Dynamin-related Protein 1 Oligomerization in Solution Impairs Functional Interactions with Membrane-anchored Mitochondrial Fission Factor.

Mitochondrial fission is a crucial cellular process mediated by the mechanoenzymatic GTPase, dynamin-related protein 1 (Drp1). During mitochondrial division, Drp1 is recruited from the cytosol to the outer mitochondrial membrane by one, or several, integral membrane proteins. One such Drp1 partner protein, mitochondrial fission factor (Mff), is essential for mitochondrial division, but its mechanism of action remains unexplored. Previous studies have been limited by a weak interaction between Drp1 and Mff in vitro. Through refined in vitro reconstitution approaches and multiple independent assays, we show that removal of the regulatory variable domain (VD) in Drp1 enhances formation of a functional Drp1-Mff copolymer. This protein assembly exhibits greatly stimulated cooperative GTPase activity in solution. Moreover, when Mff was anchored to a lipid template, to mimic a more physiologic environment, significant stimulation of GTPase activity was observed with both WT and ΔVD Drp1. Contrary to recent findings, we show that premature Drp1 self-assembly in solution impairs functional interactions with membrane-anchored Mff. Instead, dimeric Drp1 species are selectively recruited by Mff to initiate assembly of a functional fission complex. Correspondingly, we also found that the coiled-coil motif in Mff is not essential for Drp1 interactions, but rather serves to augment cooperative self-assembly of Drp1 proximal to the membrane. Taken together, our findings provide a mechanism wherein the multimeric states of both Mff and Drp1 regulate their collaborative interaction.

Phosphoproteomics of collagen receptor networks reveals SHP-2 phosphorylation downstream of wild-type DDR2 and its lung cancer mutants.

Collagen is an important extracellular matrix component that directs many fundamental cellular processes including differentiation, proliferation and motility. The signalling networks driving these processes are propagated by collagen receptors such as the ß1 integrins and the DDRs (discoidin domain receptors). To gain an insight into the molecular mechanisms of collagen receptor signalling, we have performed a quantitative analysis of the phosphorylation networks downstream of collagen activation of integrins and DDR2. Temporal analysis over seven time points identified 424 phosphorylated proteins. Distinct DDR2 tyrosine phosphorylation sites displayed unique temporal activation profiles in agreement with in vitro kinase data. Multiple clustering analysis of the phosphoproteomic data revealed several DDR2 candidate downstream signalling nodes, including SHP-2 (Src homology 2 domain-containing protein tyrosine phosphatase 2), NCK1 (non-catalytic region of tyrosine kinase adaptor protein 1), LYN, SHIP-2 [SH2 (Src homology 2)-domain-containing inositol phosphatase 2], PIK3C2A (phosphatidylinositol-4-phosphate 3-kinase, catalytic subunit type 2α) and PLCL2 (phospholipase C-like 2). Biochemical validation showed that SHP-2 tyrosine phosphorylation is dependent on DDR2 kinase activity. Targeted proteomic profiling of a panel of lung SCC (squamous cell carcinoma) DDR2 mutants demonstrated that SHP-2 is tyrosine-phosphorylated by the L63V and G505S mutants. In contrast, the I638F kinase domain mutant exhibited diminished DDR2 and SHP-2 tyrosine phosphorylation levels which have an inverse relationship with clonogenic potential. Taken together, the results of the present study indicate that SHP-2 is a key signalling node downstream of the DDR2 receptor which may have therapeutic implications in a subset of DDR2 mutations recently uncovered in genome-wide lung SCC sequencing screens.

Affinity Purification and Functional Characterization of Dynamin-Related Protein 1.

Purification of dynamin-related proteins is complicated by their oligomeric tendencies. In this chapter, we describe an established purification regime to isolate the mitochondrial fission protein Drp1 using bacterial expression. Key attributes of dynamins include their ability to hydrolyze GTP and self-assemble into larger polymers under specific conditions. Therefore, the GTPase activity of Drp1 should be examined to confirm isolation of functional protein, and we describe a conventional colorimetric assay to assess enzyme activity. To determine the ability of Drp1 to self-assemble, we induce Drp1 polymerization through addition of a non-hydrolyzable GTP analogue. A sedimentation assay provides a quantitative measure of polymerization that complements a qualitative assessment through visualization of Drp1 oligomers using negative-stain electron microscopy (EM). Importantly, we highlight the caveats of affinity tags and the influence that these peptide sequences can have on Drp1 function given their proximity to functional domains.

Steric interference from intrinsically disordered regions controls dynamin-related protein 1 self-assembly during mitochondrial fission.

The self-assembling, mechanoenzymatic dynamin superfamily GTPase, dynamin-related protein 1 (Drp1), catalyzes mitochondrial and peroxisomal fission. Distinct intrinsically disordered regions (IDRs) in Drp1 substitute for the canonical pleckstrin homology (PH) domain and proline-rich domain (PRD) of prototypical dynamin, which cooperatively regulate endocytic vesicle scission. Whether the Drp1 IDRs function analogously to the corresponding dynamin domains however remains unknown. We show that an IDR unique to the Drp1 GTPase (G) domain, the 'extended 80-loop', albeit dissimilar in location, structure, and mechanism, functions akin to the dynamin PRD by enabling stable Drp1 mitochondrial recruitment and by suppressing Drp1 cooperative GTPase activity in the absence of specific partner-protein interactions. Correspondingly, we find that another IDR, the Drp1 variable domain (VD), in conjunction with the conserved stalk L1N loop, functions akin to the dynamin PH domain; first, in an 'auto-inhibitory' capacity that restricts Drp1 activity through a long-range steric inhibition of helical inter-rung G-domain dimerization, and second, as a 'fulcrum' for Drp1 self-assembly in the proper helical register. We show that the Drp1 VD is necessary and sufficient for specific Drp1-phospholipid interactions. We further demonstrate that the membrane-dependent VD conformational rearrangement essential for the alleviation of Drp1 auto-inhibition is contingent upon the basal GTP hydrolysis-dependent generation of Drp1 dimers from oligomers in solution. IDRs thus conformationally couple the enzymatic and membrane activities of Drp1 toward membrane fission.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro.

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Cryo-EM Studies of Drp1 Reveal Cardiolipin Interactions that Activate the Helical Oligomer.

Dynamins are mechano-chemical GTPases involved in the remodeling of cellular membranes. In this study, we have investigated the mechanism of dynamin-related protein 1 (Drp1), a key mediator of mitochondrial fission. To date, it is unclear how Drp1 assembles on the mitochondrial outer membrane in response to different lipid signals to induce membrane fission. Here, we present cryo-EM structures of Drp1 helices on nanotubes with distinct lipid compositions to mimic membrane interactions with the fission machinery. These Drp1 polymers assemble exclusively through stalk and G-domain dimerizations, which generates an expanded helical symmetry when compared to other dynamins. Interestingly, we found the characteristic gap between Drp1 and the lipid bilayer was lost when the mitochondrial specific lipid cardiolipin was present, as Drp1 directly interacted with the membrane. Moreover, this interaction leads to a change in the helical structure, which alters G-domain interactions to enhance GTPase activity. These results demonstrate how lipid cues at the mitochondrial outer membrane (MOM) can alter Drp1 structure to activate the fission machinery.