Supporting Information

Wiley-VCH 2007
69451 Weinheim, Germany

The Structural Basis of Glycosidase Inhibition by Five-Membered Iminocyclitols

Matthew E. C. Caines, Susan M. Hancock, Chris A. Tarling, Tanja M. Wrodnigg, Robert V. Stick, Arnold E. Sttz, Andrea Vasella, Stephen G. Withers* and Natalie C. J. Strynadka*

Experimental Section Enzyme kinetics and inhibition: All kinetic studies were performed in sodium citrate/phosphate buffer (50 mM, pH 5.0) at 25C. Reaction progress was followed using a continuous spectrophotometric assay to monitor the release of the 2,4-dinitrophenolate ion from 2,4-dinitrophenyl !-D-lactoside at 400 nm. The final enzyme concentration depended on the inhibitor under analysis and varied from 10-500 nM. In all cases a total reaction volume of 1000 mL was used. Reactions were initiated by the addition of enzyme and initial rates were calculated for substrate concentrations ranging from 0.2 ! KM to 5 ! KM. The data were fitted to the MichaelisMenten equation in Grafit 4.0.[1] Inhibitor identification: Inhibitors were identified from a medium-throughput screen of a library of approximately 500 potential glycosidase inhibitors assembled in the Withers laboratory. This screen was performed on a Beckman Coulter BiomekFX liquid handler with an integrated plate reader operating in UV/Vis absorbance mode. The screen, run in duplicate in 384 well plates, was based on a micro-format of the conditions described above and compounds showing statistically reduced activity were investigated further using full enzyme kinetic analysis on conventional instrumentation. Approximate Ki values were determined using a fixed concentration of substrate, 2,4-dinitrophenyl !-D-lactoside (0.5 mM), and inhibitor concentrations ranging from 0.2 to 5 ! Ki value ultimately determined. A horizontal line drawn through 1/ Vmax in a Dixon plot of these data (1/V vs [I]) intersects the experimental line at an inhibitor concentration equal to -Ki. Crystallization, data collection and refinement: Sitting drops of 1 L of protein (10 mg.mL-1), containing Triton X-100 (0.1% v/v), and 1 L of well solution were suspended at 21C over a well solution of PEG 3350 (20% w/v), NaCl (0.175 M) and Tris-HCl (0.1 M, pH 8.5). Crystals belonging to the space group P21, with two molecules per asymmetric unit, grew under these conditions over a period of one week. All soaking experiments were carried out in solutions of PEG 3350 (25% w/v), NaCl (0.2 M), Triton X-100 (0.05% v/v), Tris-HCl (0.1 M, pH 8.5) and glycerol (10% v/v), containing either cellobiose-like isofagomine 1b, cellobiose-like imidazole 2b, or iminocyclitol 3b at 5 mM for 1 h, 10 mM for 10 min and 15 mM for 30 min, respectively, before flashcooling by plunging into liquid nitrogen. X-ray data were collected at 100 K using a nitrogen stream. Data for cellobiose-like isofagomine 1b and cellobiose-like imidazole 2b soaked crystals were collected at Beamline 8.2.2 of the Advanced Light Source, Berkeley, USA, using an ADSC Q315 CCD detector. Data for iminocyclitol 3b soaked crystals were collected on an in-house rotating anode X-ray generator, using a Mar345 detector. The 1

data were processed using MOSFLM[2] and the CCP4 suite of programs.[3] The inhibitor complexes with EGC were solved using the native EGC structure as the initial source of phase information. All structures were refined by iterative refinement using REFMAC5[4] and manual rebuilding using Coot.[5] 5% of the reflections were excluded for calculation of Rfree. Non-crystallographic symmetry restraints were used throughout refinement. Energy minimized ligand models and their refinement restraints were created using the PRODRG server.[6] Ligands were subsequently fitted to active site difference electron density and further rounds of restrained refinement were carried out. All figures were prepared using PyMOL.[7]

Figures

Figure S1. Stereo diagrams illustrating the enzyme-inhibitor interactions displayed by: a) 1b; b) 2b and c) 3b. Only interactions in the 1 subsite are depicted, using molecule B of the asymmetric unit. The bound inhibitors are shown as ball-and -stick representations in yellow. Water molecules are represented by light-blue spheres.

Tables
EGC1b Data collection Space group Cell dimensions a, b, c () ", !, # ( ) Wavelength () [a] Resolution range () [a] Rsym (%) [a] <I/$(I)> [a] No. unique observations [a] No. total observations [a] Completeness (%) [a] Multiplicity P21 53.5, 92.9, 94.5 90.0, 98.3, 90.0 0.980 53.00-1.50 (1.58-1.50) 8.9 (45.6) 17.0 (3.5) 146070 (21265) 1066732 (155646) 100.0 (100.0) 7.3 (7.3) EGC2b P21 53.8, 93.2, 94.4 90.0, 98.4, 90.0 0.980 65.94-1.80 (1.90-1.80) 6.9 (39.0) 23.5 (4.3) 85295 (12409) 621954 (89817) 100.0 (100.0) 7.3 (7.2) EGC3b P21 53.8, 93.3, 94.5 90.0, 98.3, 90.0 1.542 18.33-1.85 (1.95-1.85) 4.8 (34.6) 20.3 (3.9) 72622 (8650) 290395 (33486) 91.9 (75.4) 4.0 (3.9)

Refinement Rwork / Rfree (%) 17.7 / 19.8 2 B-factors ( ) Protein 15.1 Ligand 13.3 Water 26.7 R.M.S. deviations Bond lengths () 0.006 Bond angles () 1.068 [a] Numbers in parentheses represent the value in the highest resolution shell.

17.4 / 20.5 17.3 14.9 26.9 0.007 1.111

18.0 / 21.2 20.3 19.2 27.6 0.009 1.196

Table S1. Data collection and refinement statistics.

References [1] R. J. Leatherbarrow, 4.0.21 ed., Erithacus Software Ltd., 2004. [2] A. G. W. Leslie, Acta Crystallogr., Sect. D: Biol. Crystallogr. 1999, 55, 1696-1702. [3] Collaborative Computational Project Number 4, Acta Crystallogr., Sect. D: Biol. Crystallogr. 1994, 50, 760-763. [4] G. N. Murshudov, A. A. Vagin, A. Lebedev, K. S. Wilson, E. J. Dodson, Acta Crystallogr., Sect. D: Biol. Crystallogr. 1999, 55, 247-255. [5] P. Emsley, K. Cowtan, Acta Crystallogr., Sect. D: Biol. Crystallogr. 2004, 60 , 2126-2132. [6] A. W. Schuttelkopf, D. M. van Aalten, Acta Crystallogr., Sect. D: Biol. Crystallogr. 2004, 60 , 1355-1363. [7] W. L. DeLano, 0.99 ed., DeLano Scientific, San Carlos, CA, USA, 2002.