Book of Abstracts: Albany 2007
June 19-23 2007
Intracellular Transport and Kinesin Superfamily Proteins: Structure Dynamics and Function
The intracellular transport is fundamental for cell morphogenesis, functioning and survival. To elucidate this mechanism we have identified and characterized kinesin superfamily proteins, KIFs, using molecular cell biology, molecular genetics, biophysics, X ray crystallography and cryoelectron microscopy (1-3). KIF1A and KIF1B beta transport synaptic vesicle precursors and play essential roles on neuronal function and survival (5,8,15). KIF1B alpha and KIF5s transport mitochondria(4,9). KIF17 conveys NMDA type glutamate receptors, important for memory and learning, in dendrites through the interaction with scaffolding protein complex containing mLin10 (Mint1)(13). AMPA type glutamate receptors are transported by KIF5s via GRIP1-GluR2 interaction (16). KIF5s also transport specific mRNAs with a large protein complex composed of 42 proteins (21). KIF2A, a unique middle motor domain KIF, plays a significant role in brain wiring by depolymerizing microtubules in growth cones and controlling extension of axonal branches (17,19). KIF3 complex composed of KIF3A, KIF3B and KAP3 is fundamental for left-right determination of our body through formation of monocilia in the ventral node which rotate and generate leftward flow of extra embryonic fluid, nodal flow (10,25,26). This nodal flow conveys vesicular parcels containing Sonic hedgehog and retinoic acid secreted from node cells by the trigger with FGF signaling toward left and determines left-right asymmetry (24,26). Conditional gene targeting study of KAP3, an associated protein of KIF3 motor complex revealed that KIF3 suppresses tumorigenesis by transporting N cadherin - ??catenin complex from cytoplasm which works as a transcriptional factor in the nucleus with T cell factor and enhances cell proliferation as a signaling molecule down stream of Wnt canonical pathway from Golgi to plasma membrane(23). KIF4 binds poly ADP ribose polymerase 1(PARP1)?and works as a molecular switch to control activity dependent neuronal survival during brain development (27). Thus, KIFs play a number of significant roles not only on intracellular transport, but also on higher brain functions, brain wiring, fundamental developmental events such as left-right asymmetry, tumorigenesis and activity dependent neuronal survival during brain development.
Concerning the mechanism of motility we discovered that KIF 1A is a unique monomeric motor and revealed high resolution structures of motor domain at atomic level by cryoelectron microscopy combined with X-ray crystallography of 5 different nucleotide binding states during ATP hydrolysis and elucidated the mechanism of processive movement of kinesin motors on the microtubules. Because previously identified motors function as dimers such as kinesin, dynein and myosin, the prevailing hypothesis for motor movement was the hand over hand model, which means a motor needs two legs to move as humans do. However, we discovered simplest monomeric motor KIF1A (single leg) and demonstrated how the single monomeric KIF1A motor molecule can move processively on microtubules by biased Brownian movement using biophysical approaches such as single molecule motility assay and optical trapping combined with cryoelectron microscopy and X-ray crystallography of five critical transitional states during ATP hydrolysis (11,12,14,18,20). Atomic structures of AMP/PCP (preisomerization, strong binding) state, AMP/PNP (prehydrolysis, strong binding) state, ADP/AlFx (early ADP/Pi, strong binding) state, ADP vanadate (late ADP/Pi, active detaching) state and ADP (weak binding) state were solved and it was shown that KIF1A uses two microtubule-binding loops in an alternating manner to change its interaction with microtubules during ATP hydrolysis cycles; loop 11 is extended to bind helix 11? of tubulin in protofilament in the AMP-PNP state, then loop 11 dissociates from tubulin at ADP vanadate state in which KIF1A actively detaches from microtubules and at ADP state loop 12 extends and K-loop in loop 12 binds flexible C-terminus of tubulin (E-hook) which allows Brownian movement of KIF1A (14,18,20). Then after ADP release KIF1A moves to the microtubule?s plus end by ~3nm on average on binding to the microtubule (18). Thus, we showed that ATP hydrolysis is used to actively detach from microtubules and cause plus end bias and movement is mainly based on Brownian motion.
References and Footnotes
Graduate School of Medicine,