Crystallization and X-ray structure of full-length recombinant human butyrylcholinesterase

Abstract

Human butyrylcholinesterase (BChE) has been shown to function as an endogenous scavenger of diverse poisons. BChE is a 340 kDa tetrameric glycoprotein that is present in human serum at a concentration of 5 mg l(-1). The well documented therapeutic effects of BChE on cocaine toxicity and organophosphorus agent poisoning has increased the need for effective methods of producing recombinant therapeutic BChE. In order to be therapeutically useful, BChE must have a long circulatory residence time or associate as tetramers. Full-length recombinant BChE produced in Chinese hamster ovary (CHO) cells or human embryonic kidney cells has been shown to associate as monomers, with a shorter circulatory residence time than the naturally occurring tetrameric serum protein. Based on the preceding observation as well as the need to develop novel methodologies to facilitate the mass production of therapeutic recombinant BChE, studies have been initiated to determine the structural basis of tetramer formation. Towards these ends, full-length monomeric recombinant BChE has been crystallized for the first time. A 2.8 A X-ray structure was solved in space group P42(1)2, with unit-cell parameters a = b = 156, c = 146 A.

Figure 1

The two crystal morphologies of full-length monomeric BChE. (a) Form I crystals are smaller (less than 0.2 mm on the largest face) low-resolution crystals obtained in solutions buffered at acidic pH, while (b) form II crystals are larger crystals (greater than 0.8 mm on the largest face) that diffract to higher resolution.

Figure 2

Cartoon of the BChE dimer showing that each monomer has the typical fold. Each monomer is colored from blue at the N-terminus to red at the C-terminus.

Figure 3

A 2F _o − F _c electron-density map at 1σ indicates the presence of residues 530–534. The electron-density map is in aquamarine.

Figure 4

The crystallographic tetramers of full-length recombinant BChE (a) are not mediated by the tetramerization domain as is the case in the cocrystal structure of the tetramerization domain of acetylcholinesterase with PRAD (b), PDB code 1vzj (Dvir et al., 2004 ▶), which instead has two monomers with C-termini oriented away from the other two monomers.

Figure 5

Sequence of the 40-residue tetramerization domains of human AChE and BChE.