NQ-Flipper: recognition and correction of erroneous asparagine and glutamine side-chain rotamers in protein structures

Abstract

The current Protein Data Bank (PDB) contains about 40 000 protein structures with approximately half a million incorrect atom positions resulting from erroneously assigned asparagine (Asn) and glutamine (Gln) rotamers. These errors affect applications in protein structure analysis, modeling and docking and therefore the detection, correction and prevention of such errors is highly desirable. We present NQ-Flipper, a web service based on mean force potentials to automatically detect and correct erroneous Asn and Gln rotamers. The service accepts protein structure files formatted in PDB style or PDB codes. For an Asn/Gln side-chain amide NQ-Flipper computes the total interaction energy with the surrounding atoms as the sum of pairwise atom-atom interaction energies. The energy difference between the original and the alternative rotamers identifies the correct configuration of the amide group. The web service lists the interaction energies of all Asn/Gln residues found in a PDB file and shows the structure and offending residues in an interactive 3D viewer. The corrected protein structure is available for download in various compression formats. The web service is accessible at http://flipper.services.came.sbg.ac.at.

Figure 1.

NQ-Flipper analysis of Asn/Gln rotamers. We provide an example of the application of NQ-Flipper to acutohaemolysin (PDB code 1mc2) determined at 0.85 Å resolution (21). The PDB file contains a single chain (identifier A) of 122 amino acid residues. The molecule contains eleven Asn/Gln residues. Three of these residues are flagged as incorrect rotamers which is confirmed by a detailed examination based on simple physico-chemical principles. The structure is determined to a very high resolution where errors in rotamer configuration may seem unlikely. However, the result shown here is quite common. Generally protein structures, even at very high resolution, contain several incorrect rotamers. (a) The protein backbone C-alpha trace with unfavorable rotamers rendered as a ball-and-stick model. The crystal structure contains two heterogeneous groups termed IPA. (b) Table listing all Asn/Gln rotamers and their respective Δε-values as displayed on the NQ-Flipper web page. Coordinates were derived for alternate location indicator A. The software detects three Asn residues with unfavorable Δε-scores, i.e., rotamers with Δ ε > v where the threshold value is set to v = 6 (the default). These incorrect rotamers are highlighted in red and include Asn-1114 (Δ ε = + 43.3), Asn-1088 (Δ ε = + 39.9) and Asn-1020 (Δ ε = + 12.3). One residue, Gln-1133 (Δ ε = -5.1), is considered ‘ambiguous’ since its Δε-score is in the range where both rotamers are occupied with non-negligible probability (| Δ ε | < 6). Such residues of multiple occupancy are colored orange. All remaining Asn/Gln residues have Δε-scores below -6 indicating correct rotamers. Residue Asn-1016 (Δ ε =-24.0) shown in blue is in the vicinity of an isopropanol buffer molecule, IPA. The minimum distance between atoms from the IPA molecule and the respective amide group in Asn-1016 is 5.7 Å. The residue is colored blue to indicate that it is close to a heterogeneous group so that some important interactions may not be included in the score—although even in such cases a score greater than v generally indicates an incorrect rotamer. Figure 1c and d provide a detailed view of two of the incorrect rotamers in the conformation R₁, as found in the PDB file, interacting with their respective chemical environment. The atoms are colored by atom type: carbon, gray; nitrogen, blue; oxygen, red. The dashed lines indicate distances in Å between atoms. (c) In the original conformation residue Asn-1020 (Δ ε = + 12.3) is unable to participate in a hydrogen bond network. In the alternative conformation R₂ the amide nitrogen and oxygen atoms swap positions enabling hydrogen bond formation between the amide nitrogen and the carbonyl oxygen of Asn-1016, the amide oxygen and the side-chain nitrogen of Lys-1015 and a nitrogen of the guanidinium group of Arg-1118. Note that by flipping the amide group several ‘anti-hydrogen bonds’ of very high energy are replaced by genuine hydrogen bonds of very low energy. (d) Residue Asn-1088 (Δ ε = + 39.9), where the amide nitrogen and oxygen atoms have unfavorable interactions with the respective main chain nitrogen and oxygen atoms of Ser-1074. The amide oxygen of this residue additionally interacts unfavorably with Gln-1093, a correct rotamer since it forms a hydrogen bond to the carbonyl group of Gly-1086. Flipping Asn-1088 to rotamer R₂ results in the formation of three hydrogen bonds. Figure a, c and d were generated using PyMOL (http://pymol.sourceforge.net).