LIPRIN-ALPHA 调控驱动蛋白 KIF1A 的 结构生物学研究

       细胞内物质运输对于神经元发育、成熟和功能发挥至关重要。神经元具有长轴突和短树突的极性结构特点， 并发展出了高度精细和有效的运输系统。 神经元的运输系统由众多的运输机器组成。 Kinesin 超家族的驱动蛋白就属于这样的运输机器，在细胞内沿着微管的货物运输中起关键作用。Kinesin 驱动蛋白能够水解 ATP，以正向运动或反向运动的方式转运各种货物， 包括线粒体、 mRNA、囊泡、溶酶体等。
这些负责货物运输的驱动蛋白的突变会导致严重的神经系统疾病。 此外， kinesin 存在一种自抑制状态，这种自抑制状态能够降低其 A TP 酶的活性，并调节货物的转运。然而，对于 kinesin 如何维持这种自抑制状态仍然有许多谜团。
       KIF1A/UNC-104 属于 kinesin-3 亚家族。 KIF1A 是一种在神经元内特异性表达、沿着微管运动的驱动蛋白。它负责突触小泡前体在轴突内的正向运输和致密核心囊泡向神经末梢的转运。另一方面，liprin-α 是一种在神经元突触区域聚集的支架蛋白，可以调节  KIF1A 介导的货物运输，但其背后的分子机制仍不清楚。
       在这项研究中，我们纯化出全长 KIF1A 蛋白， 并用负染观察到部分 KIF1A 呈现为折叠紧密的单体状态。另外，我们通过免疫共沉淀确定了 KIF1A  与  liprin-α2上介导两者结合的区域。接下来，我们将利用冷冻电镜技术解析全长  KIF1A 的结构，揭示其自抑制的机理，并进一步解释  KIF1A  与  liprin-α 的结合机制，以最终阐明神经元中调节突触囊泡转运的分子机制。

       Intracellular  trafficking  is  essential  for  neuronal  development,  maturation  and function.  Neuron  is  characterized  by  its  polarized  structure  with  long  axon  and  short dendrites,  and  develops  a  more  delicate  and  efficient  transport  system.  The  transport system  in neurons  is  composed of numerous and various transport machines. The  motor proteins of  kinesin superfamily  are  such  transport machines, which  play  a key role in intracellular cargo transport along microtubules. These kinesin motors  can hydrolyze ATP to deliver a variety of cargos including mitochondria, mRNA, vesicles, lysosomes and so on, in an anterograde or retrograde manner. The mutations in these cargo transport motor proteins  may  result in serious neurological diseases.  Moreover,  kinesin  proteins enable an auto-inhibited conformation to reduce its ATPase activity and regulate cargo transport.
However,  it  still  largely  remains  unknown  about  how  kinesin  proteins  maintain  this autoinhibition  state.  KIF1A/UNC-104 belongs to kinesin-3  subfamily. Previous  studies revealed  that KIF1A is a neuron-specific microtubule-based motor protein in charge of anterograde axonal transport of synaptic vesicle precursors.  KIF1A is also responsible for
the trafficking of dense-core vesicle  towards the  nerve terminals.  Meanwhile,  liprin-α  is a scaffolding protein enriched at neuron synapses, which was found to regulate the cargo transport of KIF1A. But the mechanism underlying this modulation is still unclear .
       During this research,  we have purified full-length KIF1A and performed negative staining  to  observe  that  part  of  these  KIF1A  proteins  exist  as  folded  monomers.  In addition, we used co-immunoprecipitation to identify the binding regions on both KIF1A
and liprin-α2.  Next,  we will take  advantage of Cryo-EM technology to solve  the  structure of full-length KIF1A,  explain the autoinhibition of it,  and  further  explore the  binding mechanism  of  KIF1A  and  liprin-α,  in  order  to  finally  shed  light  on  the  regulatory mechanism of the synaptic vesicle transport in neurons.

[1] HIROKAWA N, NODA Y. Intracellular transport and kinesin superfamily proteins, KIFs: structure, function, and dynamics [J]. Physiol Rev, 2008, 88(3): 1089-118.

[2] HIROKAWA N, NIWA S, TANAKA Y. Molecular motors in neurons: transport mechanisms and roles in brain function, development, and disease [J]. Neuron, 2010, 68(4): 610-38.

[3] GUILLAUD L, EL-AGAMY S E, OTSUKI M, et al. Anterograde Axonal Transport in Neuronal Homeostasis and Disease [J]. Front Mol Neurosci, 2020, 13: 556175.

[4] SWEENEY H L, HOLZBAUR E L F. Motor Proteins [J]. Cold Spring Harb Perspect Biol, 2018, 10(5).

[5] NABB A T, FRANK M, BENTLEY M. Smart motors and cargo steering drive kinesin-mediated selective transport [J]. Mol Cell Neurosci, 2020, 103: 103464.

[6] SLEIGH J N, ROSSOR A M, FELLOWS A D, et al. Axonal transport and neurological disease [J]. Nat Rev Neurol, 2019, 15(12): 691-703.

[7] D V R, S R T, P S M. Identification of a novel force-generating protein, kinesin, involved in microtubule-based motility [J]. Cell, 1985, 42(1).

[8] LAWRENCE C J, DAWE R K, CHRISTIE K R, et al. A standardized kinesin nomenclature [J]. J Cell Biol, 2004, 167(1): 19-22.

[9] H M, M S, K K, et al. All kinesin superfamily protein, KIF, genes in mouse and human [J]. Proceedings of the National Academy of Sciences of the United States of America, 2001, 98(13).

[10] HIROKAWA N, TANAKA Y. Kinesin superfamily proteins (KIFs): Various functions and their relevance for important phenomena in life and diseases [J]. Exp Cell Res, 2015, 334(1): 16-25.

[11] HIROKAWA N, NODA Y, TANAKA Y, et al. Kinesin superfamily motor proteins and intracellular transport [J]. Nat Rev Mol Cell Biol, 2009, 10(10): 682-96.

[12] TORRES J Z, SUMMERS M K, PETERSON D, et al. The STARD9/Kif16a kinesin associates with mitotic microtubules and regulates spindle pole assembly [J]. Cell, 2011, 147(6): 1309-23.

[13] HOEPFNER S, SEVERIN F, CABEZAS A, et al. Modulation of receptor recycling and degradation by the endosomal kinesin KIF16B [J]. Cell, 2005, 121(3): 437-50.

[14] OKADA Y, YAMAZAKI H, SEKINE-AIZAWA Y, et al. The neuron-specific kinesin superfamily protein KIF1A is a unique monomeric motor for anterograde axonal transport of synaptic vesicle precursors [J]. Cell, 1995, 81(5): 769-80.

[15] GUEDES-DIAS P, HOLZBAUR E L F. Axonal transport: Driving synaptic function [J]. Science, 2019, 366(6462).

[16] MAEDER C I, SAN-MIGUEL A, WU E Y, et al. In vivo neuron-wide analysis of synaptic vesicle precursor trafficking [J]. Traffic, 2014, 15(3): 273-91.

[17] GONDRE-LEWIS M C, PARK J J, LOH Y P. Cellular mechanisms for the biogenesis and transport of synaptic and dense-core vesicles [J]. Int Rev Cell Mol Biol, 2012, 299: 27-115.

[18] RIZALAR F S, ROOSEN D A, HAUCKE V. A Presynaptic Perspective on Transport and Assembly Mechanisms for Synapse Formation [J]. Neuron, 2021, 109(1): 27-41.

[19] HALL D H, HEDGECOCK E M. Kinesin-related gene unc-104 is required for axonal transport of synaptic vesicles in C. elegans [J]. Cell, 1991, 65(5): 837-47.

[20] YONEKAWA Y, HARADA A, OKADA Y, et al. Defect in synaptic vesicle precursor transport and neuronal cell death in KIF1A motor protein-deficient mice [J]. J Cell Biol, 1998, 141(2): 431-41.

[21] KONDO M, TAKEI Y, HIROKAWA N. Motor protein KIF1A is essential for hippocampal synaptogenesis and learning enhancement in an enriched environment [J]. Neuron, 2012, 73(4): 743-57.

[22] PENNINGS M, SCHOUTEN M I, VAN GAALEN J, et al. KIF1A variants are a frequent cause of autosomal dominant hereditary spastic paraplegia [J]. Eur J Hum Genet, 2020, 28(1): 40-9.

[23] NICITA F, GINEVRINO M, TRAVAGLINI L, et al. Heterozygous KIF1A variants underlie a wide spectrum of neurodevelopmental and neurodegenerative disorders [J]. J Med Genet, 2020.

[24] BARKUS R V, KLYACHKO O, HORIUCHI D, et al. Identification of an axonal kinesin-3 motor for fast anterograde vesicle transport that facilitates retrograde transport of neuropeptides [J]. Mol Biol Cell, 2008, 19(1): 274-83.

[25] LO K Y, KUZMIN A, UNGER S M, et al. KIF1A is the primary anterograde motor protein required for the axonal transport of dense-core vesicles in cultured hippocampal neurons [J]. Neurosci Lett, 2011, 491(3): 168-73.

[26] HUO L, YUE Y, REN J, et al. The CC1-FHA tandem as a central hub for controlling the dimerization and activation of kinesin-3 KIF1A [J]. Structure, 2012, 20(9): 1550-61.

[27] KLOPFENSTEIN D R, TOMISHIGE M, STUURMAN N, et al. Role of phosphatidylinositol (4, 5) bisphosphate organization in membrane transport by the Unc104 kinesin motor [J]. Cell, 2002, 109(3): 347-58.

[28] OKADA Y, HIROKAWA N. A processive single-headed motor: kinesin superfamily protein KIF1A [J]. Science, 1999, 283(5405): 1152-7.

[29] OKADA Y, HIROKAWA N. Mechanism of the single-headed processivity: diffusional anchoring between the K-loop of kinesin and the C terminus of tubulin [J]. Proc Natl Acad Sci U S A, 2000, 97(2): 640-5.

[30] HIROKAWA N, NITTA R, OKADA Y. The mechanisms of kinesin motor motility: lessons from the monomeric motor KIF1A [J]. Nat Rev Mol Cell Biol, 2009, 10(12): 877-84.

[31] TOMISHIGE M, KLOPFENSTEIN D R, VALE R D. Conversion of Unc104/KIF1A kinesin into a processive motor after dimerization [J]. Science, 2002, 297(5590): 2263-7.

[32] SOPPINA V, VERHEY K J. The family-specific K-loop influences the microtubule on-rate but not the superprocessivity of kinesin-3 motors [J]. Mol Biol Cell, 2014, 25(14): 2161-70.

[33] ZHANG K, FOSTER H E, RONDELET A, et al. Cryo-EM Reveals How Human Cytoplasmic Dynein Is Auto-inhibited and Activated [J]. Cell, 2017, 169(7): 1303-14 e18.

[34] YANG S, TIWARI P, LEE K H, et al. Cryo-EM structure of the inhibited (10S) form of myosin II [J]. Nature, 2020, 588(7838): 521-5.

[35] LEE J R, SHIN H, CHOI J, et al. An intramolecular interaction between the FHA domain and a coiled coil negatively regulates the kinesin motor KIF1A [J]. EMBO J, 2004, 23(7): 1506-15.

[36] REN J, WANG S, CHEN H, et al. Coiled-coil 1-mediated fastening of the neck and motor domains for kinesin-3 autoinhibition [J]. Proc Natl Acad Sci U S A, 2018, 115(51): E11933-e42.

[37] STUCCHI R, PLUCINSKA G, HUMMEL J J A, et al. Regulation of KIF1A-Driven Dense Core Vesicle Transport: Ca(2+)/CaM Controls DCV Binding and Liprin-alpha/TANC2 Recruits DCVs to Postsynaptic Sites [J]. Cell Rep, 2018, 24(3): 685-700.

[38] HAMMOND J W, CAI D, BLASIUS T L, et al. Mammalian Kinesin-3 motors are dimeric in vivo and move by processive motility upon release of autoinhibition [J]. PLoS Biol, 2009, 7(3): e72.

[39] MILLER K E, DEPROTO J, KAUFMANN N, et al. Direct observation demonstrates that Liprinalpha is required for trafficking of synaptic vesicles [J]. Curr Biol, 2005, 15(7): 684-9.

[40] HSU C C, MONCALEANO J D, WAGNER O I. Sub-cellular distribution of UNC-104(KIF1A) upon binding to adaptors as UNC-16(JIP3), DNC-1(DCTN1/Glued) and SYD-2(Liprin-alpha) in C. elegans neurons [J]. Neuroscience, 2011, 176: 39-52.

[41] WAGNER O I, ESPOSITO A, KöHLER B, et al. Synaptic scaffolding protein SYD-2 clusters and activates kinesin-3 UNC-104 in C. elegans [J]. Proc Natl Acad Sci U S A, 2009, 106(46): 19605-10.

[42] SHIN H, WYSZYNSKI M, HUH K H, et al. Association of the kinesin motor KIF1A with the multimodular protein liprin-alpha [J]. J Biol Chem, 2003, 278(13): 11393-401.

[43] ATHERTON J, HUMMEL J J, OLIERIC N, et al. The mechanism of kinesin inhibition by kinesin-binding protein [J]. Elife, 2020, 9.

[44] WOZNIAK M J, MELZER M, DORNER C, et al. The novel protein KBP regulates mitochondria localization by interaction with a kinesin-like protein [J]. BMC Cell Biol, 2005, 6: 35.

[45] NIWA S, TANAKA Y, HIROKAWA N. KIF1Bbeta- and KIF1A-mediated axonal transport of presynaptic regulator Rab3 occurs in a GTP-dependent manner through DENN/MADD [J]. Nat Cell Biol, 2008, 10(11): 1269-79.

[46] OZYAVUZ CUBUK P. Goldberg-Shprintzen Syndrome Associated with a Novel Variant in the KIFBP Gene [J]. Mol Syndromol, 2021, 12(4): 240-3.

[47] SOLON A L, TAN Z, SCHUTT K L, et al. Kinesin-binding protein remodels the kinesin motor to prevent microtubule binding [J]. Sci Adv, 2021, 7(47): eabj9812.

[48] KEVENAAR J T, BIANCHI S, VAN SPRONSEN M, et al. Kinesin-Binding Protein Controls Microtubule Dynamics and Cargo Trafficking by Regulating Kinesin Motor Activity [J]. Curr Biol, 2016, 26(7): 849-61.

[49] MIYOSHI J, TAKAI Y. Dual role of DENN/MADD (Rab3GEP) in neurotransmission and neuroprotection [J]. Trends Mol Med, 2004, 10(10): 476-80.

[50] WADA M, NAKANISHI H, SATOH A, et al. Isolation and characterization of a GDP/GTP exchange protein specific for the Rab3 subfamily small G proteins [J]. J Biol Chem, 1997, 272(7): 3875-8.

[51] TANAKA M, MIYOSHI J, ISHIZAKI H, et al. Role of Rab3 GDP/GTP exchange protein in synaptic vesicle trafficking at the mouse neuromuscular junction [J]. Mol Biol Cell, 2001, 12(5): 1421-30.

[52] PATEL M R, SHEN K. RSY -1 is a local inhibitor of presynaptic assembly in C. elegans [J]. Science, 2009, 323(5920): 1500-3.

[53] SUDHOF T C. The presynaptic active zone [J]. Neuron, 2012, 75(1): 11-25.

[54] SCHOCH S, GUNDELFINGER E D. Molecular organization of the presynaptic active zone [J]. Cell Tissue Res, 2006, 326(2): 379-91.

[55] WU X, CAI Q, SHEN Z, et al. RIM and RIM-BP Form Presynaptic Active-Zone-like Condensates via Phase Separation [J]. Mol Cell, 2019, 73(5): 971-84 e5.

[56] SPANGLER S A, HOOGENRAAD C C. Liprin-alpha proteins: scaffold molecules for synapse maturation [J]. Biochem Soc Trans, 2007, 35(Pt 5): 1278-82.

[57] ZHEN M, JIN Y. The liprin protein SYD-2 regulates the differentiation of presynaptic termini in C. elegans [J]. Nature, 1999, 401(6751): 371-5.

[58] KITTELMANN M, HEGERMANN J, GONCHAROV A, et al. Liprin-alpha/SYD-2 determines the size of dense projections in presynaptic active zones in C. elegans [J]. J Cell Biol, 2013, 203(5): 849-63.

[59] DAI Y, TARU H, DEKEN S L, et al. SYD-2 Liprin-alpha organizes presynaptic active zone formation through ELKS [J]. Nat Neurosci, 2006, 9(12): 1479-87.

[60] OWALD D, FOUQUET W, SCHMIDT M, et al. A Syd-1 homologue regulates pre- and postsynaptic maturation in Drosophila [J]. J Cell Biol, 2010, 188(4): 565-79.

[61] LENIHAN J A, SAHA O, HEIMER-MCGINN V, et al. Decreased Anxiety-Related Behaviour but Apparently Unperturbed NUMB Function in Ligand of NUMB Protein-X (LNX) 1/2 Double Knockout Mice [J]. Mol Neurobiol, 2017, 54(10): 8090-109.

[62] GOODWIN P R, JUO P. The scaffolding protein SYD-2/Liprin-alpha regulates the mobility and polarized distribution of dense-core vesicles in C. elegans motor neurons [J]. PLoS One, 2013, 8(1): e54763.

[63] LI L, TIAN X, ZHU M, et al. Drosophila Syd-1, liprin-α, and protein phosphatase 2A B' subunit Wrd function in a linear pathway to prevent ectopic accumulation of synaptic materials in distal axons [J]. J Neurosci, 2014, 34(25): 8474-87.

[64] EDWARDS S L, YORKS R M, MORRISON L M, et al. Synapse-Assembly Proteins Maintain Synaptic Vesicle Cluster Stability and Regulate Synaptic Vesicle Transport in Caenorhabditiselegans [J]. Genetics, 2015, 201(1): 91-116.

[65] EDWARDS S L, MORRISON L M, YORKS R M, et al. UNC-16 (JIP3) Acts Through SynapseAssembly Proteins to Inhibit the Active Transport of Cell Soma Organelles to Caenorhabditis elegans Motor Neuron Axons [J]. Genetics, 2015, 201(1): 117-41.

[66] LIANG M, XIE X, PAN J, et al. Structural basis of the target-binding mode of the G proteincoupled receptor kinase-interacting protein in the regulation of focal adhesion dynamics [J]. J Biol Chem, 2019, 294(15): 5827-39.

[67] SALA K, CORBETTA A, MINICI C, et al. The ERC1 scaffold protein implicated in cell motility drives the assembly of a liquid phase [J]. Sci Rep, 2019, 9(1): 13530.

[68] WENTZEL C, SOMMER J E, NAIR R, et al. mSYD1A, a mammalian synapse-defective-1 protein, regulates synaptogenic signaling and vesicle docking [J]. Neuron, 2013, 78(6): 1012-23.

[69] CHENG Y, GRIGORIEFF N, PENCZEK P A, et al. A primer to single-particle cryo-electron microscopy [J]. Cell, 2015, 161(3): 438-49.

[70] ADRIAN M, DUBOCHET J, LEPAULT J, et al. Cryo-electron microscopy of viruses [J]. Nature, 1984, 308(5954): 32-6.

[71] CHENG Y. Single-particle cryo-EM-How did it get here and where will it go [J]. Science, 2018, 361(6405): 876-80.

[72] EARL L A, FALCONIERI V, MILNE J L S, et al. Cryo-EM: beyond the microscope [J]. Current Opinion in Structural Biology, 2017, 46: 71-8.

[73] MURATA K, WOLF M. Cryo-electron microscopy for structural analysis of dynamic biological macromolecules [J]. Biochim Biophys Acta Gen Subj, 2018, 1862(2): 324-34.

[74] MUNK C, MUTT E, ISBERG V, et al. An online resource for GPCR structure determination and analysis [J]. Nat Methods, 2019, 16(2): 151-62.

[75] SHEN J, ZHANG D, FU Y , et al. Cryo-EM structures of human bradykinin receptor-Gq proteins complexes [J]. Nat Commun, 2022, 13(1): 714.

[76] MARTYNOWYCZ M W, SHIRIAEVA A, GE X, et al. MicroED structure of the human adenosine receptor determined from a single nanocrystal in LCP [J]. Proc Natl Acad Sci U S A, 2021, 118(36).

[77] DANELIUS E, GONEN T. Protein and Small Molecule Structure Determination by the CryoEM Method MicroED [J]. Methods Mol Biol, 2021, 2305: 323-42.

[78] YAO H, SONG Y, CHEN Y, et al. Molecular Architecture of the SARS-CoV -2 Virus [J]. Cell, 2020, 183(3): 730-8 e13.

[79] TURK M, BAUMEISTER W. The promise and the challenges of cryo-electron tomography [J]. FEBS Lett, 2020, 594(20): 3243-61.

[80] NI T, FROSIO T, MENDONCA L, et al. High-resolution in situ structure determination by cryoelectron tomography and subtomogram averaging using emClarity [J]. Nat Protoc, 2022, 17(2): 421-44.

[81] DANDEY V P, WEI H, ZHANG Z, et al. Spotiton: New features and applications [J]. J Struct Biol, 2018, 202(2): 161-9.

[82] NAKANE T, KOTECHA A, SENTE A, et al. Single-particle cryo-EM at atomic resolution [J]. Nature, 2020, 587(7832): 152-6.

[83] STRACK R. Cryo-EM goes atomic [J]. Nat Methods, 2020, 17(12): 1175.

[84] NAYDENOVA K, JIA P, RUSSO C J. Cryo-EM with sub-1 Å specimen movement [J]. Science, 2020, 370(6513): 223-6.

[85] KIM H U, JUNG H S. Cryo-EM as a powerful tool for drug discovery: recent structural based studies of SARS-CoV -2 [J]. Appl Microsc, 2021, 51(1): 13.

[86] KOIFMAN N, TALMON Y. Cryogenic Electron Microscopy Methodologies as Analytical Tools for the Study of Self-Assembled Pharmaceutics [J]. Pharmaceutics, 2021, 13(7).

[87] CAO Y, WANG J, JIAN F, et al. Omicron escapes the majority of existing SARS-CoV -2 neutralizing antibodies [J]. Nature, 2021.

[88] 李敏,杨谦.一种高效构建同源重组 DNA 片段的方法--融合 PCR [J].中国生物工程杂志, 2007, (08): 53-8.

[89] LI S, XIE T, LIU P, et al. Structural insights into the assembly and substrate selectivity of human SPT -ORMDL3 complex [J]. Nat Struct Mol Biol, 2021, 28(3): 249-57.

[90] NITTA R, OKADA Y, HIROKAWA N. Structural model for strain-dependent microtubule activation of Mg-ADP release from kinesin [J]. Nat Struct Mol Biol, 2008, 15(10): 1067-75.

[91] NITTA R, KIKKAWA M, OKADA Y, et al. KIF1A alternately uses two loops to bind microtubules [J]. Science, 2004, 305(5684): 678-83.

[92] HISANAGA S, MUROFUSHI H, OKUHARA K, et al. The molecular structure of adrenal medulla kinesin [J]. Cell Motil Cytoskeleton, 1989, 12(4): 264-72.

[93] JUMPER J, EVANS R, PRITZEL A, et al. Highly accurate protein structure prediction with AlphaFold [J]. Nature, 2021, 596(7873): 583-9.

[94] TUNYASUVUNAKOOL K, ADLER J, WU Z, et al. Highly accurate protein structure prediction for the human proteome [J]. Nature, 2021, 596(7873): 590-6.

[95] SCARFF C A, CARRINGTON G, CASAS-MAO D, et al. Structure of the shutdown state of myosin-2 [J]. Nature, 2020, 588(7838): 515-20