Two Histidines in an α‐Helix: A Rigid Co²⁺‐Binding Motif for PCS Measurements by NMR Spectroscopy

Abstract

Pseudocontact shifts (PCS) generated by paramagnetic metal ions present valuable long-range information in the study of protein structural biology by nuclear magnetic resonance (NMR) spectroscopy. Faithful interpretation of PCSs,h owever,r equires complete immobilization of the metal ion relative to the protein, which is difficult to achieve with synthetic metal tags.Weshowthat two histidine residues in sequential turns of an a-helix provide abinding site for aCo 2+ ion, whichp ositions the metal ion in au niquely well-defined and predictable location. Exchange between the bound and free cobalt is slow on the timescale defined by chemical shifts, but the NMR resonance assignments are nonetheless readily transferred from the diamagnetic to the paramagnetic NMR spectrum by an I z S z -exchange experiment. The double-histidine-Co 2+ motif offers as traightforward, inexpensive,a nd convenient wayofg enerating precision PCSs in proteins.

Three-Dimensional Protein Structure Determination Using Pseudocontact Shifts of Backbone Amide Protons Generated by Double-Histidine Co2+-Binding Motifs at Multiple Sites

“…Protein Expression and Purification. In principle, a dHis motif alone is sufficient to allow purification by affinity chromatography on a Ni-NTA column, 28,40 The ERp29-C samples with dHis motif were titrated with metal ions in equimolar ratio except for Mu4, where a five-fold excess of CoCl2 was needed to observe PCSs. There was no evidence for alternative binding sites for Co 2+ ions in ERp29, as PREs were evident only in the vicinity of the dHis motif.…”

“…metal position for each model, which is in agreement with the location expected for the helical dHis-Co 2+ motif, where the histidine residues are located in positions i and i+4. 28 For each model, metal coordinates were determined by a least squares fit to match the restraints and these coordinates were used for  tensor fits. The restraints correspond to the average distances found in the ubiquitin E24H/A28H and A28H/D32H mutants, using the Co 2+ ion position determined from PCSs and modeling the histidine side chain conformations.…”

“…The restraints correspond to the average distances found in the ubiquitin E24H/A28H and A28H/D32H mutants, using the Co 2+ ion position determined from PCSs and modeling the histidine side chain conformations. 28 To assess the contribution of the PCS data to the structure calculation, the performance of each PCS dataset was analysed individually. All four PCS datasets contributed to the convergence of the GPS-Rosetta modelling ( Figure S5).…”

“…The fold of ERp29-C comprises five -helices and therefore is ideally suited for assessing the performance of the dHis-Co 2+ motif in generating PCSs, as the dHis-Co 2+ motif is more readily installed in -helices than in other secondary structure elements. 28 In the present work, the dHis motifs were installed in four different helices, in analogy to previous GPS-Rosetta calculations, where lanthanide tags were installed in different helices. 15 The Rosetta algorithm is one of the most successful methods for de novo protein structure prediction.…”

“…Although Co 2+ ions generate smaller PCSs than some of the lanthanide ions, the dHis-Co 2+ motif features a number of advantages, including exceptional localization of the metal ion in relatively close proximity of the protein backbone, compatibility with naturally occurring cysteine residues in the protein, and straightforward sample preparation. 28 To evaluate the performance of the dHis-Co 2+ motif for PCS-aided 3D structure determination, we used the C-terminal domain of the rat protein ERp29 (ERp29-C) as a model system. ERp29 is a chaperone associated with the endoplasmic reticulum.…”