Protein Crystal Engineering of YpAC-IV using the Strategy of Excess Charge Reduction

Abstract

The class IV adenylyl cyclase from Yersinia pestis has been engineered by site-specific mutagenesis to facilitate crystallization at neutral pH. The wild-type enzyme crystallized only below pH 5, consistent with the observation of a carboxyl-carboxylate H bond in a crystal contact in the refined structure 2FJT. Based on that unliganded structure at 1.9 A resolution, two different approaches were tested with the goal of producing a higher-pH crystal needed for inhibitor complexation and mechanistic studies. In one approach, Asp 19, which forms the growth-limiting dicarboxyl contact in wild-type triclinic crystals, was modified to Ala and Asn in hopes of relieving the acid-dependence of that crystal form. In the other approach, wild-type residues Met 18, Glu 25, and Asp 55 were (individually) changed to lysine to reduce the protein's excess negative charge in hopes of enabling growth of new, higher-pH forms. These 3 sites were selected based on their high solvent exposure and lack of intraprotein interactions. The D19A and D19N mutants had reduced solubility and did not crystallize. The other 3 mutants all crystallized, producing several new forms at neutral pH. One of these forms, with the D55K mutant, enabled a product complex at 1.6 A resolution, structure 3GHX. This structure shows why the new crystal form required the mutation in order to grow at neutral pH. This approach could be useful in other cases where excess negative charge inhibits the crystallization of low-pI proteins.

Figure 1

Wild-type triclinic crystals (cell 33.5, 35.5, 72.0 Å), typical clusters of thin plates, growing at pH 4.6. The largest single crystallite is about 100 microns wide and 5 microns thick. The large (001) face is growth-limiting, and the difficulty of growing thicker crystals (which, for this form, corresponds to stronger diffraction) was a major obstacle to solving the structure.

Figure 2

Packing in the ab plane of the triclinic crystals. Molecules in this plane have a hexagonal-close-packed arrangement, such that each molecule has 6 neighbors in the plane. The dimers in this view are seen end-on, so that behind each visible A-chain is a hidden B-chain (related by noncrystallographic symmetry). Thus the long axis of the dimer is along the crystal's c direction. Asp A19 in the central molecule is shown projecting outward (toward the viewer). Each Asp A19 forms a hydrogen bond to Asp B19 in the next higher layer of the crystal, by the contact shown in Figure 3.

Figure 3

Stereo view showing the dicarboxyl hydrogen bond in the c-directed crystal contact in the wild-type structure 2FJT. This type of hydrogen bond can occur only at low pH, when one of the two carboxylates is protonated. The 2Fo-Fc electron density map is shown contoured at 1.1 sigma.

Figure 4

Ribbon representation of the dimer showing the locations of the mutations. All were observed to be on exterior convex regions of the protein surface in the wild-type structure.

Figure 5

Monoclinic crystal of the D55K mutant growing at pH 6.5. Width of the field is about 200 μm. Although this crystal has many surface satellites, it has a thickness of over 25 μm and consists mostly of a single unit. Similar and slightly better (with larger single domains) crystals were soaked with substrate analogs to generate active-site complexes diffracting to about 1.6 Å.

Figure 6

Stereo view showing the hydrogen bond from the mutated Lys 55 residue (left) to Asp 156 on a neighboring molecule. This bond could not have occurred in wild-type. The molecule on the right has two carboxylates in the region (Asp 156 and Glu 153), hence this crystal contact would be unlikely to occur at neutral pH without the mutation. 2Fo-Fc electron density is shown contoured at 1.3 sigma.

Figure 7