Crystallization of the class IV adenylyl cyclase from Yersinia pestis

Abstract

The class IV adenylyl cyclase from Yersinia pestis has been cloned and crystallized in both a triclinic and an orthorhombic form. An amino-terminal His-tagged construct, from which the tag was removed by thrombin, crystallized in a triclinic form diffracting to 1.9 A, with one dimer per asymmetric unit and unit-cell parameters a = 33.5, b = 35.5, c = 71.8 A, alpha = 88.7, beta = 82.5, gamma = 65.5 degrees. Several mutants of this construct crystallized but diffracted poorly. A non-His-tagged native construct (179 amino acids, MW = 20.5 kDa) was purified by conventional chromatography and crystallized in space group P2(1)2(1)2(1). These crystals have unit-cell parameters a = 56.8, b = 118.6, c = 144.5 A, diffract to 3 A and probably have two dimers per asymmetric unit and VM = 3.0 A3 Da(-1). Both crystal forms appear to require pH below 5, complicating attempts to incorporate nucleotide ligands into the structure. The native construct has been produced as a selenomethionine derivative and crystallized for phasing and structure determination.

Figure 1

15% SDS–PAGE analysis of the purification of YpAC2 on a Ni–agarose column. Cells from a 2 l culture (5 g wet weight) were suspended in 50 ml of the lysis buffer. Lane 1, 2 µl French press extract; lane 2, 2 µl 20 000g supernatant; lane 3, 2 µl flowthrough on Ni–NTA resin; lane 4, 8 µl wash with lysis buffer (no imidazole); lane 5, 8 µl wash with 10 mM imidazole; lane 6, 8 µl (16 µg protein) wash with 20 mM imidazole; lane 7, 2.5 µl (16 µg protein) eluate with 200 mM imidazole, fraction 1; lane 8, 2.5 µl (2.8 µg protein) eluate with 200 mM imidazole, fraction 2; lane 9, molecular-weight markers. Relevant molecular-weight markers (kDa) are labeled. YpAC2 is indicated.

Figure 2

Photograph of two typical clusters of self-nucleated triclinic thin-plate crystals. Crystal thicknesses are about 4 µm. The crystal that gave the data set in Table 2 ▶ was similar but was cultivated to a thickness of 25 µm. Analysis of morphology and diffraction patterns indicates that the large slow-growing faces of these crystals correspond to the (001) plane.

Figure 3

Photograph of orthorhombic crystals of the native enzyme. The faces of these crystals index are (110) and (011). The longest dimension of the largest crystal is 350 µm. The higher symmetry and easier cultivation of these crystals makes them preferable for structure determination despite their weaker diffraction.

Figure 4

Adenylyl cyclase activity analysis of the purified YpAC2 as a function of temperature. Assays using radiolabeled ATP substrate and monitoring formation of labeled cAMP were as described by Reddy et al. (2001 ▶).