African Swine Fever Virus Protein p54 Interacts with the Microtubular Motor Complex through Direct Binding to Light-Chain Dynein

Alonso, Covadonga; Miskin, James; Hernáez, Bruno; Fernandez-Zapatero, Patricia; Soto, Lourdes Diaz; Cantó, Carmen; Rodrı́guez-Crespo, Ignacio; Dixon, Linda K.; Escribano, José M.

doi:10.1128/jvi.75.20.9819-9827.2001

Cited by 176 publications

(186 citation statements)

References 62 publications

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“…Higher apparent affinity of DYNLL binding to dimeric protein fragments was first noted by studying DYNLL2 and myoVa interactions (2) and later attributed to avidity (15). This hypothesis questioned the original proposal of heterodimeric cargo adapter role of DYNLL on dynein and myoVa motor complexes (6,7,21,(55)(56)(57)(58).…”

Section: Fine-tuning Of Affinities Is Achieved Through Diversity Of Bmentioning

confidence: 85%

Affinity, Avidity, and Kinetics of Target Sequence Binding to LC8 Dynein Light Chain Isoforms

Radnai

Rapali

Hódi

et al. 2010

Journal of Biological Chemistry

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LC8 dynein light chain (DYNLL) is a highly conserved eukaryotic hub protein with dozens of binding partners and various functions beyond being a subunit of dynein and myosin Va motor proteins. Here, we compared the kinetic and thermodynamic parameters of binding of both mammalian isoforms, DYNLL1 and DYNLL2, to two putative consensus binding motifs (KXTQTX and XG(I/V)QVD) and report only subtle differences. Peptides containing either of the above motifs bind to DYNLL2 with micromolar affinity, whereas a myosin Va peptide (lacking the conserved Gln) and the noncanonical Pak1 peptide bind with K d values of 9 and 40 M, respectively. Binding of the KXTQTX motif is enthalpy-driven, although that of all other peptides is both enthalpy-and entropy-driven. Moreover, the KXTQTX motif shows strikingly slower off-rate constant than the other motifs. As most DYNLL partners are homodimeric, we also assessed the binding of bivalent ligands to DYNLL2. Compared with monovalent ligands, a significant avidity effect was found as follows: K d values of 37 and 3.5 nM for a dimeric myosin Va fragment and a Leu zipper dimerized KXTQTX motif, respectively. Ligand binding kinetics of DYNLL can best be described by a conformational selection model consisting of a slow isomerization and a rapid binding step. We also studied the binding of the phosphomimetic S88E mutant of DYNLL2 to the dimeric myosin Va fragment, and we found a significantly lower apparent K d value (3 M). We conclude that the thermodynamic and kinetic fine-tuning of binding of various ligands to DYNLL could have physiological relevance in its interaction network.

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Section: Fine-tuning Of Affinities Is Achieved Through Diversity Of Bmentioning

confidence: 85%

Affinity, Avidity, and Kinetics of Target Sequence Binding to LC8 Dynein Light Chain Isoforms

Radnai

Rapali

Hódi

et al. 2010

Journal of Biological Chemistry

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“…Although in our complex the corresponding T157 is still spatially close to F73, the pi-cation interaction is no longer possible and polar interactions involve the side chains of N10 and H68. Importantly, mutational analyses showed that binding activity was maintained in mutants preserving the T-SQT motif indicating that T157 is essential for binding [12]. Remarkably, a threonine is also present in the nNOS peptide in the equivalent position.…”

Section: Discussionmentioning

confidence: 99%

Structural models of DYNLL1 with interacting partners: African swine fever virus protein p54 and postsynaptic scaffolding protein gephyrin

2010

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Keywords:Dynein light chain African swine fever virus protein p54 Gephyrin NMR Protein-protein interaction a b s t r a c t DYNLL1, the smallest dynein light chain, interacts with different cargos facilitating their cellular transport. Usually the sequence recognized in the targets is homologous to the GIQVD or the KXTQT motifs with a glutamine that is important for binding. Here we add two new examples of DYNLL1 targets that can be classified into these two groups: ASFV p54 and gephyrin. Using NMR we demonstrate the direct interaction between DYNLL1 and two peptides derived from their interacting sequences. We model the structure of both complexes and show that the overall binding mode is preserved as in other complexes despite differences at the residue-specific interactions. Structured summary:MINT-8058152: DYNLL1 (uniprotkb:P63167) and gephyrin (uniprotkb:Q9NQX3) bind (MI:0407) by nuclear magnetic resonance (MI:0077) MINT-8058141:DYNLL1 (uniprotkb:P63167) and p54 (uniprotkb:Q4TWM1) bind (MI:0407) by nuclear magnetic resonance (MI:0077)

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“…Although the sequences responsible for p53, PTHrP, and Rb interaction with MTs/dynein remain largely undefined, short sequences from several other proteins have been identified, using the yeast two-hybrid system and proteomics approaches, which confer interaction with the dynein light chains (DLC), LC8 and Tctex-1. These sequences variously include the consensus motifs KSTQT (single letter amino acid code) and GIQVD for LC8, as well as less well-defined motifs for Tctex-1 interaction, including SKCSR (Raux et al, 2000;Alonso et al, 2001;Poisson et al, 2001;RodriguezCrespo et al, 2001;Mueller et al, 2002;Martinez-Moreno et al, 2003;Sugai et al, 2003;Lo et al, 2005). DLC-ASs have been suggested to mediate cargo association with dynein motor complexes to effect nuclear targeting, so that they could potentially be exploited for delivery of therapeutics to This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E07-01-0030) on June 13, 2007. cell nuclei (Cohen et al, 2005;Mastrobattista et al, 2006a,b); however, this has never been directly demonstrated.…”

Section: Introductionmentioning

confidence: 99%

“…Rather, it has been reported that DLC association can sequester proteins in the cytoplasm, inhibiting nuclear entry of protein (Ninomiya et al, 2005) or localization at the nuclear membrane (Bouillet and Strasser, 2002). Given the requirement for viral trafficking to the nucleus/perinuclear MTOC and the presence of LC8 binding DLC-ASs in numerous viral proteins, it has been conjectured that LC8 DLC-AS-containing proteins could direct viral particles to this site, although direct evidence for this is lacking (Alonso et al, 2001;Rodriguez-Crespo et al, 2001;Dohner et al, 2005;Greber and Way, 2006). We set out to examine nuclear localization of the rabies virus phosphoprotein (P-protein [RPP]) in live cells.…”

mentioning

confidence: 99%

Dynein Light Chain Association Sequences Can Facilitate Nuclear Protein Import

Moseley

Roth

Dejesus

et al. 2007

MBoC

105

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Nuclear localization sequence (NLS)-dependent nuclear protein import is not conventionally held to require interaction with microtubules (MTs) or components of the MT motor, dynein. Here we report for the first time the role of sequences conferring association with dynein light chains (DLCs) in NLS-dependent nuclear accumulation of the rabies virus P-protein. We find that P-protein nuclear accumulation is significantly enhanced by its dynein light chain association sequence (DLC-AS), dependent on MT integrity and association with DLCs, and that P-protein-DLC complexes can associate with MT cytoskeletal structures. We also find that P-protein DLC-AS, as well as analogous sequences from other proteins, acts as an independent module that can confer enhancement of nuclear accumulation to proteins carrying the P-protein NLS, as well as several heterologous NLSs. Photobleaching experiments in live cells demonstrate that the MT-dependent enhancement of NLS-mediated nuclear accumulation by the P-protein DLC-AS involves an increased rate of nuclear import. This is the first report of DLC-AS enhancement of NLS function, identifying a novel mechanism regulating nuclear transport with relevance to viral and cellular protein biology. Importantly, this data indicates that DLC-ASs represent versatile modules to enhance nuclear delivery with potential therapeutic application. INTRODUCTIONNuclear protein transport is central to normal and aberrant cellular development and physiology, as well as the infectious cycles of intracellular pathogens Hearps and Jans, 2006). The ability to exploit the cellular factors involved in nuclear targeting is of great therapeutic value as it permits the design of vehicles for the efficient delivery of drugs/therapeutic genes to the nuclear compartment (Chan and Jans, 2002; Mastrobattista et al., 2006a,b). All trafficking across the nuclear membrane occurs through the nuclear membrane embedded nuclear pore complex (NPC). The movement of molecules Ͼ45 kDa generally requires active transport, where proteins conventionally interact via a nuclear localization sequence (NLS) with import proteins (importins) which mediate docking to and transit through the NPC . Current understanding of this process focuses largely on these events at the NPC because investigation of nuclear import has conventionally used permeabilized cell systems with associated disruption of cytoplasmic structures. Thus, it is only through recent studies with live cells that it has become clear that nuclear import can involve additional levels of regulation in the cytoplasm involving the microtubule (MT) cytoskeleton (Giannakakou et al., 2000;Lam et al., 2002;Roth et al., 2007), a network composed of ϩ/Ϫ polarized polymers of tubulin with the negative end at the MT organizing center (MTOC) located near the nucleus of nonpolarized cells. MTs are suggested to act as tracks for cargo delivery by molecular motors including the multiprotein complex motor, dynein, which mediates net movement of cargoes toward the MTOC (retrograde movement),...

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African Swine Fever Virus Protein p54 Interacts with the Microtubular Motor Complex through Direct Binding to Light-Chain Dynein

Cited by 176 publications

References 62 publications

Affinity, Avidity, and Kinetics of Target Sequence Binding to LC8 Dynein Light Chain Isoforms

Affinity, Avidity, and Kinetics of Target Sequence Binding to LC8 Dynein Light Chain Isoforms

Structural models of DYNLL1 with interacting partners: African swine fever virus protein p54 and postsynaptic scaffolding protein gephyrin

Dynein Light Chain Association Sequences Can Facilitate Nuclear Protein Import

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