Fig. 3.

Peptic coverage of AGR2 protein in the seven-point deuteration reaction time course. A, Gene structure of the recombinant AGR2 protein. The panel highlights the functional motifs within recombinant human AGR2; the poly-His-tag used for nickel affinity purification; an N-terminal intrinsically disordered region; a dimerization motif; a thioredoxin fold, the Reptin docking site, and the ER retention site. B, Coomassie Blue staining of purified His-AGR2 showing the major band at 20 kDa under reducing SDS-PAGE condition. C, Peptic peptides recovered from ligand-free mature AGR2 protein (with the N-terminal 20 amino acids containing the hydrophobic ER leader sequence removed). The proteolytic peptide fragments can be grouped into four regions based on extents of recovery after mass spectrometry; (i) The N-terminal region containing the poly his-tag and amino acids Arg21-Leu52 containing the intrinsically disordered region that plays a negative regulatory role in dimer stability (23) (7); (ii) a central grouping from Ile53 to Asn108, containing the beginning of the dimerization motif (60-EALYK-64), the CxxS thioredoxin fold, and the Reptin binding motif (104-FVLLNLVY-111); (iii) a third pepsin resistant region from amino acids 109–130; and (iv) a region of a cluster of overlapping peptic fragments including the specific peptide binding domain (this study) and the degenerate KTEL endoplasmic reticulum retention site.