|
InDrosophila testis, myosin VI plays a special role, distinctfrom its motor function, by anchoring components to the unusual actin-basedstructures （cones） that are required for spermatid individualization.For this, the two calmodulin （CaM） light-chain molecules of myosinVI are replaced by androcam （ACaM）, a related protein with 67% identityto CaM. Although ACaM has a similar bi-lobed structure to CaM, withtwo EF hand-type Ca2+ binding sites per lobe, only onefunctional Ca2+ binding site operates in the amino-terminus.To understand this light chain substitution, we used hydrogen–deuteriumexchange mass spectrometry （HDX-MS） to examine dynamic changes inACaM and CaM upon Ca2+ binding and interaction with thetwo CaM binding motifs of myosin VI （insert2 and IQ motif）. HDX-MSreveals that binding of Ca2+ to ACaM destabilizes its N-lobebut stabilizes the entire C-lobe, whereas for CaM, Ca2+ binding induces a pattern of alternating stabilization/destabilizationthroughout. The conformation of this stable holo-C-lobe of ACaM seemsto be a “prefigured” version of the conformation adoptedby the holo-C-lobe of CaM for binding to insert2 and the IQ motifof myosin VI. Strikingly, the interaction of holo-ACaM with eitherpeptide converts the holo-N-lobe to its Ca2+-free, morestable, form. Thus, ACaM in vivo should bind the myosin VI light chainsites in an apo-N-lobe/holo-C-lobe state that cannot fulfill the Ca2+-related functions of holo-CaM required for myosin VI motorassembly and activity. These findings indicate that inhibition ofmyosin VI motor activity is a precondition for transition to an anchoringfunction.
|
