on an Abimed AMS 422 multiple peptide synthesizer. A microtiter plate with 96 wells was used. Each well was filled with 20 mg of TentaGel S RAM resin from Rapp Polymere, Germany (the resin had a derivatization degree of 0.25 mmol/g) suspended in a 3:7 mixture of dimethylacetamide:dichloromethane. To obtain sufficient yields for our studies, 8 wells were used for each of the desired peptomers, giving a maximum theoretical yield of 40 μmol for each peptomer. For chain elongation with natural peptide residues, Fmoc-protected amino acids with free carboxyl groups were used, and coupling was achieved with standard DIC/HOBT chemistry. Chain elongation with N-substituted peptoid residues was achieved by reacting the free amine-groups first with bromoacetic acid-dinitrophenylester and then with the desired primary amine. For coupling of the residue N-terminal to a peptoid residue, the amino acids to be added were pre-activated as anhydrides with 0.5 eq of DIC as described in the literature (36Ast T. Heine N. Germeroth L. Schneider-Mergener J. Wenschuh H. Tetrahedron Lett. 1999; 40: 4317-4318Crossref Scopus (45) Google Scholar). Cleavage of the complete peptomers from the resin, as well as cleavage of side-chain protecting groups, was achieved by adding 500 μl of a solution of 1% (w/v) phenol, 2% (v/v) water, 5% (v/v) dichloromethane, and 3% (v/v) triisobutylsilane in trifluoroacetic acid to each well. The eight separate reaction vessels for each compound were pooled and diluted with a hundred times excess of cold diethylether to facilitate precipitation of the reaction product. The suspensions were centrifuged with a Heraeus Megafuge 1.0 R, using a BS4402/A rotor at 14,000 rpm for 5 min. The supernatants were discarded, and the pellets were resuspended in cold ether. This procedure was repeated three times. All peptomers were analyzed by reversed phase-high performance liquid chromatography on an analytical Vydac C18 column using a linear gradient of 5-60% acetonitrile:water (0.05% (v/v) trifluoroacetic acid) for 20 min at 1.2 ml/min flow rate (detection at 214 nm) and MALDI-TOF mass spectrometry using α-cyano-4-hydroxycinnamic acid as matrix (LaserTec BenchTop II mass spectrometer, PE Biosystems, Weiterstadt, Germany) and purified by preparative high performance liquid chromatography on a preparative Vydac C18 column if necessary. Biacore Measurements—Biacore measurements were carried out on a Biacore system using CM5 chips. VASP-(1-115) was coupled to the chip using standard protocols provided by the manufacturer, at pH 3.0 in 10 mm glycine buffer. Putative ligands were measured in seven steps, at concentrations: 15.625 μm, 31.25 μm, 62.5 μm, 125.0 μm, 250.0 μm, 500.0 μm, and 1 mm. Compounds were dissolved in standard Biacore HBS-EP buffer, containing 0.01 m HEPES, 015 m NaCl, 3 mm EDTA, and 0.005% polysorbate 20 (v/v) at pH 7.4. Binding curves were fitted using the program Origin 5.0 by Microcal. NMR Titrations—NMR spectra were recorded at 300 K, using a Bruker DRX 600 spectrometer in standard configuration, with a triple resonance probe equipped with triple axis self-shielded gradient coils. Uniformly 15N-labeled VASP EVH1 in phosphate buffer (20 mm KH2PO4, 50 mm KCl, 0.2 mm NaN3, 10% D2O; pH 6.0) was used at a concentration of 0.1 mm. Peptomers were dissolved in the same buffer (with the pH adjusted to 6.0 before titrations) and added stepwise to the protein samples. After each addition of ligand, a 15N-HSQC spectrum with eight scans was recorded. Measurements of K D were all calculated from CSPs of the EVH1 domain residue Ala75, which showed large perturbations in response to all ligands titrated in this work. Data were processed using the XWIN-NMR program (version 2.6) of Bruker Analytik GmbH (Rheinstetten, Germany) and the AZARA program (version 2.1) of W. Boucher. Assignment was carried out on Silicon Graphics O2 workstations, using the interactive program ANSIG 3.3 (37Kraulis P.J. Biochemistry. 1994; 33: 3515-3531Crossref PubMed Scopus (289) Google Scholar, 38Kraulis P.J. J. Magn. Reson. 1989; 24: 627-633Google Scholar). AZARA and ANSIG are both available by anonymous file transfer protocol from ftp.bio.cam.ac.uk in the directory ftp/pub. Modeling of Complexes of VASP EVH1 with Peptomers—Complexes of the VASP EVH1 domain with peptomer ligands were modeled in a 250-ps molecular dynamics simulation at 300 K using Amber 5.0 (39Case D.A. Pearlman D.A. Caldwell J.W. III T.E.C. Ross W.S. Simmerling C.L. Darden T.A. Merz K.M. Stanton R.V. Cheng A.L. Vincent J.J. Crowley M. Ferguson D.M. Radmer R.J. Seibel G.L. Singh U.C. Weiner P.K. Kollman P.A. Amber 5. 5th ed. University of California, San Francisco1997Google Scholar). Simulations were performed under periodic boundary conditions using explicit water molecules. The periodic boundary conditions box was equilibrated by using constant pressure dynamics. The use of the SHAKE option constraining all heavy atom-hydrogen bond lengths allowed a step width of 2 fs. The non-bonded cutoff was set to 12 Å, and the non-bonded pair list was updated every 10 fs. During the calculation only those residues that showed CSPs greater than 0.1 ppm were allowed to move. The starting structure for each complex was obtained by docking of the FPXφP motif of the peptide onto the EVH1 domain surface with the same contacts as reported in the crystal structure of Mena EVH1 (20Prehoda K.E. Lee D.J. Lim W.A. Cell. 1999; 97: 471-480Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). The structures of the complexes after 250 ps were minimized until a root mean square deviation gradient of 0.005 was reached. The AMBER program package is available from www.amber.ucsf.edu/amber/amber.html. We have shown previously that substitution of either of the proline residues at positions 5 or 8 in the ActA peptide 1SFEFPPPPTEDEL13 by any other natural amino acid results in loss of EVH1 binding (22Ball L.J. Kühne R. Hoffmann B. Häffner A. Schmieder P. Volkmer-Engert R. Hof M. Wahl M. Schneider-Mergener J. Walter U. Oschkinat H. Jarchau T. EMBO J. 2000; 19: 4903-4914Crossref PubMed Scopus (99) Google Scholar). However, by replacing these prolines with non-natural N-substituted peptoid building blocks, we found it was possible to create peptomer ligands that maintained moderate EVH1-binding affinities (90-400 μm) comparable to some of the natural ligands (20-400 μm). Fig. 1a shows a 13-residue sequence from the third ActA repeat, which we used as the starting point for our studies. The most highly conserved residues of this peptide, Phe4, Pro5, Pro8, and Leu13, as identified from previous SPOT amino acid substitution scans (22Ball L.J. Kühne R. Hoffmann B. Häffner A. Schmieder P. Volkmer-Engert R. Hof M. Wahl M. Schneider-Mergener J. Walter U. Oschkinat H. Jarchau T. EMBO J. 2000; 19: 4903-4914Crossref PubMed Scopus (99) Google Scholar) (shaded in the figure) were chosen for substitution analysis by N-substituted peptoids (Fig. 1b). In addition, the two very highly conserved prolines were investigated simultaneously using double substitution analysis (Fig. 1c). Single Amino Acid Substitutions—To study the effects of single substitutions (Fig. 1b), each of the abovementioned residues were substituted in turn against the 24 commercially available peptoid building blocks shown in Table I, whereas all other residues were left unchanged (see Fig. 1a). The amino group of the new substituent is in each case incorporated into the peptide backbone producing a sarcosine-derived structure for substituted residues. The row labeled “WT” shows the binding of the native ActA peptide as a control.Table IStructures of the 24 primary amines used in the SPOT synthesis and the following solid-phase synthesis of peptomers Substitution of the FPxφP-core-flanking position Leu13 with these peptoid building blocks lead only to very weak spots on the membrane, indicating the importance of leucine at this position. Single substitutions of Phe4 for peptoid building block 7 (see Table I), Pro5 for peptoid building blocks 4, 7-10, and 21, and Pro8 for peptoid building blocks 4, 7-10, 14, and 23, all produced peptomers that bound to the VASP EVH1 domain. It is clear from our previous work and that of others, that a large aromatic side chain, Phe or Trp, at position 4 is essential for EVH1 binding (19Niebuhr K. Ebel F. Frank R. Reinhard M. Domann E. Carl U.D. Walter U. Gertler F.B. Wehland J. Chakraborty T. EMBO J. 1997; 16: 5433-5444Crossref PubMed Scopus (332) Google Scholar, 22Ball L.J. Kühne R. Hoffmann B. Häffner A. Schmieder P. Volkmer-Engert R. Hof M. Wahl M. Schneider-Mergener J. Walter U. Oschkinat H. Jarchau T. EMBO J. 2000; 19: 4903-4914Crossref PubMed Scopus (99) Google Scholar). This can be rationalized by the structural data available for EVH1 complexes that show that a large, flat side chain at this Ca position is required in the peptide to intercalate the exposed aromatic side chains (Tyr16, Trp23, Phe79; VASP numbering) of the VASP EVH1 domain surface (20Prehoda K.E. Lee D.J. Lim W.A. Cell. 1999; 97: 471-480Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 21Fedorov A.A. Fedorov E. Gertler F. Almo S.C. Nat. Struct. Biol. 1999; 6: 661-665Crossref PubMed Scopus (99) Google Scholar, 22Ball L.J. Kühne
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