The assembly of helical and β-sheet peptide blocks containing reactive chain ends results inhighly branched chain architectures (‘locked-in folds') mimicking native tertiary structures.This molecular kit strategy allows to bypass the protein folding problem in protein de novodesign and gives access to protein mimetics of high thermodynamic stability. The validity ofthis concept is exemplified for the design and synthesis of locked-in folds mimicking the zincfinger and MHC folding motif

Lehmann, Christian

Mathieu, Marc

Razaname, Alain

Tuchscherer, Gabriele

Summary: The assembly of helical and β-sheet peptide blocks containing reactive chain ends results in highly branched chain architectures (‘locked-in folds') mimicking native tertiary structures. This molecular kit strategy allows to bypass the protein folding problem in protein de novo design and gives access to protein mimetics of high thermodynamic stability. The validity of this concept is exemplified for the design and synthesis of locked-in folds mimicking the zinc finger and MHC folding motif

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Letters in Peptide Science, 4 (1997) 95-100 95 ESCOM LIPS 164 Engineering of zinc finger and MHC motifs to locked-in tertiary folds Marc Mathieu, Christian Lehmann,  Alain Razaname and Gabriele Tuchscherer* Institute of Organic Chemistry, University of Lausanne, BCH-Dorigny, CH-IO15 Lausanne, Switzerland Received 27 December 1996 Accepted 22 January 1997 Keywords: Protein design; Locked-in folds; Chemoselective l gation; Zinc finger motif; MHC motif; Protein mimetics; Non-native architectures SUMMARY The assembly of helical and [3-sheet peptide blocks containing reactive chain ends results in highly branched chain architectures ('locked-in folds') mimicking native tertiary structures. This molecular kit strategy allows to bypass the protein folding problem in protein de novo design and gives access to protein mimetics of high thermo- dynamic stability. The validity of this concept is exemplified for the design and synthesis of locked-in folds mimick- ing the zinc finger and MHC folding motifs. INTRODUCTION The construction of novel proteins from scratch is hampered by the low probability of linear poly- peptides to fold into a unique, stable tertiary structure [1]. Since the complex folding mechan- ism has yet to be unraveled [2], we proposed some years ago a conceptually different approach in protein de novo design to bypass this well-known folding problem: the template assembled synthetic proteins (TASP) concept [3,4]. As a key element, a topological template [5,6] serves as a built-in device to induce and reinforce intramolecular interactions of the covalently attached amphi- pathic peptide blocks, thus leading to predeter- mined packing topologies such as (x-helical bundle or ~-sheet TASP molecules. As a general feature, TASP molecules adopt a globular structure show- ing cooperative denaturation behaviour similar to natural proteins [7,8]. Recent progress in the synthetic methodology for assembling peptides allows us now to access the full potential of the TASP concept. For example, in separating struc- tural and functional domains of native proteins, we have recently established the use of templates (RAFT) as scaffolds for the assembly of receptor binding loops [9]. Here, we extend the TASP concept for the construction of multibranched, oligocyclic peptides, termed locked-in tertiary folds. These conformationally trapped folding units bypass the complex folding pathway of linear polypeptides and are thought to exhibit unique structural and physicochemical prop- erties. *To whom correspondence should be addressed. 0929-5666/$ 6.00 + 1.00 9 1997 ESCOM Science Publishers B.V. 96 Fig. 1. Engineering ofpeptides: molecular kit (a-helices, ~- sheets) for the construction of locked-in tertiary folds. (a) Zinc finger derived [313c~ folding unit with the appropriate g ometry for Zn(II) complexation. (b) MHC derived [~C~lC~ 2 folding unit mimetic. X,X'; Y,Y', Z,Z': functional groups (-CHO, -ONH2; -SH, -CHBr) for covalently attaching the individual building blocks (cq, ~, antigenic peptide) via chemoselective l gation techniques [12-14]. RESULTS AND DISCUSSION Design of a zinc finger locked-in fold <flflo~> According to the novel approach, the envisioned folding motif is set up by covalently linking the individual helical or ~-sheeted peptide blocks at both chain ends via linkers onto templates to result in highly branched packing arrangements ('locked-in folds' (LIFs), Fig. 1). In contrast o regularly folded polypeptides, intramolecular long-range interactions are enforced here by trap- ping each secondary structure block at both chain ends, resulting in a conformationally restricted chain architecture with increased thermodynamic stability. In particular, the large number of alter- native folding pathways and tertiary assemblies iconsiderably excluded within the depicted locked- in motifs. The present concept is exemplified first for the design and chemical synthesis of a zinc finger (Zif) derived locked-in folding unit of type ~.  So far, the Zif folding module has been the sub- ject of numerous studies for delineating structure- function relationships of this DNA-binding pro- tein [10]. The consensus sequence Xaa3-Cys-Xaa2_4-Cys-Xaal2His-Xaa3_4-His-Xaa4 (Xaa denoting any amino acid) has been shown to fold in the presence of Zn(II) into a 1313c~ folding unit [11] which binds via its helical part to a three base pairs subsite of the DNA. Multimeric zinc finger proteins, consisting of several Zif modules, are considered to play a key role in controlling gene expression [10]. In applying the concept of locked-in folds, the [3[3c~ framework of the Zif motif suggests the use of a topological template as a mimetic of the structural part and the covalent trapping of the bifunctional helical block as shown in Fig. l a. To this end, the helix is covalently attached at both chain ends to the cyclic 14-mer template via amide bonds or by chemoselective ligation procedures [12-15] to result in a locked-in fold of type <[313a>. It has been demonstrated earlier that cyclic peptide templates as depicted in Fig. 1 and Scheme 1 adopt preferentially an anti- parallel [3-sheet structure directing the e-NH 2 side chains of the lysine residues as attachment sites towards the same face of the template plane [5,6]. Superposition of the X-ray structure of DNA- bound Zif 268 and the conformationally relaxed zinc finger LIF reveals a nearly perfect match of the two molecules (Ref. 15, not shown here). Most notably, after the incorporation of cysteine and histidine residues in the template and helix, respectively (Scheme 1), the Zn(II) complexation site could be readily accommodated in the LIF molecule. 97 Template: H 2 NiLys(Dde)-Pro-Gly-Cys(Tr)-Asp(O t Bu)-Arg(Pmc)-Ala-Lys(Dde)-Pro-Gly-Phe-Ala-Cys(Trt)-Ala-Sasrin 1. cleavage from resin (5% TFA) 2. cyclization (PyBOP, DMF) 3. -Dde (10% H2NNH 2 in DMF) Helix: Pme O tBu Pmc Pmc t Bu Suc-Phe-Ser-Arg-Ser-Asp-G•u-Leu-Thr(Me•Me`}•pr•)-Arg-His-••e-Arg-••e-His-Thr-G•y-Lys-G•y-Sasrin t Bu t Bu O tBu Trt Trt Boc I. cSeavage from resin (5% TFA) 2. attachment o template (PyBOP, DMF) 3. deprotection and ring opening (95% TFA) zinc finger LIF Scheme l. Synthesis of the zinc finger molecule <13[3cc>. The orthogonally protected 14-mer template isprepared by Fmoc strategy based on solid phase peptide synthesis and cyclized in solution as described elsewhere [5]. In parallel, the helical block is cleaved from the Sasrin-resin to result in a fully protected peptide containing carboxy functions at both chain ends (Suc= HOOC-(CH2)- CO-). qJPro is used as a temporary protection of threonine to increase the solvation of the helical block during the condensation reaction to the template in solution. The target molecule <1313c~> is obtained after simultaneous side-chain deprotection and ~Pro ring opening. a 1 1 I : I ,",1 3 II ii i I I !  i I I l i | I I i I I I i I ~ I i I ! I ~0  i I I I  i I I I  i I I I  i I I I  .5  i I i I i I I I i I I I i I I I i I I I i I I I i I I I ,~ , 0 rain b 2.0 1.0 i 0 -1.0 - 2.0 ~ 1 )  , , ,~ .4  u I ,9 .Io * e 9 7 eTez  I I i I 200 220 245 nm Fig. 2. (a) Reverse-phase HPLC profile of the condensation reaction of the helical block a to the 14-mer template (Scheme 1) after 8 h of reaction; h template; 2: helical block; 3: target product <[313a> (dashed lines indicate the profile at t = 0). (b) CD spectra of the Zn(II) complexed (1) and metal free (2) <J313c~>. The spectrum of the single helix block is shown for comparison (3). c=6 x 10 -5 raM; 10 mM aqueous phosphate buffer, pH 7; inset: ESI-MS of <~ct>. 98 Template: Trt Boc Dde Trt Dde Boc H 2 N-Ala-Cys-Lys-Pro-Gly-Lys-Cys-Ala-Ala-Lys-Pro-Gly-Lys-Ala-COOH Helix: o~ 1 1. cyclization (PyBOE DMF) 2. - Dde (10% H2NNH2 in DMF) 3. + Fmoc-NHOCH2COOH 4. -Boc (95% TFA) 5. + Boe-NHOCH2COOH 6. - Fmoc (20% pipendine) OtBu Trt OtBu Trt Ac-Ser-Phe-Glu-Lys-GIn-Ala-Ala-Leu-Ala-Asn-Ile-Ala-VaI-Asp-Lys-Ala-Asn- Lys-CONH 2 t Boc Ser(tBu) Trt Boc Boc Ser(tBu) 1. -Boc, Bu, Trt (95% TFA) 2. oxidation with NalO 4 (---~CHO) 3. attachment o~ 1 (via oxime bonds) 1. -[3oc (95% TFA) Helix: o~ 2 OtBu Pmc OtBu tBu Pmc tBu Ac-Pro-Leu-Lys-Glu-GIn-Arg-Arg-Ala-Ala-VaI-Asp-Thr-Tyr-Lys-Arg- Lys-Asn-Tyr-CONH 2 tBu Boc Ser(tBu) Trt Pmc I~u Dde Boc Ser(tBu) 1. -Boc, Bu, Trt (95% TFA) 2. oxidation with NalO 4 (--'*CliO) 3. attachment o~ 2 (via oxime bonds) Scheme 2. Synthesis of the MHC LIF molecule <13[3cz~et2>, After cyclization and functionalization f the template molecule prepared by SPPS, sequential condensation f the helical c~] and ct 2 blocks via oxime bond formation in solution results in the target molecule <[313c~1ct2>. Theattachment si es in the constituent blocks for the oxime bond formation are depicted in bold letters. As shown, the functional groups of the helix are obtained by IO 4 oxidation of the serine residues attached to the e-NH 2 group of the lysine residues [10]; the aminooxy functions in the template molecule were fixed to the orthogonally protected lysine side chains using aminooxy acetic acid derivatives. As potential attachment si e for the antigenic peptide an orthogonal protected (Dde) lysine residue was incorporated in helix c~ 2. 99 Synthesis and conformational properties of <flfla> The chemical synthesis (Scheme 1) was achieved by preparing the individual building blocks, i.e. the template molecule and the helical segment, by stepwise solid phase synthesis [16]. After cleavage from the resin and HPLC purification, the side- chain protected peptides (containing carboxylic groups at both chain ends) were reacted in aque- ous solution by a one-step condensation of the helical 18-mer peptide with the cyclic template via amide bond formation. To increase the solubility of the fully protected helical block, Thr s (Scheme 1) was protected as a dimethyl pseudo-proline (~Pro) derivative [17]. Due to the pronounced structure disrupting effect of UdPro, the 18-mer peptide was readily soluble in a variety of organic solvents; in addition, the condensation reaction of the helix to the template proceeded to completion within a few hours (Fig. 2a, molecular weight 4015, see inset 2b), pointing to the absence of aggregation effects of the fully protected helical block. As suggested by molecular modeling studies [15], the zinc finger LIF preserves the essential structural and functional features of the native Zif molecule, as documented by the absorption and CD spectra nd by the Zn(II) complexation prop- erties (Fig. 2b). Contrary to the native linear polypeptide, <[313cz> retains some of its secondary structure lements even in the absence of the zinc complex, reflecting the onset of tertiary interac- tions in <131~o~> induced by C- and N-capping of the helix block on the template. In the search for Zif derived mimetics with novel DNA binding properties, the incorporation of non-proteinogenic building blocks offers a wide range for structural and functional modifications; moreover, the assembly to tertiary locked-in folds by a single condensation step allows for the facile access of a large number of analogues, including LIF libraries. Design and synthesis of an MHC derived locked-in fold In applying similar strategies as for zinc finger LIF, the more complex MHC folding motif [18] was engineered according to our molecular kit approach in condensing two helical blocks mim- icking parts of the A-chain (a~: 53-70) and B- chain (c~2: 66-83) helix of native MHC II to a [3- sheet emplate. By fixing both helical chain ends covalently to the template, a locked-in fold of the type <[~aa> mimicking some of the structural features of the native molecule is obtained (Fig. lb). As for the zinc finger LIE the chemical synthesis of the MHC locked-in fold was achieved by a two-step approach (Scheme 2). First, a cyclic 14-mer peptide xhibiting two pairs of orthogonal- ly protected aminooxy groups as attachment sites and the helical blocks (containing aldehyde groups at the C- and N-terminus) were prepared by SPPS. In a second step, the sequential condensation of the two helices to the template was achieved via oxime bond formation [12,14]. The target mol- ecule <[313a~(x2> was obtained in high yields and its chemical integrity was confirmed by ESI-MS (theoretical nd obtained molecular weight: 6179). The elucidation of the conformational nd bio- structural properties of the target molecule is currently in progress. CONCLUSIONS The concept of locked-in folds offers an elegant way to bypass the notorious folding problem in protein de novo design. Due to the reduced con- formational space, the multibranched chain archi- tectures exhibit increased thermodynamic stability as exemplified for the zinc finger locked-in fold, thus serving as versatile scaffolds for mimicking protein function. Further elaboration of today's methodologies in chemoselective ligation and orthogonal protection techniques will further expand the scope of the present approach. Con- sequently, the efficient chemical synthesis of pro- tein-like molecules may substantially contribute to our understanding of the complex interplay be- tween protein structure and function, with inter- esting prospects for the design of therapeutically interesting target molecules. 100 ACKNOWLEDGEMENTS One of  the authors  (G.T.) is grateful for f inan-  cial support  granted by the Agassiz Foundat ion ,  Lausanne,  Switzer land. We thank  Prof. Manf red  Mut ter  for helpful  discussions. REFERENCES 1 a. Richardson, J.S. and Richardson, D.C., Trends Bio- chem. Sci., 14 (1989) 304. b. Bryson, J.W., Betz, S.E, Lu, H.S., Suich, D.J., Zhou, H.X., O'Neil, K.T. and DeGrado, W.E, Science, 270 (1995) 935. e. Vuilleumier, S. and Mutter, M., Angew. Chem., Int. Ed. Engl., 28 (1989) 535. d. Olofsson, S. and Baltzer, L., Folding Des., 1 (1996) 347. e. Broo, K., Brive, L., Lundh, A.-C., Ahlberg, E and Baltzer, L., J. Am. Chem. Soc., 118 (1996) 8172. f. Struthers, M.D., Cheng, R.E and Imperiali, B., Science, 271 (I996) 342. 2 a. Creighton, T.E., Proteins, Freeman, New York, NY, U.S.A., 1984. b. Borman, S., Chem. Eng. News, 74 (1996) 29. 3 Mutter, M. and Tuchseherer, G., Makromol. Chem. Rapid Commun., 9 (1988) 437. 4 Mutter, M., Tuehscherer, G, Miller, C., Altmann, K.H., Carey, R.I., Wyss, D.E, Labhardt, A.M. and Rivier, J.E., J. Am. Chem. Sot., 114 (1992) 1463. 5 Dumy, E, Eggleston, I.M., Cervigni, S.E., Sila, U., Sun, X. and Mutter, M., Tetrahedron Lett., 36 (1995) 1255. 6 Dumy, R, Eggleston, I.M., Esposito, G., Nicula, S. and Mutter, M., Biopolymers, 39 (1996) 297. 7 Mutter, M. and Tuchscherer, G., In Symposium in Print, Highlights in Bioorganic Chemistry, Pure Appl. Chem., 68 (1996) 2153. 8 Tuchscherer, G. and Mutter, M., J. Pept. Sci., 1 (1995) 3. 9 Mutter, M., Dumy, E, Garrouste, E, Lehmann, C., Mathieu, M., Peggion, C., Peluso, S., Razaname, A. and Tuchscherer, G., Angew. Chem., Int. Ed. Engl., 35 (1996) 1482. 10 a. Choo, Y. and Klug, A., Proc. Natl. Acad. Sci. USA, 91 (1994) 11163. b. Jamieson, A.C., Wang, H. and Kim, S.-H., Proc. Natl. Acad. Sci. USA, 93 (1996) 12834. c. Frankel, A.D., Berg, J.M. and Pabo, C.O., Proc. Natl. Acad. Sci. USA, 84 (1987) 4841. d. Michael, S.F., Kilfoil, V.J., Schmidt, M.H., Amann, B.T. and Berg, J.M., Proc. Natl. Acad. Sci. USA, 89 (1992) 4796. e. Desjarlais, J.R. and Berg, J.M., Proc. Natl. Acad. Sci. USA, 90 (1993) 2256. f. Rebar, E. and Pabo, C.O., Science, 263 (1994) 671. 11 Pavletich, N.E and Pabo, C.O., Science, 252 (1991) 809. 12 Rose, K., J. Am. Chem. Soc., 116 (1994) 30. 13 a. Liu, C.E and Tam, J.E, Proc. Natl. Acad. Sci. USA, 91 (1994) 6584. b. Dawson, EE. and Kent, S.B.H., J. Am. Chem. Soc., 115 (1993) 7263. 14 Tuchseherer, G., Tetrahedron Lett., 34 (1993) 8419. 15 Mutter, M., Lehmann, C., Mathieu, M., Rohwedder, B. and Tuchscherer, G., Proc. Natl. Acad. Sci. USA, manu- script submitted. 16 Stewart, J.M. and Young, J.D., Solid Phase Peptide Synthesis, Pierce Chemical Co., Rockford, IL, U.S.A., 1984. 17 W6hr, T., Wahl, E, Nefzi, A., Rohwedder, B., Sato, T., Sun, X. and Mutter, M., J. Am. Chem. Soc., 118 (1996) 9218. 18 Stern, L.J., Brown, J.H., Jardetzky, T.S., Gorga, J.C., Urban, R.G., Strominger, J.L. and Wiley, D.C., Nature, 368 (1994) 215. 

Engineering of zinc finger and MHC motifs to locked-in tertiary folds

Letters in Peptide Science, 4 (1997) 95–100 95*To whom correspondence should be addressed.ESCOMLIPS 164Engineering of zinc finger and MHC motifs tolocked-in tertiary foldsMarc Mathieu, Christian Lehmann, Alain Razaname and Gabriele Tuchscherer*Institute of Organic Chemistry, University of Lausanne, BCH-Dorigny, CH-1015 Lausanne, SwitzerlandReceived 27 December 1996Accepted 22 January 1997Keywords: Protein design; Locked-in folds; Chemoselective ligation; Zinc finger motif; MHC motif;Protein mimetics; Non-native architecturesSUMMARYThe assembly of helical and β-sheet peptide blocks containing reactive chain ends results in highly branchedchain architectures (‘locked-in folds’) mimicking native tertiary structures. This molecular kit strategy allows tobypass the protein folding problem in protein de novo design and gives access to protein mimetics of high thermo-dynamic stability. The validity of this concept is exemplified for the design and synthesis of locked-in folds mimick-ing the zinc finger and MHC folding motifs.INTRODUCTIONThe construction of novel proteins from scratchis hampered by the low probability of linear poly-peptides to fold into a unique, stable tertiarystructure [1]. Since the complex folding mechan-ism has yet to be unraveled [2], we proposed someyears ago a conceptually different approach inprotein de novo design to bypass this well-knownfolding problem: the template assembled syntheticproteins (TASP) concept [3,4]. As a key element,a topological template [5,6] serves as a built-indevice to induce and reinforce intramolecularinteractions of the covalently attached amphi-pathic peptide blocks, thus leading to predeter-mined packing topologies such as α-helical bundleor β-sheet TASP molecules. As a general feature,TASP molecules adopt a globular structure show-ing cooperative denaturation behaviour similar tonatural proteins [7,8]. Recent progress in thesynthetic methodology for assembling peptidesallows us now to access the full potential of theTASP concept. For example, in separating struc-tural and functional domains of native proteins,we have recently established the use of templates(RAFT) as scaffolds for the assembly of receptorbinding loops [9]. Here, we extend the TASPconcept for the construction of multibranched,oligocyclic peptides, termed locked-in tertiaryfolds. These conformationally trapped foldingunits bypass the complex folding pathway oflinear polypeptides and are thought to exhibitunique structural and physicochemical prop-erties.0929-5666/$ 6.00 + 1.00 © 1997 ESCOM Science Publishers B.V.96RESULTS AND DISCUSSIONH HCC<ββα >Y X'X'Y'XX ααββY'YZ'Z<ββα α >1 2abFig. 1. Engineering of peptides: molecular kit (α-helices, β-sheets) for the construction of locked-in tertiary folds. (a) Zincfinger derived ββα folding unit with the appropriate geometryfor Zn(II) complexation. (b) MHC derived ββα1α2 folding unitmimetic. X,X'; Y,Y', Z,Z': functional groups (-CHO, -ONH2;-SH, -CHBr) for covalently attaching the individual buildingblocks (α1, α2, antigenic peptide) via chemoselective ligationtechniques [12–14].Design of a zinc finger locked-in fold <bba>According to the novel approach, the envisionedfolding motif is set up by covalently linking theindividual helical or β-sheeted peptide blocks atboth chain ends via linkers onto templates toresult in highly branched packing arrangements(‘locked-in folds’ (LIFs), Fig. 1). In contrast toregularly folded polypeptides, intramolecularlong-range interactions are enforced here by trap-ping each secondary structure block at both chainends, resulting in a conformationally restrictedchain architecture with increased thermodynamicstability. In particular, the large number of alter-native folding pathways and tertiary assemblies isconsiderably excluded within the depicted locked-in motifs.The present concept is exemplified first for thedesign and chemical synthesis of a zinc finger(Zif) derived locked-in folding unit of type ββα.So far, the Zif folding module has been the sub-ject of numerous studies for delineating structure–function relationships of this DNA-binding pro-tein [10]. The consensus sequenceXaa3-Cys-Xaa2-4-Cys-Xaa12His-Xaa3-4-His-Xaa4(Xaa denoting any amino acid) has been shown tofold in the presence of Zn(II) into a ββα foldingunit [11] which binds via its helical part to a threebase pairs subsite of the DNA. Multimeric zincfinger proteins, consisting of several Zif modules,are considered to play a key role in controllinggene expression [10]. In applying the concept oflocked-in folds, the ββα framework of the Zifmotif suggests the use of a topological template asa mimetic of the structural part and the covalenttrapping of the bifunctional helical block as shownin Fig. 1a. To this end, the helix is covalentlyattached at both chain ends to the cyclic 14-mertemplate via amide bonds or by chemoselectiveligation procedures [12–15] to result in a locked-infold of type <ββα>. It has been demonstratedearlier that cyclic peptide templates as depicted inFig. 1 and Scheme 1 adopt preferentially an anti-parallel β-sheet structure directing the ε-NH2 sidechains of the lysine residues as attachment sitestowards the same face of the template plane [5,6].Superposition of the X-ray structure of DNA-bound Zif 268 and the conformationally relaxedzinc finger LIF reveals a nearly perfect match ofthe two molecules (Ref. 15, not shown here).Most notably, after the incorporation of cysteineand histidine residues in the template and helix,respectively (Scheme 1), the Zn(II) complexationsite could be readily accommodated in the LIFmolecule.97H  N-Lys(Dde)-Pro-Gly-Cys(Tr)-Asp(O  Bu)-Arg(Pmc)-Ala-Lys(Dde)-Pro-Gly-Phe-Ala-Cys(Trt)-Ala-Sasrin2 t1. cleavage from resin (5% TFA)2. cyclization (PyBOP, DMF)3. - Dde (10% H  NNH   in DMF)2 2Template:Helix:zinc finger LIFPPGG1. cleavage from resin (5% TFA)2. attachment to template (PyBOP, DMF)3. deprotection and ring opening (95% TFA)Suc-Phe-Ser-Arg-Ser-Asp-Glu-Leu-Thr(            pro)-Arg-His-Ile-Arg-Ile-His-Thr-Gly-Lys-Gly-SasrinΨMe,MePmcO  ButO  ButButButPmc ButPmcTrt Trt Boc<ββα>Scheme 1. Synthesis of the zinc finger molecule <ββα>. The orthogonally protected 14-mer template is prepared by Fmoc strategybased on solid phase peptide synthesis and cyclized in solution as described elsewhere [5]. In parallel, the helical block is cleavedfrom the Sasrin-resin to result in a fully protected peptide containing carboxy functions at both chain ends (Suc = HOOC-(CH2)-CO-). ΨPro is used as a temporary protection of threonine to increase the solvation of the helical block during the condensationreaction to the template in solution. The target molecule <ββα> is obtained after simultaneous side-chain deprotection and ΨProring opening.a bFig. 2. (a) Reverse-phase HPLC profile of the condensation reaction of the helical block α to the 14-mer template (Scheme 1) after8 h of reaction; 1: template; 2: helical block; 3: target product <ββα> (dashed lines indicate the profile at t = 0). (b) CD spectraof the Zn(II) complexed (1) and metal free (2) <ββα>. The spectrum of the single helix block is shown for comparison (3). c = 6× 10−5 mM; 10 mM aqueous phosphate buffer, pH 7; inset: ESI-MS of <ββα>.98PPGGα1α21. -Boc (95% TFA)1. -Boc, Bu, Trt (95% TFA)3. attachment α (via oxime bonds)22. oxidation with NaIO   ( CHO)4H  N-Ala-Cys-Lys-Pro-Gly-Lys-Cys-Ala-Ala-Lys-Pro-Gly-Lys-Ala-COOH2Ac-Pro-Leu-Lys-Glu-Gln-Arg-Arg-Ala-Ala-Val-Asp-Thr-Tyr-Lys-Arg- Lys-Asn-Tyr-CONH2O ButTrtPmcPmcButPmcO But But ButHelix: α2<ββα >16. - Fmoc (20% piperidine)1. cyclization (PyBOP, DMF)4. -Boc (95% TFA)2. - Dde (10% H  NNH   in DMF)2 23. + Fmoc-NHOCH  COOH25. + Boc-NHOCH  COOH21. -Boc, Bu, Trt (95% TFA)43. attachment α (via oxime bonds)12. oxidation with NaIO   ( CHO)Template:α1 O ButBut Trt BocO But TrtAc-Ser-Phe-Glu-Lys-Gln-Ala-Ala-Leu-Ala-Asn-Ile-Ala-Val-Asp-Lys-Ala-Asn-Lys-CONH2TrtBoc Ser(       )ButBocBoc Ser(       )But DdeBut Boc Ser(       )But<ββα  α  >1 2Trt DdeDdeBocTrtBoc Ser(       )ButHelix:Scheme 2. Synthesis of the MHC LIF molecule <ββα1α2>. After cyclization and functionalization of the template moleculeprepared by SPPS, sequential condensation of the helical α1 and α2 blocks via oxime bond formation in solution results in thetarget molecule <ββα1α2>. The attachment sites in the constituent blocks for the oxime bond formation are depicted in bold letters.As shown, the functional groups of the helix are obtained by IO−4 oxidation of the serine residues attached to the ε-NH2 groupof the lysine residues [10]; the aminooxy functions in the template molecule were fixed to the orthogonally protected lysine sidechains using aminooxy acetic acid derivatives. As potential attachment site for the antigenic peptide an orthogonal protected (Dde)lysine residue was incorporated in helix α2.99Synthesis and conformational properties of <bba>The chemical synthesis (Scheme 1) was achievedby preparing the individual building blocks, i.e.the template molecule and the helical segment, bystepwise solid phase synthesis [16]. After cleavagefrom the resin and HPLC purification, the side-chain protected peptides (containing carboxylicgroups at both chain ends) were reacted in aque-ous solution by a one-step condensation of thehelical 18-mer peptide with the cyclic template viaamide bond formation. To increase the solubilityof the fully protected helical block, Thr8 (Scheme1) was protected as a dimethyl pseudo-proline(ΨPro) derivative [17]. Due to the pronouncedstructure disrupting effect of ΨPro, the 18-merpeptide was readily soluble in a variety of organicsolvents; in addition, the condensation reaction ofthe helix to the template proceeded to completionwithin a few hours (Fig. 2a, molecular weight4015, see inset 2b), pointing to the absence ofaggregation effects of the fully protected helicalblock.As suggested by molecular modeling studies[15], the zinc finger LIF preserves the essentialstructural and functional features of the native Zifmolecule, as documented by the absorption andCD spectra and by the Zn(II) complexation prop-erties (Fig. 2b). Contrary to the native linearpolypeptide, <ββα> retains some of its secondarystructure elements even in the absence of the zinccomplex, reflecting the onset of tertiary interac-tions in <ββα> induced by C- and N-capping ofthe helix block on the template.In the search for Zif derived mimetics withnovel DNA binding properties, the incorporationof non-proteinogenic building blocks offers a widerange for structural and functional modifications;moreover, the assembly to tertiary locked-in foldsby a single condensation step allows for the facileaccess of a large number of analogues, includingLIF libraries.Design and synthesis of an MHC derived locked-infoldIn applying similar strategies as for zinc fingerLIF, the more complex MHC folding motif [18]was engineered according to our molecular kitapproach in condensing two helical blocks mim-icking parts of the A-chain (α1: 53–70) and B-chain (α2: 66–83) helix of native MHC II to a β-sheet template. By fixing both helical chain endscovalently to the template, a locked-in fold of thetype <ββαα> mimicking some of the structuralfeatures of the native molecule is obtained (Fig.1b). As for the zinc finger LIF, the chemicalsynthesis of the MHC locked-in fold was achievedby a two-step approach (Scheme 2). First, a cyclic14-mer peptide exhibiting two pairs of orthogonal-ly protected aminooxy groups as attachment sitesand the helical blocks (containing aldehyde groupsat the C- and N-terminus) were prepared by SPPS.In a second step, the sequential condensation ofthe two helices to the template was achieved viaoxime bond formation [12,14]. The target mol-ecule <ββα1α2> was obtained in high yields and itschemical integrity was confirmed by ESI-MS(theoretical and obtained molecular weight: 6179).The elucidation of the conformational and bio-structural properties of the target molecule iscurrently in progress.CONCLUSIONSThe concept of locked-in folds offers an elegantway to bypass the notorious folding problem inprotein de novo design. Due to the reduced con-formational space, the multibranched chain archi-tectures exhibit increased thermodynamic stabilityas exemplified for the zinc finger locked-in fold,thus serving as versatile scaffolds for mimickingprotein function. Further elaboration of today’smethodologies in chemoselective ligation andorthogonal protection techniques will furtherexpand the scope of the present approach. 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Engineering of zinc finger and MHC motifs to locked-in tertiary folds

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