To study the role of B insert in the Drp1:CL interaction, we made three naturally occurring splice variants of human Drp1 that differ in the length of the B insert as follows: Variant 1, the fulllength 736-residue protein; Variant 2 lacking 26 residues in B insert; and Variant 3 lacking 37 residues in B Insert. The three isoforms were purified without affinity tags. All three isoforms eluted as a mixture of dimers and tetramers after gel filtration and showed virtually no contaminating proteins. Since the Talazoparib supply results presented thus far indicate that specific binding of Drp1 to CL is essential to stimulate its GTPase activity, we examined the effect of mutations in B insert on CL-dependent GTP hydrolysis. As shown above, the Drp1-4KA mutant had a reduced capacity to interact with CL. Determination of the GTPase activity of this mutant in the presence or absence of CL containing vesicles shows that the reduced affinity for CL correlates perfectly with a reduced capacity of the CL-containing liposomes to stimulate its GTPase activity. Importantly, the basal GTPase activity of the 4KA mutant was unaffected. Reintroduction of each of the lysine pairs separately into B insert both led to similar partial restoration of cardiolipin-stimulated GTPase hydrolysis although the values obtained with the WT protein were not reached in either case. Based on these data, we conclude that all four lysines present in B insert of Drp1 participate in lipid association and stimulation of GTPase activity. In agreement with our observations, it has recently been seen that addition of anionic lipids containing liposomes to a Drp1 construct lacking the B insert does not increase the GTP hydrolysis rate. In summary, our results strongly suggest that the interaction of Drp1 B insert with CL is intimately linked to Drp1 functional activation. In contrast to our findings, Strack and Cribbs observed that the B insert is dispensable for mitochondrial recruitment, association with Mff and basal. In this regard it is quite possible that the mechanisms of Drp1 recruitment and activation in the MOM vary depending on the cell context. CL dependent Drp1 binding and activation may be relevant in cellular processes where CL has been proposed to be transported from the mitochondrial inner membrane to the MOM such as apoptosis and mitophagy. Classical dynamins are known to undergo stimulated GTPase activity upon lipid-induced self-assembly, an event that has also been described for some dynamin-like proteins. Thus, we decided to investigate structural changes occurring in Drp1 upon interaction with CL. Far-UV CD spectra for the protein alone in solution or in the presence of liposomes with or without CL showed minor differences in the secondary structure of the protein. A common theme to emerge from the study of dynamins is that oligomerization plays an important role in regulating their functional state. It had previously been shown that stimulated dynamin GTPase activity was highly cooperative in the presence of PIP2 containing vesicles, which reflects higher order dynamin self-assembly.