CheShift-2 resolves a local inconsistency between two X-ray crystal structures

Since chemical shifts provide important and relatively accessible information about protein structure in solution, a Web server, CheShift-2, was developed for structure interrogation, based on a quantum mechanics database of C-13 (alpha) chemical shifts. We report the application of CheShift-2 to a local inconsistency between two X-ray crystal structures (PDB IDs 1IKN and 1NFI) of the complex between the p65/p50 heterodimer of NF kappa B and its inhibitor I kappa B alpha. The availability of NMR resonance assignments that included the region of the inconsistency provided an opportunity for independent validation of the CheShift-2 server. Application of the server showed that the C-13 (alpha) chemical shifts measured for the Gly270-Pro281 sequence close to the C-terminus of I kappa B alpha were unequivocally consistent with the backbone structure modeled in the 1IKN structure, and were inconsistent with the 1NFI structure. Previous NOE measurements had demonstrated that the position of a tryptophan ring in the region immediately N-terminal in this region was not consistent with either structure. Subsequent recalculation of the local structure in this region, based on the electron density of the deposited structure factors for 1IKN, confirmed that the local backbone structure was best modeled by 1IKN, but that the rotamer of Trp258 is consistent with the 1NFI structure, including the presence of a hydrogen bond between the ring N epsilon H of Trp258 and the backbone carbonyl group of Gln278. The consensus between all of these measures suggests that the CheShift-2 server operates well under circumstances in which backbone chemical shifts are available but where local plasticity may render X-ray structural data ambiguous.