Rational Design of Orthogonal Receptor-Ligand Combinations

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[ I S ] The density of 2 in the absence of solvent is just 0.87 Mgm--' compared to Fig. 1. Regulated intracellular dimerization with cell-permeable synthetic ligands
1.856 Mgm in the corresponding dihydrate 1. The density was not deter- named CIDs.
mined cxprrimcntally because of the tendency of the crystals to lose solvent
rapidly synthetically in a rational way to remove their intrinsic immuno-
suppressive and toxic properties. This requires modifying the
[16] As noted by 'I reviewer and revealed by Figure 1 a, there is considerable distor- calcineurin-binding (but not the immunophilin-binding) do-
tion of the thermal ellipsoids associated with carbon atoms 2,3,5.6,2',3',5' and main of these naturally occurring CIDs. FKBP12 and CyP bind
6' o l the 4.4'-bipyridine ligands. This observation is consistent with slight twist- their ligands with high affinity (K,, = 0 . 5 n ~and 5 n ~r,espec-
ing of the the pyridine moieties and either static or dynamic disorder between tively), are monomeric, have no discernible effect when ex-
pressed in cells, and their small size (12 kDa and 18 kDa, respec-
two orient11tions. tively) facilitates the incorporation of their cDNA into
expression vectors. A shortcoming is that the natural im-
[I71 C-H" F and C . . F contacts, 2.342 and 3.429(9) A, respectively. are well munophilins are expressed at high levels in many cells and can
therefore diminish the potency of CID ligands toward im-
within ranges expected for significant C-H ' . . F interactions. 1.Shimoni, H. 1. munophilin fusion proteins by forming nonproductive recep-
Carrell. J. P Glusker, M. M. Coombs, J. Am. Chrm. Soc. 1994, ff6. 8162. tor-ligand complexes (Fig. 2a). The resulting loss in specificity
is expected to be especially troublesome in whole organisms,
[IS] Compounds with comparable or larger pores have recently been reported but where cells expressing the fusion proteins may be relatively few
cither cations (T. J. McCarthy, T. A. Tanzer, M. G. Kanatzidis, J. Am. Chem. in number, thus magnifying the buffering effect of natural im-
So( 1995. 117. 1294) or anions are necessarily present in the pores (B. F. munophilins.
Ahrdhams. U. F. Hoskins, D. M. Michall, R. Robson, A'urure, 1994,369, 727).
Fig. 2. Improved receptor-ligand pairs for the regulated inlracellular protein asso-
Rational Design of Orthogonal ciation with synthetic ligands a) Possible protein associations occurring in cells
Receptor-Ligand Combinations** expressing wild-type immunophilins as dimerization domains. b) Protein associa-
tions predicted to occur in cells by using modified ClDs and iinniunophilin~with
Peter J. Belshaw, Joseph G. Schoepfer, compensatory mutations.
Karen-Qianye Liu, Kim L. Morrison, and
Stuart L. Schreiber* To solve this problem we envisioned the creation of new re-
ceptor-ligand pairs with interacting surfaces that ensure a high
Signal transduction in cells occurs primarily by two mecha- degree of specificity. Immunophilin ligands were designed to
contain substituents that clash sterically with amino acid
nisms, one involving allostery and the other involving proximi- sidechains in the immunophilin receptor, thereby abolishing
their interaction with endogenous immunophilins. Compensa-
ty.['] The role of ligand-induced allosteric change in proteins has tory mutations in the receptor were sought that would remove
the offending interaction, thereby creating a unique receptor
been appreciated for many years, and has led to the synthesis of that would be used as a dimerization domain (Fig. 2b). Herein,
we report the implementation of this strategy using CyP-CsA as
numerous molecules that either promote or inhibit the allosteric a test system. In addition to providing an effective solution to
the specific research problem outlined above, we propose that
change required to transduce information in cells. More recent- the creation of new receptor-ligand pairs in this manner will
result in new experimental systems for testing our understand-
ly, cell biological studies have illuminated the role of regulated ing of molecular recognition involving protein receptors.

protein dimerization or oligomerization as a means of informa-

tion transfer. Such an event can promote a proximal relation-

ship between an enzyme and its substrate or a receptor and its

ligand, thereby facilitating a molecular interaction leading to a

signaling evcnt. Examples include the dimerization of growth

factor receptors,[*]oligomerization of antigen and

dimerization of transcription factors.[41These insights have cre-

ated new opportunities for chemists to synthesize molecules

with two protein-binding surfaces and thereby to induce protein

association. Such chemical inducers of dimerization (CIDs)

have the potential to activate many cellular processes, including

ones of biological and medical significance. We recently de-

scribed a method to inducibly control the association of proteins

in cells.[', 61 This was accomplished through the expression of

chimeric proteins in mammalian cells, consisting of a dimeriza-

tion domain fused to a protein or protein domain of interest. By

treating these cells with a synthetic, cell-permeable C I D that

binds to thc dimerization domain, self-association of the protein

occurs and a signal is transmitted (Fig. 1).

Although in theory many receptor-ligand systems can be

used, the immunophilins FKBP12 and cyclophilin A (CyP) and

their ligands FK506 and cyclosporin A (CsA) were selected for

this purpose. The ligands are cell permeable and can be modified

[*I Prof S L Schreiber, P. J. Belshaw. Dr. J. G. Schoepfer, K-Q. Liu,

K.L Morrison
Howard 11ughes Medical Institute, Department of Chemistry
Harvard Uniwrsity, Cambridge, MA 02138 (USA)

TCkfaX' + (617) 495-0751

e-mail . bclshawfn slsiris.harvard edu

[*"I This research was supported by a grant from the National Institute of General

Medical Sciences (GM-52067). We thank theNSERCfor a postgraduateschol-
arahip awarded to P. J. B., and the Swiss National Science Foundation, the

Roche Kcsearch Foundation, and Ciba-Geigy Jubilaums Stiftung for fellow-
ship) to J G S

An,qw Cliem. Inr Ed. Engl. 1995. 34, No. 19 A, VCH Ver-lu~sge.s~~llschmrrhfiH, D-694if Weinheim, 1995 O571~-0833/95;34fY-ZIZY$ / O flOT .Z5X 2129

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The high-resolution X-ray structure of the CyP-CsA com- group of the modified ligand and the phenyl ring of P h e l l 3 of

p l e ~ [ ' . *s~erved as the starting point for the design of modified the receptor. Removal of this ring (Phell3 to Ala) should relieve

ligands. The primary hydrophobic interaction between CyP and the steric clash (Fig. 3C), yet it should also create a considerable

CsA involves the sidechain of MeVal11 of CsA and a deep volume of unoccupied space. To minimize these voids and max-

pocket on the receptor that is lined with the hydrophobic imize binding energy, two secondary mutations were designed

residues Phe60, Met61, AlalOl, Phell3, and Leu122 (Fig. 3A). wherein nearby sidechains were increased in size. A Ser99 to Thr

Several CsAs with modifications at residue 11 have been synthe- mutation (Fig. 3D) adds an extra methylene group and a

sized previously.[91Binding assays showed that an increase in CysllS to Met mutation (Fig. 3E) adds two methylenes. Com-

the size of the MeValll sidechain abrogated binding to CyP puter models were generated with MacroModel4.0 by perform-

(MeIlelI, Me-allo-Ilel I), and conversely that compounds with ing Monte Carlo substructure minimizations with all torsions

smaller sidechains (MeAlal 1) still bind with substantial affinity. specified for the sidechains of residues MeIlel1, Phe60, Met61,
Our approach would lead to an increase in buried hydrophobic
Met1 15, and Thr99. All other atoms within 6 A of these atoms

surface, and as hydrophobic interactions can provide a signifi- were considered but were assigned a 100 k J A - ' movement

cant contribution to the free energy of binding,["] this pocket penalty. These calculations indicated that the conformer re-

was selected for modification. quired for efficient packing was the one least frequently ob-

For simplicity, the first target selected was MeIlelICsA. A served for threonine residues in protein X-ray structures.["]

hypothetical model of MeIlellCsA bound to CyP (Fig. 3B) sug- These modeling studies also predicted that the two secondary

gests there should be a steric clash between the strategic methyl mutants could simultaneously assume their preferred conforma-

tions, thus the triple mu-

tant was also investigated

(Fig. 3F). These mutant pro-

teins were prepared by PCR

mutagenesis using the mega-

primer method['21 and veri-

fied by sequencing.

Our synthetic route begins

with the readily available

CsA, and produces a common

intermediate for the synthesis

of compounds with modi-

fications at residues 1 and 11

(Scheme 1). This plan is par-

ticularly attractive since posi-

tion 1 is the site we have used

previously to prepare dimeric

versions of CsA, named

( C S A ) ~ . [ ' ~TIhus, by using

modified amino acids at posi-

tions 1 and 11, we should be

able to simplify the synthesis

of CIDs with greater specifici-

ty than (CsA)2. CsA was ring-

opened and degraded by first

converting it to i s ~ - C s A [ ' ~ ~

and then subjecting the result-

ing amine to a modified Ed-

man degradation sequence." ']

This procedure excises the

MeBmt amino acid, produc-

ing a linear peptide that was

protected at its amino- and

carboxy-termini. The selec-

tion of the MEM ester was es-

sential for the subsequent re-

duction,[161as all other com-

binations of common esters

and reducing agents failed.

Following reduction, another

acid-catalvzed N to 0 shift

Fig. 3. Graphical representation of the binding interfaces between Cyp and CsA and vanants. The solid blue surface and white and an N-acylation produced
mesh represent the solvent-accessible surfaces of CsA and Cyp. respectively (nonpolar hydrogens omitted). The contact the target intermediate from

sidechains of Cyp are represented as tubular bonds and viewed from the insidc of the protein; the lowest proti-usion ofsolid blue which CsAs with modifica.
corresponds to the sidechain of residue 1I of CsA. A) Cyp-CsA crystal structure. B) Hypothetical model ofCyp-MeIlelICsA tions at residues 1 and/or 11
complex. C) Cyp(F113A)-MeIlellCsA complex. D) Cyp(S99T, FI I.iA)-MellellCsA complex. E) Cyp(F113A. C115M)- are accessible. T W O cycles of
MeIlel lCsA complex. F) Cyp(S99T, FI 13A. CI 15M)-Mellel lCsA complex. Graphical representations were created by using deprotection and coupling

GRASP [23]

yielded a linear, protected undecapeptide that was deprotected COMMUNICATIONS
and cyclized to give MeIlel1CsA. This unprecedented cycliza-
tion between residues 10and 11 proceeds with good yield and no although we cannot rule out a disturbance of the
detectable racemization. The choice of coupling agent and base local structure due to the Met. Interestingly, the
proved to be critical for efficient cyclization.[''1 triple mutant CyP(S99T, F113A, C115M) binds
MeIlel 1CsA more poorly than the double mutant
CyP experiences an approximate twofold enhancement in flu- CyP(S99T, F113A), and the free energy difference
orescence upon binding CsA. This effect provides a convenient between these two mutants is approximately the
assay for the determination of Kd values.['8-201Using this fluo- same as the difference between CyP(F113A) and
rescence assay. the binding constants for MeIlellCsA and CsA CyP(F113A, C115M). This indicates that the desta-
with the wild-type and mutant CyP proteins were determined bilizing effect of the C115M mutation is indepen-
and are shown in Table 1. The data show that the extra methyl dent of the S99T mutation. All of the mutants show
group of MeIlel1 CsA dramatically decreases binding to CyP. good binding to CsA with affinity generally increas-
ing as the predicted void volume decreases.

We have successfully generated new high affinity
receptor-ligand pairs through structure-guided ra-
tional design. By selecting other residues with large,
hydrophobic, conformationally constrained side-
chains as replacements for MeIlel1 and by judicious
introduction of point mutations, we may be able to
extract even greater free energy gains from the hy-
drophobic effect. In addition to this goal, we intend
to synthesize dimeric versions of these new ligands
in order to induce the dimerization of'target proteins
in vivo, thereby assessing the effect of endogenous
immunophilins in these types of experiments. The
generation of "bumps" and compensatory "holes" provides a
controlled system for studying molecular recognition involving
protein receptors. Through the iterative interplay of synthesis,
molecular and structural biology, and calorimetry and/or other
physical methods, the contribution of specific molecular inter-
actions to the free energy of binding can be assessed

Experimental Procedure

lable I . Binding constants K , [ n ~o]f CsA-based ligands for wild-type and mutant Fluorescence measurements were made on a Hitachi F2000 fluorescence spectrom-
('yPr. eter following a 10 min incubation with Iigand at 20 :C. Excitation was at 280 nm,
10 nm bandwidth and 331100 filter. Emissions were scanned at 300-400 nm, 10 nm
bandwidth. Proteins were diluted to 3 m L in Tris-buffered saline pH 7.4 at about
100 nM (for&> IOnM) o r 2 0 0 - 5 0 0 n ~(for K , <lOnM). Small aliquots ofligand in
50% ethanol were added between successive measurements such that the total
volume added was < 1.5%.

An equation relating the observed fluorescence change (A&,,.), the total fluores-
(Ace,),cence change at saturation
the protein concentration (E,,),the ligand concen-

Protein MeIlel lCsA CsA tration (fJ. and the Kd can be derived from consideration of tight binding kinetics

and the assumption that Fob=, ( H ) 8 J E o [Eq. (a)].

hCyP(wild-type) > 3000 [a] 5*1 +AFmr = [ ( E , 1, + KJ ~ { ( E D+ lo+ &Iz - 4 ( L ) ( ~ J ) i ' 2(1AF,,d/2) (a )
hCyP(F113A) 53+9 55+15
hCyP(F113A. ( ' l l 5 M ) 75+13 19+7 The fluorescence change at 338 nm (after adjustment for fluorcscence of the hgand
hCyP(S99T. Fl l.3A) 2k0.5 solution alone) was plotted vs the concentration of the hgand. The data for each
hCyP(SYY1. F l l i A . C115M) 4+1 5+1 determination of K , (t 14 measurements) were fitted to Equation (a) with
7fl Kaleidagraph 3. 0. All fits have r >0.99.

[a] This \,aIuc represents the lowest possible value, as precise determination is hin- Physical data for all compounds are in accord with the expected structures and are
dered by Ihc solubility 01' the hgand. available upon request from the authors.

Mutation of Phel13 to Ala has an even more dramatic effect, 1: To a solution of IsoCsA [14](2.886 g. 2.401 mmol) in pyridine. (144 mL) triethyl-
restoring the binding affinity to within an order of magnitude of amine (7.2 mL) and methyl isothiocyanate (7.2 mL) were added. The solution was
the wild-type receptor. The double mutant CyP(S99T, F113A) heated under nitrogen a t 50 'C for 30 min. More triethylamine (2.9 mL) and methyl
binds MeIlel lCsA with an affinity of 2 n ~T.hus, by adding a isothiocyanate (2.9 mL) were added and the reaction mixture was heated for an
substituent to CsA and by creating a binding pocket for it on its additional 30 min. Following evaporation at room temperature (RT), the residue
receptor using site-directed mutagenesis, a new receptor-ligand was dissolved in chorobutdne/trifluoroacetic acid (TFA) (9111. stirred at 10°C for
pair results that binds with two- to threefold greater affinity 1 h, evaporated, and the residue was precipitated three times from EtOAclhexane to
than the wild-type system. The other double mutant, give the crude precursor of 1. To this crude product (2.4g) in dichloromethane
CyP(F113A. C115M), has less affinity for MeIlellCsA than the (DCM) (100 mL) was added fert-butyoxycarbonyl (Boci anhydride (654 mg,
F113A single mutant, indicating that this mutation is slightly 3.0 mmol) and diisopropylethylamine (DIPEA) (1.4 mL. 8.00 mmol). After 4h at
destabilizing. This appears to be due to an interfering contact RT, MEMCI (570 pL, 5 mmol) was added and the mixture was stirred overnight.
between the terminal methyl of MI15 and the terminal methyl The solution was diluted with DCM (200 mL), washed with sodium bisulfate (1 M).
of the MeIlel1 sidechain. Models show that this interaction is water, sodium bicarbonate (sat.). water, and brine (50 mL each). The organic layer
slightly closer than any other contact in the binding pocket, was dried (Na,SO,), filtered. and evaporated. The residue a a s chromatographed
(silica gel, DCM/MeOH (9812, 95/5) to afford 1 (1.933 g, 66 74 from IsoCsA).

2 : To a solution of 1 (975 mg, 0.795 mmol) in DCM (10 m L ) . 1 mL of 2~ lithium
horohydride in T H F was added dropwise at O T . The reaction mixture was stirred
overnight at room temperature and an additional 0.5 mL of lithium borohydride
solution was added. After 2 4 h the reaction mixture wah cooled to O ' C and

COMMUNICATIONS

quenched with a solution of citric acid. The mixture was diluted with DCM [16] R. E. Ireland, W. J. Thompson, Tefruhedron Left. 1979, 4705.
(100 mL) and washed with sodium blcarbonate (sat.). water. and brine (25 mL 1171 L. A. Carpino, E A. El. J. Org. Chem. 1994, 59, 695.
each). The organic layer was dried (Na,SO,), evaporated. and the residue was [18] R. E. Handschumacher, M. W. Harding, J. Rice, R. J. Drugge, D. W. Speicher.
dissolved in DCM/MeOH (98/2) and filtered through a plug of silica gel. Evdpora-
tion gave the crude precursor of 2 (773 mg). To this crude product (746 mg) in dry Science 1984. 226. 544.
T H F (40 mL) was added methanesulfonic acid (300 pL). After stirring the mixture [19] H. Husi. M. Zurini, A n d . Biochem. 1994, 222, 251.
at RT for 72 h under nitrogen, pyridine (400 pL), DIPEA (1.5 mL), and acetic [20] L. D. Zydowsky. F. A. Etzkorn, H. Y Chang, S. B. Ferguson, L. A. Stolz, S. 1.
anhydride were added at O'C. After 2 h the solution was diluted with DCM
(lOOmL), washed with sodium bisulfate (1 M), water. sodium bicarbonate (sat.), Ho. C. T. Walsh, Protein Sci. 1992. I , 1092.
water, and brine (25 mL each). The organic layer was dried (Na,SO,), filtered, [21] W. D. Lubell, T. F. Jamison, H. Rapoport, 1 Org. Chem. 1990, 55. 3511.
evaporated. and chromatographed (silica gel, DCM/MeOH (98/2)) to afford 2 [22] J. Coste, E. Frerot, P. Jouin, J. Org. Chem. 1994. SY, 2437.
(360 mg, 40 % based on 1). [23] A. Nicholls. K. A. Sharp, B. Honig, Pror. Sfruct. Funcr. Genet. 1991, if. 283.

3: To 2 (60 mg, 0.051 mmol) in DCM (1 mL), TFA (0.5 mL) was added dropwise Strong Binding of Paraquat and Polymeric
and stirred for 1 h a t 0 C. The reaction mixture was diluted with DCM (50 mL) and Paraquat Derivatives by Basket-Shaped Hosts**
washed three times with sodium bicarbonate (sat.) then water and brine (10 mL
each). The organic layer was dried (Na,SO,), evaporated, and redissolved in DCM Albertus P. H. J. Schenning, Bas de Bruin,
(0.5 mL). Boc-(2S,3R,4R,6E)-3-hydoxy-4-methyl-2-(methy~dmino)-6-octenoiaccid Alan E. Rowan,* Huub Kooijman, Anthony L. Spek,
(Boc-MeBmt) [21](18.7 mg, 0.062 mmol), bromotripyrrolidinophosphonium hexa- and Roeland J. M. Nolte
fluorophosphate (PyBroP)[22] (27 mg, 0.060 mmol) and DIPEA (27 pL) were
added at 0 ° C under nitrogen. After 2 h a t 0°C the reaction was allowed to warm to Clip-shaped host molecules of type 1 can bind uncharged
RT, diluted with DCM (50 mL), washed with sodium bisulfate (1 M), water, sodium aromatic guest molecules, for example resorcinol, by TI-% stack-
bicarbonate (sat.), water, and brine (20 mL each). The organic layer was dried ing and hydrogen bonding interactions."] Basket-shaped
(Na,SO,), filtered, evaporated and chromatographed (flash silica gel, EtOAciace- derivates of 1 containing crown ether moieties (compounds of
tone (9/l)) to give 3 (56 mg, 81 "A). type 2) are, in addition, able to bind alkali metal ions and pro-
tonated amines.12] We report here on the binding affinities of
4: To 3 (27 mg, 0.022 mmol) in DCM (1 mL), TFA (0.5 mL) was added dropwise these host molecules towards charged aromatic compounds,
and stirred for 1 h at 0 'C. diluted with DCM (50 mL) and washed three times with such as paraquat 3 and the polymeric paraquat derivatives 4 and
with sodium bicarbonate (sat.). water and brine (10 mL each). The organic layer
was dried (Na,SO,), filtered and evaporated. To the residue in DCM (30 pL), M.<;I=e;>.o2p" 2a, R = C6H5
Fmoc-N-Melle (12 mg, 0.032 mmol). PyBroP (14 mg, 0.032 mmol) and DIPEA b , R = CH,C,H,
(15 pL) were added and the mixture was stirred for 6 h at O'C and 30 min at RT. Me0e O M e
Fmoc-N-MeIle (5 mg, 0.013 mmol), PyBroP (5 mg, 0.011 mmol) were added again
at 0 "C and allowed to warm to R T overnight. The mixture was diluted with DCM
(50 mL), washed with sodium bisulfate (1 M). water, sodium bicarbonate (sat.),
water, brine (20 mL each). The organic layer was dried (Na,SO,). filtered. evapo-
rated, and chromatographed (silica gel, EtOAchcetone 9: I) to afford 4 (19 mg.
60 Yo).

5: To 4 (4.6mg, 0.0028 mmol) in THF/water (10:l. 200 pL), DBU ( 3 pL) and
lithium bromide (2 mg) were added. After stirring overnight at RT. DBU (4 pL) and
lithium bromide (3 mg) were added again After 5 h. the reaction was quenched with
acetic acid (20 pL) and purified by reverse-phase HPLC (BeckmanODS ultrasphere
5 p 10 mm x 25 cm, 0.1 % TFAiMeCN 70/30 +10/90 in 30 min, 70 C, 3 runs) to
yield the peptide precursor of 5 (2.1 mg, 60%). A solution ofthis peptide precursor
(1.2 mg, 970 nmol), AOP[17] (4 mg, 0.009 mmol) and 2,6-lutidine (4 pL) in DCM
(1.2 mL) was stirred for 48 h at RT. The reaction mixture was quenched with acetic
acid (30 pL), the DCM evaporated, and the residue dissolved in acetonitrile and
purified by reverse-phase HPLC (Beckman ODS ultrasphere 5 p 10 mm x 25 cm.
0.1 % TFAiMeCN 70/30 +10/90 in 30 min. 70 -C) to afford the pure cyclic peptide
5 (0.65 mg, 55 YO).

Received: June 9, 1995 [ZX0801E]
German version: Angew. Chem. 1995. 107, 2313-2317

-Keywords: cyclophilin * cyclosporin * immunophilins protein

dimerization

- 5 . There is currently a great deal of interest in paraquat-binding,
which has resulted in the design and construction of new molec-
[ l ] D. J. Austin, G. R. Crabtree, S. L. Schreiber, Chem. d Bid. 1994, I . 131 ular structures, as exemplified by the elegant work by Stoddart
[2] M. A. Lemmon, J. Schlessinger. Trendv Binchem. Sci. 1994, 19. 459. et al. on catenands and r o t a x a n e ~ . [W~ ]e describe here that com-
[3] A. C. Chan, D. M. Desai, A. Weiss, Annu. Rer. Immunof. 1994, 12. 555.
[4] W. H. Landschulz, P. F. Johnson, S. L. McKnight, Science 1988, 240, 1759. [*I Dr. A. E. Rowan, DipLChem. A. P. H. J. Schenning. DipLChem. B. de Bruin,
[5] D. M. Spencer, T. J. Wandless, S. L. Schreiber, G . R. Crabtree, Science 1993,
Prof. Dr. R. J. M. Nolte
262. 1019. Department of Organic Chemistry, NSR Center
[6] M. N. Pruschy, D. M. Spencer. T. M. Kapoor, H. Miyaki, G. R. Crabtree, S. University of Nijmegen
Toernooiveld. NL-6525 ED Nijmegen (The Netherlands)
S. L. Schreiber. Chem. & B i d . 1994, I . 163. Telefax: Int. code +(go) 553450
[7] H. Ke, D. Mayrose, P. J. Belshaw. D . G. Alberg, S. 1.Schreiber. Z. Y. Chang.
Dr. H. Kooijman, Dr. A. L. Spek
F. A. Etzkorn, S. Ho. C. T. Walsh, Structure (London) 1994. 2, 33. Bijvoet Center for Biomolecular Research, Crystal and Structural Chemistry
[XI G . Pflugl. J. Kallen, T. Schirmer. J. N. Jansonius, M . G. Zurini, M . D. Walkin- Utrecht University (The Netherlands)

shaw, Nature 1993, 361, 91. [**I This work was supported by the Netherlands Foundation for Chemical Re-

[9] V. F. Quesniaux. M. H Schreier, R. M. Wenger, P. C. Hiestand, M. W. Hard- search (SON) with financial aid from the Netherlands Organization for Scien-
ing, M. H. V. Van Regenmortel, Eur. 1 Immunol. 1987, 17, 1359. tific Research (NWO)

[lo] T. Clackson, J. A. Wells, Science 1995, 267, 383.
[ l l ] J. S. Richardson, D. C. Richardson in Predicfioir of prolein strucmre and !he

principles of protein conformution, Vol. X I I I ; (Ed. G . D. Fasman), Plenum.
New York, 1989.
[12] G. Sarkar, S. S. Sommer, BioTechniquPs 1990, 8,404.
[13] P. J. Bekhdw, S. L. Schreiber, Chem. d Biol.. submitted.
[14] R. Oliyai, V. .I. Stella, Phurm. Rex 1992. Y, 617.
[15] A. Ruegger, M. Kuhn, H. Lichti, H. R. Looali, R. Huguenin, C . Quiquerez,
W. A. Von, Hell'. Chim. Acto. 1976, 59. 1075.

21 32 (CI VCH Verlugsgesdlschuft mhH. 0 4 9 4 5 1 WcJinheim.1995 0570-0N33/95/3419-2132$ 10.00f ,2510 A n g w . Chern. h t . Ed. Engl. 1995. 34, N o . i Y