Altered antigen structure is known to affect epitope generation but less is known about how human autoantigens may display different conformations to the immune system. In order to gain better understanding into the initiation and propagation of autoimmune responses, we investigated changes in antigen structure which occur under biologically relevant conditions. Nucleophosmin (NPM1) is an abundant human tumor autoantigen and has important roles in cellular proliferation and differentiation. Previously, we had described how nucleophosmin from liver tumor samples was biochemically distinct and proposed that an amino-terminal deletion mutant, M7-NPM, modeled the behavior of the tumor protein. In our present work, we have utilized deuterium exchange mass spectrometry (DXMS) to investigate the structural differences between wild-type NPM1 and M7-NPM, and found that M7-NPM had reduced solvent exposure of the entire core oligomerization domain, while wild-type NPM1 demonstrated surprising plasticity at a key monomer-monomer interface called the β-hairpin latch. We also show here that removing interactions at the β-hairpin interface destabilizes nucleophosmin oligomers and alters recognition by granzyme B. Finally, we demonstrate that oxidant signaling in differentiating vascular smooth muscle cells leads to destabilization and/or dissociation of NPM1 oligomers and relocalization to mitochondria.