#motor proteins

Cytoskeleton

The AAA+ domain in dynein is responsible for:

Structural analysis of dynein heavy chain shows tail responsible for dimerization and cargo adaptor binding, followed by six AAA+ domains arranged as heterohexameric ring with central pore. AAA1 bears conserved Walker A P-loop GXXXXGKT and Walker B hhhhDE motif essential for ATP binding and hydrolysis, AAA2-AAA4 modulate allosteric communication, AAA5-AAA6 stabilize ring and interact with stalk and strut. Upon ATP hydrolysis at AAA1, conformational changes propagate around ring altering interface between AAA5 and stalk buttress, shifting coiled-coil registry and linker docking. This generates measurable force of several piconewtons per stroke. Mutagenesis of lysine in Walker A or glutamate in Walker B abolishes motility, dominant negative in cells. Cargo attachment mediated by N-terminal tail associating with intermediate chain, light intermediate chain, light chains LC8, Tctex, Roadblock that bind adaptors like BICD, HOOK, Spindly. No interaction with actin filaments, no direct tubulin polymerization regulation, distinguishing AAA ring as force generating engine analogous to other ring-translocases adapted for cytoskeletal motor function.

Ref: Carter et al. Nature 2011; AAA+ AAA1 Walker A/B hydrolyzes ATP generating 4-5 pN force.

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Cytoskeleton

How does dynein achieve movement along microtubules?

Dynein stepping mechanism contrasts with kinesin neck-linker model, employing large AAA ring plus linker swing. In absence of nucleotide, microtubule-binding domain tightly bound, linker straight docking at AAA5. ATP binding to AAA1 induces closure of AAA1-AAA2 interface, pulling buttress from AAA5 that shifts stalk coiled-coil registry from alpha to beta, weakening microtubule affinity and detaching motor. Hydrolysis allows linker undocking from AAA5 and migration toward AAA2 site forming pre power stroke bent conformation. Forward diffusion and microtubule rebinding triggers release of phosphate, conformational relaxation pulling AAA2-AAA3, generating strain that drives linker back toward AAA5, producing power stroke that moves tail forward. ADP release completes cycle. Thus movement specifically results from coordinated ADP release coupled linker rotation and registry-controlled microtubule affinity switching. No myosin binding occurs, GTP irrelevant because catalytic pocket specific for adenine, oligomeric filament formation unnecessary because dimeric motor processive via alternating heads anchored to dynactin cargo complex. Integration with cell cycle kinases, calcium signaling and mechanical cues ensures coordinated remodeling during growth, migration and differentiation.

Ref: Roberts Cell 2013; ATP binding detaches dynein, ADP release plus linker rotation powers stroke.

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Cytoskeleton

Dynein movement along microtubules requires:

Dynein mechanochemistry is powered by Mg-ATP, with intracellular concentration typically one to three millimolar far above Km around twenty to fifty micromolar, supporting near maximal velocity in vivo. Cycle involves ATP binding to AAA1 causing microtubule release, hydrolysis triggering primer stroke of linker into bent conformation, microtubule reattachment forward, then phosphate and ADP release driving power stroke returning linker to straight conformation. Without ATP supply motor enters rigor bound to microtubule, transport stalls. Metabolic poisons depleting ATP, such as sodium azide plus deoxyglucose, arrest retrograde organelle movement within minutes, reversible upon washout. Phosphorylation of stalk modulates cargo affinity but not basal stepping, myosin II interaction irrelevant because actin and microtubule systems use distinct ATPases, nuclear envelope attachment mediates nucleokinesis only in specialized migrations requiring additional adaptors. Hence requirement for constant ATP reflects dependence of AAA1 catalytic turnover to reload each step, similar to myosin and kinesin ATPases but with larger ring architecture.

Ref: Shpetner J Cell Biol; dynein ATPase Km ~30μM, continuous Mg-ATP needed for stepping cycle.

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Cytoskeleton

What is the function of the linker domain in dynein?

Linker domain is a four helix bundle lever that spans AAA ring, mechanically linking tail to motor. In post power stroke conformation it is straight, docked near AAA5, positioning cargo forward. ATP binding to AAA1 induces closure of ring and steric clash pushing linker off AAA5 toward AAA2 docking site, forming bent pre power stroke state that primes motor. This repositioning stores elastic strain. Upon microtubule rebinding and ADP release, linker snaps back toward AAA5, executing forceful swing that drags cargo complex relative to microtubule by several nanometers. Because length exceeds 10 nanometers, small conformational changes at AAA1 amplified into large displacement. It contains no microtubule-binding motifs; those reside in stalk tip microtubule-binding domain. It never contacts actin filaments. Artificial insertion of flexible hinges or deletion uncouples ATPase activity from translocation, yielding motors that hydrolyze ATP futilely without movement, proving linker functions as transmission rod converting hydrolysis energy into directed mechanical work rather than scaffolding.

Ref: Schmidt Nature 2012; linker as lever converting ATP hydrolysis into mechanical work in dynein.

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Cytoskeleton

Which of the following statements about dynein heavy chains is TRUE?

Dynein heavy chains define motor identity through massive size and AAA ATPase ring arrangement unique among cytoskeletal motors distinguishable from kinesin and myosin smallest motors. Each 530 kDa heavy chain encodes six AAA plus repeats arranged in asymmetric ring with seventh pseudo AAA domain closing structure. AAA1 through AAA4 contain P loop Walker A motifs capable of nucleotide binding AAA1 major catalytic site hydrolyzing ATP for movement AAA2 through AAA4 bind ATP regulatory maintaining ring closure and coordinating allostery. AAA5 AAA6 structural no nucleotide hydrolysis. Linker arching over ring converts ring conformational wave to power stroke directed toward minus end. Unlike myosin containing actin binding cleft in motor domain dynein heavy chains possess separate stalk MTBD projecting from AAA4 no actin interaction ever. They do not polymerize into filaments but operate as obligate dimers via tail dimerization domain. Statements claiming actin binding or filament formation incorrect and inconsistent with biochemical assays. True distinguishing characteristic presence of ATPase domains within hexameric AAA ring converting ATP chemical energy to minus end movement and force generation for transport.

Ref: Roberts et al., Cell 2013 – Dynein heavy chain has ATPase domains within AAA+ ring true statement architecture.

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Cytoskeleton

What is the function of the dynactin complex in dynein activity?

Although dynein can bind microtubules and hydrolyze ATP alone in vitro motility assays in vivo most physiological functions require dynactin 1 MDa multiprotein complex discovered as activator of vesicle transport from squid axoplasm. Structure comprises Arp1 actin related protein filament 8 copies forming short filament 37 nm rod barbed end capped by CapZ pointed end by Arp11 p62 p25 p27 complex shoulder arm containing p150Glued DCTN1 with N terminal CAP Gly plus basic microtubule binding domains and long coiled coil dimer interacting with dynein intermediate chain via CC1 box. Dynactin performs dual roles: binding diverse cargo adaptors BicD2 Hook3 Spindly Ninein that select vesicles nuclei kinetochores via coiled coil cargo binding domains and tethering dynein to microtubules via p150Glued MTBD increasing processivity from limited hundred nanometer runs to several microns run length up to 10 microns. Cargo binding relieves dynein autoinhibition converting weak diffusive complex into high force super complex containing two dynein dimers for faster movement. Therefore dynactin links dynein to cargo and enhances processivity not inhibiting.

Ref: Schroer, Annu Rev Cell Dev Biol 2004 – Dynactin function linking dynein to cargo and enhancing processivity.

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Cytoskeleton

What happens to dynein when ATP binds to its AAA1 domain?

Nucleotide dependent affinity switching allows dynein stepping without dragging cargo backward and enables high force production. In high affinity state stalk MTBD binds tightly to microtubule lattice resisting detachment under piconewton loads up to 7 pN corresponding to apo or ADP bound heavy chain state with stalk helices registry CC1 CC2 in specific alignment. When ATP enters AAA1 pocket AAA ring closure pulls AAA5 and buttress subdomains sliding stalk coiled coils by half heptad altering MTBD helices arrangement converting to low affinity conformation reducing binding energy about tenfold causing rapid detachment within milliseconds from track enabling motor to diffuse forward and execute priming stroke where linker bends away from ring. Hydrolysis restores high affinity allowing reattachment a few tubulin dimers toward minus end about 8 nm displacement. This detach mechanism parallels myosin where ATP binding also dissociates rigor actomyosin linkage though dynein uses AAA ring allostery rather than direct cleft opening of actin binding site. Without transient detachment upon ATP binding motor would remain locked unable to step processively essential for transport under load.

Ref: Imamula et al., Cell 2007 – Detachment upon ATP binding to AAA1 causes dynein release.

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Cytoskeleton

Which domain of dynein contains the microtubule-binding region?

Dynein heavy chain organization subdivided into distinct functional modules arranged linearly from N to C terminus: tail stem region for cargo adaptor binding and dimerization via intermediate chain interactions, motor ring core, and stalk linker elements bridging. Tail contains dimerization domain plus binding sites for light intermediate chains controlling cargo selectivity not microtubule binding. Central motor ring of six AAA domains AAA1 to AAA6 forming closed asymmetric ring plus protruding antiparallel coiled coil stalk emerging between AAA4 small and large subdomains after second coiled coil sequence. At tip of stalk small globular microtubule binding domain MTBD consisting of six helices H1 to H6 that directly contacts alpha beta tubulin at intradimer interface between protofilaments. Conformational changes in ring driven by AAA1 ATP hydrolysis cause sliding of coiled coil helices within stalk altering MTBD affinity registry from high to low switching. Linker domain connects tail to ring and bends during power stroke converting ring closure to directional movement. Cargo binding domain separate near N terminus distinct from stalk. Therefore domain containing microtubule binding region is stalk domain linking motor ring to track.

Ref: Gibbons et al., J Cell Biol 2005; Carter 2013 – Dynein stalk domain contains microtubule-binding region.

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Cytoskeleton

The power stroke of dynein is initiated by:

Unlike kinesin or myosin where ATP binding induces neck linker docking lever swing generating force dynein power stroke triggered specifically by ATP hydrolysis chemistry within AAA1 site coupling ring closure to linker movement distinct mechanism. Cycle stages: apo or ADP state linker straight spanning ring from tail to opposite side AAA4-5 high affinity microtubule binding strong attachment. ATP binding closes ring but microtubule already detached due to previous allosteric signaling via stalk. Hydrolysis of ATP to ADP Pi inside AAA1 produces large conformational change transmitted via AAA ring helical shifts to linker causing linker to bend from straight to pre power stroke curved configuration storing elastic strain and priming microtubule binding stalk forward position. Pi release closes stalk registry favoring high affinity binding and force generation as linker straightens power stroke dragging cargo toward minus end up to 32 nm power stroke size. ADP release resets motor cycle. Experiments using vanadate ADP Pi analogues trap pre power stroke state showing absolute requirement of hydrolysis rather than just binding to initiate force providing efficiency.

Ref: Carter et al., Science 2011 – Dynein power stroke initiated by hydrolysis of ATP in AAA1 domain mechanism.

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Cytoskeleton

What is the primary function of dynein in cellular transport?

Cytoplasmic dynein 1 is sole minus end directed microtubule motor powering retrograde intracellular transport complementary to kinesin superfamily anterograde transport systems establishing bidirectional highways. Structural features include two identical 530 kDa heavy chains dimerized at tail each with six concatenated AAA ATPase domains forming asymmetric ring linker that swings across ring coiled coil stalk ending in microtubule binding domain MTBD and cargo binding tail associated with intermediate light intermediate and light chains LC8 Tctex. Motor moves toward microtubule minus ends anchored near nucleus centrosome stepping 8 to 32 nm due to variable power stroke size and flexible stalk allowing large steps under load. Functions include transport of endosomes lysosomes autophagosomes mRNA nuclei centrosome positioning Golgi clustering perinuclearly and retrograde axonal transport of neurotrophic growth factors and injury signals. It does not transport cargo toward plus ends that is kinesin role; does not directly stabilize or crosslink microtubules independent of transport though cortical anchoring contributes to pulling forces on spindle. Its minus direction ensures inward delivery and centralization.

Ref: Roberts et al., Nat Rev Mol Cell Biol 2011 – Cytoplasmic dynein minus end directed transport primary function.

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Cytoskeleton

What regulates kinesin-1 activation?

Kinesin-1 regulation avoids gratuitous ATP consumption and traffic jams by autoinhibition mechanism evolutionarily conserved ensuring motor only active when bound to cargo. In cargo free state heavy chain tail IAK motif folds back contacting motor head switch regions blocking microtubule stimulated ADP release maintaining ATPase low about 10 fold inhibition. Light chains also contribute to folded globular conformation compact. Cargo adaptors such as JNK interacting proteins JIP1-3 FEZ1 Milton TRAK bind light chains or tail sterically unfolding molecule to extended active state increasing microtubule affinity 20 fold and ATPase rate. ATP hydrolysis itself occurs in motor domain P loop driving stepping not in stalk domain which serves purely structural coiled coil dimerization role linking heads. Thus activation triggered by cargo binding induced unfolding not stalk ATP hydrolysis. ATP hydrolysis at motor provides energy for movement. This regulatory paradigm ensures motor travels only when loaded linking ATP cycles to cargo availability for processive anterograde movement failure leads to organelle mislocalization neurodegeneration and developmental defects.

Ref: Verhey & Hammond, Nat Rev Mol Cell Biol 2009 – Kinesin-1 activation by cargo binding unfolding mechanism.

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Cytoskeleton

The stalk domain of kinesin-1 is responsible for:

Domain architecture of kinesin-1 reveals clear division of labor essential for processive dimer motor function. N terminal globular motor domain about 340 amino acids contains P loop ATPase site switch I II microtubule binding interface and neck linker for mechanochemistry converting ATP hydrolysis to mechanical step. Immediately C terminal lies neck coiled coil ensuring dimerization of motor domains upon folding critical for hand over hand coordination followed by elongated stalk extending about 50 nm formed by interrupted coiled coil heptad repeats left handed supercoils stabilizing heavy chain dimer through hydrophobic packing of a and d positions leucine zipper like. Disruption of stalk by mutation prevents dimer formation and abolishes processivity yielding monomeric non processive motor. Central hinge regions provide flexibility for autoinhibited folding where C terminal tail interacts with motor domains to block ATPase until cargo binding unfolds. C terminal globular tail binds kinesin light chains via heptad interactions adapting to diverse vesicles mitochondria mRNA. Thus stalk responsible for dimerization via coiled coil interaction not ATP hydrolysis.

Ref: Hirokawa & Noda, Physiol Rev 2008 – Kinesin-1 stalk coiled coil mediated dimerization domain function.

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