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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Keywords: serpin/1-antitrypsin/interleukin-1ß/protease inhibitors
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Introduction |
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It has been shown that short peptides corresponding to the serpin RSL sequences are poor inhibitors of serine proteases (McRae et al., 1980). 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, 1992
). 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., 1991
; Lawrence et al., 1994
; Wilczynska et al., 1995
; Stratikos and Gettins, 1997
; Wilczynska et al., 1997
). 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
1-antitrypsin, a serpin, retained significant specific inhibitory activity towards elastase, its primary target in vivo (Wolfson et al., 1991
). 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 1-antitrypsin (Glu-Ala-Ile-Pro-Ile-Met-Ser-Ile-Pro-Pro-Glu, P5-P5') replaced residues 5053 from IL-1ß, and the activity of the construct (Wolfson et al., 1991
, 1996
) as well as its binding to the IL-1ß receptor (Wolfson et al., 1993
). 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.
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Materials and methods |
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Recombinant IL-1ß and AT-IL were prepared using the vectors pMKIL and pMKAT, respectively, as described previously (Wolfson et al., 1991). 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., 1989). pGP1-2 carries a gene encoding kanamycin resistance (kan) and the temperature-sensitive
repressor gene cI-857 behind the lac promoter (Tabor and Richardson, 1985
). The T7 RNA polymerase gene is also present behind the
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 68 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 0.51 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 48 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., 1987; Wolfson et al., 1991
). 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 4575% 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 25300 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 SDSPAGE. 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., 1984; Bax and Subramanian, 1986
). 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 I
.
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Two-dimensional 2QF and 3QF COSY (Rance et al., 1983; Boyd et al., 1985
), 2Q COSY (Braunschweiler et al., 1983
), NOESY(Kumar et al., 1980
), and TOCSY (Bax and Davis, 1985
) were acquired for unlabeled AT-IL samples. NOESY mixing times were 100150 ms. Isotropic mixing in the TOCSY experiment was achieved with DIPSI-2 (Shaka et al., 1988
) 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., 1992
; Lippens et al., 1995
).
15N-separated NMR
The 15N carrier frequency in all experiments was placed at 120 p.p.m. A 2D HSQC experiment (Bodenhausen and Ruben, 1980) was recorded with water suppression achieved by a 1 ms purge pulse (Messerle et al., 1989
). A 3D NOESY-HMQC experiment was acquired with spin lock water suppression (Fesik and Zuiderweg, 1988
; Marion et al., 1989a
; Messerle et al., 1989
) and a mixing time of 110 ms. The 3D TOCSY-HSQC experiment (Marion et al., 1989b
) 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., 1991
) was recorded with a 19 ms delay for 15NHß coupling evolution. A 3D HMQC-NOESY-HMQC experiment (Frenkiel et al., 1990
; 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., 1989
). 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., 1990a) and constant-time HNCO and HN(CO)CA (Bax and Ikura, 1991
; Grzesiek and Bax, 1992
) 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., 1990) and HCCH-COSY (Bax et al., 1990
; Kay et al., 1990b
) 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 dimension (Kay et al., 1990a
; Grzesiek and Bax, 1993
). The spectrum was centered at 63.5 and 177.0 p.p.m. in the 13C
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, 1994) was recorded using gradients for water suppression (Kay, 1993
; P.Schmieder, personal communication). The 13C carrier frequency was set to 64 p.p.m. The HCACO-TOCSY experiment (Kay et al., 1992
) 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, 1987) 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., 1994) 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., 1989c) 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., 1991). 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., 1983
). 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
angle restraints were used in the calculations (see below), five
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., 1991
).
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., 1989Torda 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 MaxwellBoltzmann distribution, and dynamics were performed at 300 K for 3000 steps (
t = 1 fs) without restraints. This was followed by a long (100 000 step) dynamics simulation with time-average NOE restraints and
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 cistrans 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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Results |
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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 II. 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, 1981
). The absence of more than one distinguishable minor population suggests that proline cistrans isomerism is limited to a single site in the isolated RSL peptide.
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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 CH 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 1
). 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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Structures of the AT-IL chimera generated by XPLOR reveal the diversity of allowed RSL conformations (Figure 8). 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., 1990a
) with 15N{1H} NOE values approaching 0.2.
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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., 1997). Temperature gradients measured for both AT-IL and the RSL peptide over the range 1030°C are graphed in Figure 9
. 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., 1990b
), 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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Discussion |
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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., 1986), and results in a reduced temperature coefficient in solution (Heinz et al., 1992
). 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
-turn motifs, respectively. The
-turn motif was observed with some frequency in MD simulations (Figure 10
). 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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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 serpinprotease complex partitions between substrate and inhibitory pathways (Cooperman et al., 1993). 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, 1985
; Gettins and Harten, 1988
; Bruch et al., 1988
; Sancho et al., 1995
). 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., 1991
).
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 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., 1992
). A high resolution structure of
1AT in its active form was reported recently (Elliott et al., 1996
) 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
1-AT (Song et al., 1995
) reveals the RSL as a distorted helix, somewhat resembling that of an engineered antichymotrypsin (Wei et al., 1994
) incorporating the P3-P3' sequence from
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., 1991; Perkins et al., 1992
; Hopkins et al., 1993
; Lawrence et al., 1994
). The current model also holds that the inhibitory complex contains a covalent serpin-protease linkage in which the RSL is cleaved (Lawrence et al., 1995
; Olson et al., 1995
; Wilczynska et al., 1995
), and that formation of this complex involves a structural rearrangement within the active site of the protease (Plotnick et al., 1996
), as well as transposition of the protease across the face of the inhibitor (Stratikos and Gettins, 1997
; Wilczynska et al., 1997
).
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, 1989). 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, 1991
). 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.
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Acknowledgments |
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Notes |
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2 Present address: Department of Chemistry and Biochemistry, Northwestern University, Evanston IL 60208-3113, USA
3 To whom correspondence should be addressed
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References |
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Received September 8, 1998; revised December 1, 1998; accepted December 8, 1998.