Members of the myosin superfamily of molecular motors are large mechanochemical ATPases that are implicated in an ever-expanding array of cellular functions. This review focuses on mammalian nonmuscle myosin-2 (NM2) paralogs, ubiquitous members of the myosin-2 family of filament-forming motors. Through the conversion of chemical energy into mechanical work, NM2 paralogs remodel and shape cells and tissues. This process is tightly controlled in time and space by numerous synergetic regulation mechanisms to meet cellular demands. We review how recent advances in structural biology together with elegant biophysical and cell biological approaches have contributed to our understanding of the shared and unique mechanisms of NM2 paralogs as they relate to their kinetics, regulation, assembly, and cellular function.

The loss of function of AAA (ATPases associated with diverse cellular activities) mechanoenzymes has been linked to diseases, and small molecules that activate these proteins can be powerful tools to probe mechanisms and test therapeutic hypotheses. Unlike chemical inhibitors that can bind a single conformational state to block enzyme function, activator binding must be permissive to different conformational states needed for mechanochemistry. However, we do not know how AAA proteins can be activated by small molecules. Here, we focus on valosin-containing protein (VCP)/p97, an AAA unfoldase whose loss of function has been linked to protein aggregation-based disorders, to identify druggable sites for chemical activators. We identified VCP ATPase Activator 1 (VAA1), a compound that dose-dependently stimulates VCP ATPase activity up to ~threefold. Our cryo-EM studies resulted in structures (ranging from ~2.9 to 3.7 Å-resolution) of VCP in apo and ADP-bound states and revealed that VAA1 binds an allosteric pocket near the C-terminus in both states. Engineered mutations in the VAA1-binding site confer resistance to VAA1, and furthermore, modulate VCP activity. Mutation of a phenylalanine residue in the VCP C-terminal tail that can occupy the VAA1 binding site also stimulates ATPase activity, suggesting that VAA1 acts by mimicking this interaction. Together, our findings uncover a druggable allosteric site and a mechanism of enzyme regulation that can be tuned through small molecule mimicry.

Neurolastin is a dynamin family GTPase that also contains a RING domain and exhibits both GTPase and E3 ligase activities. It is specifically expressed in the brain and is important for synaptic transmission, as neurolastin knockout animals have fewer dendritic spines and exhibit a reduction in functional synapses. Our initial study of neurolastin revealed that it is membrane-associated and partially co-localizes with endosomes. Using various biochemical and cell-culture approaches, we now show that neurolastin also localizes to mitochondria in HeLa cells, cultured neurons, and brain tissue. We found that the mitochondrial localization of neurolastin depends upon an N-terminal mitochondrial targeting sequence and that neurolastin is imported into the mitochondrial intermembrane space. Although neurolastin was only partially mitochondrially localized at steady state, it displayed increased translocation to mitochondria in response to neuronal stress and mitochondrial fragmentation. Interestingly, inactivation or deletion of neurolastin\'s RING domain also increased its mitochondrial localization. Using EM, we observed that neurolastin knockout animals have smaller but more numerous mitochondria in cerebellar Purkinje neurons, indicating that neurolastin regulates mitochondrial morphology. We conclude that the brain-specific dynamin GTPase neurolastin exhibits stress-responsive localization to mitochondria and is required for proper mitochondrial morphology.