Crystallization and preliminary crystallographic analysis of Mycoplasma arthritidis-derived mitogen complexed with peptide/MHC class II antigen

Abstract

Mycoplasma arthritidis-derived mitogen (MAM), a bacterial superantigen, has been crystallized in complex with its human receptor, major histocompatibility complex (MHC) class II antigen, by the hanging-drop vapor-diffusion method. Crystals were obtained under three conditions, with ammonium sulfate, phosphate salt and PEG 8000 as the precipitant. The crystals grown under these conditions all belong to space group I222, with the same unit-cell parameters: a = 137.4, b = 178.2, c = 179.6 A. Diffraction data were collected to 3.3 and 3.4 A resolution from crystals of native and selenomethionylated MAM-MHC complexes, respectively. Self- and cross-rotation function calculations suggest the presence of two complex molecules in the asymmetric unit, resulting in a V(M) of 4.0 and a solvent content of 69%. An interpretable electron-density map was produced using a combination of molecular replacement and SAD phasing.

Figure 1

Profiles of chromatography and mass spectrometry of recombinant MAM. (a) Profile of gel-filtration chromatography of recombinant MAM on a Waters Protein Pak 300SW column. The positions were labeled for the Bio-Rad molecular-weight standard, which was run under the same conditions on the same column. (b) Electrospray ionization (ESI) mass spectra of recombinant MAM. (c) Deconvoluted ESI mass spectra of recombinant MAM, showing the molecular weight detected. The molecular weight calculated for the sequence is 25 704 Da.

Figure 2

Native crystals of the MAM–HA–DR1 complex grown under (a) sodium potassium phosphate condition and (b) ammonium sulfate condition.

Figure 3

Projection of the κ = 180° section of the self-rotation function, showing the presence of a non-crystallographic twofold axis at spherical polar coordinates (ϕ = 0, ψ = 45, κ = 180°).