Pitfalls in the interpretation of structural changes in mutant proteins from crystal structures
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- Pokkuluri, P.R., Yang, X., Londer, Y.Y. et al. J Struct Funct Genomics (2012) 13: 227. doi:10.1007/s10969-012-9147-1
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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.
KeywordsCrystal contactsCrystal packing effectsCytochrome c7Geobacter sulfurreducensSingle mutant structuresStructural changes
Nuclear magnetic resonance
Protein Data Bank
Periplasmic cytochrome A
Root mean square deviation
X-ray diffraction data collected from crystals provide detailed structures of molecules that are valid in the context of that particular crystal packing environment. The molecule in a crystal adopts a conformation in which the inter-atomic interactions between self and neighboring molecules are optimized. The coordinates deposited in the Protein Data Bank (PDB) represent the protein that makes up the asymmetric unit (the smallest component that can be used to build up the crystal by mathematical operations). Understanding the effects of a mutation on the native protein structure requires careful consideration of the influence of the crystal lattice, rather than simple examination of only one molecule (i.e., the coordinates deposited in the PDB). The user needs to generate the neighboring molecules by using the symmetry operators provided in the PDB and a crystallographic program. This consideration is especially important for non-specialist users of coordinates deposited in the PDB.
The importance of considering the crystal lattice in understanding structural changes in mutants is demonstrated by a series of crystal structures of single mutants at Met58 in a small tri-heme cytochrome c7 from Geobacter sulfurreducens (Gs). PpcA (periplasmic cytochrome A) is one of the most abundant proteins in the periplasm of Gs . PpcA is part of a family of five tri-heme cytochromes c7 encoded by the genome of Gs. We previously determined the structures of all five proteins by X-ray crystallography [2, 3]. In addition, the solution structure of fully reduced PpcA was also reported .
PpcA is a protein with 71 residues that contains three covalently bound hemes. It is one of the smallest c-type cytochromes with the highest ratio of hemes to amino acids, approximately 1 heme per 24 amino acids. The purified PpcA can reduce Fe(III), U(VI), Cr(VI) and other metal ions . Gs can respire using various electron donors and electron acceptors . PpcA is thought to be directly responsible for the reduction of soluble metal species, such as U(VI), which can enter the periplasm . Furthermore, PpcA is believed to serve as an intermediate carrier of electrons obtained from oxidation of the electron donor acetate to the terminal reductase(s) localized in the outer membrane of the organism for extracellular reduction of insoluble Fe(III) oxide [1, 5, 6].
Desulfuromonas acetoxidans (Da) is another delta proteobacteria closely related to Gs. Da has a homologous cytochrome c7 (Dac7), the structure of which has been determined first in solution by NMR [7, 8] and later by X-ray crystallography . The sequence identity between PpcA and Dac7 is 46 % . The sequence identity is largely confined to the heme binding motifs, CXXCH, in which the two cysteines are covalently attached to the heme groups through a thioether bond, and the histidine serves as the proximal ligand to the heme Fe. The hemes are numbered I, III and IV in keeping homology with well-studied tetra-heme cytochromes c3 .
Materials and methods
Site directed mutagenesis
For mutagenesis, QuikChange Site-Directed Mutagenesis Kit (Stratagene) was used in accordance with the manufacturer’s instructions. Oligonucleotides were synthesized by MWG Biotech (High Point, NC, USA). PpcA expression vector pCK32  was used as a template. The presence of desired mutations was confirmed by DNA sequencing performed by MWG Biotech.
Expression and purification of PpcA mutants
E. coli strain BL21(DE3) containing the plasmid pEC86  was co-transformed with the expression vector containing the PpcA mutants (M58S, M58N, M58D, M58K). Transformed E. coli cells were grown in 2xYT medium containing 34 μg/mL chloramphenicol and 100 μg/mL ampicillin. Protein expression was induced with 10 μM IPTG. PpcA mutants were purified by cation-exchange chromatography followed by gel-filtration chromatography, as described for the wild-type cytochrome [2, 11]. The purity of the proteins was evaluated by SDS-PAGE (15 %), stained with Coomassie blue. The correct heme incorporation in the mutants was confirmed by determination of molecular mass by electrospray ionization mass spectrometry performed in the HHMI Biopolymer Laboratory and W.M. Keck Foundation Biotechnology Resource Laboratory at Yale University.
All PpcA mutants reported here were crystallized in the same conditions as the native protein in presence of the additive deoxycholic acid  by hanging-drop vapor-diffusion method at room temperature. The precipitant solution was high concentrations (3.5–4.0 M) of ammonium sulfate (pH adjusted with ammonium hydroxide). Protein concentrations were 30 mg/mL for M58S, 60 mg/mL for M58D, 50 mg/mL for M58N, 50 mg/mL for M58K. Precipitant solutions were as follows: M58S: 3.8 M ammonium sulfate pH 6.0; M58N: 4.0 M ammonium sulfate pH 6.0; M58D: 3.6 M ammonium sulfate pH 6.5; M58K: 4.0 M ammonium sulfate pH 5.7. In each case, X-ray diffraction quality crystals were obtained in presence of 0.25 % deoxycholic acid in the drop.
Cryoprotection was achieved by transferring the crystals to the reservoir solution containing 28 % (w/v) sucrose for 20–30 s prior to plunging directly into liquid nitrogen. X-ray diffraction data sets on each mutant to a resolution of about 2 Å were collected at the Structural Biology Center’s 19BM beam line at the Advanced Photon Source (Argonne National Laboratory, Lemont, IL). Data sets were processed with HKL2000 .
Structure determination and refinement
Crystallographic parameters of M58 mutants
a = b (Å)
Resolution range (Å)
No. of atoms [mean B-factor (Å2)]
RMS deviations from ideal geometry
Bond lengths (Å)
Bond angles (°)
Results and discussion
M58 mutants had similar expression yields (3 mg/L of culture) and identical UV–visible spectra in both oxidized and reduced forms when compared with the native protein (data not shown). All M58 mutants required deoxycholate for crystallization; they produced well-diffracting crystals that were isomorphous with the native crystals with a maximum observed deviation in the c-axis of about 0.8 % (see Table 1). Therefore, the mutant structures were determined by rigid-body refinement of the native coordinates (PDB code: 1OS6, with the side chain of 58 truncated at CB) against the respective mutant data set.
Overall protein structure
The overall structure of the native protein, PpcA is shown as a ribbon drawing of the Cα carbons (Fig. 1). The three hemes together with a few hydrophobic amino acids form the hydrophobic core of the protein. PpcA contains an N-terminal two strand β-sheet followed by helices and loops, and also a deoxycholate molecule required for crystallization [2, 3]. The site of mutation, methionine 58 is rendered as a stick model in Fig. 1. The side chain of M58 in PpcA was well defined in the electron density and was found to form van der Waals interactions with heme III. In the observed conformation, the sidechain of methionine 58 limits the surface exposure of heme III. To examine the consequences of mutation at this site, we replaced it with serine (the amino acid found at the equivalent position in Dac7; see Ref. ), and with asparagine and aspartic acid (same size but one is neutral and the other is negatively charged), and a positively charged lysine.
Comparison of native and mutant structures
Deviations in Cα positions (Å) observed in regions 1 and 2 between the native and the M58 single mutant structures
Crystal packing effects on molecular structure
Implications for the non-expert
In the present case of M58 mutants of PpcA, insufficient thought could lead to the erroneous conclusion that the changes observed in region 2 (which affect the heme binding residues of a distant heme and cause shifts in that heme position) are linked directly to the changes at the mutation site (region 1). In reality, the deviations observed in both regions 1 and 2 upon comparison of crystal structures result from a combination of the original changes at the mutation site and the effects of slight rearrangement of the crystal packing. Interestingly, the side chain of mutated residue M58 is not directly in the crystal contact. It is likely that there may not be any significant changes in region 2 of the mutant proteins in solution.
Evaluation of the influence of a mutation on the structure of a protein molecule as derived from crystal structures requires consideration of effects on the whole crystal lattice. The mutation can influence not only the neighboring residues in the molecule (primary effect), but also residues of neighboring molecules in the crystal (secondary effect). The structural changes caused by the primary effect can cause secondary effects; together, they result in a new minimum energy conformation for the crystal. It is therefore impossible to isolate the effects of the mutation when structural changes occur at or near the interfaces of molecules in the crystal. In case of the M58 mutants of PpcA, the crystals are essentially isomorphous with the native protein crystal. If the primary and secondary effects of the mutation are large, then the mutant protein mostly likely will not crystallize in the same lattice as the native protein. Erroneous conclusions could be drawn without a careful consideration of this fact, especially by non-specialists.
The M58 mutant structures were determined with support from U.S. Department of Energy Office of Biological and Environmental Research program under contract no. DE-AC02-06CH11357. The structural analysis of the mutants is partially supported by the division of Chemical Sciences, Geosciences, and Biosciences, Office of Basic Energy Sciences of the U.S. Department of Energy program under contract no. DE-AC02-06CH11357 and partially by the National Institutes of Health grant GM094585. The Structural Biology Center beam lines at APS were supported by U.S. Department of Energy Office of Biological and Environmental Research program under contract no. DE-AC02-06CH11357. Former students in the laboratory, J. Erickson, V. Orshonsky, L. Orshonsky are acknowledged for their contribution in protein purification and crystallization.