Lysozyme contamination facilitates crystallization of a heterotrimeric cortactin-Arg-lysozyme complex

Abstract

Crystallization of contaminating proteins is a frequently encountered problem for macromolecular crystallographers. In this study, an attempt was made to obtain a binary cocrystal structure of the SH3 domain of cortactin and a 17-residue peptide from the Arg nonreceptor tyrosine kinase encompassing a PxxPxxPxxP (PxxP1) motif. However, cocrystals could only be obtained in the presence of trace amounts of a contaminating protein. A structure solution obtained by molecular replacement followed by ARP/wARP automatic model building allowed a 'sequence-by-crystallography' approach to discover that the contaminating protein was lysozyme. This 1.65 Å resolution crystal structure determination of a 1:1:1 heterotrimeric complex of Arg, cortactin and lysozyme thus provides an unusual `caveat emptor' warning of the dangers that underpurified proteins harbor for macromolecular crystallographers.

Figure 1

Identification of a contaminating protein. (a) SDS–PAGE (12%) analysis of cortactin SH3 purified by SEC. 10 µl of cortactin SH3 domain at 2 mg ml⁻¹ concentration (a total of 20 µg) was loaded onto the gel. (b) Crystals of the heterotrimeric cortactin SH3–Arg peptide–lysozyme complex that formed in 1.0 M sodium citrate pH 6.0. These crystals only formed on pre-incubation of Arg PxxP1 peptide with cortactin SH3 domain. (c) Stereoview showing electron density for the initial ARP/wARP solution, allowing us to ‘sequence by crystallogaphy’. Residues KSFGRCE can clearly be identified. (d) Overloaded SDS–PAGE (15%) analysis of cortactin SH3. Lane L, lysozyme control; lane CL, 10 µl cortactin SH3 domain at 20 mg ml⁻¹ concentration (a total of 200 µg); this is the same protein crystallization sample shown in (a); lane C, 10 µl cortactin SH3 domain at 20 mg ml⁻¹ concentration (a total of 200 µg) obtained by following an optimized purification protocol; lane M, molecular-weight markers (labelled in kDa).

Figure 2

Cortactin SH3–Arg PxxP–lysozyme complex structure. (a) Overall view of the cortactin SH–Arg PxxP1–lysozyme heterotrimeric complex structure. Cortactin is shown in blue, Arg in yellow and lysozyme in green. (b) Electrostatic potential of the surface of cortactin. Arg binds to the hydrophobic PxxP1 binding surface of the SH3 domain. Lysozyme packs against both a hydrophobic patch on cortactin and Arg PxxP1. (c) Close-up view of the interaction between Arg PxxP1 and the cortactin SH3 domain, highlighting residues discussed in the text. The images were generated with CCP4mg (Potterton et al., 2003 ▶).