The Crystal Structure of a Novel, Inactive, Lysine 49 PLA2 from Agkistrodon acutus Venom

Abstract

The crystal structure of acutohaemolysin, a lysine 49 phospholipase A2 protein with 1010 non-hydrogen protein atoms and 232 water molecules, has been determined ab initio using the program SnB at an ultrahigh resolution of 0.8 Å. The lack of catalytic activity appears to be related to the presence of Phe 102 , which prevents the access of substrate to the active site. The substitution of tryptophan for leucine at residue 10 interferes with dimer formation and may be responsible for the additional loss of hemolytic activity. The ultrahigh resolution of the experimental diffraction data permits alternative conformations to be modeled for disordered residues, many hydrogen atoms to be located, the protonation of the N⑀2 atom in the catalytic residue His 48 to be observed experimentally, and the density of the bonding electrons to be analyzed in detail.

Snake Venom Phospholipases A2: A Novel Tool Against Bacterial Diseases

“…The existence of a consensus sequence Tyr 25 -Gly-Cys-Asn-Cys-Gly-X-X-X-Arg-Gly 35 implies that this region is functionally homologous among all family members. Compared with the neighboring region containing Cys 27 -Asn-CysGly 30 , the electron density of Val 31 -Gly-Gly 33 is quite poor, indicating that these residues have relatively high thermal motion [56]. In connection with the Asp 49 PLA 2 activity, Scott et al [57] proposed that the backbone amide of Gly 30 stabilizes the oxyanion of the substrate tetrahedral intermediate.…”

“…They cause plasma membrane perturbation that promotes a heavily increase of the cytosolic Ca 2+ concentration leading to cell degeneration of muscle cells and neuromuscular junction [140]. PLA 2 s employ a conserved catalytic triad consisting of His 48 , Tyr 52 , and Asp 99 to hydrolyze substrate into free fatty acids and lysophospholipids [56]. It is believed that His 48 and Asp 99 are involved in the nucleophilic attack of a water molecule at the sn-2 position of the phospholipid substrate.…”

“…Identical interactions are observed in myotoxins as a consequence of a coupled conformational change involving Gly 30 and neighboring residues of the Ca +2 -binding loop and residues 115-125 [41]. The buried conformation adopted by Lys 122 in the ligand-bound form leads to increased hydrogen bonding between the C-terminal region (residues [115][116][117][118][119][120][121][122][123][124][125] 54], and the potential acylation of these toxins via autocatalysis [55], or via an interrupted catalytic cycle that fails to release a free fatty acid [56] which may enhance membrane perturbation. This would be the key toxic event, allowing an uncontrolled influx of ions (i.e.…”

Frontier example in experimental charge density research: Experimental electrostatics of proteins

“…This study has demonstrated the feasibility of the method and was discussed in Part I. The structures of trypsin [36] and of a snake venom phospholipase [37], both at 0.8 Å resolution, were also analyzed recently using the program MoPro; they display clear bonding electron density.…”

Macromolecular refinement at subatomic resolution with interatomic scatterers

“…Previously, refinement at subatomic resolution using multipolar models has been reported for crambin (Fernandez-Serra et al, 2000;0.54 Å), trypsin (Schmidt et al, 2003; 0.80 Å), phospholipase (Liu et al, 2003; 0.80 Å; for the resolution higher than 0.86 Å the data completeness is below 50%) and scorpion toxin (Housset et al, 2000;0.96 Å). The corresponding models were extracted from the PDB (Bernstein et al, 1977;Berman et al, 2000).…”