Protein–Protein Interactions

DOI: 10.1002/anie.200503965

Discovery of Low-Molecular-Weight Ligands forthe AF6 PDZ Domain**

Mangesh Joshi, Carolyn Vargas, Prisca Boisguerin,Annette Diehl, Gerd Krause, Peter Schmieder,Karin Moelling, Volker Hagen, Markus Schade,* andHartmut Oschkinat*

Despite their central role in most regulatory processes anddisease mechanisms, protein–protein interactions (PPIs)remain largely unconquered ground for drug discovery andchemical-tool generation. In many cases, these interactionsare mediated by protein interaction domains like Src homol-ogy 2 (SH2), Src homology 3 (SH3), WW domain, andpostsynaptic density/discs large/zona occludens-1 (PDZ).[1]

PDZ domains may be considered “drugable” because of ashallow ridge on their surface which is, however, not a propercavity. Hence, they are good test cases for the development ofPPI inhibitors.

PDZ domains comprise � 90 residues and occur morethan 450 times in approximately 250 human proteins. In mostcases, they recognize the C terminal four to seven residues ofmembrane receptors and ion channels,[2–4] although they arealso known to bind to internal sequence motifs.[5] Consistingof a six-stranded b-sheet flanked by two a helices, PDZ

[*] Dr. M. SchadeCombinature Biopharm AGRobert-Roessle-Strasse 10, 13125 Berlin (Germany)Fax: (+49)30-94894-038E-mail: schade@combinature.com

M. Joshi, C. Vargas, Dr. A. Diehl, Dr. G. Krause, Dr. P. Schmieder,Dr. V. Hagen, Prof. Dr. H. OschkinatLeibniz-Institut f=r Molekulare Pharmakologie FMPRobert-Roessle-Strasse 10, 13125 Berlin (Germany)Fax: (+49)30-94793-169E-mail: oschkinat@fmp-berlin.de

Dr. K. MoellingInstitut f=r Medizinische VirologieUniversitAt Z=rich8028 Z=rich (Switzerland)

Dr. P. BoisguerinInstitut f=r Medizinische ImmunologieCharitD-UniversitAtsmedizin BerlinHessische Strasse 3–4, 10115 Berlin (Germany)

[**] This work was supported by grants from the European Community(QLK3-2000-00924-SH2 Libraries) and the Deutsche Forschungs-gemeinschaft (DFG) (OS 106/4-2). The authors thank DietmarLeitner for assistance in NMR spectroscopic measurements, ArvidSoderhall and Jens Laettig for their help in molecular dynamics(MD) simulations, Viviane Uryga-Polowy for the MSmeasurements,and Daniel Geißler for helpful discussions. The structure of theAF6 PDZ domain is deposited in the protein data bank (PDB) underaccession code 1XZ9 and the AF6 PDZ-5 f complex under accessioncode 2EXG.

Supporting information for this article is available on the WWWunder http://www.angewandte.org or from the author.

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domains bind cognate peptides in an extended conformationwhereby the terminal carboxy group is hydrogen bonded withthe backbone amides of the highly conserved GLGF loop.[6–8]

The protein AF6 (ALL-1 fusion partner on chromosome 6),also known as s-afadin,[9] contains one type II PDZ domain(abbreviated: AF6 PDZ), two N-terminal Ras-association(RA) domains, one forkhead-association (FHA) domain, andone dilute (DIL) domain. AF6 PDZ mediates interactionswith a subset of ephrine-receptor protein-tyrosine kin-ases,[10, 11] the poliovirus receptor-related protein PRR2/nectin,[12] the junctional adhesion molecule (JAM),[13] andthe breakpoint-cluster-region protein (BCR).[14] It bindstarget peptides with comparatively low affinities in the 20–150 mm range.[4] When the C-terminal valine of full-lengthBCR is mutated to Ala, binding to full-length AF6 isabrogated in various cell-based assays.[14]

Herein, we report the identification of novel, low-molecular-weight ligands for the AF6 PDZ domain byNMR spectroscopy-based screening and chemical synthesis.These compounds are active in competition assays andrepresent building blocks for the design of tight-bindingcompetitors. Furthermore, we have determined the solutionstructure of AF6 PDZ in complex with the ligand of highestaffinity. The synthetic ligand induces the formation of ahydrophobic subpocket in AF6 PDZ that is absent inpublished structures of both apo and peptide-bound PDZdomains. This unexpected ligand–subpocket interaction rede-fines the proteinBs drugability and opens the door to small-molecule modulators for the entire family of PDZ domains.

We selected NMR spectroscopy-based screening as the hitdiscovery method of choice for the identification of weaklybinding small organic frameworks because of its highsensitivity towards weakly binding ligands and unique struc-tural support for a fragment-based lead-generation approach.In the first round of screening, a library of 5000 syntheticcompounds was screened against uniformly 15N-labeledprotein by using 2D 1H-15N-heteronuclear single quantumcorrelation (HSQC) experiments. Three chemically distinctclasses of binders were identified as indicated by the examplesin Figure 1a. Comparison of the three compounds with thestructure of the conserved C-terminal valine of naturalpeptide ligands (Figure 1b) revealed a consistent pharmaco-phore pattern of one moiety showing at least one hydrogenbond acceptor (red), a moiety showing one hydrogen bonddonor (yellow), and a hydrophobic core of variable size.Compound 3 occurs in two tautomeric forms in aqueoussolution (Figure 1a) and in organic solvents (Figure 1c), asobserved by NMR spectroscopy.

In the second round of screening, initial structure–activityrelationships (SAR) were obtained by assaying analogueswithin the remaining 15000 compounds of our fragmentlibrary and further commercially available compounds. Eachof the three hit classes showed consistent SAR (data notshown). Hit compound 3was selected for further optimizationbecause of its comparatively high binding affinity andchemical tractability.

Derivatives of compound 3 were synthesized by conden-sation of 2-thioxo-4-thiazolidinone with a series of aldehydesaccording to the procedures described earlier[15,16] (Fig-

ure 1c). Some of the condensation products 4 (all Z isomers)were reduced to yield the racemates 5, generating a moreflexible linkage between the two ring systems. Normal-phaseHPLC and a chiral column (Chiralpak IA, 5 mm, 250 E4.6 mm2, Chiral Technologies Europe) were used to separatethe enantiomers of 5 f. Although this attempt was initiallysuccessful, interconversion produced the racemate again.Aldehydes were chosen to sample hydrophobic moieties ofvariable sizes in the R1 position of 4 or 5, ranging from a smallisopropyl group to various substituted and unsubstitutedaromatic ring systems (Table 1).

To monitor the binding activity of the 4 and 5 compoundseries initially, the chemical shift perturbation (CSP) of Leu25HN was used. This residue is in the GLGF loop, in contactwith the five-membered ring of the ligands (see below), andshould hence be in a similar chemical environment in allinvestigated complexes. In the 4 compound series, thederivatives with a 3-thienyl (4a), iPr (4b), C6H5 (4c), or 4-MeC6H4 (4d) substituent at the R1 position showed weakchemical-shift changes for Leu25 HN. Compounds 4a and 4c

Figure 1. a) Novel ligands for the AF6 PDZ domain as identified byNMR spectroscopy-based screening (H-bond acceptors, red; H-bonddonors, yellow). b) Structure of the conserved C-terminal valine ofnatural peptide ligands. c) Synthesis of 2-thioxo-4-thiazolidinone deriv-atives (for structures of R1, see Table 1). Different tautomers owing todifferent solvents are shown in a) and c).

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showed dissociation constants of 460� 32 and 580� 62 mm,respectively. Chemical-shift changes below 0.02 ppm wereobserved for derivatives with 3-MeC6H4 (4e), the bulkyindolyl group (4 i), and 4-PhC6H4 (4j) as substituents and werecategorized as nonbinding. Interestingly, the 4-CF3C6H4 (4 f)-,3-CF3C6H4 (4g)-, and 4-BrC6H4 (4h)-substituted derivativesshowed binding.

Significantly, improved binding to AF6 PDZ wasobserved when compound 4 f was reduced to 5 f. The para-substitution of the phenyl ring in 5 in the R1 position by atrifluoromethyl (CF3) is important for binding. In the 5compound series, however, the 3-CF3C6H4-substituted deriv-ative (5g) showed negligible CSPs. Taken together, a five-membered ring system with hydrogen bond acceptingcapacity is critical for activity, and 5 with 4-CF3C6H4

substitution in the R1 position (5 f) showed the tightestbinding of the compounds investigated. By using the racemicmixture of 5 f, we determined a dissociation constant of100 mm. Assuming that only one of the enantiomers bindspreferentially, its dissociation constant may be approximatedto 50 mm, which is in the same range as that of the naturalpeptide ligands.

To demonstrate that 5 f binds competitively to theAF6 PDZ domain, a series of 15N-filtered 1D spectra withincreasing amounts of 5 f were recorded on a samplecontaining equimolar concentrations of U-15N-labeledAF6 PDZ and the natural peptide ligand[10,4] (NH2-IQS-VEV-COOH). Figure 2 shows the result of the titration experi-ment, in which only signals from the 14N and carbon-boundprotons appear. The peptide signal at d= 8.1 ppm, whichvanishes in the presence of the protein, re-emerges withincreasing 5 f concentration. In addition, the signals at d=

8.3 ppm and d= 7.6 ppm revert back to the “unbound” state(black arrows). The other signals of the peptide could not beobserved as well because of the overlap with the aromaticprotons of the protein or 5 f (gray arrows). This experimentproves that the peptide ligand is ejected from the proteinBsbinding groove by 5 f.

To understand the binding mode of compounds 5 atatomic resolution, we determined the solution structure of thecomplex between AF6 PDZ and 5 f (see the Supporting

Information). The structure is well defined by 11.8 inter-residual NOEs per amino acid. The ensemble of the 20lowest-energy structures has a mean a-carbon root-mean-square deviation (rmsd) of 0.31� 0.09 J for the well-struc-tured regions (residues 11–28, 40–70, 77–95) and 1.21�0.23 J for all heavy atoms (Table 2). The overall structureof the AF6 PDZ domain in complex with 5 f is similar to otherPDZ structures.[6, 8, 17] The protein has six b strands (bA to bF)and two a helices (aA, aB) (see the Supporting Information).

The binding mode of 5 f complexed to AF6 PDZ isdefined by 11 intermolecular NOEs obtained from 2D 13C-F2-filtered NOESY and 2D 13C-F2-filtered HMQC-NOESYspectra. Since the signal of 5-H of 5 f overlaps with thewater signal, only NOEs to 8-H/12-H and 9-H/11-H, whichare degenerate in the bound state, were extracted from thespectra. All 11 intermolecular NOEs occur between thesedegenerate protons, 8-H/12-H and 9-H/11-H, and the side-chain protons of Met23, Leu25, Ile27, Ala80, Met83, andThr84. As the thiazolidinone ring did not show NOErestraints, its location was determined by computationaldocking of the whole molecule utilizing all observed NMRspectroscopic data. By enforcing the 11 intermolecular NOErestraints, we docked the R and S enantiomers of 5 f into thelowest-energy AF6 PDZ structure in a constrained MDsimulation lasting for 2 ns using Amber 8.[20] The tautomericform of 3 (Figure 1) was chosen for the simulations as the

Table 1: Structure-activity relationship of 4 and 5 compound series.

Compd Compd[a] R1 CSP[b] of Leu25HN

Dissociationconstant [mm]

4 5[a] 4 5[a]

4a 5a 3-thienyl 0.20 0.17 460�32 –4b – iPr 0.27 – – –4c 5c C6H5 0.17 0.21 680�58 576�404d – 4-MeC6H4 0.14 – – –4e – 3-MeC6H4 <0.02 – – –4 f 5 f 4-CF3C6H4 0.50 0.88 348�30 100�124g 5g 3-CF3C6H4 0.21 <0.02 – –4h 5h 4-BrC6H4 0.24 – – –4 i – 3-indolyl <0.02 – – –4 j – 4-PhC6H4 <0.02 – – –

[a] Analyzed as racemic mixtures: (�) compound not synthesized or value notdetermined. [b] Chemical shift perturbation (CSP) in ppm units. CSPs<0.02 ppm are considered inactive.

Figure 2. Competition experiment with the AF6 PDZ domain,

NH2-IQSVEV-COOH peptide ligand, and 5 f. 1) 100 mm peptide and

100 mm AF6 PDZ domain; 2–8) Spectra recorded at 100, 200, 400, 600,800, 1000, and 1400 mm concentrations of 5 f, respectively; 9) 1H NMRspectrum of the peptide (100 mm).

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experiments were done in aqueous solution. During docking,the backbone of the protein was kept rigid and the side chainsof the residues were restrained by using the same distanceconstraints as in the Cyana structure calculation. The totalenergy obtained for the complex was significantly higher forthe S enantiomer than for the R enantiomer, suggesting thatthe R enantiomer of 5 f is the active binder.

The final complex structure shows the 4-CF3C6H4 group of5 f embedded in a deep hydrophobic pocket surrounded bythe side chains of residues Met23, Leu25, Ile27, Ala80,Met83, Val90, and Leu92 (Figure 3b and c). N3 and O4 of 5 fform H-bonds with the backbone HNs of residues Gly24 andLeu25, respectively, which belong to the conserved GLGFloop (GMGL in the case of AF6 PDZ). The SH group isinvolved in hydrophobic interactions. It also explains thelower affinity of compounds 5a and 5c as compared to 5 fbecause of incomplete occupancy of the proteinBs hydro-phobic pocket. The weak binding of 5g can be attributed tosteric hindrance because the meta-substituted ring wouldrequire a larger hydrophobic cavity for binding.

To better understand the binding mode of 5 f, wecompared the structures of the AF6 PDZ domain in theligand-free (see the Supporting Information) and ligand-bound forms. Although the superposition of the structureswith the lowest Cyana target function show a good overallagreement with an a-carbon rmsd of 1.25 J for the b strands,three areas of significant deviation located in aA, aB, and bEwere observed. Upon binding of 5 f, the Ca atom of Gln76 atthe beginning of aAwas displaced by 3.3 J, which results inthe widening of the peptide-binding groove (Figure 4a). Inaddition, closer inspection of the superimposed structuresreveals significant side-chain rearrangements of residuesMet23, Leu25, and Met83 and a backbone rearrangementfor the residue Ala80 in the ligand-binding groove toaccommodate the bulky 4-CF3C6H4 group of 5 f. The side

chains of the other hydrophobic residues lining this pocket(Ile27, Val90, and Leu92) are virtually unchanged. As aconsequence, 5 f triggers an induced-fit rearrangement toshape a new hydrophobic pocket without unbound AF6 PDZ.

To gain insight into the possible interaction between 5 fand other classes of PDZ domains and to aid the structure-

Table 2: Structural statistics of AF6 PDZ-5 f complex.

Restraints

total no. of experimental restraints 1518total no. of NOE restraints 1440intraresidue (i= j) 428sequential (j i�j j=1) 390medium-range (2�j i�j j�5) 151long-range (j i�j j>5) 471no. of H-bond restraints 14no. of dihedral angle restraints (TALOS) 64average inter-residual NOEs per residue 12no. of NOE violations >0.3 N 0no. of dihedral angle violations >58 0

f�y Space (residues)[a,b]

most favored regions [%] 70.3additionally allowed regions [%] 22.5generously allowed regions [%] 6.3disallowed regions [%] 1.0

rmsd values[c,d]

Ca 0.31�0.09heavy atoms 1.21�0.23

[a] Residues considered: 11–95. [b] From Procheck-NMR.[18] [c] Residuesconsidered: 11–17, 25–29, 40–46, 60–66, 69–72, and 77–95. [d] Calcu-lated by using MOLMOL.[19]

Figure 3. a) Surface representation of the AF6 PDZ domain withoutligand, 1XZ9. Surface coloring indicates hydrophobic (yellow) andhydrophilic (green) areas. b) AF6 PDZ domain in complex with 5 f.Surface coloring indicates hydrophobic (yellow) and hydrophilic(green) areas. The hydrogen bonding interaction between the PDZdomain and 5 f are shown as yellow-dotted lines. c) Schematicrepresentation of the AF6 PDZ-5 f interaction. Hydrogen bonds areshown as green-dotted lines. Hydrophobic interactions are highlightedby red line fences. The Figure was generated by using the programLigplot.[21]

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based extension of 5 f for developing specific ligands, wecompared the structure of AF6 PDZ in the ligand-boundform to the crystal structures of the syntrophin (1QAV)[5] anderbin (1MFG)[22] PDZ domains in complex with theirrespective ligands. AF6 PDZ in the ligand-bound form super-imposes rather well with the two other PDZ domains withrmsd values of 1.7 and 1.5 J, respectively. In the structure ofthe syntrophin PDZ domain, the residues homologous toMet23, Leu25, Ile27, Ala80, Met83, Thr84, and Leu92 adoptsimilar positions (Figure 4b). Only Leu149 undergoes a slightside-chain rearrangement to accommodate the 4-CF3C6H4

group of 5 f, suggesting that it may interact similarly with5 f. Indeed, weaker binding of 5 f to the syntrophin PDZdomain (Kd= 270� 21 mm) was observed in an NMR spec-troscopic screening experiment. In the syntrophin PDZdomain, His141 narrows the peptide-binding groove at thehomologous position of Gln76 in AF6 PDZ, suggesting that

variation of 5 f specifically designed tointeract with either His141 or Gln76 maybe helpful in developing specific ligandsfor either of the two domains.

In the structure of the erbin PDZdomain, the bulky side chain of Phe1293,which is homologous to Leu25 of ourAF6 PDZ construct, severely clashes withthe CF3 moiety of 5 f (Figure 4c). As theconformational freedom of the Phe1293side chain is limited, specificity may becontrolled by the CF3 group or relatedmoieties. Other residues including thehomologues of Ile27 and Ala80, whichplay an important role in the recognitionof hydrophobic residues in the �2 posi-tion with respect to the C-terminal resi-due of cognate peptides, show similarorientations, thus being less-promisinginteraction points for the design of spe-cific ligands.

By using NMR spectroscopic screen-ing, we identified three chemically dis-tinct classes of compounds binding to thechallenging PPI target AF6 PDZ. Weimproved a 2-thioxo-4-thiazolidinonederivative to become a 100-mm Kd ligandwith a molecular weight of 291 Da, anddetermined the solution structure of theR enantiomer complexed with AF6 PDZ.The 3D structure reveals a new hydro-phobic subpocket formed throughinduced-fit binding of 5 f. This findingredefines the drugability of PDZ domainsand discloses 5-aryl-2-thioxo-4-thiazolidi-nones and related frameworks as promis-ing candidates for the development ofpotent and selective small-molecule mod-ulators of individual domains from thelarge PDZ family.

Experimental SectionAll NMR spectroscopic experiments were performed at 295 K onBruker DRX600 and DMX750 spectrometers in standard configu-ration by using triple-resonance probes equipped with self-shieldedgradient coils. Two samples of 1.5 mm U-15N, 13C-labeled AF6 PDZwith 5 f (1:1 protein/ligand) at pH 6.5 in Na phosphate buffer solution(20 mm) with NaCl (50 mm) and dimethyl sulfoxide (DMSO; 10%v/v) either in D2O (99.98% v/v) or H2O/D2O (90:10% v/v).

Backbone chemical-shift assignments for the complex wereachieved as described earlier.[4] Side-chain assignments were obtainedfrom 3D HCCH-COSY, HCCH-TOCSY (100 ms), and 13C-editedNOESY (80 ms) experiments recorded on a sample in D2O (99.98%v/v); and from 15N-edited NOESY (80 ms) spectra recorded on asample in H2O/D2O (90:10% v/v).[23]

Data were processed by using XWIN-NMR (version 1.3, BrukerBioSpin GmbH, Rheinstetten, Germany) and analyzed by usingSPARKY.[24] Structures were calculated from the restraints listed inTable 2 using CYANA.[25] The 20 lowest-energy structures withoutdistance violations greater than 0.3 J and no angle violations greater

Figure 4. a) Comparison of AF6 PDZ in ligand-free (blue) and ligand-bound (red) state(stereoview). b) Comparison of AF6 PDZ ligand-bound (red) state and syntrophin PDZ domainbound to nNOS b-finger, 1QAV, blue, n-NOS b-finger not shown (stereoview). c) Comparisonof AF6 PDZ ligand-bound (red) state and erbin PDZ domain bound to Erbb2 receptor peptide,1MFG, blue, peptide not shown (stereoview).

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than 58 were accepted into the final ensemble. A total of 64 dihedral-angle restraints were obtained from backbone chemical shifts by usingTALOS.[26] Furthermore, 14 hydrogen-bond restraints were identifiedfrom the NOE pattern in the 3D 13C-edited NOESY spectrum.

15N-labeled AF6 PDZ at 50 mM concentration in a 20 mm Naphosphate buffer solution containing NaCl (50 mm), and [D]6DMSO(10% v/v) at pH 7.0 was used for screening experiments. Ligandbinding was detected at 300 K by acquiring 1H-15N HSQC spectra inthe presence and absence of compounds. Compounds were initiallytested at 400 mm each in mixtures of 16 compounds, with subsequentdeconvolution to mixtures of 4 compounds at 400 mm each and then toindividual compounds. Spectra were acquired with 16 scans and 128points in the indirect dimension on a Bruker DRX600 spectrometerequipped with a cryoprobe. Chemical-shift mapping of the bindingsite was achieved by comparing the shifts of protein alone to those ofthe protein in presence of ligands. Chemical shifts were quantified byusing the formula Dd= [(DH)2 + (DN/5)2]

1=2 , in which Dd is theweighted chemical-shift change, and DH and DN are the chemicalshift changes in the proton and the nitrogen dimensions, respectively.Dissociation constants were obtained for selected compounds bymonitoring the chemical shift changes as a function of ligandconcentration. Data were fit by using a one-site binding model. Anonlinear least-square optimization was performed by varying thevalues of Kd and the chemical shift of the fully saturated protein.

Received: November 8, 2005Revised: January 26, 2006Published online: May 3, 2006

.Keywords: inhibitors · ligand effects · NMR spectroscopy ·PDZ domains · protein structures

[1] T. Pawson, P. Nash, Science 2003, 300, 445 – 452.[2] Z. Songyang, A. S. Fanning, C. Fu, J. Xu, S. M. Marfatia, A. H.

Chishti, A. Crompton, A. C. Chan, J. M. Anderson, L. C.Cantley, Science 1997, 275, 73 – 77.

[3] B. Z. Harris, W. A. Lim, J. Cell Sci. 2001, 114, 3219 – 3231.[4] U. Wiedemann, P. Boisguerin, R. Leben, D. Leitner, G. Krause,

K. Moelling, R. Volkmer-Engert, H. Oschkinat, J. Mol. Biol.2004, 343, 703 – 718.

[5] B. J. Hillier, K. S. Christopherson, K. E. Prehoda, D. S. Bredt,W. A. Lim, Science 1999, 284, 812 – 815.

[6] D. A. Doyle, A. Lee, J. Lewis, E. Kim, M. Sheng, R. MacKinnon,Cell 1996, 85, 1067 – 1076.

[7] M. Cabral, J. H. Petosa, M. J. Sutcliffe, S. Raza, O. Byron, F. Poy,S. M. Marfatia, A. H. Chishti, R. C. Liddington, Nature 1996,382, 649 – 652.

[8] J. Schultz, U. Hoffmuller, G. Krause, J. Ashurst, M. J. Macias, P.Schmieder, J. Schneider-Mergener, H. Oschkinat, Nat. Struct.Biol. 1998, 5, 19 – 24.

[9] R. Prasad, Y. Gu, H. Alder, T. Nakamura, O. Canaani, H. Saito,K. Huebner, R. P. Gale, P. C. Nowell, K. Kuriyama, Cancer Res.1993, 53, 5624 – 5628.

[10] B. Hock, B. BPhme, T. Karn, T. Yamamoto, K. Kaibuchi, U.Holtrich, S. Holland, T. Pawson, H. Rubsamen-Waigmann, K.Strebhardt, Proc. Natl. Acad. Sci. USA 1998, 95, 9779 – 9784.

[11] M. Buchert, S. Schneider, V. Meskenaite, M. T. Adams, E.Canaani, T. Baechi, K. Moelling, C. M. Hovens, J. Cell Biol.1999, 144, 361 – 371.

[12] K. Takahashi, H. Nakanishi, M. Miyahara, K. Mandai, K. Satoh,A. Satoh, H. Nishioka, J. Aoki, A. Nomoto, A. Mizoguchi, Y.Takai, J. Cell Biol. 1999, 145, 539 – 549.

[13] K. Ebnet, C. U. Schulz, M. K. Meyer Zu Brickwedde, G. G.Pendl, D. Vestweber, J. Biol. Chem. 2000, 275, 27979 – 27988.

[14] G. Radziwill, R. A. Erdmann, U. Margelisch, K. Moelling, Mol.Cell. Biol. 2003, 23, 4663 – 4672.

[15] G. Bruno, L. Constantino, C. Curinga, R. Maccari, F. Manforte,F. NicolQ, R. OttanR, M. G. Vigorita, Bioorg. Med. Chem. 2002,10, 1077 – 1084.

[16] S. Kukolya, S. Draheim, B. Graves, D. Hunden, J. Pfeil, R.Cooper, J. Ott, F. Counter, J. Med. Chem. 1985, 28, 1896 – 1903.

[17] N. L. Stricker, K. S. Christopherson, B. A. Yi, P. J. Schatz, R. W.Raab, G. Dawes, D. E. Bassett Jr, D. S. Bredt, M. Li, Nat.Biotechnol. 1997, 15, 336 – 342.

[18] R. A. Laskowski, J. A. Rullmann, M. W. MacArthur, R. Kap-tein, J. M. Thornton, J. Biomol. NMR 1996, 8, 477 – 486.

[19] R. Koradi, M. Billeter, K. WSthrich, J. Mol. Graphics 1996, 14,29 – 32.

[20] D. A. Case, T. A. Darden, T. E. Cheatham III, C. L. Simmerling,J. Wang, R. E. Duke, R. Luo, K. M. Merz, B. Wang, D. A.Pearlman, M. Crowley, S. Brozell, V. Tsui, H. Gohlke, J.Mongan, V. Hornak, G. Cui, P. Beroza, C. Schafmeister, J. W.Caldwell, W. S. Ross, P. A. Kollman, AMBER 8, University ofCalifornia, San Francisco, 2004.

[21] M. S. Wallace, R. A. Laskowski, J. M. Thornton, Protein Eng.1995, 8, 127 – 134.

[22] G. Birrane, J. Chung, J. A. Ladias, J. Biol. Chem. 2003, 278,1399 – 1402.

[23] V. Kanelis, J. D. Forman-Kay, L. E. Kay, IUBMB Life 2001, 52,291 – 302.

[24] T. D. Goddard, D. G. Kneller, SPARKY 3, University ofCalifornia, San Francisco 2003.

[25] P. Guntert, C. Mumenthaler, K. WSthrich, J. Mol. Biol. 1997,273, 283 – 298.

[26] G. Cornilescu, F. Delaglio, A. Bax, J. Biomol. NMR 1999, 13,289 – 302.

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