Competition between two high- and low-affinity protein-binding sites in myosin VI controls its cellular function

Abstract

Myosin VI is involved in many cellular processes ranging from endocytosis to transcription. This multifunctional potential is achieved through alternative isoform splicing and through interactions of myosin VI with a diverse network of binding partners. However, the interplay between these two modes of regulation remains unexplored. To this end, we compared two different binding partners and their interactions with myosin VI by exploring the kinetic properties of recombinant proteins and their distribution in mammalian cells using fluorescence imaging. We found that selectivity for these binding partners is achieved through a high-affinity motif and a low-affinity motif within myosin VI. These two motifs allow competition among partners for myosin VI. Exploring how this competition affects the activity of nuclear myosin VI, we demonstrate the impact of a concentration-driven interaction with the low-affinity binding partner DAB2, finding that this interaction blocks the ability of nuclear myosin VI to bind DNA and its transcriptional activity in vitro. We conclude that loss of DAB2, a tumor suppressor, may enhance myosin VI–mediated transcription. We propose that the frequent loss of specific myosin VI partner proteins during the onset of cancer leads to a higher level of nuclear myosin VI activity.

Binding partners regulate unfolding of myosin VI to activate the molecular motor

“…We extracted an association rate constant of 1.72 µM -1 s -1 and dissociation rate constant of 3.3 s -1 , giving a Kd of 1.9 µM. This is consistent with the Equilibrium Dissociation constant previously derived from titrations [6]. The second phase in all three traces was also fitted to a single exponential function ( Figure 2C), however the derived rate constants (average 2.1 s -1 ) were independent of NDP52 concentration ( Figure 2D).…”

“…NDP52 is a dimeric protein ( Figure 3B) and therefore capable of dimerizing MVI with one CBD bound to each monomer. However, tDab2, which also has been previously shown to be able to dimerize MVI [6], is monomeric ( Figure 3B). This finding reinforces our previous conclusion that dimerization is an intrinsic property of MVI.…”

“…As with NDP52, tDab2 sequestered the CBD, preventing the interaction between the two domains ( Figures 1B and 1C), suggesting that Dab2 is also able to disrupt the intramolecular back-folding in both isoforms. However, tDab2 was not as efficient as NDP52, which is consistent with its weaker affinity for MVI [6].…”

“…To address whether back-folding is a generic feature of MVI, independent of the isoform, we investigated the conformation of LI-MVI. We utilised a previously employed FRET-based assay [4,6,20] by titrating Alexa555-CBD against FITC-MVI814-1091 (containing the LI) to assess whether there is an interaction between the N-and C-terminal tail domains, as would occur in a back-folded state. A significant concentration-dependent change in FRET was measured, indicating that the two domains are in close proximity ( Figure 1B).…”

“…Unfolding of the NI isoform subsequently exposes dimerization sites, leading to protein oligomerization [4], similar to the LI isoform with binding partners [6]. There is a debate as to whether MVI dimerizes intrinsically or through a binding partner mediator [4,13,[22][23][24].…”