MMP-inhibitor interaction: the solution structure of the catalytic domain of Human Matrix metalloproteinase -3 with different inhibitors Luis A. Alcaraz 1,2, Lucia Banci 1,3, Ivano Bertini 1,3, Francesca Cantini 1, Antonio Donaire 4, Leonardo Gonnelli 1 Magnetic Resonance Center (CERM) University of Florence, Via L. Sacconi 6, 50019 Sesto Fiorentino, Italy (1); From Instituto de Biologia Molecular y Celular, Universidad Miguel Hernandez, Edificio Torregaitan, Elche (Alicante), Spain (2); Department of Chemistry - University of Florence, Via della Lastruccia 3, 50019 Sesto Fiorentino, Italy (3); Department of Inorganic Chemistry, Universidad de Murcia, Campus Universitario de Espinardo, Apdo. 4021, 30071 Murcia, Spain (4). Address correspondence to: Prof. Ivano Bertini Magnetic Resonance Center, University of Florence Via L. Sacconi, 6 50019 Sesto Fiorentino, ITALY Fax: +39 055 4574271 Tel: +39 055 4574272 E-mail: ivanobertini@cerm.unifi.it Supplementary Material
Figure S1. Schematic representation of the active-site of MMPs interacting with a polipeptide substrate (A) and with the inhibitor NNGH (B). Numbering is according to MMP3 sequence. A B
Figure S2. RMSD values per residue to the mean structure for the backbone (filled squares) and all heavy atoms (open circles) of the REM family of 30 conformers of MMP3-NNGH. The secondary structure elements of MMP3-NNGH are also reported at the top. RMSD (Å) 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 β1 α1 β2 β3 β4 β5 α2 α3 100 120 140 160 180 200 220 240 Residue Number
Figure S3. 15 N R 1 and R 2 relaxation data and heteronuclear NOEs of the MMP3-NNGH measured at 600 MHz and 298 K. The secondary structure elements of MMP3-NNGH are also reported at the top. The fits and their uncertainties were calculated by means of the Levenberg-Marquardt algorithm and a Monte Carlo approach, respectively. β1 α1 β2 β3 β4 β5 α2 α3 R 2 [s -1 ] NOE 1.2 1.0 0.8 0.6 0.4 0.2 0.0 20 16 12 8 4 0 2.5 2.0 R 1 [s -1 ] 1.5 1.0 0.5 0.0 100 120 140 160 180 200 220 240 Residue number
Figure S4. Parameters characterizing the overall and internal mobility of MMP3-NNGH adduct within the Lipari-Szabo model. The backbone amide order parameters (S 2 ), the effective correlation time for motions faster than the overall tumbling rate (τ e ), and the conformational exchange contribution to R 2 are shown. The secondary structure elements are reported at the top. β1 α1 β2 β3 β4 β5 α2 α3 8 6 S 2 t e (ps) K ex 4 2 0 5 4 3 2 1 0 1.0 0.8 0.6 0.4 100 120 140 160 180 200 220 240 Residue number
Figure. S5. Combined chemical shift differences between: A) MMP3-NNGH and MMP3-MLC88; B) MMP3-NNGH and MMP3-InhVII. The secondary structure elements are reported at the top. 1.0 A β1 α1 β2 β3 β4 β5 α2 α3 0.8 Combined δ (ppm) 0.6 0.4 0.2 0.0 100 120 140 160 180 200 220 240 Residue Number 1.0 B β1 α1 β2 β3 β4 β5 α2 α3 Combined δ (ppm) 0.8 0.6 0.4 0.2 0.0 100 120 140 160 180 200 220 240 Residue Number
Figure. S6. Representative structures of the adducts of MMP3 with the inhibitors MLC88 (A) and InhVII (B) for the lowest energy clusters obtained from Autodock and Xplor-NIH calculations. The total Xplor-NIH energies calculated considering the energetic contributions deriving from the intra-inhibitor atoms and inter protein-inhibitor interactions together with the standard deviation within the family structures of the cluster are also shown. A Cluster 1 (-87 ± 4 kcal mol - 1 ) Cluster 2 (-83± 3 kcal mol - 1 ) B Cluster 1 (-106 ± 6 kcal mol - 1 ) Cluster 2 (-100± 7 kcal mol - 1 )
Supplementary Table S1. NMR experiments performed on MMP3-NNGH at 298 K for spectral assignment and structure determination. Experiments Dimension of acquired data (nucleus) Spectral width (ppm) n a t 1 t 2 t 3 F 1 F 2 F 3 [ 1 H- 1 H]-NOESY b 1024( 1 H) 2048( 1 H) 16 16 128 1 H- 15 N-HSQC c 64( 15 N) 2048( 1 H) 34 14.6 8 1 H- 13 C-HSQC c 128 2048 40 16 4 CBCA(CO)NH c 128( 13 C) 40( 15 N) 1024( 1 H) 87 40 16 16 CBCANH c 128( 13 C) 40( 15 N) 1024( 1 H) 87 40 16 16 (h)cch-tocsy c 230( 13 C) 64( 13 C) 1024( 1 H) 78.8 78.8 14 24 13 C-NOESY-HSQC b 240( 13 C) 64( 1 H) 2048( 1 H) 80 14 14 24 15 N-NOESY-HSQC b 180( 1 H) 40( 15 N) 2048( 1 H) 14.6 34 14.6 24 HNHA c 256( 1 H) 40( 15 N) 1024( 1 H) 15 40 15 16 15 N R 1 c 256( 15 N) 1676 ( 1 H) 36 14 16 HNCA 96 32 1024 30 30 14 32 HNCOCA 64 32 1024 30 30 14 32 15 N R 2 c 256( 15 N) 1676( 1 H) 36 14 16 Filtered noesy b 512( 1 H) 4096( 1 H) 17 17 240 steady-state heteronuclear 256( 15 N) 1676( 1 H) 36 14 64 NOEs c a Number of acquired scans. b These experiments were acquired on a 900 MHz spectrometer with mixing time of 100ms ( 15 N) or 120ms ( 13 C). c Data acquired on 500MHz or 800MHz spectrometers. All the triple resonance cryo-probes (TXI 5-mm) used were equipped with Pulsed Field Gradients along the z-axis. In R 1, R 2 and heteronuclear NOE experiments the water signal was suppressed with the water flip-back scheme. R 2 rates were measured using a refocusing time of 450 µs. All 2D and 3D spectra were then processed using the standard Bruker software (XWINNMR) and analyzed with the Sparky program
Supplementary Table S2. 1 H, 15 N and 13 C resonance assignment of MMP3-NNGH at 298 K. The assignment of the side chain resonances was performed through the analysis of the 3D (H)CCH-TOCSY spectrum together with the 15 N- NOESY-HSQC and 13 C- NOESY-HSQC spectra. Proton resonances were calibrated with respect to the signal of 2,2- dimethylsilapentane-5-sulfonic acid (DSS). Nitrogen chemical shifts were referenced indirectly to the 1 H standard using a conversion factor derived from the ratio of NMR frequencies. Carbon resonances were calibrated using the signal of dioxane at 69.3 ppm as secondary reference. N (HN) CA (HA) CB (HB) G88 (4.23, 4.72) I89 116.3 (8.54) (3.79) (1.96) QG1 1.034 QG2 0.81 P90 61.21 (4.08) 28.74 (1.93, 2.22) CD 46.39 CG 24.16 HD1 3.23 HD2 3.288 QG 1.839 K91 111.8 (7.04) 57.86 (3.46) 29.82 (2.16) CD 24.24 CE 47.07 CG 19.06 QD 1.79 QE 3.42 QG 0.85 W92 123.5 (8.215) 53.7 (4.34) 22.17 (3.20, 3.57) HD1 7.59 HE1 10.01 HZ2 7.94 NE1 129.4 R93 119.6 (7.912) 52.9 (3.878) 26.46 (1.45) CD 37.36 CG 30.2 HD1 3.40 HD2 2.909 HG1 1.683 HG2 1.8 K94 116.7 (7.473) 53.49 (4.224) 33.34 (2.5) CD 26.78 CE 43.13 CG 18.85 HE1 2.90 HE2 2.67 HG1 1.28 HG2 0.99 QD 1.80 T95 105.3 (7.993) 58.56 (4.253) 66.91 (4.148) CG2 18.63 QG2 1.278 H96 119.8 (7.027) 51.49 (4.693) 26.68 (2.92, 2.88) L97 127.5 (8.166) 51.23 (4.991) (1.05) HG 0.69 QD1-0.43 QD2-0.136 T98 110.8 (9.242) 55.45 (5.548) 70.33 (4.018) QG2 0.75 Y99 117.5 (8.563) 51.87 (5.536) 39.38 (2.67, 1.68) QD 6.275 QE 7.211 R100 119.2 (8.158) 51.74 (4.277) 32.05 (1.96, 2.09) CD 39.58 CG 30.13 HG1 1.5 HG2 1.68 QD 3.22 I101 128.2 (8.32) 58.67 (4.71) 34.39 (1.54) CD1 11.32 CG1 25.45 CG2 14.5 QD1 0.86 QG1 1.35 QG2 0.1771 V102 129.8 (9.476) 63.28 (3.292) 30 (1.941) CG1 20.68 CG2 18.37 QG1 0.89 QG2 0.86 N103 116.1 (7.577) 56.26 (4.685) 36.37 (2.82, 3.05) HD21 6.80 HD22 7.45 ND2 112.9 Y104 111.9 (8.049) 57.08 (4.092) 37.85 (2.91) T105 (8.42) (3.992) (1.947) HG1 0.83 QG2 0.31 P106 (4.507) D107 121.7 (8.827) 53.85 (4.066) 38.72 (2.91, 2.61) L108 116.5 (6.929) 48.4 (4.776) 44.57 (1.47, 1.07) CD1 21.84 CG 23.56 HG 0.44 QD1 1.01 QD2 0.41 P109 59.4 (4.383) 29.23 (1.95, 2.34) CD 50.87 CG 25.24 K110 123.8 (8.586) 57.93 (3.795) 29.47 (1.25, 1.34) CD 26.51 CE 39.17 CG 21.6 HD1 1.14 HD2 1.02 HE1 2.61 HE2 2.52 HG1 0.79 HG2 0.55 D111 113.6 (8.457) 53.9 (4.222) 37.16 (2.69, 2.55) A112 122.4 (7.263) 51.54 (4.21) 16.29 (1.47) V113 121.7 (7.472) 62.78 (3.639) 28.67 (1.941) CG1 18.85 QG1 0.41 QG2 0.59 D114 119.3 (8.497) 55.71 (4.073) 37.15 (2.58, 2.51) S15 112.5 (8.001) 58.81 (4.12 60.58 (3.64,3.44) A16 124.1 (7.637) 52.95 (3.945) 15.54 (1.32) V117 115.7 (7.948) 64.3 (3.135) 28.74 (2.385) CG1 19.47 CG2 19.4 QG1 0.63 QG2 0.41 E118 118 (8.337) 57.52 (3.585) 26.97 (1.95, 2.07) CG 33.99 HG1 2.10 HG2 2.36 K119 119.1 (8.235) 56.88 (3.819) 29.52 (2.09,1.85) CD 23.17 CE 39.09 CG 26.19 HD1 1.054 HD2 1.07 HE1 2.35 HE2 2.40 HG1 0.90 HG2 1.22 A120 123.3 (8.034) 52.65 (4.503) 16.33 (0.75) L121 115 (7.64) 54.93 37.99 (1.45, 0.64) CD2 18.43 CG 22.59 HG -0.65 QD1 0.39 QD2-0.62 K122 118.9 (7.786) 56.27 (3.974) 29.37 (2.02, 1.90) CD 26.07 CE 37.48 CG 22.34 HG1 1.39 HG2 1.52 QD 1.63 QE 2.78 V123 115.1 (7.35) 63.56 (3.46) 28.43 (1.362) CG1 21.29 CG2 18.67 QG1 0.64QG2 0.79 W124 117.2 (6.582) 54.84 (4.848) 27.18 (3.09, 2.92) HD1 7.47 HE1 9.89 HZ2 7.04 NE1 126.2 E125 121.4 (8.43) (4.728) (1.99) QG 2.916 E126 113.4 (7.742) (4.095) (2.04, 2.27) QG 2.55 V127 106.2 (7.02) (4.803) (2.58) QG1 1.11 QG2 0.8 T128 108.9 (7.3) 57.47 (5.422) 69.52 (4.203) CG2 21.76 QG2 1.437 P129 (5.336) (2.06) L130 118.5 (7.259) 52.19 (4.395) 39.83 (1.59, 1.50) CD1 22.17 CD2 20.89 CG 23.83 HG 1.27 QD1 0.43 QD2 0.1565
T131 110.2 (8.008) 57.39 (4.432) 69.65 (4.027) CG2 18.73 QG2 0.9978 F132 117.3 (8.215) 53.47 (5.643) 40.73 (2.75, 2.73) QD 6.964 QE 6.747 S133 116.7 (8.67) 54.63 (4.744) 62.78 (3.80, 3.58) R134 125.1 (8.546) (4.601) (1.42 1.24) HE 6.11 QD 2.74 L135 125.2 (8.455) 50.2 (4.585) 42.7 (1.15, 1.50) CD1 22.33 CD2 20.13 CG 24.62 HG 0.7837 QD1-0.171 QD2-0.594 Y136 118.9 (9.205) 55.59 (4.331) 36.32 (2.93, 3.15) HD2 7.072 HE1 6.809 E137 116.6 (7.491) 51.81 (4.684) 30.1 (1.99, 1.82) CG 32.34 HG1 1.98 HG2 2.07 G138 108.3 (8.504) 41.94 (3.95, 3.66) E139 119 (8.168) 53.63 (4.195) 26.98 (2.02) CG 33.4 HG1 2.138 HG2 2.067 A140 131.5 (7.986) 46.96 (4.366) 19.01 (1.1) D141 121.8 (8.273) 56.66 (4.436) 38.64 I142 123.5 (8.436) 57.99 (4.375) 36.38 (1.787) CD1 10.49 CG1 24.03 CG2 14.07 QD1 0.80 QG2 0.89 M143 126.4 (7.234) 50.58 (4.218) 30.85 (1.97, 1.85) QE 2.574 I144 128.6 (9.171) 58.34 (5.3) 36.84 (1.762) CD1 13.55 CG1 26.2 CG2 14.33 HG12 1.70 HG13 1.07 QD1 0.58 QG2 0.77 S145 118.2 (8.516) 54.57 (4.963) 63.59 (3.17, 3.67) F146 120 (9.43) 53.71 (5.231) 39.14 (2.65, 2.74) A147 125.5 (9.266) 48.39 (4.768) 20.57 (0.98) V148 111.5 (8.166) (4.731) (2.124) QG1 0.79 QG2 0.83 R149 117.3 (9.616) 52.52 (4.705) 26.51 (2.25, 2.10) HG1 1.54 HG2 1.82 QD 3.179 E150 124.7 (7.552) 55.47 (4.066) 27.8 (2.01, 1.94) CG 34 HG1 2.263 HG2 2.533 H151 123.7 (9.291) 60.4 (4.205) 31.87 (2.95) HD2 6.763 G152 109 (8.071) 53.35 (3.88) D153 115.6 (6.822) (4.694) 41.46 (2.71, 2.91) F154 115.6 (7.814) (4.056) QD 6.43 Y155 116.8 (6.776) (4.456) QE 7.06 P156 (3.92) (2.75) F157 122.7 (8.505) (3.99) (2.998) QD 6.601 QE 7.202 D158 115.5 (7.869) 50.8 (4.027) 37.57 (2.67) G159 110.3 (8.857) 42.01 (3.33, 4.33) P160 (3.946) G161 119.7 (11.33) 40.6 (3.86) N162 118.1 (8.798) 52.88 (3.841) 36.51 (3.16, 2.56) HD21 6.902 HD22 7.462 ND2 114.1 V163 125.1 (9.962) 60.52 (3.51) 29.12 (2.206) CG1 18 CG2 16.72 QG1 1.126 QG2 0.8457 L164 125.5 (8.447) (4.575) (1.27) HG 1.75 QD1 0.312 QD2 0.1345 A165 114.5 (7.438) (3.14) 15.65 (1.68) H166 115.6 (9.072) 51.48 (4.457) 27.09 (2.92) HD2 8.201 A167 121.5 (8.013) (4.568) 16.44 (2.09) Y168 123.8 (7.979) 55.34 (4.391) 36.04 (2.77, 2.62) QD 6.68 QE 6.238 A169 119.1 (7.442) (4.7) (-0.268) P170 60.77 (4.138) 29.36 (1.85) CD 47.46 CG 24.68 QD 3.511 QG 1.713 G171 109.5 (6.209) 43.64 (4.67, 3.11) P172 60.39 (4.716) 29.26 (1.94) CD 48.37 CG 24.53 HD1 3.45 HD2 3.74 QG 1.77 G173 109.7 (8.756) 42.78 (3.76) I174 131.8 (8.904) 58.77 (4.031) 35.92 (1.731) CD1 10.44 CG1 24.46 CG2 14.63 HG12 1.32 HG13 1.04 QD1 0.71 QG2 0.78 N175 117.5 (14.09) 53.65 (4.392) 33.18 (2.27, 2.065) HD21 6.77 HD22 7.376 ND2 112.2 G176 120.4 (8.098) 43.28 (4.66) D177 123.8 (7.76) 51.86 (4.41) 38.25 (2.59, 2.45) A178 122.4 (8.21) (4.938) (1.15) H179 120.3 (9.191) (4.857) F180 123.5 (8.818) 54.58 (4.749) 36.75 (3.06) HD2 7.074 HE2 6.81QD 6.43 QE 6.572 D181 121.8 (8.115) 51.03 (4.233) 36.54 (2.71, 2.75) D182 128.3 (9.797) 50.55 (5.399) 38.27 (2.37, 2.62) D183 123.8 (9.226) (4.73) (2.73 2.57)
E184 115.9 (7.203) 50.44 (4.831) 23.49 (2.35) Q185 126.3 (8.38) 50.55 (4.378) (1.64 1.85) HE21 7.44 HE22 6.765 NE2 111.7 W186 131.2 (9.587) 54.22 (4.924) 27.37 (3.14) HD1 7.421 HE1 9.35 NE1 126 T187 110.9 (8.738) 55.92 (4.754) 70.24 (4.387) CG2 19.23 QG2 0.9601 K188 120 (9.053) 54.1 (4.389) 30.51 (1.84, 1.72) CD 26.65 CE 39.34 CG 21.75 HD1 1.57 HD2 1.585 HE1 2.82 HE2 2.88 HG1 1.307 HG2 1.396 D189 120.4 (7.39) 49.83 (4.742) 39.14 (2.68, 3.30) T190 128 (7.77) 61.36 (4.592) 66.88 (3.936) CG2 18.76 QG2 1.112 T191 114.6 (8.176) 61.05 (4.041) 67.11 (4.058) CG2 18.98 HG21 1.146 G192 112.1 (7.266) 42.37 (3.4 3.85) T193 123.7 (8.534) 59.8 (3.54) 65.06 (2.645) CG2 21.63 QG2 0.0539 N194 126.1 (8.221) (4.704) (2.78) HD21 7.705 HD22 6.968 ND2 112.4 L195 128.1 (7.944) (4.404) 39.2 (2.62, 2.63) HG 1.109 QD1 0.8375 QD2-0.343 F196 119.1 (8.332) 58.42 (3.93) 34.62 (3.12, 3.28) QD 7.11 QE 6.54 L197 119.8 (8.616) 55.34 (2.985) 40.12 (1.60, 2.56) HG 1.476 QD1 0.8412 QD2 0.509 V198 114.7 (7.174) 63.14 (3.884) 29.18 (2.206) CG1 24.69 CG2 21.31 QG1 1.123 QG2 0.7268 A199 121.8 (9.472) 53.19 (3.97) 13.86 (1.07) A200 120.7 (8.816) 53.98 (3.872) 14.26 (0.95) H201 119.8 (7.663) (4.167) (3.87) E202 117.4 (9.195) 56.3 (3.805) 26.56 (1.69, 0.96) CG 31.07 QG 2.33 I203 117.6 (9.016) 59.12 (3.801) 32.84 (1.868) CD1 6.942 CG1 25.03 CG2 15.52 QD1 0.3846 QG1 1.276 QG2 0.4894 G204 108.3 (7.474) 43.05 (2.29 3.67) H205 119.7 (7.178) (4.389) (2.77, 4.21) S206 118.3 (8.209) 58.75 (4.647) 60.4 (3.91, 3.80) L207 109 (8.19) 55.29 (3.791) (1.75) CD1 24.55 CD2 19.54 CG 25.91 QD2-0.26 HG 1.849 QD1 0.781 G208 109.3 (8.394) 55.84 (5.736) L209 122.5 (8.804) (5.26) (1.56) HG 1.417 QD1 0.972 QD2 0.5462 F210 125.1 (8.505) (4.829) H211 107.1 (5.297) (3.44) S212 110.9 (6.821) (4.677) 64.7 (4.04, 3.07) A213 127.3 (8.793) 48.99 (4.255) 16.79 (1.64) N214 121.4 (8.821) (4.529) (3.15) HD21 6.608 T215 117.2 (8.029) 61.06 (2.368) 65.65 (3.725) CG2 18.73 QG2 0.8115 E216 121.7 (8.581) (4.451) (2.1) A217 123.2 (7.833) 49.5 (4.179) 16.49 (1.4) L218 131.8 (11.49) 54.19 (4.411) 39.89 (1.61, 1.35) CD2 21.59 CG 24.15 HG 1.588 QD1 0.9787 QD2 1.128 M219 110.9 (8.016) (4.577) (2.6) QE 0.599 Y220 128.2 (8.066) 53.31 (4.853) 36.15 (2.88) QD 7.873 QE 7.671 P221 58.81 (3.553) 36.12 (0.78, 1.70) CG 24.48 QG 0.7482 L222 119.5 (7.501) 52.25 (4.443) 40.16 (1.33, 1.67) CD1 20.54 CD2 19.15 CG 23.61 HG 0.2039 QD1 0.9782 QD2 0.809 Y223 128 (9.054) 56.74 (4.592) 34.3 (2.21, 2.71) H224 127.5 (8.157) 55 (4.28) 35.32 (2.87) HD2 6.96 S225 119.1 (7.832) 55.85 (3.911) 61.56 (3.99, 3.74) L226 122.1 (7.816) 51.83 (4.329) 39.23 (1.57, 1.42) QQD 0.7595 T227 120.8 (7.908) 58.8 (4.277) 67.1 (4.101) QG2 1.216 D228 123.3 (7.968) 50.22 (4.63) 38.27 (2.57, 2.48) L229 126.5 (8.896) 54.12 (4.037) 39.27 (1.49, 1.66) CD1 22.9 CD2 21.1 CG 24.21 HG 1.586 QD1 0.9031 QD2 0.8175 T230 111.4 (8.361) 61.71 (4.065) 66.35 (4.167) CG2 19.14 QG2 1.176 R231 119.4 (7.34) 53.13 (4.672) 28.08 (1.92, 1.504) CD 40.41 CG 24.17 HG1 1.389 HG2 1.54 QD 3.041 F232 120.9 (7.271) 57.38 (4.122) 37.26 (2.79, 3.059) HE2 6.505 R233 126.2 (7.067) 51.71 (3.843) 30.19 (1.37, 1.58) CD 40.63 CG 22.98 HD1 2.995 HD2 3.004 QG 1.279 L234 121.9 (8.331) 52.41 (4.413) 40.5 CD1 22.64 CD2 18.29 CG 24.62 QD1 0.179 QD2 0.192 S235 118 (8.664) 56.28 (3.742) 63.14 (4.71, 3.91)
Q236 122.4 (9.095) 55.15 (4.412) 25.6 (2.07, 1.95) CG 31.23 QG 2.34 D237 117.9 (8.062) 55.28 (4.279) 39.87 (2.55) D238 117.7 (8.035) 56.13 (3.898) 36.5 (2.97) I239 119.1 (7.968) 62.71 (3.592) 36.47 (1.684) CD1 10.45 CG1 27.8 CG2 14.23 QD1 0.6474 QG1 0.8007 QG2 0.7681 N240 118.4 (9.111) 53.27 (4.361) 34.85 (2.85, 2.77) HD21 7.63 HD22 6.965 ND2 111.6 G241 108.2 (8.193) 44.43 (3.782) I242 123.3 (8.532) 60.5 (4.272) 28.98 (2.09) CD1 14.82 CG1 24.81 CG2 19.86 QD1 0.76 QG1 1.031 QG2 1.228 Q243 120.9 (8.279) (4.655) HE21 6.725 HE22 7.666 QG 2.956 S244 117.3 (8.029) (4.528) L245 118.1 (6.737) 54.62 (4.093) 40.45 (1.62, 1.47) CD1 22.48 CD2 21.41 CG 24.4 HG 1.469 QD1 0.7247 QD2 0.7793 Y246 113 (7.58) 53.02 (2.845) 40.57 (3.15, 3.45) QD 7.997 QE 7.068 G247 110 (8.313) 41.86 (4.2 3.97) P248 QG 1.819 NNGH QB 3.256 HG 2.227 HNA 11.43 HP2 7.349 QM 3.888 HP6 7.106 QP3 6.649 QQD 0.9211
Supplementary Table S3. Statistical analysis of the energy minimized structures of MMP3- NNGH. MMP3-NNGH a <MMP3-NNGH> Total number of meaningful NOE upper distance 2060 constraints Intra-protein NOEs b : Intra-residue 194 Inter-residue Sequential ( i-j = 1) 584 Medium-range ( i-j < 4) 652 Long-range ( i-j > 5) 630 Total meaningful dihedral angle restraints 164 Phi 100 Psi 64 Inhibitor c -protein NOEs 17 Intra-inhibitor NOEs d 1 Hydrogen bond derived restraints 52 Intra-protein 51 Protein-inhibitor e 1 RMS violations per meaningful distance constraint (Å): Intraresidue 0.009± 0.005 0.0052 Sequential 0.017± 0.002 0.0148 Medium range 0.019± 0.002 0.0193 Long range 0.023± 0.003 0.0170 RMS violations per meaningful dihedral angle constraints ( ): Phi 4.166 ± 1.218 4.8754 Psi 0.441 ± 0.735 0.7890 Average number of violations per conformer: Phi 5.000 ± 1.590 6.0 Psi 0.367 ± 0.547 1.0 NOE violations between 0.1 Å and 0.3 Å 25.1 ± 4.64 19.0 NOE violations larger than 0.3 Å 0.0 0.0 Average RMSD to the mean (Å) Secondary structure elements (backbone atoms) 0.49 ± 0.06 Secondary structure elements (all heavy atoms) 1.12 ± 0.10 Loop regions (backbone atoms) 0.81 ± 0.10 Loop regions (all heavy atoms) 1.45 ± 0.10 NNGH atoms 1.16 ± 0.29 Target Function (Å 2 ) 1.21 ± 0.14 Structural analysis f % of residues in most favourable regions 76.2 73 % of residues in allowed regions 21.6 26.1 % of residues in generously allowed regions 2.0 0.9 % of residues in disallowed regions 0.2 0.0 Completeness of NOE s within 4 Å g 60% 58% Completeness of NOE s within 5 Å 41% 42% H-bond energy (kcal mol -1 ) 0.7± 0.11 WHAT IF structure Z-scores h 1st generation packing quality -2.6 ± 0.3-2.0 Ramachandran plot appearance -3.0 ± 0.5-3.1 χ1/χ2 rotamer normality -3.2 ± 0.4-3.1
Backbone conformation -2.4 ± 0.9-1.0 a Structure calculations were performed with the program CYANA 2.1. A total of 400 random conformers were subjected to 10000 steps of a simulated annealing process. The 30 conformers with the lowest target function were refined through restraint energy minimization (REM) with the Amber 8.0 package. Values of 32 kcal mol -1 Ã -2 and 50 kcal mol -1 rad -2 were used as force constants for the NOE and torsion angle restraints, respectively. The data are calculated over the 30 conformers representing the NMR structure and on the energy minimized mean structure. The mean value and the standard deviation are also given b Number of meaningful constraints for each class. c These NOEs are given in Table 2 of the manuscript. The CYANA library corresponding to NNGH was obtained using the program Molmol. d Stereospecific assigned NOE between the protons HNA and HP2 of the NNGH inhibitor. e Hydrogen bond between Leu164 NH and the sulfonyl oxygen O2 of NNGH. f As it results from the Ramachandran plot analysis performed with PROCHECK. g Calculated considering amide and alpha protons g The Z-scores is reported considering the secondary structure elements h A Z-score is defined as the deviation from the average value for this indicator observed in a database of high-resolution crystal structures, expressed in units of the standard deviation of this database-derived average. Typically, Z-scores below a value of -3 are considered poor, those below - 4 are considered bad.