A single point mutation changes the crystallization behavior of Mycoplasma arthritidis-derived mitogen

Abstract

Mycoplasma arthritidis-derived mitogen (MAM) functions as a conventional superantigen (SAg). Although recombinant MAM has been crystallized by the hanging-drop vapour-diffusion method, the crystals diffracted poorly to only 5.0 A resolution, with large unit-cell parameters a = 163.8, b = 93.0, c = 210.9 A, beta = 93.7 degrees in the monoclinic space group P2(1). Unit-cell content analysis revealed that as many as 24 molecules could be present in the asymmetric unit. Systematic alanine mutagenesis was applied in order to search for mutants that give crystals of better quality. Two mutants, L50A and K201A, were crystallized under the same conditions as wild-type MAM (MAMwt). Crystals of the L50A mutant are isomorphous with those of MAMwt, while a new crystal form was obtained for the K201 mutant, belonging to the cubic space group P4(1)32 with unit-cell parameters a = b = c = 181.9 A. Diffraction data were collected to 3.6 and 2.8 A resolution from crystals of the MAM L50A and K201A mutants, respectively. Molecular-replacement calculations suggest the presence of two molecules in the asymmetric unit for the MAM K201A mutant crystal, resulting in a VM of 5.0 A Da(-1) and a solvent content of 75%. An interpretable electron-density map for the MAM K201A mutant crystal was produced using the molecular-replacement method.

Figure 1

Crystals of the wild-type MAM and two MAM alanine mutants (MAM_K201A and MAM_L50A). (a) Plate-shaped MAM_wt; (b) MAM_L50A; (c) MAM_K201A; (d) MAM_K201A; (e) MAM_wt after seeding with the diamond-shaped MAM_K201A crystals.

Figure 2

Diffraction images from crystals of (a) MAM_L50A and (b) MAM_K201A.