Pitfalls in the interpretation of structural changes in mutant proteins from crystal structures

Abstract

PpcA is a small protein with 71 residues that contains three covalently bound hemes. The structures of single mutants at residue 58 have shown larger deviations in another part of the protein molecule than at the site of the mutation. Closer examination of the crystal packing has revealed the origin of this unexpected structural change. The site of mutation is within Van der Waals distance from another protein molecule related by a crystallographic twofold axis within the crystal. The structural changes occurred at or near the mutation site have led to a slight adjustment of the surface residues in contact. The observed deviations between the native and the mutant molecular structures are derived from the new crystal packing even though the two crystals are essentially isomorphous. Without careful consideration of the crystal lattice a non-expert looking at only the coordinates deposited in the Protein Data Bank could draw erroneous conclusion that mutation in one part of the molecule affected the structure of the protein in a distant part of the molecule.

Fig. 1

Structure of PpcA showing protein as a cartoon of C_α tracing along with the heme cofactors and the deoxycholic acid moiety required for crystallization. Side chain of methionine 58 is shown in pink as a stick representation. The two regions of change (region 1 and 2) are also indicated. The figure was prepared by using the program PyMOL

Fig. 2

Native (carbons in green) and M58D (carbons in gray) mutant structures. The two structures were overlapped using all C_α carbons. For both structures, O atoms are in red, N atoms are in blue and S atoms are in yellow. Note the higher deviations at Asp26 compared to the changes at 58, the site of mutation. The figure was prepared by using the program PyMOL

Fig. 3

The two protein molecules related by a crystallographic twofold axis in the native PpcA crystal. The molecule generated by crystallographic symmetry is shown in gray. The contacts between residues 58 and 25 are indicated by broken lines with the corresponding distances shown in Å. The regions 1 and 2 are indicated. The figure was prepared by using the program Chain [15]

Fig. 4

Close-up view of the interactions between the symmetry-related molecules of the native protein crystal as shown in Fig. 3. The symmetry-related molecule is at the top. The C atoms for the symmetry-related molecule are in cyan; C atoms for the molecule in the asymmetric unit are in green. For both molecules, O atoms are in red, N atoms are in blue and the S atoms are in yellow. Note the close packing of the two molecules. Any changes caused by mutation at residue 58 will have an impact on the symmetry-related molecule due to close packing. The figure was prepared by using the program PyMOL