Solution structure and dynamics of a serpin reactive site loop using interleukin 1ß as a presentation scaffold

C.C. Arico-Muendel1, A. Patera2, T.C. Pochapsky3, M. Kuti and A.J. Wolfson4

Department of Chemistry, Brandeis University, Waltham, MA 02454-9110 and 4 Department of Chemistry, Wellesley College, Wellesley, MA 02181-8284, USA


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 Abstract
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 Materials and methods
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Human interleukin-1ß (IL1ß) was used as a presentation scaffold for the characterization of the reactive site loop (RSL) of the serpin {alpha}1-antitrypsin (A1AT), the physiological inhibitor of leukocyte elastase. A chimeric protein was generated by replacement of residues 50–53 of IL1ß, corresponding to an exposed reverse turn in IL1ß, with the 10-residue P5-P5' sequence EAIPMSIPPE from A1AT. The chimera (antitrypsin-interleukin, AT-IL) inhibits elastase specifically and also binds the IL1ß receptor. Multinuclear NMR characterization of AT-IL established that, with the exception of the inserted sequence, the structure of the IL1ß scaffold is preserved in the chimera. The structure of the inserted RSL was analyzed relative to that of the isolated 10-residue RSL peptide, which was shown to be essentially disordered in solution. The chimeric RSL was also found to be solvent exposed and conformationally mobile in comparison with the IL1ß scaffold, and there was no evidence of persisting interactions with the scaffold outside of the N- and C-terminal linkages. However, AT-IL exhibits sigificant differences in chemical shift and NOE patterns relative to the isolated RSL that are consistent with local features of non-random structure. The proximity of these features to the P1-P1' residues suggests that they may be responsible for the inhibitory activity of the chimera.

Keywords: serpin/{alpha}1-antitrypsin/interleukin-1ß/protease inhibitors


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 Abstract
 Introduction
 Materials and methods
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The `canonical' inhibitors of serine proteases are small proteins with highly conserved structure of their protease binding regions. These are short, external sequences, termed reactive site loops (RSLs), that mimic the geometry of substrate molecules but continue to bind the protease when cleaved. Members of the serpin family of serine protease inhibitors differ from this archetype. Compared with the canonical inhibitors, serpins are larger (400 versus 30–180 residues) with longer RSLs (10–13 versus 5–8 residues). The longer length of the serpin RSL is implicated in some of the unusual features of serpins, including polymerization, reversible conversion to an inactive form (the `latent state') and irreversible deactivation following cleavage of the RSL. The structural basis for these transformations, which appears to involve the facile incorporation of the RSL loop into the main ß-sheet of the molecule, has been elucidated over the last decade by biochemical and crystallographic methods (Loebermann et al., 1984Go; Carrell et al., 1991Go; Mottonen et al., 1992Go).

It has been shown that short peptides corresponding to the serpin RSL sequences are poor inhibitors of serine proteases (McRae et al., 1980Go). This indicates that, despite the local nature of the binding interaction, which involves predominantly three residues to each side to the cleavage site (the P3-P3' sequence), there exist important long-range forces that stabilize the inhibitory RSL conformation. In the canonical inhibitors, these are believed to include both covalent (disulfide bond) and noncovalent (hydrogen bond and salt bridge) interactions between the RSL and the protein core (Bode and Huber, 1992Go). Such interactions are much less evident in the case of serpins. For these molecules, inhibition has been linked to the conformational plasticity described above (Carrell et al., 1991Go; Lawrence et al., 1994Go; Wilczynska et al., 1995Go; Stratikos and Gettins, 1997Go; Wilczynska et al., 1997Go). We have explored the scope of these long-range interactions in both types of inhibitors by measuring the activity of chimeric proteins in which RSL sequences were substituted into a loop region of a foreign host. The protein chosen for this role was the cytokine interleukin-1ß (IL-1ß), which shows structural homology with a number of canonical inhibitors such as that for soybean trypsin inhibitor. To our surprise, the construct containing the RSL from {alpha}1-antitrypsin, a serpin, retained significant specific inhibitory activity towards elastase, its primary target in vivo (Wolfson et al., 1991Go). This finding was unexpected because it suggested that inhibitory activity could be conferred on a serpin RSL in a quasi-canonical context, and therefore by a mechanism much less delocalized than that operating in the parent molecule. A detailed understanding of this behavior in terms of structure is thus of considerable interest.

Previous reports have described the engineering of the chimeric protein, in which the 10-residue RSL from {alpha}1-antitrypsin (Glu-Ala-Ile-Pro-Ile-Met-Ser-Ile-Pro-Pro-Glu, P5-P5') replaced residues 50–53 from IL-1ß, and the activity of the construct (Wolfson et al., 1991Go, 1996Go) as well as its binding to the IL-1ß receptor (Wolfson et al., 1993Go). We now report a structural study of the chimeric protein, designated antitrypsin-interleukin (AT-IL), in solution by NMR spectroscopy. This work confirms that the ß-barrel fold of IL-1ß is preserved in the chimeric protein. The inserted RSL appears to be a relatively independent structural feature which does not interact strongly with other regions of AT-IL. Although the RSL sequence is conformationally dynamic relative to the protein scaffold, there is evidence of significant local structure that may be implicated in the inhibitory activity of the chimera.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Materials

Recombinant IL-1ß and AT-IL were prepared using the vectors pMKIL and pMKAT, respectively, as described previously (Wolfson et al., 1991Go). Escherichia coli AR-58 and TB-1 cells were obtained from New England Biolabs. T4 DNA ligase, Klenow fragment, restriction enzymes and buffers were purchased from Promega. Isotopically enriched 15NH4Cl and 13C glucose were purchased from CIL (Cambridge, MA). Uniformly 13C enriched proline was the generous gift of Dr David LeMaster (Northwestern University); 13C/15N enriched proline was purchased from CIL.

Construction of vector pSP3950 for specific amino acid labeling

A CsCl preparation of the plasmid pGP1-2 was obtained from E.coli K38 cells following standard procedures (Sambrook et al., 1989Go). pGP1-2 carries a gene encoding kanamycin resistance (kan) and the temperature-sensitive {lambda} repressor gene cI-857 behind the lac promoter (Tabor and Richardson, 1985Go). The T7 RNA polymerase gene is also present behind the {lambda}PL promoter. An aliquot of the plasmid preparation was digested with the restriction enzymes BamHI and EcoRI, which excises the T7 RNA polymerase gene. The desired 3.95 kb fragment containing the kan and cI-857 genes purified electrophoretically and isolated using a Gene Clean kit (Bio101, Inc.). Blunt-ended DNA was prepared from the fragment using Klenow fragment and the blunt ends joined using T4 DNA ligase. This new vector pSP3950 was then transformed along with pMKAT, which encodes the AT-IL construct behind the lac promoter, into E.coli strain TB-1 (a proline auxotroph). Resistance to ampicillin and kanamycin was conferred by pMKAT and pSP3950, respectively.

Expression of unlabeled and uniformly isotope-labeled samples of AT-IL

Appropriately transformed E.coli strain AR-58 cells were grown overnight in 1 liter LB medium from a single colony culture. When the OD660 reached 1.0, protein expression was induced by adding an equal volume of medium at 55°C degrees, and continuing growth at 6–8 h at 42°C. Uniform labeling with 15N was accomplished by 4% inoculation with a single colony LB culture into 500 ml of unlabeled M9 minimal medium. The M9 culture was grown at 30°C to OD660 > 1.0, at which time the cells were collected by centrifugation and resuspended in 1 liter M9 at 42°C with 15NH4Cl as the sole nitrogen source. The resuspended culture was allowed to grow for approximately 8 h at 42°C. For uniformly 13C/15N labeled samples, the protocol was modified slightly to accommodate the slower growth rate of the cells in the isotopically enriched medium. Thus, a single colony culture was grown at 30°C in 25 ml LB to OD660 > 2, then spun down and resuspended in 50 ml M9 medium containing 15NH4Cl and 13C-U-glucose as the sole nitrogen and carbon sources, respectively. After acclimating for 0.5–1 h, the medium was diluted to 600 ml and grown to OD660 > 1 (~20 h). At this point the cells were heat shocked with 400 ml of labeled M9 at 55°C and grown for another 4–8 h at 42°C.

Expression of 15N and/or 13C proline labeled AT-IL

A single colony culture of E.coli strain TB-1 doubly transformed with pMKAT and pSP3950 cells was grown overnight in 50 ml LB at 30°C. The cells were centrifuged and resuspended in 600 ml of defined medium containing unlabeled proline. The culture was grown at 30°C to OD660 > 1.0. The cells were then spun down and resuspended in 600 ml of defined medium containing labeled proline. After acclimatization for 1 h at 30°C, the cells were heat shocked by the addition of another 400 ml defined labeled medium at 55°C, raising the temperature of the growth medium to 42°C. The cells were then maintained for 8 h at 42°C prior to harvesting.

Protein purification

AT-IL was purified using a modified version of literature procedures (Meyers et al., 1987Go; Wolfson et al., 1991Go). Cell pellets were resuspended in pH 8 buffer (10 mM Tris, 1 mM EDTA; 60 ml/15 g cells) with 1 mg/ml lysozyme and 0.5% toluenesulfonyl chloride. The suspension was sonicated and the soluble fraction treated with ammonium sulfate, the AT-IL precipitating in the 45–75% fraction at 25°C. The precipitate was dissolved in a minimal volume pH 5.7 buffer containing 25 mM ammonium acetate and 14 mM ß-mercaptoethanol, and the solution was dialyzed against the same buffer. The AT-IL was then purified chromatographically on an S-Sepharose column using a linear gradient of 25–300 mM ammonium acetate in a total elution volume of 600 ml. Fractions containing AT-IL were identified from the absorption at 280 nm. Protein purity was verified by SDS–PAGE. Samples for NMR spectroscopy were prepared by exchanging the protein into 50 mM sodium phosphate buffer, pH 5.5, in either 9:1 H2O:D2O or 99.9% D2O. The buffers were degassed extensively with argon and treated with sodium azide (0.02%) to extend the lifetimes of the samples. Final concentrations were roughly 2.5, 1.5 and 0.8 mM for the unlabeled, 15N labeled and uniformly 15N, 13C labeled samples of AT-IL, respectively. The concentration of an unlabeled Il-1ß sample was 3 mM.

NMR spectroscopy

All spectra were acquired at 25°C on a Bruker AMX-500 spectrometer equipped with a Bruker Acustar three-channel pulsed field gradient amplifier and an x,y,z three-axis gradient inverse detection triple resonance probe. Carrier frequencies were set to 500.13, 50.68 and 125.76 MHz for 1H, 15N and 13C, respectively. 1H and 13C were referenced to 2,2-dimethyl-2-silapentane-5-sulfonate sodium salt (DSS) at 0 p.p.m. 15N shifts were referenced indirectly to liquid NH3 using the 1H resonance of H2O (Live et al., 1984Go; Bax and Subramanian, 1986Go). Heteronuclear correlation experiments were acquired in inverse detection mode, using low power GARP decoupling of the heteronuclei during acquisition. Acquisition parameters for individual NMR experiments are listed in Table IGo.


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Table I. Acquisition parameters for NMR experiments applied to AT-IL
 
Homonuclear NMR

Two-dimensional 2QF and 3QF COSY (Rance et al., 1983Go; Boyd et al., 1985Go), 2Q COSY (Braunschweiler et al., 1983Go), NOESY(Kumar et al., 1980Go), and TOCSY (Bax and Davis, 1985Go) were acquired for unlabeled AT-IL samples. NOESY mixing times were 100–150 ms. Isotropic mixing in the TOCSY experiment was achieved with DIPSI-2 (Shaka et al., 1988Go) composite pulse spin-locking. Unless otherwise noted, the 1H frequency was placed at the water resonance and the water signal was attenuated by low power presaturation. In order to characterize cross-relaxation of RSL resonances with solvent water and to minimize signal loss due to saturation transfer from water, a NOESY experiment incorporating flip-back pulses was performed as well (Piotto et al., 1992Go; Lippens et al., 1995Go).

15N-separated NMR

The 15N carrier frequency in all experiments was placed at 120 p.p.m. A 2D HSQC experiment (Bodenhausen and Ruben, 1980Go) was recorded with water suppression achieved by a 1 ms purge pulse (Messerle et al., 1989Go). A 3D NOESY-HMQC experiment was acquired with spin lock water suppression (Fesik and Zuiderweg, 1988Go; Marion et al., 1989aGo; Messerle et al., 1989Go) and a mixing time of 110 ms. The 3D TOCSY-HSQC experiment (Marion et al., 1989bGo) was recorded with an isotropic mixing time of 32.6 ms using a 7.9 kHz RF spin lock field. A 3D HNHB experiment (Archer et al., 1991Go) was recorded with a 19 ms delay for 15N–Hß coupling evolution. A 3D HMQC-NOESY-HMQC experiment (Frenkiel et al., 1990Go; Ikura et al., 1990a) was recorded with a 110 ms mixing time. A heteronuclear two-dimensional 15N{1H} steady-state NOE experiment was performed using a published pulse sequence (Kay et al., 1989Go). Broad band saturation of 1H was accomplished by applying a train of 120° nonselective proton pulses separated from each other by 20 ms for a total time of 3 s.

13C/15N separated NMR of uniformly labeled AT-IL

HNCA (Kay et al., 1990aGo) and constant-time HNCO and HN(CO)CA (Bax and Ikura, 1991Go; Grzesiek and Bax, 1992Go) experiments were recorded following published pulse sequences. Off-resonance carbonyl decoupling was accomplished with phase-modulated shaped pulses. For all experiments, the 15N carrier frequency was set to 120 p.p.m. The 13C carrier frequency was set to 61.6 and 55.0 p.p.m. for the HNCA experiment and HN(CO)CA experiments, respectively, and to 175.17 p.p.m. for the HNCO experiment.

HCCH-TOCSY (Bax et al., 1990Go) and HCCH-COSY (Bax et al., 1990Go; Kay et al., 1990bGo) experiments of uniformly 13C/15N labeled AT-IL were performed with a 0.8 mM sample dissolved in D2O, using off-resonance presaturation to attenuate the residual water signal. The carrier frequencies were set to 2.74 (1H) and 41.86 p.p.m. (13C). Three loops of the DIPSI-3 mixing sequence were applied in the HCCH-TOCSY experiment, yielding a total mixing time of 21 ms at a frequency of 7.8 kHz.

13C separated NMR of proline labeled AT-IL

A 2D HSQC experiment was obtained with carrier frequencies set to 45.86 p.p.m. (13C) and 4.78 p.p.m. (1H), respectively. HCCH-TOCSY experiments were recorded with mixing times of 7.6 and 22.8 ms. The carrier frequencies were set to 3.18 (1H) and 45.86 p.p.m. (13C). A 3D NOESY-HMQC experiment was recorded with a mixing time of 80 ms. The carrier frequencies were at 2.58 and 45.86 p.p.m. in the 1H and 13C dimensions, respectively. A 3D HCACO spectrum was recorded with constant-time acquisition in the 13C{alpha} dimension (Kay et al., 1990aGo; Grzesiek and Bax, 1993Go). The spectrum was centered at 63.5 and 177.0 p.p.m. in the 13C{alpha} and 13CO dimensions, respectively, whereas in the 1H dimension the spectrum was centered around the water frequency.

The HACA(CO) (N) experiment (Olejniczak and Fesik, 1994Go) was recorded using gradients for water suppression (Kay, 1993Go; P.Schmieder, personal communication). The 13C carrier frequency was set to 64 p.p.m. The HCACO-TOCSY experiment (Kay et al., 1992Go) was recorded as a two-dimensional spectrum with CO evolution and gradient water suppression. The 13C carrier was initially set to 64 p.p.m., and subsequently moved to 46 p.p.m. during the DIPSI sequence. The t1 dimension (13CO) was centered at 177 p.p.m.

12C filtered NMR of proline labeled AT-IL

Two-dimensional NOESY and TOCSY spectra of 13C proline-labeled AT-IL were recorded using a J half-filter (Bax and Weiss, 1987Go) to identify correlations originating at protons bound to 13C. The filter delay was set to 3.45 ms. The NOESY mixing time was set to 40 ms. In the TOCSY experiment, isotropic mixing was achieved with 12 loops of the DIPSI-2 sequence, resulting in a 50 ms mixing time at a field strength of 6.9 kHz.

A 3D 13C separated, 12C filtered HMQC-NOESY experiment was performed by appending double half-filters (Gemmecker et al., 1994Go) to the pulse sequence of Majumdar and Zuiderweg (1993). The half-filter delays were 1.78 and 2.0 ms, and the corresponding purge pulses were 1.0 and 1.5 ms. The NOESY mixing time was set to 110 ms. Carrier frequencies were 45.86 (13C) and 4.78 p.p.m. (1H).

Characterization of elastase-cleaved AT-IL

A 1H, 15N-HSQC experiment was performed as described above on 0.15 ml of 1 mM uniformly labeled AT-IL in 90% H2O/10% D2O 50 mM phosphate buffer (pH 5.5). After completion of the experiment, the sample was diluted to 5 ml with 50 mM phosphate buffer (pH 7.5) and incubated for 4 h at room temperature with ~10 mg of fresh porcine elastase (Sigma). The sample was reconcentrated using centrifugal filtration, and the buffer exchanged using a spin column filled with Biorad P-2 gel equilibrated with the pH 5.5 NMR sample buffer. A second 1H, 15N-HSQC experiment was then performed under identical conditions to the first in order to identify residues which were affected by the presence of elastase.

Data processing

Analysis of spectra was done using FELIX version 2.3 (Biosym). Homonuclear 2D experiments were transformed with Gaussian or 45° shifted sine bell window functions; heteronuclear spectra were processed with unshifted squared sine bells in the acquisition dimension and 70° shifted sine bells in the indirectly detected dimensions, applied over an additional 10% of the data size in order to minimize signal loss. Time-domain convolution (Marion et al., 1989cGo) was applied in the case of heteronuclear experiments to attenuate the residual water signal. Signals from indirect dimensions recorded with constant-time evolution were doubled in length by linear prediction prior to Fourier transformation.

Structure and dynamics calculations

Structural calculations were performed using XPLOR 3.1 operating on a Silicon Graphics Iris Indigo 2 workstation (Brunger, 1993). The NMR-generated structure of IL1ß (PDB entry 6I1B) was used as a starting point for the calculations (Clore et al., 1991Go). The IL1ß structure was imported into Quanta (Molecular Simulations, Inc.) and the sequence modified appropriately to generate the AT-IL sequence. The structure of IL1ß was retained in the starting coordinate set for residues which are conserved in both structures, while the starting coordinates of the added RSL residues were generated using the regularization feature of Quanta. The resulting structure was exported as an XPLOR format.pdb file, and an appropriate XPLOR format.psf file was generated using standard amino acid topology and parameters (topallhdg.pro and parallhdg.pro in the XPLOR library). NOE constraints were obtained from 2D NOESY, 3D 15N-separated NOESY-HSQC and 3D 13C-separated, 12C-filtered spectra. Fifty-two NOEs which were identified for the sequence of residues from Phe46 to Val58 were ultimately used as input for structural calculations. NOE-based distance restraints were assigned as a function of NOE intensity (strong, 3 Å; medium, 4 Å; weak, 5 Å). For NOE-derived restraints involving methyl or methylene groups not assigned stereospecifically, a pseudoatom correction factor of either 2.4 (methyl) and 1 Å (methylene) was added to the maximum allowed distance. For NOEs involving symmetry related aromatic ring protons, a pseudoatom correction factor of 2.4 Å was added (Wüthrich et al., 1983Go). For all NOE restraints, atoms were allowed close approach to within 2 Å, and a separation to 0.5 Å above the restraint distance, without penalty. Although no {phi} angle restraints were used in the calculations (see below), five {chi}1 dihedral angle restraints (on Gln48, Glu I, Ile iii, Ile vii, Glu x, Asp54 and Lys55) that were obtained by comparison of spectra from HNHB and short-mixing time TOCSY-HSQC experiments were used in the structural calculations (Archer et al., 1991Go). {chi}1 angles were assigned as (±)-gauche/anti and were permitted to vary ±30° without penalty. Harmonic restraints were used to maintain the structure of IL1ß in the conserved residues, except for Gln48, Gly49, Asp54 and Lys55, which were restrained by NOEs. Simulated annealing molecular dynamics were performed at 1500 K for 20 000 3 fs steps followed by 10 000 3 fs steps of cooling to 100 K, using the protocol of Nilges et al. (1988).

As a further stage of structural analysis, equilibrium molecular dynamics simulations of AT-IL were performed which incorporated time-average NOE restraints (Torda et al., 1989GoTorda et al., 1990). A modification of the protocol of O'Donoghue (input file tasa.inp in the XPLOR 3.1 manual) was used. The dynamics simulations were performed in a low-dielectric continuum. After minimization, initial atomic velocities were randomly assigned from a Maxwell–Boltzmann distribution, and dynamics were performed at 300 K for 3000 steps ({Delta}t = 1 fs) without restraints. This was followed by a long (100 000 step) dynamics simulation with time-average NOE restraints and {chi}1 dihedral restraints active for a total of 100 ps. Restraints were applied only in the RSL and adjoining amino acid residues (Ser45 to Ala59). The remainder of the structure was allowed to evolve without structural restraints.

Conformational analyses of the RSL as a function of proline cis–trans isomerism were performed using the CHARMm program and force field. Interstrand distance restraints derived from NOEs observed in the AT-IL construct were used to constrain the ends of the 21-residue peptide corresponding to the sequence from Ser45 to Ala59 in AT-IL into the geometry they occupy in the chimeric protein. Residues within the RSL (from Val47 to Lys55) remained unconstrained. Starting structures were generated with appropriate proline conformations and were minimized for 9999 steps of steepest-descents minimization. These were then heated from 0 to 300 K in 300 1 fs steps, after which the structures were subjected to 5000 1 fs steps of dynamics, followed by another 1000 steps of minimization.


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Analysis of the isolated RSL peptide

Sequence-specific resonance assignment of the isolated RSL peptide, Ac-EAIPMSIPPE-NH2, was accomplished using a combination of 2D COSY, TOCSY and ROESY methods and is summarized in Table IIGo. An additional set of peaks integrating to approximately 10% of the major population was also identified. This value is similar to that expected for the cis conformer of a sequence isomerizing about an X-Pro peptide bond (Grathwohl and Wüthrich, 1981Go). The absence of more than one distinguishable minor population suggests that proline cis–trans isomerism is limited to a single site in the isolated RSL peptide.


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Table II. 1H Chemical shifts (p.p.m.) of the RSL peptide, Ac-(EAIPMSIPPE)-NH2 (298 K, pH 5.55)
 
The chemical shifts of NH and C{alpha}H resonances in the isolated RSL are almost all within 0.1 p.p.m. of random coil values (Bundi and Wüthrich, 1979). Two exceptions were Ile iii and Ile vii C{alpha}H, whose shifts are probably biased by the location of these two residues preceding prolines. Interresidue ROE correlations in the major population of the RSL peptide were limited to the peptide backbone. The strongest were C{alpha}H-NH(ii,i+1) interactions, found throughout the sequence. In addition, weak NH-NH(i,i+1) ROEs were identified between Glu1 and Ala2, Ala2 and Ile3, and Ser6 and Ile7. The data are thus consistent with a predominantly extended backbone in which the N-terminus is subject to somewhat greater conformational averaging than the C-terminal region.

Analysis of the chimeric protein AT-IL

Resonance assignments NOE-based heteronuclear 3D methods were used to make sequential assignments of the chimeric AT-IL. NH and C{alpha}H assignments in AT-IL were obtained from NOESY-HSQC and TOCSY-HSQC experiments for all but three residues in the IL-1ß scaffold: Ile56, Ser114 and Ile143. All of these exhibited very weak crosspeaks in the 15N HSQC spectrum (Figure 1Go). Complete assignments for most of the side chains were then made using TOCSY-HSQC, HNHB, HNCA, HCCH-COSY and HCCH-TOCSY spectra.



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Fig. 1. 15N HSQC spectrum of AT-IL, 25°C, pH 5.6. Correlations in the IL1ß scaffold are labelled according to their residue assignment in the protein sequence, using the one-letter amino acid code. Correlations assigned to RSL residues are labelled with a roman numeral according to their position in the RSL sequence.

 
The NOE-based strategy described above yielded only partial assignments for the RSL insert of AT-IL. Residues at the N-terminus of the RSL exhibit strong C{alpha}H-NH(i,i+1) NOEs, allowing the straightforward assignment of backbone resonances for Glu i, Ala ii, Ile iii and Pro iv. Interresidue NOEs for the middle and C-terminal end of the RSL were considerably weaker. The assignment process was also complicated by the broadening and doubling of several of the loop resonances, including Glu I, Ile iii and Met v, in the 15N edited spectra (see below). Extension of the sequential assignments through the length of the RSL required the use of through-bond correlations observed in HNCA and HN(CO)CA spectra of uniformly 13C, 15N-labeled AT-IL (Figure 2Go).



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Fig. 2. Sequential assignment of residues in the RSL via inter- and intra-residue NH-C{alpha} correlations. Shown are alternating 2D strip plots of HNCOCA and HNCA spectra at the 15N shift of the source amide. Amide 1H shifts are listed along the x-axes and 13C{alpha} shifts along the y.

 
The RSL contains three prolines (iv, viii and ix). In order to obtain complete side chain assignments of these structurally important residues, we prepared a sample of AT-IL that was specifically 13C, 15N enriched at proline. Nonetheless, the side chain 13C chemical shifts of the three residues turned out to be nearly degenerate, making assignments using the HCCH-TOCSY experiment ambiguous. However, the 13CO signals of the three prolines are well dispersed. We were thus able to resolve most of the proline C{alpha}H resonances in an HCACO experiment and complete the side chain assignments using the HCACO-TOCSY experiment (Figure 3Go) (Kay et al., 1992Go). Sequence-specific assignments for Pro iv and Pro ix were verified using HNCO correlations from Met v and Glu x. We were also able to confirm the H{alpha}/C{alpha} assignments of Pro viii from a 2D HACA(CO) (N) experiment, which is specific for residues preceding proline (Olejniczak and Fesik, 1994Go). When applied to our 13C, 15N-proline-labelled sample, this experiment revealed the Pro viii–Pro ix correlation uniquely.



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Fig. 3. 2D HCACO-TOCSY spectrum of AT-IL incorporating uniformly 13C-labeled proline. Proline 1H side chain correlations (F2) are separated by 13CO shift (F1).

 
Chemical shift analysis The 15N HSQC spectrum of AT-IL (Figure 1Go) closely resembles that recorded for IL-1ß (Driscoll et al., 1990aGo), providing strong evidence that overall structure of the chimera is unaltered relative to native IL-1ß. Except in the neighborhood of the inserted loop, C{alpha}H chemical shift differences between AT-IL and IL-1ß are generally less than 0.05 p.p.m., and even the C{alpha}H of Glu i (Glu50 in IL-1ß) is displaced by less than 0.1 p.p.m. The 13C{alpha} shift differences are larger, but only two residues outside the region of the RSL show shift differences exceeding 1 p.p.m. These residues (Glu128 and Thr137) are also located in a surface-exposed loop of IL-1ß, suggesting that the shift deviations are due to very localized structural changes. Within the RSL, the NH and C{alpha}H chemical shifts in AT-IL differ only slightly from those in the peptide control for several residues (Figure 4Go). On the other hand, the P1'-P3' residues (Ser vi, Ile vii and Pro viii) exhibit distinctly larger deviations from the isolated peptide, indicating that conformational biases induced by the scaffold may exist in this region.






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Fig. 4. (Top row) Differences in C{alpha} and H{alpha} chemical shifts between AT-IL and IL1ß (Clore et al., 1990bGo). Note that AT-IL shifts were reported at 25°C and IL1ß shifts were reported at 36°C. (Bottom row) Chemical shift differences between the AT-IL RSL and the isolated RSL peptide. (A) Differences in {delta}C{alpha}H; (B) differences in {delta}NH.

 
Further characterization of the RSL is obtained from the HSQC spectrum following treatment of 15N labelled AT-IL with porcine elastase. Numerous peaks corresponding to residues within the RSL or adjacent to it are shifted or disappear; perturbations to other signals are small (Figure 5Go). It is apparent that cleavage of AT-IL by elastase is specific and limited to the RSL, in accordance with our previous findings (Wolfson et al., 1991Go).



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Fig. 5. Superimposed 15N HSQC spectra of AT-IL before (broken lines, multiple contours) and after (single solid lines) treatment with elastase. Correlations for residues in the RSL or adjacent to it are indicated, as well as some additional residues that are not affected (e.g. E113) for reference.

 
NOE and coupling constant analysis of structure and dynamics The antiparallel ß-barrel fold of the IL-1ß scaffold is clearly delineated by sequential and long-range NOEs (Driscoll et al., 1990bGo). Likewise, NOE data for AT-IL can be readily superimposed on the secondary structure of IL-1ß (Driscoll et al., 1990bGo) as shown in Figure 6Go. The observed NOEs are completely compatible with the 12-stranded antiparallel ß-sheet topology of the parent protein. It is, however, somewhat unclear of the degree to which the loop insertion disrupts the ß-sheet structure of the adjacent strands, IV and V. In wild-type IL-1ß, these strands, extending from residue 40 to 62, are linked by a short loop, and 13 long range NOEs typical of antiparallel ß-structure were found to extend as far as between Val47 and Val58 (Driscoll et al., 1990bGo). In AT-IL, a C{alpha}H-C{alpha}H(i-j) NOE was identified between Phe42 and Leu62; a second NOE of this type seen between Met44 and Leu60 in IL-1ß could not be confirmed due to resonance overlap. Other correlations seen in IL-1ß include the C{alpha}H-NH(i-j) NOE between Val58 and Ser45, and the NH-NH(i-j) NOE between Ser45 and Ala59. The clear absence of an NOE between the C{alpha}H of Phe46 and the NH of Ala59 in AT-IL seems to indicate that the ß-sheet is disrupted between the Ser45–Ala59 and the Phe46–Val58 pairings. However, NOEs were still observed between the side chain protons of Phe46 and Val58, indicating that these residues remain in close proximity.



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Fig. 6. Long range C{alpha}H-C{alpha}H(i, j), NH-NH(i, j) and C{alpha}H-NH(i, j) NOEs observed in AT-IL superimposed on the secondary structure of IL1ß. For comparison, see figure 5Go in Driscoll et al. (1990b). Solid lines indicate NOEs observed in AT-IL. Dotted lines indicate NOEs which are observed in IL1ß but not in AT-IL due to the disruption of the ß-sheet structure by the insertion of the RSL.

 
For the RSL insert itself, all observed interresidue NOEs are sequential; no long-range interactions with the scaffold or within the RSL were observed. Most NOEs in the RSL are C{alpha}H-NH(i-i+1) correlations, although one very weak NH-NH NOE was found between Met v and Ser vi in both NOESY-HSQC and HMQC-NOESY-HMQC spectra. A three-dimensional 13C-separated, 12C-filtered HSQC-NOESY experiment using the selectively labeled 13C, 15N-proline AT-IL sample revealed a number of interresidue NOEs between the prolines and the adjacent isoleucine side chains, NOEs that would otherwise be obscured by intense intra-residue signals (Figure 7Go).



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Fig. 7. Plane corresponding to the chemical shift of several proline C{delta} carbons (50.8 p.p.m.) from a 3D 13C-separated, 12C-filtered HSQC-NOESY spectrum of proline-labeled AT-IL showing inter-residue NOEs between proline C{delta}H protons and nearby unlabeled side chains. The 13C-labeled 1H shifts (proline C{delta}H) are resolved in F2, while those of the unlabeled (12C-attached) protons are resolved in F3.

 
Values of 3JNH-C{alpha}H measured in a series of J-modulated 1H-15N HSQC experiments (Neri et al., 1990Go) for residues in the RSL were in the intermediate range (6–7 Hz) and thus likely to reflect conformational averaging of the {phi} torsional angle. These values were not employed in subsequent structural calculations or dynamics simulations. On the other hand, the HNHB and TOCSY-HSQC experiments indicated a measurable bias in side chain conformation for several residues in the RSL, particularly for Ile iii, Ile vii, and other residues near the loop termini. Thus, five {chi}1 dihedral angle constraints were extracted for use in the structural calculations.

Structures of the AT-IL chimera generated by XPLOR reveal the diversity of allowed RSL conformations (Figure 8Go). Overall, reversal of the peptide backbone is accomplished by the proline residues, with the other amino acid residues adopting conformations that are generally extended. The modest definition of the loop sequence is, to an extent, an artifact of the NOE method, which is biased towards internal regions where the density of NOEs is higher. To confirm that the loop is indeed mobile, we recorded additional, heteronuclear relaxation experiments that are sensitive to backbone dynamics. The 15N{1H} NOE experiment, in particular, is sensitive to backbone motion for a protein the size of AT-IL. For the majority of residues throughout the protein, 15N{1H} NOE values fall in the range between 0.5 and 1.0, indicating a high degree of order, as expected in well structured regions of the protein core. Exceptions are found at the chain termini and at certain loop regions, especially at the RSL insertion point. Here, negative 15N{1H} NOEs were found for Ala ii and the overlapped Met v residues consistent with a high degree of backbone flexibility. This region is also extensively disordered in IL-1ß (Clore et al., 1990aGo) with 15N{1H} NOE values approaching 0.2.




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Fig. 8. (A) Structure of the AT-IL construct. The IL1ß structure (PDB entry 6I1B) was used to generate the coordinates of residues 1–47 and 56–153, which were then fixed by harmonic restraints. The position of the RSL insert is indicated at the positions of Ala iii, Pro iv and Pro ix. Numbering of residues past the insert in the sequence is consistent with the numbering of the unmodified interleukin 1ß. NOEs were used to restrain residues 48–55 (IL1ß sequence numbering) as well as the RSL insert. (B) Superposition of 20 structures extracted at 1.25 ps intervals from 300 K molecular dynamics simulations of AT-IL in which the RSL and nearby residues were restrained using time averaged NOEs, while the remainder of the structure was unconstrained. For a complete description of the simulations, see text. Residues 45–58 are shown, as well as the RSL insert.

 
These findings indicate that the structure(s) giving rise to the inhibitory activity of AT-IL is probably transient, and localized within short stretches of amino acid residues. The sequence from Ile iii to Pro ix, in particular, contains several conformationally constrained residues (two isoleucines and three prolines), with a high density of sequential NOEs. It was thus of interest to determine the extent to which conformers generated by cis–trans isomerism of the X-Pro amide bonds might be present. As noted above, several residues in and near the RSL, including Phe46, Gly49, Glu i, Ile iii, Met v and Val58, show broadening and splitting of their NMR signals in a fashion that could be consistent with this behavior. It is also observed for Arg4, Ser5, Lys92 and Met 95, which are nearby in the three-dimensional structure of the scaffold (Finzel et al., 1989Go; Priestle et al., 1989Go; Clore et al., 1991Go). However, such signal doubling was also observed in the corresponding residues of IL-1ß (Driscoll et al., 1990bGo), and attributed to slow conformational exchange of the loop between residues 86 and 99 (IL-1ß numbering, Clore et al., 1990aGo). Two features of the NMR spectra also argue against the involvement of a significant cis-proline population in the chimera RSL. First, NOE correlations to the residue following the prolines are exclusively those expected for a trans geometry (C{alpha}H-C{delta}H). Second, the characteristic upfield C{gamma} chemical shift (<24 p.p.m.) and downfield Cß chemical shift (>33 p.p.m.) seen in cis-prolines are not observed for any of the RSL prolines in AT-IL. However, Pro91, which maintains a cis-conformation in IL-1ß, is seen to exhibit these shifts in AT-IL.

In order to further investigate proline conformations in the RSL, we performed a series of molecular dynamics simulations on the 10-residue RSL sequence in which the ends were constrained according to the experimentally determined ß-sheet conformation of the full length protein. These simulations were performed for the eight possible combinations of RSL X-Pro bond rotamers. Of the eight final structures, only three appeared to accommodate the observed NH-NH NOE between Met v and Ser vi: ttt, tct and ctc (corresponding to prolines iv, viii and ix, respectively). Moreover, many of the structures containing cis-prolines appeared to require close contacts between internal residues of the loop and the core of the protein if these conformations were to be maintained in the chimera. No non-sequential NOEs representing such interactions were detected (see above), again consistent with the idea that the primary conformation of the RSL is all-trans in the AT-IL construct.

Amide NH temperature dependence in AT-IL and the RSL peptide Temperature gradients of amide NH chemical shifts offer a site-specific probe for solvent accessibility in structurally well-ordered peptides and proteins. Thus, large dependencies (>6 ppb/°C in water) correlate with predominant hydrogen bonding to solvent, whereas smaller coefficients reflect sequestration from solvent and/or the formation of internal hydrogen bonds. In the case of unconstrained peptides and protein fragments, this analysis has been shown to be unreliable for the assessment of solvent exposure, although a small gradient may still be a useful indicator of structure in general if accompanied by a significant deviation of the NH chemical shift from random coil values (Andersen et al., 1997Go). Temperature gradients measured for both AT-IL and the RSL peptide over the range 10–30°C are graphed in Figure 9Go. For the peptide, the coefficients are uniformly large, in agreement with the observed random coil chemical shifts. In the case of AT-IL, small coefficients of were measured for three residues at the periphery of the loop, Phe46, Gln48 and Lys55. These residues are assigned to ß-sheet strands IV and V in IL-1ß (Driscoll et al., 1990bGo), and their amides may be shielded by internal hydrogen bonding. Unexpectedly, however, Ser vi in AT-IL also exhibits a significantly reduced temperature dependence (–3.0 ppb/°C) that is not observed in the isolated RSL peptide. The NH of this residue is also shifted upfield by 0.26 p.p.m. in AT-IL (see above).



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Fig. 9. Bar graph comparison of temperature gradients of NH chemical shifts in the AT-IL RSL and in the free RSL peptide. AT-IL gradients are shown in the top half in black; peptide dependencies are shown below shaded. Dependencies are graphed as –{Delta}{delta}/{Delta}T in ppb/K.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The present work culminates a study of the context dependence of RSL inhibitory activity. From the observation that the cytokine IL1ß shows significant structural homology with soybean trypsin inhibitor (STI), chimeric proteins were constructed in which the RSL's from STI, turkey ovomucoid inhibitor (TOI) and {alpha}1-AT were spliced into the exposed loop linking ß-sheet strands IV and V of IL1ß. The chimeras were then subjected to functional assays corresponding to both parent molecules (Wolfson et al., 1993Go). Like AT-IL, all three chimeras retained nearly wild-type IL1ß receptor binding activity. Incorporation of the RSLs conferred sensitivity to protease cleavage on the chimeras at the P1-P1' site. Unexpectedly, AT-IL exhibited measurable inhibitory activity in excess of the isolated RSL peptide against its primary physiological target, elastase (McRae et al., 1980Go; Wolfson et al., 1991Go, 1996Go). Antitrypsin has a core architecture very different from STI or IL1ß. Its RSL is also longer than that of STI (resulting in a net addition of seven amino acid residues to the chimeric protein) and its inhibitory mechanism is fundamentally distinct from that of the canonical protease inhibitors. Our objective was to probe the structural basis for elastase inhibition in AT-IL along the following lines: first, what alterations, if any, had occurred to the scaffold as a result of the large RSL insertion; and second, what kind of structure had been induced in the RSL itself. We can summarize our results as follows: (i) the NMR spectrum of AT-IL closely resembles that of IL1ß for most backbone and side chain resonances, with a high level of chemical shift and NOE homology except in the neighborhood of the inserted RSL. Cross-strand NOE's seen in IL1ß are found essentially up to the RSL–scaffold junction, indicating that the insertion causes minimal disruption to the scaffold. (ii) 1H and 15N NOE information, along with backbone NH-{alpha}CH coupling constants, are consistent with an RSL sequence that is mobile but predominantly extended, with chain reversals defined by the proline residues. This architecture is thus broadly in agreement with the extended chain seen in the X-ray crystal structure of intact {alpha}1-antitrypsin (Elliott et al., 1996Go), and is inconsistent with the irregular helix found in an engineered antichymotrypsin (Wei et al., 1994Go). (iii) Despite the overall flexibility of the AT-IL RSL, comparison with the isolated 10-mer peptide reveals evidence of localized structure in the chimera RSL. There is no evidence for a significant population of cis-proline conformers in AT-IL, although this is observed in the RSL peptide. In addition, NH-NH ROE's observed between Glu i and Ala ii, and between Ala ii and Ile iii, in the 10-mer peptide are not detectable in the chimeric protein. In contrast, AT-IL does exhibit a weak NH-NH NOE between Met v and Ser vi that is not seen in the peptide. Finally, the amide NH of Ser vi shows an anomalous, strongly depressed temperature dependence of the chemical shift. These results suggest that many conformations are excluded, whereas others are stabilized in the context of the chimeric protein.

The observation of a low temperature dependence for the NH of Ser vi is probably the most unexpected result of this study. Such gradients are commonly observed for amides that are buried from solvent in the hydrophobic core of a protein, or otherwise involved in stable hydrogen bonding that protects them from interactions with solvent. It is possible to suggest some possible H-bonding partners for the NH of Ser vi within the RSL. One is the hydroxyl group of the serine; an intraresidue hydrogen bond of this kind has been identified at the P1' Asp of the canonical elatase inhibitor eglin c (Bode et al., 1986Go), and results in a reduced temperature coefficient in solution (Heinz et al., 1992Go). The latter workers suggested that the rigidity induced by this hydrogen bond could play a role in conferring inhibitory activity on eglin c, so it is tempting to propose a similar mechanism in AT-IL. Alternative hydrogen bonding schemes involving other amino acid residues are also conceivable. Thus, interactions with the carbonyls of Ile iii or Pro iv could occur in ß- or {gamma}-turn motifs, respectively. The {gamma}-turn motif was observed with some frequency in MD simulations (Figure 10Go). However, some caution is required when interpreting temperature dependencies of NH shifts. Following the analysis of Andersen et al. (1997), the low temperature dependence of Ser vi NH may be explained as a consequence of opposing chemical shift tendencies upon heating (a downfield shift reflecting denaturation superimposed on an upfield shift corresponding to a weakened hydrogen bond). Regardless of whether Ser vi NH is internally hydrogen bonded, however, it is apparent by comparison with the control peptide that some form of structure is present in the RSL insert. From the lack of evidence for interactions between Ser vi and the protein core, it follows that this structure must be induced over a considerable distance by the constraint on the N' and C' termini of the RSL.



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Fig. 10. A conformation of AT-IL accessible from the all-trans configuration of the three prolines in the RSL as obtained from dynamics simulations on the peptide fragment (not the entire construct shown in Figure 8AGo, see Materials and methods). The conformation shown allows hydrogen bonding (shown as a dotted line) between Ser vi NH and Pro iv CO.

 
That Ser vi occupies the P1' site strongly suggests that structure involving this residue contributes to the inhibitory activity of AT-IL. However, the basis for this activity is subtle and not solely attributable to constraint of the residues that flank the scissile amide bond. In a separate publication (Wolfson et al., 1996Go), we reported the kinetics of inhibition in a series of AT-IL analogues in which the length of the RSL insert was varied. Thus, the six-residue insert PMSIPP was a significantly poorer elastase inhibitor, whereas the MS insert was inactive. Moreover, the rate of RSL hydrolysis also declined with loop length, suggesting that the loss of inhibition was due to poorer binding. Deletion of important binding contacts, and/or alteration of the residual RSL structure, could account for this behavior.

Beyond their relevance to protein engineering, our results bear on the continuing debate over the inhibitory mechanism of full-length serpins. We briefly summarize features of the current model that are pertinent here. Several studies have shown that greater than stoichiometric amounts of serpin are required for complete inactivation of a protease; this has been interpreted in terms of a suicide mechanism in which, following the initial binding event, the serpin–protease complex partitions between substrate and inhibitory pathways (Cooperman et al., 1993Go). The latter leads to a stable inactivated complex, whereas the former leads to inhibitor cleavage and restoration of protease activity. The hydrolyzed serpin is also stabilized relative to the active form, so that (unlike for the canonical inhibitors), cleavage is an irreversible process (Carrell and Owen, 1985Go; Gettins and Harten, 1988Go; Bruch et al., 1988Go; Sancho et al., 1995Go). A reversibly inactivated form, termed the latent state, has been identified for plasminogen activator inhibitor (PAI) in vivo, and is induced in other serpins by treatment with mild denaturants (Carrell et al., 1991Go).

Over the last decade, X-ray studies of serpins in these three states have allowed this behavior to be rationalized in structural terms. The first report by Loeberman et al. (1984) of cleaved {alpha}1-antitrypsin showed the newly formed ends from the cleaved RSL to be 70 Å apart, with the N-terminal sequence inserted as a strand of the main ß-sheet of the molecule. The stability of this rearrangement was demonstrated in the structure of latent PAI, where the RSL was incorporated in sheet A without cleavage (Mottonen et al., 1992Go). A high resolution structure of {alpha}1AT in its active form was reported recently (Elliott et al., 1996Go) and confirmed that the unbroken RSL projected as a solvent exposed loop at one end of the molecule, leaving sheet A with only four strands. The portion of the sequence between P5 and P4', which form hydrogen bonds with the core of the protein, is relatively unconstrained. The P3-P3' sequence approximates the canonical conformation, whereas between P3 and P8 the backbone is extended. In contrast, a lower resolution structure of {alpha}1-AT (Song et al., 1995Go) reveals the RSL as a distorted helix, somewhat resembling that of an engineered antichymotrypsin (Wei et al., 1994Go) incorporating the P3-P3' sequence from {alpha}1AT.

These studies suggest that the RSL conformation in active serpins may be somewhat plastic, but that the canonical geometry appropriate for protease binding is accessible. On the other hand, inhibitory activity requires at least partial insertion of the RSL into sheet A, as shown by adding peptides corresponding to the inserted sequence (which presumably block insertion by forming a binary complex) or by introducing mutations into the RSL that hinder insertion (Carrell et al., 1991Go; Perkins et al., 1992Go; Hopkins et al., 1993Go; Lawrence et al., 1994Go). The current model also holds that the inhibitory complex contains a covalent serpin-protease linkage in which the RSL is cleaved (Lawrence et al., 1995Go; Olson et al., 1995Go; Wilczynska et al., 1995Go), and that formation of this complex involves a structural rearrangement within the active site of the protease (Plotnick et al., 1996Go), as well as transposition of the protease across the face of the inhibitor (Stratikos and Gettins, 1997Go; Wilczynska et al., 1997Go).

In these models, therefore, the RSL is primarily envisioned as a passive `bait' sequence that mimics a substrate; inhibition is attained only within the context of the rearranged complex. Our results suggest that this view should be qualified. We propose that the RSL, if appropriately constrained, possesses intrinsic inhibitory activity that can affect the partitioning ratio between substrate and inhibitor. Constraints on the N- and C-termini are supported by the restrictions of some of the internal residues, such as Ile and Pro; such residues are common in other serpin RSLs (Huber and Carrell, 1989Go). There is further experimental evidence from intact serpins that the uncleaved serpin RSL has a preferred structure in solution; namely, from NMR experiments which show little difference in mobility between the RSL and serpin core (Hood and Gettins, 1991Go). Our model system represents a very preliminary approach to replicating this behavior. For serpins in vivo, on the other hand, the degree of inhibitory activity attainable may have evolved over time to become significant, representing an additional level of control in the mechanism of protease inhibition.

Supplementary material

1H, 15N and 13C chemical shift assignments for AT-IL are available upon request from the corresponding author.


    Acknowledgments
 
This work was supported by grants from the Johnson and Johnson Focused Giving Program (T.C.P.) and the National Science Foundation (A.W.). C.A.M. acknowledges support from a National Institutes of Health post-doctoral fellowship. T.C.P. acknowledges support from the NSF Young Investigators and the Camille and Henry Dreyfus Teacher-Scholar programs.


    Notes
 
1 Present address: Department of Chemistry, PRAECIS Pharmaceuticals, Inc., 1 Hampshire Street, Cambridge, MA 02139, USA Back

2 Present address: Department of Chemistry and Biochemistry, Northwestern University, Evanston IL 60208-3113, USA Back

3 To whom correspondence should be addressed Back


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Received September 8, 1998; revised December 1, 1998; accepted December 8, 1998.