Delineation of the TRAK binding regions of the kinesin-1 motor proteins.
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ABSTRACT:
SUBMITTER: Randall TS
PROVIDER: S-EPMC3853714 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Almost 25 years of kinesin research have led to the accumulation of a large body of knowledge about this widespread superfamily of motor and nonmotor proteins present in all eukaryotic cells. This review covers developments in kinesin research with an emphasis on structural aspects obtained by X-ray crystallography and cryoelectron microscopy 3-D analysis on kinesin motor domains complexed to microtubules.
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Project description:[Figure: see text].
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Project description:The human genome encodes 45 kinesin motor proteins that drive cell division, cell motility, intracellular trafficking and ciliary function. Determining the cellular function of each kinesin would benefit from specific small-molecule inhibitors. However, screens have yielded only a few specific inhibitors. Here we present a novel chemical-genetic approach to engineer kinesin motors that can carry out the function of the wild-type motor yet can also be efficiently inhibited by small, cell-permeable molecules. Using kinesin-1 as a prototype, we develop two independent strategies to generate inhibitable motors, and characterize the resulting inhibition in single-molecule assays and in cells. We further apply these two strategies to create analogously inhibitable kinesin-3 motors. These inhibitable motors will be of great utility to study the functions of specific kinesins in a dynamic manner in cells and animals. Furthermore, these strategies can be used to generate inhibitable versions of any motor protein of interest.
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Project description:Kinesins and myosins hydrolyze ATP, producing force that drives spindle assembly, vesicle transport and muscle contraction. How do motors do this? Here we discuss mechanisms of motor force transduction, based on their mechanochemical cycles and conformational changes observed in crystal structures. Distortion or twisting of the central ?-sheet - proposed to trigger actin-induced Pi and ADP release by myosin, and microtubule-induced ADP release by kinesins - is shown in a movie depicting the transition between myosin ATP-like and nucleotide-free states. Structural changes in the switch I region form a tube that governs ATP hydrolysis and Pi release by the motors, explaining the essential role of switch I in hydrolysis. Comparison of the motor power strokes reveals that each stroke begins with the force-amplifying structure oriented opposite to the direction of rotation or swing. Motors undergo changes in their mechanochemical cycles in response to small-molecule inhibitors, several of which bind to kinesins by induced fit, trapping the motors in a state that resembles a force-producing conformation. An unusual motor activator specifically increases mechanical output by cardiac myosin, potentially providing valuable information about its mechanism of function. Further study is essential to understand motor mechanochemical coupling and energy transduction, and could lead to new therapies to treat human disease.
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Project description:In neurons, the proper distribution of mitochondria is essential because of a requirement for high energy and calcium buffering during synaptic neurotransmission. The efficient, regulated transport of mitochondria along axons to synapses is therefore crucial for maintaining function. The trafficking kinesin protein (TRAK)/Milton family of proteins comprises kinesin adaptors that have been implicated in the neuronal trafficking of mitochondria via their association with the mitochondrial protein Miro and kinesin motors. In this study, we used gene silencing by targeted shRNAi and dominant negative approaches in conjunction with live imaging to investigate the contribution of endogenous TRAKs, TRAK1 and TRAK2, to the transport of mitochondria in axons of hippocampal pyramidal neurons. We report that both strategies resulted in impairing mitochondrial mobility in axonal processes. Differences were apparent in terms of the contribution of TRAK1 and TRAK2 to this transport because knockdown of TRAK1 but not TRAK2 impaired mitochondrial mobility, yet both TRAK1 and TRAK2 were shown to rescue transport impaired by TRAK1 gene knock-out. Thus, we demonstrate for the first time the pivotal contribution of the endogenous TRAK family of kinesin adaptors to the regulation of mitochondrial mobility.
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Project description:Dynein and kinesin motor proteins transport cellular cargoes toward opposite ends of microtubule tracks. In neurons, microtubules are abundantly decorated with microtubule-associated proteins (MAPs) such as tau. Motor proteins thus encounter MAPs frequently along their path. To determine the effects of tau on dynein and kinesin motility, we conducted single-molecule studies of motor proteins moving along tau-decorated microtubules. Dynein tended to reverse direction, whereas kinesin tended to detach at patches of bound tau. Kinesin was inhibited at about a tenth of the tau concentration that inhibited dynein, and the microtubule-binding domain of tau was sufficient to inhibit motor activity. The differential modulation of dynein and kinesin motility suggests that MAPs can spatially regulate the balance of microtubule-dependent axonal transport.
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Project description:The regulation of molecular motors is an important cellular problem, as motility in the absence of cargo results in futile adenosine triphosphate hydrolysis. When not transporting cargo, the microtubule (MT)-based motor Kinesin-1 is kept inactive as a result of a folded conformation that allows autoinhibition of the N-terminal motor by the C-terminal tail. The simplest model of Kinesin-1 activation posits that cargo binding to nonmotor regions relieves autoinhibition. In this study, we show that binding of the c-Jun N-terminal kinase-interacting protein 1 (JIP1) cargo protein is not sufficient to activate Kinesin-1. Because two regions of the Kinesin-1 tail are required for autoinhibition, we searched for a second molecule that contributes to activation of the motor. We identified fasciculation and elongation protein zeta1 (FEZ1) as a binding partner of kinesin heavy chain. We show that binding of JIP1 and FEZ1 to Kinesin-1 is sufficient to activate the motor for MT binding and motility. These results provide the first demonstration of the activation of a MT-based motor by cellular binding partners.
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Project description:Mitochondrial distribution and motility are recognized as central to many cellular functions, but their regulation by signaling mechanisms remains to be elucidated. Here, we report that reactive oxygen species (ROS), either derived from an extracellular source or intracellularly generated, control mitochondrial distribution and function by dose-dependently, specifically, and reversibly decreasing mitochondrial motility in both rat hippocampal primary cultured neurons and cell lines. ROS decrease motility independently of cytoplasmic [Ca2+], mitochondrial membrane potential, or permeability transition pore opening, known effectors of oxidative stress. However, multiple lines of genetic and pharmacological evidence support that a ROS-activated mitogen-activated protein kinase (MAPK), p38?, is required for the motility inhibition. Furthermore, anchoring mitochondria directly to kinesins without involvement of the physiological adaptors between the organelles and the motor protein prevents the H2O2-induced decrease in mitochondrial motility. Thus, ROS engage p38? and the motor adaptor complex to exert changes in mitochondrial motility, which likely has both physiological and pathophysiological relevance.
| S-EPMC5710826 | biostudies-literature
Project description:BackgroundThe Gram-negative bacterium Haemophilus influenzae is a glutathione auxotroph and acquires the redox-active tripeptide by import. The dedicated glutathione transporter belongs to the ATP-binding cassette (ABC)-transporter superfamily and displays more than 60% overall sequence identity with the well-studied dipeptide (Dpp) permease of Escherichia coli. The solute binding protein (SBP) that mediates glutathione transport in H. influenzae is a lipoprotein termed GbpA and is 54% identical to E. coli DppA, a well-studied member of family 5 SBP's. The discovery linking GbpA to glutathione import came rather unexpectedly as this import-priming SBP was previously annotated as a heme-binding protein (HbpA), and was thought to mediate heme acquisition. Nonetheless, although many SBP's have been implicated in more than one function, a prominent physiological role for GbpA and its partner permease in heme acquisition appears to be very unlikely. Here, we sought to characterize five representative GbpA homologs in an effort to delineate the novel GbpA-family of glutathione-specific family 5 SBPs and to further clarify their functional role in terms of ligand preferences.ResultsLipoprotein and non-lipoprotein GbpA homologs were expressed in soluble form and substrate specificity was evaluated via a number of ligand binding assays. A physiologically insignificant affinity for hemin was observed for all five GbpA homologous test proteins. Three out of five test proteins were found to bind glutathione and some of its physiologically relevant derivatives with low- or submicromolar affinity. None of the tested SBP family 5 allocrites interacted with the remaining two GbpA test proteins. Structure-based sequence alignments and phylogenetic analysis show that the two binding-inert GbpA homologs clearly form a separate phylogenetic cluster. To elucidate a structure-function rationale for this phylogenetic differentiation, we determined the crystal structure of one of the GbpA family outliers from H. parasuis. Comparisons thereof with the previously determined structure of GbpA in complex with oxidized glutathione reveals the structural basis for the lack of allocrite binding capacity, thereby explaining the outlier behavior.ConclusionsTaken together, our studies provide for the first time a collective functional look on a novel, Pasteurellaceae-specific, SBP subfamily of glutathione binding proteins, which we now term GbpA proteins. Our studies strongly implicate GbpA family SBPs in the priming step of ABC-transporter-mediated translocation of useful forms of glutathione across the inner membrane, and rule out a general role for GbpA proteins in heme acquisition.
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Project description:Protein translocation is an important cellular process. SecA is an essential protein component in the Sec system, as it contains the molecular motor that facilitates protein translocation. In this study, a bioinformatics approach was applied in the search for possible lipid-binding helix regions in protein translocation motor proteins. Novel lipid-binding regions in Escherichia coli SecA were identified. Remarkably, multiple lipid-binding sites were also identified in other motor proteins such as BiP, which is involved in ER protein translocation. The prokaryotic signal recognition particle receptor FtsY, though not a motor protein, is in many ways related to SecA, and was therefore included in this study. The results demonstrate a possible general feature for motor proteins involved in protein translocation.
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