Metal binding to porcine pancreatic elastase: calcium or not calcium

Abstract

Porcine pancreatic elastase has been crystallized at slightly acidic pH under two similar but slightly different conditions. Diffraction data were collected at a wavelength of 1.5 A Ê to a maximum resolution of 1.7 A Ê . Both difference electrondensity maps and anomalous difference electron-density maps suggest that in crystals grown from a sodium sulfate solution PPE binds Na + in its metal-binding site. In contrast, PPE binds Ca 2+ in crystals grown from a solution containing sodium citrate and calcium chloride. This observation is in contradiction to most PPE structures reported in the PDB. In addition to the metal-binding site, up to three other binding sites, which appear to be anion-binding sites, could be identi®ed based on the observed anomalous intensity differences.

Crystal structure of human ISG20, an interferon‐induced antiviral ribonuclease

“…In order to identify Mn 2+ ions unambiguously, data collection at wavelengths on both sides of the K‐absorption edge of Mn is usually necessary. However, the heights of these two peaks were much greater than those of the peaks of the sulfur atoms of the Met and Cys residues, and therefore two Mn 2+ ions can reasonably be assigned to these positions [27]. In addition to these two Mn 2+ ions, another Mn 2+ ion associated with a slightly smaller, diffuse peak was found between the side chains of Asp90 and His93 (not shown).…”

Crystallization and Preliminary Diffraction Studies of Porcine Pancreatic Elastase in Complex with a Novel Inhibitor

“…The calculated V M [23] is 2.1 Å 3 /Da, which corresponds to a solvent content of ~ 41% and one molecule per asymmetric unit. Crystals of native PPE [18,19] and PPE/inhibitor complexes [24][25][26] grown under the same experimental conditions show similar cell parameters. The initial calculated maps showed electron density near the catalytic triad, which corresponds to the inhibitor.…”

“…JM54 was incubated with PPE for 30 minutes at room temperature (inhibitor in a 10 molar excess, final concentration of 10% DMSO). The crystallization trials were set up at 293 K using the sitting drop vapour-diffusion method, and were based on previously known crystallization conditions [18,19]. PPE co-crystallized with the inhibitor JM54 in 200 mM sodium sulfate and 100 mM sodium acetate at pH 5.1 yielded good quality crystals.…”

“…The X-ray data were integrated and scaled using MOSFLM [20] and SCALA programs [21]. Since the crystals obtained for the enzyme:inhibitor complex were isomorphous to the native ones and belonged to the same space group, the structure was solved by Fourier synthesis [18,19]. A rigid-body refinement step was performed with Refmac [22] yielding an R-work and R-free of 27.7 and 30.5%, respectively.…”

The endoproteinase furin contains two essential Ca2+ions stabilizing its N-terminus and the unique S1 specificity pocket

“…However, this latter technique might be of limited use if the atoms in question have very similar atomic numbers, if the real occupancy is unclear and/or if the diffraction data are not well resolved. The widespread use of tunable synchrotron radiation has given rise to several studies where optimized anomalous scattering data for the element in question have been used to calculate anomalous difference Fourier maps, showing the spatial location of the anomalously scattering atoms and facilitating several element distinctions (see, for example, Einspahr et al, 1985;Tereshko et al, 2001;Weiss et al, 2001Weiss et al, , 2002DeLa Barre & Brunger, 2003;Steinbacher et al, 2004;Ferreira et al, 2004). The first application of such a method permitted distinction between Mn 2+ and Ca 2+ ions in pea lectin crystals (Einspahr et al, 1985).…”