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1.
Protein Sci ; 33(3): e4915, 2024 Mar.
Article in English | MEDLINE | ID: mdl-38358250

ABSTRACT

Human cytomegalovirus (HCMV) is an opportunistic pathogen that infects a majority of the world population. It may cause severe disease in immunocompromised people and lead to pregnancy loss or grave disabilities of the fetus upon congenital infection. For effective replication and lifelong persistence in its host, HCMV relies on diverse functions of its tegument protein UL82, also known as pp71. Up to now, little is known about the molecular mechanisms underlying the multiple functions of this crucial viral protein. Here, we describe the X-ray structure of full-length UL82 to a resolution of 2.7 Å. A single polypeptide chain of 559 amino acids mainly folds into three ß-barrels. We show that UL82 forms a dimer in the crystal as well as in solution. We identify point mutations that disturb the dimerization interface and show that the mutant protein is monomeric in solution and upon expression in human cells. On the basis of the three-dimensional structure, we identify structural homologs of UL82 from other herpesviruses and analyze whether their functions are preserved in UL82. We demonstrate that UL82, despite its structural homology to viral deoxyuridinetriphosphatases (dUTPases), does not possess dUTPase activity. Prompted by the structural homology of UL82 to the ORF10 protein of murine herpesvirus 68 (MHV68), which is known to interact with the RNA export factor ribonucleic acid export 1 (Rae1), we performed coimmunoprecipitations and demonstrated that UL82 indeed interacts with Rae1. This suggests that HCMV UL82 may play a role in mRNA export from the nucleus similar to ORF10 encoded by the gammaherpesviruses MHV68.


Subject(s)
Cytomegalovirus , Viral Proteins , Animals , Mice , Humans , Cytomegalovirus/genetics , Cytomegalovirus/metabolism , Cell Line , Viral Proteins/genetics , Viral Proteins/metabolism
2.
Sci Rep ; 13(1): 17218, 2023 10 11.
Article in English | MEDLINE | ID: mdl-37821577

ABSTRACT

Transgenic expression of protective molecules in porcine cells and tissues is a promising approach to prevent xenograft rejection. Viruses have developed various strategies to escape the host's immune system. We generated porcine B cells (B cell line L23) expressing the human adenovirus protein E3/49K or the human cytomegalovirus protein pUL11 and investigated how human T, NK and B cell responses are affected by the expression of the viral proteins. Binding studies revealed that E3/49K and pUL11 interact with CD45 on human but not porcine peripheral blood mononuclear cells. T cell proliferation in response to L23-E3/49K cells was significantly reduced and accompanied by development of an anti-inflammatory cytokine milieu (low: TNF-alpha, IFN-gamma, IL-6; high: IL-4, IL-10). Human peripheral blood mononuclear cells which had been primed for four weeks by L23-E3/49K cells included an extended population of regulatory T cells. Cytotoxicity of effector T and natural killer cells against L23 cells was significantly reduced (40 to 50%) by E3/49K expression. B cell activation and antibody production to E3/49K expressing cells was also diminished. Surprisingly, pUL11 expression showed no effects. Reduction of human anti-pig immune responses by transgenic expression of selected viral genes may be a novel approach for protection of porcine xenografts.


Subject(s)
Killer Cells, Natural , Leukocytes, Mononuclear , Animals , Humans , Swine , Leukocytes, Mononuclear/metabolism , Ligands , Killer Cells, Natural/metabolism , Cells, Cultured , Animals, Genetically Modified , Cytomegalovirus/metabolism , Viral Proteins/genetics , Immunity
3.
J Virol ; 97(5): e0189822, 2023 05 31.
Article in English | MEDLINE | ID: mdl-37125921

ABSTRACT

Human adenoviruses (HAdVs) are widespread pathogens that generally cause mild infections in immunocompetent individuals but severe or even fatal diseases in immunocompromised patients. In order to counteract the host immune defenses, HAdVs encode various immunomodulatory proteins in the early transcription unit 3 (E3). The E3/49K protein is a highly glycosylated type I transmembrane protein uniquely expressed by species D HAdVs. Its N-terminal ectodomain sec49K is released by metalloprotease-mediated shedding at the cell surface and binds to the receptor-like protein tyrosine phosphatase CD45, a critical regulator of leukocyte activation and functions. It remained elusive which domains of CD45 and E3/49K are involved in the interaction and whether such an interaction can also occur on the cell surface with membrane-anchored full-length E3/49K. Here, we show that the two extracellular domains R1 and R2 of E3/49K bind to the same site in the domain d3 of CD45. This interaction enforces the dimerization of CD45, causing the inhibition of T cell receptor signaling. Intriguingly, the membrane-anchored E3/49K appears to be designed like a "molecular fishing rod" using an extended disordered region of E3/49K as a "fishing line" to bridge the distance between the plasma membrane of infected cells and the CD45 binding site on T cells to effectively position the domains R1 and R2 as baits for CD45 binding. This design strongly suggests that both secreted sec49K as well as membrane-anchored full-length E3/49K have immunomodulatory functions. The forced dimerization of CD45 may be applied as a therapeutic strategy in chronic inflammatory disorders and cancer. IMPORTANCE The battle between viruses and their hosts is an ongoing arms race. Whereas the host tries to detect and eliminate the virus, the latter counteracts such antiviral measures to replicate and spread. Adenoviruses have evolved various mechanisms to evade the human immune response. The E3/49K protein of species D adenoviruses mediates the inhibition of immune cell function via binding to the protein tyrosine phosphatase CD45. Here, we show that E3/49K triggers the dimerization of CD45 and thereby inhibits its phosphatase activity. Intriguingly, the membrane-anchored E3/49K seems to be designed like a "molecular fishing rod" with the two CD45 binding domains of E3/49K as baits positioned at the end of an extended disordered region reminiscent of a fishing line. The adenoviral strategy to inhibit CD45 activity by forced dimerization may be used for therapeutic intervention in autoimmune diseases or to prevent graft rejection after transplantation.


Subject(s)
Adenovirus E3 Proteins , Adenoviruses, Human , Humans , Adenoviridae/metabolism , Adenovirus E3 Proteins/chemistry , Adenovirus E3 Proteins/metabolism , Dimerization , Receptors, Antigen, T-Cell/metabolism , Leukocyte Common Antigens
4.
Biochem Biophys Res Commun ; 661: 50-55, 2023 06 18.
Article in English | MEDLINE | ID: mdl-37087798

ABSTRACT

The Gram-negative bacterium Legionella pneumophila is an accidental human pathogen that can cause a life-threatening respiratory infection called Legionellosis. In the course of infection, L. pneumophila injects more than 300 effector proteins into the host cell. The effector proteins modify the intracellular environment in order to create a stable compartment for proliferation within the host cell. The effector protein SidI has been shown to potently inhibit host translation upon translocation. SidI is able to interact with the translation elongation factor eEF1A, which has been hypothesized to be a target of SidI. A postulated glycosyltransferase domain in the C-terminal half may be responsible for the toxic effect of SidI. Here, we present the crystal structure of an N-terminal fragment of SidI containing residues 37-573. The structure is divided into three subdomains, two of which display a novel fold. The third subdomain shows close structural homology to GT-B fold glycosyltransferases. Based on structural analysis we predict that the two previously identified residues R453 and E482 assume roles in the catalytic activity of SidI. Furthermore, we show that the N-terminal fragment of SidI is able to directly interact with a postulated target, the translation elongation factor eEF1A.


Subject(s)
Legionella pneumophila , Humans , Legionella pneumophila/genetics , Glucosyltransferases/metabolism , Protein Biosynthesis , Peptide Elongation Factor 1/genetics , Bacterial Proteins/metabolism , Glycosyltransferases/metabolism
5.
Biotechniques ; 70(6): 350-354, 2021 06.
Article in English | MEDLINE | ID: mdl-34114503

ABSTRACT

Multicomponent protein complexes called inflammasomes play a major role in the innate immune system by activating proinflammatory cytokines and promoting a highly inflammatory form of programmed cell death, called pyroptosis. A hallmark of the function of the nucleotide-binding domain, leucine-rich repeat and NLRP3-mediated inflammasome assembly is the polymerization of ASC into large filaments. The ASC filaments recruit and activate procaspase-1 by induced proximity. We developed an in vitro assay for monitoring the polymerization of the pyrin domain of ASC by microscale thermophoresis. We have validated the assay by analyzing the effects of buffer conditions, mutations of ASC and the use of seeds on the polymerization behavior of ASC.


Subject(s)
CARD Signaling Adaptor Proteins/chemistry , NLR Family, Pyrin Domain-Containing 3 Protein , Pyrin Domain , Apoptosis , Inflammasomes , Polymerization
6.
Biochem Biophys Res Commun ; 527(3): 696-701, 2020 06 30.
Article in English | MEDLINE | ID: mdl-32423822

ABSTRACT

Persistence and replication of the gram-negative bacterium Legionella pneumophila in the human host cell depend on so-called effector proteins that target diverse cellular functions and modulate them in favor of the pathogen. We solved the crystal structure of the L. pneumophila effector protein MesI de novo to a resolution of 2.2 Å. The 34 kDa polypeptide chain folds into two distinct α-helical domains. The larger C-terminal domain shows similarity to tetratricopeptide repeat proteins. Using size-exclusion chromatography, we confirmed that MesI binds tightly to full-length SidI and that deletion of either the N- or the C-terminus weakens the interaction. Based on the three-dimensional structure of MesI we suggest a possible binding mode for SidI and identified two homologs of MesI within the proteome of L. pneumophila that do not bind to SidI, but may act as specific inhibitors of other yet to be identified effectors.


Subject(s)
Bacterial Proteins/chemistry , Legionella pneumophila/chemistry , Bacterial Proteins/metabolism , Crystallography, X-Ray , Host-Pathogen Interactions , Humans , Legionella pneumophila/physiology , Legionnaires' Disease/metabolism , Legionnaires' Disease/microbiology , Models, Molecular , Protein Conformation
7.
Biol Chem ; 399(12): 1421-1432, 2018 11 27.
Article in English | MEDLINE | ID: mdl-30067507

ABSTRACT

Dynamins are essential as membrane remodelers in various cellular processes, like receptor-mediated endocytosis, synaptic vesicle recycling and spermatogenesis. Moreover, dynamin is involved in the internalization of numerous viruses and in the motility of several cancer cell lines. As tools for dissecting the underlying mechanisms of these important biological processes and as potential future therapeutics, small molecules have been developed in the last two decades that modulate the functions of dynamin. In this review we give an overview of the compound classes that are currently in use and describe how they affect dynamin function.


Subject(s)
Dynamins/metabolism , Small Molecule Libraries/pharmacology , Animals , Humans , Models, Molecular , Molecular Structure , Small Molecule Libraries/chemistry
8.
Biochem Biophys Res Commun ; 482(4): 530-535, 2017 Jan 22.
Article in English | MEDLINE | ID: mdl-27865841

ABSTRACT

Survivin inhibits apoptosis in numerous tumor cell lines and has emerged as promising target for cancer therapy. The anti-apoptotic effect of survivin was attributed to a direct interaction with XIAP (X-linked inhibitor of apoptosis) and to an indirect effect, where survivin antagonizes the anti-XIAP action of Smac. The direct interaction is thought to lead to synergistic inhibition of caspase-9 and, at the same time, to enhanced stability of XIAP by reducing its auto-ubiquitination. Using recombinant proteins, we have investigated the influence of survivin on the inhibition of caspase-9 by XIAP in vitro. With a fluorescence-based assay for the apoptosome-stimulated activity of caspase-9, we show that survivin has no effect on the inhibition of caspase-9 by XIAP, neither in the presence nor in the absence of Smac. Employing analytical size exclusion chromatography (SEC) and analytical ultracentrifugation, we show that survivin does not physically interact with XIAP. We confirm in vitro that XIAP ubiquitinates itself in the presence of the appropriate recombinant enzymes and Mg2+-ATP and could show that survivin neither influences the kinetics nor the extent of XIAP's self-ubiquitination. Our results call for a revision of the current view of how survivin interferes with the mitochondrial pathway of apoptosis.


Subject(s)
Apoptosis , Caspase 9/metabolism , Inhibitor of Apoptosis Proteins/metabolism , X-Linked Inhibitor of Apoptosis Protein/metabolism , Apoptosis Regulatory Proteins , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Mitochondrial Proteins/metabolism , Protein Binding , Recombinant Proteins/metabolism , Survivin , Ubiquitination
9.
Biochem Biophys Res Commun ; 469(1): 76-80, 2016 Jan 01.
Article in English | MEDLINE | ID: mdl-26612256

ABSTRACT

Dynamin is the prototype of a family of large multi-domain GTPases. The 100 kDa protein is a key player in clathrin-mediated endocytosis, where it cleaves off vesicles from membranes using the energy from GTP hydrolysis. We have solved the high resolution crystal structure of a fusion protein of the GTPase domain and the bundle signalling element (BSE) of dynamin 1 liganded with GDP. The structure provides a hitherto missing snapshot of the GDP state of the hydrolytic cycle of dynamin and reveals how the switch I region moves away from the active site after GTP hydrolysis and release of inorganic phosphate. Comparing our structure of the GDP state with the known structures of the GTP state, the transition state and the nucleotide-free state of dynamin 1 we describe the structural changes through the hydrolytic cycle.


Subject(s)
Dynamins/chemistry , Dynamins/ultrastructure , GTP Phosphohydrolases/chemistry , GTP Phosphohydrolases/ultrastructure , Guanosine Diphosphate/chemistry , Molecular Docking Simulation , Binding Sites , Crystallography , Enzyme Activation , Protein Binding , Protein Conformation , Protein Structure, Tertiary
10.
Nature ; 525(7569): 404-8, 2015 Sep 17.
Article in English | MEDLINE | ID: mdl-26302298

ABSTRACT

The mechanochemical protein dynamin is the prototype of the dynamin superfamily of large GTPases, which shape and remodel membranes in diverse cellular processes. Dynamin forms predominantly tetramers in the cytosol, which oligomerize at the neck of clathrin-coated vesicles to mediate constriction and subsequent scission of the membrane. Previous studies have described the architecture of dynamin dimers, but the molecular determinants for dynamin assembly and its regulation have remained unclear. Here we present the crystal structure of the human dynamin tetramer in the nucleotide-free state. Combining structural data with mutational studies, oligomerization measurements and Markov state models of molecular dynamics simulations, we suggest a mechanism by which oligomerization of dynamin is linked to the release of intramolecular autoinhibitory interactions. We elucidate how mutations that interfere with tetramer formation and autoinhibition can lead to the congenital muscle disorders Charcot-Marie-Tooth neuropathy and centronuclear myopathy, respectively. Notably, the bent shape of the tetramer explains how dynamin assembles into a right-handed helical oligomer of defined diameter, which has direct implications for its function in membrane constriction.


Subject(s)
Dynamins/antagonists & inhibitors , Dynamins/chemistry , Protein Multimerization , Charcot-Marie-Tooth Disease , Crystallography, X-Ray , Dynamins/genetics , Dynamins/metabolism , Humans , Markov Chains , Models, Molecular , Molecular Dynamics Simulation , Mutant Proteins/antagonists & inhibitors , Mutant Proteins/chemistry , Mutant Proteins/genetics , Mutant Proteins/metabolism , Mutation/genetics , Myopathies, Structural, Congenital , Nucleotides , Protein Multimerization/genetics , Structure-Activity Relationship
11.
Microb Cell ; 2(5): 150-162, 2015 May 04.
Article in English | MEDLINE | ID: mdl-28357287

ABSTRACT

Inhibition of programmed cell death pathways of mammalian cells often facilitates the sustained survival of intracellular microorganisms. The apicomplexan parasite Toxoplasma gondii is a master regulator of host cell apoptotic pathways. Here, we have characterized a novel anti-apoptotic activity of T. gondii. Using a cell-free cytosolic extract model, we show that T. gondii interferes with the activities of caspase 9 and caspase 3/7 which have been induced by exogenous cytochrome c and dATP. Proteolytic cleavage of caspases 9 and 3 is also diminished suggesting inhibition of holo-apoptosome function. Parasite infection of Jurkat T cells and subsequent triggering of apoptosome formation by exogenous cytochrome cin vitro and in vivo indicated that T. gondii also interferes with caspase activation in infected cells. Importantly, parasite inhibition of cytochrome c-induced caspase activation considerably contributes to the overall anti-apoptotic activity of T. gondii as observed in staurosporine-treated cells. Co-immunoprecipitation showed that T. gondii abolishes binding of caspase 9 to Apaf-1 whereas the interaction of cytochrome c with Apaf-1 remains unchanged. Finally, T. gondii lysate mimics the effect of viable parasites and prevents holo-apoptosome functionality in a reconstituted in vitro system comprising recombinant Apaf-1 and caspase 9. Beside inhibition of cytochrome c release from host cell mitochondria, T. gondii thus also targets the holo-apoptosome assembly as a second mean to efficiently inhibit the caspase-dependent intrinsic cell death pathway.

12.
FEBS Lett ; 588(18): 3327-32, 2014 Sep 17.
Article in English | MEDLINE | ID: mdl-25064844

ABSTRACT

The NOD-like receptor NLRP1 (NLR family, pyrin domain containing 1) senses the presence of the bacterial cell wall component l-muramyl dipeptide (MDP) inside the cell. We determined the crystal structure of the LRR domain of human NLRP1 in the absence of MDP to a resolution of 1.65Å. The fold of the structure can be assigned to the ribonuclease inhibitor-like class of LRR proteins. We compared our structure with X-ray models of the LRR domains of NLRX1 and NLRC4 and a homology model of the LRR domain of NOD2. We conclude that the MDP binding site of NLRP1 is not located in the LRR domain.


Subject(s)
Acetylmuramyl-Alanyl-Isoglutamine/chemistry , Adaptor Proteins, Signal Transducing/chemistry , Apoptosis Regulatory Proteins/chemistry , Amino Acid Motifs , Binding Sites , Crystallography, X-Ray , Humans , Models, Molecular , NLR Proteins , Protein Binding , Protein Structure, Tertiary , Repetitive Sequences, Amino Acid , Structural Homology, Protein
13.
Biochemistry ; 52(13): 2319-27, 2013 Apr 02.
Article in English | MEDLINE | ID: mdl-23521171

ABSTRACT

Apoptosome assembly is highly regulated in the intrinsic cell death pathway. To better understand this step, we created an improved model of the human apoptosome using a crystal structure of full length Apaf-1 and a single particle, electron density map at ~9.5 Å resolution. The apoptosome model includes N-terminal domains of Apaf-1, cognate ß-propellers, and cytochrome c. A direct comparison of Apaf-1 in the apoptosome and as a monomer reveals conformational changes that occur during the first two steps of assembly. This includes an induced-fit mechanism for cytochrome c binding to regulatory ß-propellers, which is dependent on shape and charge complementarity, and a large rotation of the nucleotide binding module during nucleotide exchange. These linked conformational changes create an extended Apaf-1 monomer and drive apoptosome assembly. Moreover, the N-terminal CARD in the inactive Apaf-1 monomer is not shielded from other proteins by ß-propellers. Hence, the Apaf-1 CARD may be free to interact with a procaspase-9 CARD either before or during apoptosome assembly. Irrespective of the timing, the end product of assembly is a holo-apoptosome with an acentric CARD-CARD disk and tethered pc-9 catalytic domains. Subsequent activation of pc-9 leads to a proteolytic cascade and cell death.


Subject(s)
Apoptosomes/metabolism , Apoptotic Protease-Activating Factor 1/chemistry , Apoptotic Protease-Activating Factor 1/metabolism , Apoptosomes/chemistry , Cytochromes c/chemistry , Cytochromes c/metabolism , Humans , Models, Molecular , Protein Conformation
14.
Cell Signal ; 24(7): 1420-5, 2012 Jul.
Article in English | MEDLINE | ID: mdl-22446004

ABSTRACT

Apoptosomes are signaling platforms that initiate the dismantling of a cell during apoptosis. In mammals, assembly of the apoptosome is the pivotal point in the mitochondrial pathway of apoptosis, and is prompted by binding of cytochrome c to the apoptotic protease-activating factor 1 (Apaf-1) in the presence of ATP. The resulting wheel-like heptamer of seven molecules Apaf-1 and seven molecules cytochrome c binds and activates the initiator caspase-9, which in turn ignites the downstream caspase cascade. In this review we discuss the molecular determinants for the formation of the mammalian apoptosome and caspase activation and describe the related signaling platforms in flies and nematodes.


Subject(s)
Apoptosis/genetics , Apoptosomes/chemistry , Apoptotic Protease-Activating Factor 1/chemistry , Apoptotic Protease-Activating Factor 1/metabolism , Caspase 9/metabolism , Adenosine Triphosphate/metabolism , Animals , Apoptosomes/metabolism , Caspase 9/genetics , Cytochromes c/chemistry , Cytochromes c/metabolism , Mitochondria/chemistry , Mitochondria/metabolism , Protein Conformation , Protein Structure, Tertiary , Signal Transduction
15.
J Biol Chem ; 284(47): 32717-24, 2009 Nov 20.
Article in English | MEDLINE | ID: mdl-19801675

ABSTRACT

The cytosolic adaptor protein Apaf-1 is a key player in the intrinsic pathway of apoptosis. Binding of mitochondrially released cytochrome c and of dATP or ATP to Apaf-1 induces the formation of the heptameric apoptosome complex, which in turn activates procaspase-9. We have re-investigated the chain of events leading from monomeric autoinhibited Apaf-1 to the functional apoptosome in vitro. We demonstrate that Apaf-1 does not require energy from nucleotide hydrolysis to eventually form the apoptosome. Despite a low intrinsic hydrolytic activity of the autoinhibited Apaf-1 monomer, nucleotide hydrolysis does not occur at any stage of the process. Rather, mere binding of ATP in concert with the binding of cytochrome c primes Apaf-1 for assembly. Contradicting the current view, there is no strict requirement for an adenine base in the nucleotide. On the basis of our results, we present a new model for the mechanism of apoptosome assembly.


Subject(s)
Apoptosomes/metabolism , Apoptotic Protease-Activating Factor 1/chemistry , Caspases/metabolism , Adenosine Triphosphate/chemistry , Animals , Apoptosis , Caspase 9/metabolism , Cytochromes c/metabolism , Cytosol/metabolism , Enzyme Activation , Humans , Hydrolysis , Insecta , Models, Biological , Nucleotides/chemistry
17.
J Muscle Res Cell Motil ; 27(2): 115-23, 2006.
Article in English | MEDLINE | ID: mdl-16450056

ABSTRACT

The myosin cross-bridge has two essential properties: to undergo the "power stroke" and to bind and release from actin - both under control of ATP binding and hydrolysis. In the absence of ATP the cross-bridge binds to actin with high affinity: the binding of ATP causes rapid release of the cross-bridge from actin. The actin binding-site is split by a deep cleft that closes on strong binding to actin. The cleft is straddled by a short polypeptide known as the "strut". In the following we summarise the structural basis of the power stroke and the control of actin affinity and then present data on the effects on actin affinity of replacing the strut by a flexible linker.


Subject(s)
Actins/metabolism , Adenosine Triphosphate/metabolism , Mutation , Myosin Type II/metabolism , Actins/genetics , Animals , Humans , Myosin Type II/genetics , Protein Structure, Quaternary/genetics
18.
Proc Natl Acad Sci U S A ; 102(37): 13093-8, 2005 Sep 13.
Article in English | MEDLINE | ID: mdl-16141317

ABSTRACT

Here, we present the 1.9-A crystal structure of the nucleotide-free GTPase domain of dynamin 1 from Rattus norvegicus. The structure corresponds to an extended form of the canonical GTPase fold observed in Ras proteins. Both nucleotide-binding switch motifs are well resolved, adopting conformations that closely resemble a GTP-bound state not previously observed for nucleotide-free GTPases. Two highly conserved arginines, Arg-66 and Arg-67, greatly restrict the mobility of switch I and are ideally positioned to relay information about the nucleotide state to other parts of the protein. Our results support a model in which switch I residue Arg-59 gates GTP binding in an assembly-dependent manner and the GTPase effector domain functions as an assembly-dependent GTPase activating protein in the fashion of RGS-type GAPs.


Subject(s)
Dynamin I/chemistry , GTP Phosphohydrolases/chemistry , Animals , Arginine , Catalysis , Crystallization , Crystallography, X-Ray , Dynamin I/genetics , Dynamin I/metabolism , Enzyme Activation/genetics , GTP Phosphohydrolases/genetics , GTP Phosphohydrolases/metabolism , Guanosine Triphosphate/metabolism , Kinetics , Mutation , Protein Structure, Tertiary , Rats
19.
Nat Struct Biol ; 10(10): 826-30, 2003 Oct.
Article in English | MEDLINE | ID: mdl-14502270

ABSTRACT

Myosins are molecular motor proteins that harness the chemical energy stored in ATP to produce directed force along actin filaments. Complex communication pathways link the catalytic nucleotide-binding region, the structures responsible for force amplification and the actin-binding domain of myosin. We have crystallized the nucleotide-free motor domain of myosin II in a new conformation in which switch I and switch II, conserved loop structures involved in nucleotide binding, have moved away from the nucleotide-binding pocket. These movements are linked to rearrangements of the actin-binding region, which illuminate a previously unobserved communication pathway between the nucleotide-binding pocket and the actin-binding region, explain the reciprocal relationship between actin and nucleotide affinity and suggest a new mechanism for product release in myosin family motors.


Subject(s)
Actins/metabolism , Adenosine Diphosphate/metabolism , Adenosine Triphosphate/metabolism , Myosins/metabolism , Animals , Binding Sites , Dictyostelium/metabolism , Protein Binding , Protein Structure, Tertiary
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