Conformational isomers of calcineurin follow distinct dissociation pathways

In the gas-phase, ions of protein complexes typically follow an asymmetric dissociation pathway upon collisional activation, whereby an expelled small monomer takes a disproportionately large amount of the charges from the precursor ion. This phenomenon has been rationalized by assuming that upon activation, a single monomer becomes unfolded, thereby attracting charges to its newly exposed basic residues. Here, we report on the atypical gas-phase dissociation of the therapeutically important, heterodimeric calcium/calmodulin-dependent serine/threonine phosphatase calcineurin, using a combination of tandem mass spectrometry, ion mobility mass spectrometry, and computational modeling. Therefore, a hetero-dimeric calcineurin construct (62 kDa), composed of CNa (44 kDa, a truncation mutant missing the calmodulin binding and auto-inhibitory domains), and CNb (18 kDa), was used. Upon collisional activation, this hetero-dimer follows the commonly observed dissociation behavior, whereby the smaller CNb becomes highly charged and is expelled. Surprisingly, in addition, a second atypical dissociation pathway, whereby the charge partitioning over the two entities is more symmetric is observed. The presence of two gas-phase conformational isomers of calcineurin as revealed by ion mobility mass spectrometry (IM-MS) may explain the co-occurrence of these two dissociation pathways. We reveal the direct relationship between the conformation of the calcineurin precursor ion and its concomitant dissociation pathway and provide insights into the mechanisms underlying this co-occurrence of the typical and atypical fragmentation mechanisms.