The Atomic Resolution Crystal Structure of Atratoxin Determined by Single Wavelength Anomalous Diffraction Phasing

Xiaohua Lou,Qun Liu,Xiongying Tu,Jing Wang,Maikun Teng,Liwen Niu,David J Schuller,Qingqiu Huang,Quan Hao

doi:10.1074/jbc.m403863200

Abstract

By using single wavelength anomalous diffraction phasing based on the anomalous signal from copper atoms, the crystal structure of atratoxin was determined at the resolution of 1.5 A and was refined to an ultrahigh resolution of 0.87 A. The ultrahigh resolution electron density maps allowed the modeling of 38 amino acid residues in alternate conformations and the location of 322 of 870 possible hydrogen atoms. To get accurate information at the atomic level, atratoxin-b (an analog of atratoxin with reduced toxicity) was also refined to an atomic resolution of 0.92 A. By the sequence and structural comparison of these two atratoxins, Arg(33) and Arg(36) were identified to be critical to their varied toxicity. The effect of copper ions on the distribution of hydrogen atoms in atratoxin was discussed, and the interactions between copper ions and protein residues were analyzed based on a statistical method, revealing a novel pentahedral copper-binding motif.

Highlights

By using single wavelength anomalous diffraction phasing based on the anomalous signal from copper atoms, the crystal structure of atratoxin was determined at the resolution of 1.5 Å and was refined to an ultrahigh resolution of 0.87 Å
␣-Neurotoxins are small three-finger proteins, which can bind to acetylcholine receptors (AChRs)1 on the postsynaptic membrane and inhibit signal transmission across the synapse [1]
Because of the potential pharmacological significance for this protein family [4], the binding activity of ␣-neurotoxins with their acetylcholine receptors has been intensively investigated based on the crystal structure of an acetylcholine-binding protein (AChBP) [5]

Summary

EXPERIMENTAL PROCEDURES

Determination of Copper Content in Crystals—In order to determine the ratio between the copper ions and the atratoxin molecule, four crystals from the crystallization drops were dissolved into 120 ␮l of 0.1 M HCl solutions. Crystals of ϳ0.4 ϫ 0.3 ϫ 0.3 mm were soaked in a NaCl solution (pH 4.6) containing 10% ethanol, 10 mM CuCl2, and 25% (v/v) glycerol for about 20 s and subjected to cryogenic flash freezing at a temperature of 100 K Both SAD data and atomic resolution data were collected under the same cryoprotection conditions. The ABS program, based on the Ps function method [27, 28], has been written to determine the absolute configuration at a much earlier stage, i.e. immediately after the heavy atoms sites are found by a direct methods or a Patterson procedure For these three copper coordinates, hand was correctly assigned by using anomalous data at about 3.0 Å resolution cut off.

Atomic Resolution Crystal Structure of Atratoxin

RESULTS AND DISCUSSION

Data collection

Side chain hydrogen in molecule A