X-ray structure of bovine pancreatic phospholipase A₂at atomic resolution

Abstract

Using synchrotron radiation and a CCD camera, X-ray data have been collected from wild-type bovine pancreatic phospholipase A(2) at 100 K to 0.97 A resolution allowing full anisotropic refinement. The final model has a conventional R factor of 9.44% for all reflections, with a mean standard uncertainty for the positional parameters of 0.031 A as calculated from inversion of the full positional least-squares matrix. At 0.97 A resolution, bovine pancreatic phospholipase A(2) reveals for the first time that its rigid scaffolding does not preclude flexibility, which probably plays an important role in the catalytic process. Functionally important regions (the interfacial binding site and calcium-binding loop) are located at the molecular surface, where conformational variability is more pronounced. A cluster of 2-methyl-2,4-pentanediol molecules is present at the entrance of the hydrophobic channel that leads to the catalytic site and mimics the fatty-acid chains of a substrate analogue. Bovine pancreatic phospholipase A(2) at atomic resolution is compared with previous crystallographic structures and with models derived from nuclear magnetic resonance studies. Given the high structural similarity among extracellular phospholipases A(2) observed so far at lower resolution, the results arising from this structural analysis are expected to be of general validity for this class of enzymes.

A Low-barrier Hydrogen Bond Between Histidine of Secreted Phospholipase A2 and a Transition State Analog Inhibitor

“…(i) X-ray structures of several sPLA 2 -inhibitor complexes do not reveal imidazole ring flipping. 16 36 which is too far apart for a H-bond. Thus, the N d1 atom of H48 likely forms a hydrogen bond only with the transition-state analog.…”

“…Both NMR studies 20 and the recent 0.97 Å ultrahigh resolution crystal structure 36 of free bovine pancreatic sPLA 2 show that H48 and D99 form a H-bond involving the N 12 of the imidazole ring. Taken together, we conclude that the LBHB is formed between H48 and the inhibitor.…”

Phenylalanine-24 in the N-terminal region of ammodytoxins is important for both enzymic activity and presynaptic toxicity

“…The presumed IBS residues [51] of AtxA, surrounding the opening to the active-site pocket with His 48 , are shaded dark grey and pointed towards the reader. The residues identified so far by site-directed mutagenesis as being important for neurotoxicity of AtxA are indicated, and are shown in black.…”

Structural Insights on the Bacteriolytic and Self-protection Mechanism of Muramidase Effector Tse3 in Pseudomonas aeruginosa

“…Considering that ARM-and HEAT-like motifs are commonly found to mediate protein-protein interactions by their modular recognition of extended peptide regions (34), it is reasonable to speculate that the N-terminal domain of Tse3 is crucial for the proper folding and conformational stabilities of the Tse3. Although some enzymes that catalyze a hydrolytic reaction, such as phospholipase A2 and staphylococcal nuclease, bind with Ca 2ϩ in their active site as a catalytic factor (40,41), Ca 2ϩ binding and involvement of enzymatic activity are not observed in the reported T6SS toxins (15, 16, 18 -22) or the goose-type lysozyme homologues (35)(36)(37). Based on the high-resolution structure and biochemical studies presented here, we identified three Ca 2ϩ ions that bind with Tse3; two Ca 2ϩ ions bind directly in the catalytic site and adjacent to the catalytic residue Glu-250 (Figs.…”