Mycobacterium tuberculosis Mpa is an AAA+ ATPase that unfolds and translocates substrate proteins during proteasomal degradation. Mpa spontaneously assembles into a homohexamer driven by interdomain interactions, even in the absence of a nucleotide. To dissect the mechanisms underlying its oligomerization and deoligomerization, we perturbed the system using chemical and thermal denaturants and monitored the oligomeric states by far-UV CD, fluorescence, calorimetry, state-of-the-art single-particle mass photometry, and ATPase activity assays. Equilibrium chemical denaturation resulted in gradual changes in spectroscopic signals, whereas mass photometry revealed a direct transition from a hexamer to monomeric species, indicating a concerted but noncooperative pathway without any intermediate oligomeric or folded monomeric states. In contrast, thermal perturbation showed two sharp and distinct transitions, the first one corresponding to a concerted and cooperative transition from hexamer to unfolded state, which further aggregates, and the second transition to disaggregation at elevated temperatures. Both chemical and thermal unfolding processes were irreversible with respect to the reassembly of the functional oligomer. Using single-particle mass photometry complemented by spectroscopy and calorimetry, these findings establish that Mpa is an obligate oligomer and can provide insights into the oligomerization pathways of AAA+ enzymes that spontaneously hexamerize and have the potential to further illuminate evolutionary strategies underlying their assembly mechanisms.
Sahu et al. (Tue,) studied this question.