For MMP-12AHA adduct an aliquot of 2 m l of protein solution (10 mM Tris/5 mM CaCl₂/0.1 mM ZnCl₂/300 mM NaCl/200 mM AHA, pH 8) was mixed with 2 m l of reservoir buffer (0.1 M Tris·HCl/25% PEG 6000/200 mM AHA, pH 8). The final protein concentration was 8 mg/ml. Crystallization was carried out with the hanging drop vapor diffusion method at 20°C. For MMP12--NNGH adduct, 3 mM of NNGH was added to protein solution before the mixing with a reservoir buffer containing 0.1 M Tris·HCl, 20% PEG 6000, 200 mM AHA, and 1.0 M LiCl₂ at pH 8.0.

The data were measured for both data sets at the ELETTRA XRD-1 beamline (Trieste, Italy) at 100 K and the crystals used for data collection were cryo-cooled without any cryo-protectant treatment. The MMP12-NNGH crystal had a mosaicity of ~0.6° and diffracted to a maximum resolution of 1.3 Å, whereas the MMP12-AHA crystal diffracted to 1.0-Å resolution. The crystal space group for the NNGH adduct is P2₁2₁2 with a = 37.30, b = 62.63, c = 69.24 Å, and a = b = g = 90°, and one molecule in the asymmetric unit is present; for the AHA complex the space group is C2 with a = 50.92, b = 59.59, c = 53.49 Å, a = g = 90°, and b = 115.14° and one molecule in the asymmetric unit is present.

The data were processed in both cases by using the program MOSFLM (1) and scaled by using the program SCALA (2) with the TAILS and SECONDARY corrections on (the latter restrained with a TIE SURFACE command) to achieve an empirical absorption correction. Table 1 shows the data collection and processing statistics for both data sets.

The structure of the NHGH adduct was solved by using the molecular replacement technique; the model used was that of a molecule of human MMP12 (PDB ID code 1OS9), and the structure of the AHA adduct (PDB ID code 1YG3) was solved with the NNGH adduct (PDB ID code 1RMZ) as the model from where the inhibitor, all of the water molecules, and ions were omitted. The correct orientation and translation of the molecule within the crystallographic unit cell was determined with standard Patterson search techniques (3, 4) as implemented in the program MOLREP (5, 6). The anisotropic refinement was carried out by using REFMAC5 (7). In between the refinement cycles the models were subjected to manual rebuilding with XTALVIEW (8). The same program has been used to model the NNGH and AHA inhibitors. Water molecules have been added by using the standard procedures within the ARP/WARP suite (9). For the NNGH adduct 236 water molecules have been added, reaching a final R_cryst of 0.17 and an R_free of 0.21; for the AHA complex 291 water molecules have been found and hydrogen has been added in the refinement procedure, leading to a final R_cryst of 0.15 and an R_free of 0.16.

The stereochemical quality of the refined models was assessed by using the program PROCHECK (10). The Ramachandran plot for both structures is of very good quality. The stereochemistry of the refined NNGH adduct is within the standard tolerance, with RMSDs of 0.014 Å for bond lengths and 1.6° for bond angles, whereas for the AHA complex the RSMD on bond length is 0.009 Å and 1.3° on bond angles. Table 2 reports all of the refinement statistics for both models. The coordinates for the MMP12-NNGH complex have been deposited in the Protein Data Bank under the ID code 1RMZ.

NMR samples of the NNGH-MMP12 adduct were prepared by dissolving the protein in 550 m l of the solution containing 10 mM deuterated Tris, 5 mM CaCl₂, 0.1 mM ZnCl₂, 0.3 M NaCl ,and 10% D₂O in water. The final concentrations of the ¹³C/¹⁵N-enriched and ¹⁵N-enriched F171D- mutated MMP12 samples for both the structural calculation and the mobility measurements were 0.9 and 0.6 mM, respectively. The pH was adjusted to 7.2.

The NMR spectra were acquired on Bruker AVANCE 900, AVANCE 800, AVANCE 700, and DRX 500 spectrometers operating at proton nominal frequencies of 900.13, 800.13, 700.13, and 500.13 MHz, respectively. All spectrometers are equipped with a triple resonance (TXI) 5-mm probe with a z axis pulse field gradient, and the 500-MHz spectrometer is equipped with a triple resonance CRYO-probe. All NMR experiments were performed at 298 K. The samples were kept at 4°C in between measurements. All spectra were processed with the Bruker XWINNMR software packages and analyzed by the program XEASY (Eidgenössische Technische Hochschule, Zürich). The residual water signal was suppressed by different pulse schemes depending on the experiment type.

The backbone resonance assignment was obtained by the analysis of HNCA, HNCO, HN(CA)CO, CBCANH, and CBCA(CO)NH spectra at 500 MHz. The assignment of the aliphatic side-chain resonances was performed through the analysis of 3D H(C)CH-TOCSY and (H)CCHTOCSY spectra at 500 MHz, together with 3D ¹⁵N- and ¹³C-NOESY-HSQC spectra at 800 MHz. The obtained assignments are reported in Tables 3 and 4. ³J_HNHa coupling constants were determined through the HNHA experiment at 500 MHz. Good agreement of ³J_HNHa and NOE intensity ratios with CSI (11) estimates was found. Backbone dihedral j angles (Table 5) were independently derived from ³J_HNHa coupling constants through the appropriate Karplus equation (12). Backbone dihedral y angles for residue i-1 (Table 6) were also determined from the ratio of the intensities of the da _N(i-1,i) and d_Na (i,i) NOEs present on the ¹⁵N(i) plane of residue i obtained from the ¹⁵N-edited NOESY-HSQC spectrum. Chemical shift index analysis on Ha , Ca , and Cb resonances, the ³J_HNHa coupling constants, the da _N(i-1,i)/d_Na (i,i) ratios (13), and the NOEs patterns indicated the presence of three a -helices and five b -sheets.

For the assignment of the inhibitor (NNGH) resonance, 2D ¹H-¹H NOESY and 2D ¹⁵N/¹³C isotope-filtered NOESY spectra (14) were performed at 298 K and 900 MHz. The mixing time used was 120 ms. The obtained intramolecular NNGH NOE constraints and intermolecular NNGH-protein constraints are summarized in Table 7.

3D ¹⁵N- and ¹³C-enriched NOESY-HSQC cross peak intensities were integrated by using the elliptical integration routine implemented in XEASY and converted into interatomic upper distance limits by the program CALIBA. The obtained constraints are reported in Table 8.

Residual dipolar couplings have been measured in the presence of an external orienting medium constituted by a binary mixture of C₁₂E₅ (penta-ethyleneglycol dodecyl ether, ³ 98% purity, Fluka) and neat n-hexanol (puriss, Fluka), forming a stable liquid crystalline phase made of neutral aggregates (called bicelles) in the temperature range from 295 to 312 K (15). The molar ratio of C₁₂E₅ to n-hexanol was 0.96, and the C₁₂E₅/water ratio was 5 wt %. One-bond ¹H-¹⁵N coupling constants were measured at 298 K and 800 MHz by using the IPAP method (16). For the sample of MMP12 with bicelles, 1D ²H-NMR was also performed before and after the IPAP measurement. The water signal was split by ~31 Hz at 298 K in both 1D ²H-NMR spectra. A total of 111 RDC values could be measured, and they ranged from -15 to +18 Hz. Of them, only those RDC values corresponding to residues not experiencing mobility nor large RMSD (60 residues, mostly in a or b secondary structures) have been used for structure calculations.

The program DYANA (17) was used to calculate a family of 200 structures starting from randomly generated conformers in 10,000 annealing steps, using the structural constraints of Tables 5-8, whereas the selected RDC values were introduced only in the final minimization step, and the family was energy-minimized by iterative cycles of PARAMAGNETIC-DYANA with the program FANTAORIENT (www.postgenomicnmr.net) (18). The family of the best 20 structures in terms of target function was then subjected to restrained energy minimization with AMBER 6.0 (19). The agreement between calculated and observed RDC values after DYANA calculations is reported in Fig. 6. The overall quality of the solution structure determined in this work based on Ramachandran plots (10) is quite good (~85.2% of residues in most favored regions and 13.4% in additionally allowed regions with overall G factor of -0.31).

The experiments for the determination of ¹⁵N longitudinal and transverse relaxation rates and ¹H-15N NOE were recorded at 298 K and 700 MHz. ¹⁵N-enriched sample was used for this experiments. ¹⁵N R₁ relaxation rates were measured by using a sequence modified to remove cross-correlation effects during the relaxation delay. The recycle delay was 3.0 s, and the R₁ inversion recovery times were 11.4, 22.8, 34.2, 45.6, 91.2, 182.4, 364.8, 729.6, 1,300, 2,000, and 2,500 ms. For R₂ measurement, a Carr--Purcell--Meiboom--Gill (CPMG) pulse sequence with a refocusing delay of 450 m sec and 11 different delays (17, 34, 51, 68, 85, 102, 136, 170, 204, 272, and 340 msec) was used. ¹H-¹⁵N NOEs were measured with a previously reported sequence (20). The results are reported in Fig. 4 (21).

The R₂ experiments were also performed as a function of the spin-echo delay in R₂-CPMG experiments (22) by using WATERGATE at 298 K and 700 MHz. Sets of experiments were carried out at five CPMG refocusing delays (t _CPMG = 175, 300, 450, 800, and 1,100 m sec). Off-resonance rotating frame relaxation rates, R_1r ^OFF were measured as a function of effective spin-lock power at 700 MHz by using a reported pulse sequence (23). Eight measurement sets of R_1r ^OFF were recorded at different effective spin-lock power values with 35^o of tilt angle (2,442, 2,190, 1,963, 1,757, 1,573, 1,406, 1,260, and 1,123 Hz). In each measurement set, the nine different delays (i.e., the duration of the trapezoid-shaped spin lock pulse; 10, 20, 36, 60, 86, 100, 150, 200 and 300 msec) were used. Residues experiencing conformational exchange on the time scale of CPMG and R_1r ^OFF experiments are listed in Table 10.

To assess the binding properties of compounds AHA and NNGH, their abilities to inhibit the hydrolysis of fluorescence-quenched peptide substrate Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH₂ (Biomol, Plymouth Meeting, PA) were tested. The assays were performed in 50 mM Hepes buffer, containing 10 mM CaCl₂, 0.05% Brij-35, at pH 7.0, using 1 nM of MMP-12 catalytic domain and 1 m M of peptide. The enzyme was incubated at 298 K with increasing concentration of inhibitor, and the fluorescence (excitation_max 328 nm; emission_max 393 nm) was measured for 3 min after the addition of the substrate by using a Varian Eclipse fluorimeter. Fitting of rates as a function of inhibitor concentration provided an IC₅₀ value of 8 mM for AHA and 10 nM for NNGH.

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