ABSTRACT
Myosin motor proteins convert chemical energy into force and movement through their interactions with nucleotide and filamentous actin (F-actin). The evolutionarily conserved lysine-265 (K265) of the myosin-2 motor from Dictyostelium discoideum (Dd) is proposed to be a key residue in an allosteric communication pathway that mediates actin-nucleotide coupling. To better understand the role of K265, point mutations were introduced within the Dd myosin-2 M765-2R framework, replacing this lysine with alanine (K265A), glutamic acid (K265E) or glutamine (K265Q), and the functional and kinetic properties of the resulting myosin motors were assessed. The alanine and glutamic acid substitutions reduced actin-activated ATPase activity, slowed the in vitro sliding velocity and attenuated the inhibitory potential of the allosteric myosin inhibitor pentabromopseudilin (PBP). However, glutamine substitution did not substantially change these parameters. Structural modelling suggests that K265 interacts with D590 and Q633 to establish a pivotal allosteric branching point. Based on our results, we propose: (1) that the K265-D590 interaction functions to reduce myosins basal ATPase activity in the absence of F-actin, and (2) that the dynamic formation of the K265-Q633 salt bridge upon actin cleft closure regulates the activation of product release by actin filaments.
Subject(s)
Actins/metabolism , Binding Sites , Lysine/metabolism , Myosin Type II/chemistry , Myosin Type II/metabolism , Nucleotides/metabolism , Actins/chemistry , Adenosine Diphosphate/metabolism , Adenosine Triphosphatases/metabolism , Adenosine Triphosphate/metabolism , Alanine/metabolism , Allosteric Regulation , Enzyme Activation , Gene Expression , Glutamic Acid , Kinetics , Models, Molecular , Mutation , Myosin Type II/genetics , Nucleotides/chemistry , Protein Binding , Structure-Activity RelationshipABSTRACT
We determined the crystal structure of the motor domain of human non-muscle myosin 2B (NM-2B) in a nucleotide-free state and at a resolution of 2.8 Å. The structure shows the motor domain with an open active site and the large cleft that divides the 50 kDa domain in a closed state. Compared to other rigor-like myosin motor domain structures, our structure shows subtle but significant conformational changes in regions important for actin binding and mechanochemical coupling. Moreover, our crystal structure helps to rationalize the impact of myosin, heavy chain 9 (MYH9)-related disease mutations Arg709Cys and Arg709His on the kinetic and functional properties of NM-2B and of the closely related non-muscle myosin 2A (NM-2A).
Subject(s)
Myosins/chemistry , Amino Acid Sequence , Cloning, Molecular , Crystallography, X-Ray , Humans , Models, Molecular , Mutation , Myosins/genetics , Myosins/isolation & purification , Protein Structure, TertiaryABSTRACT
Myosin 1c (Myo1c) plays a key role in supporting motile events that underlie cell migration, vesicle trafficking, insulin-stimulated glucose uptake and hearing. Here, we present the crystal structure of the human Myo1c motor in complex with its light chain calmodulin. Our structure reveals tight interactions of the motor domain with calmodulin bound to the first IQ motif in the neck region. Several of the calmodulin residues contributing to this interaction are also involved in Ca(2+) binding. Contact residues in the motor domain are linked to the central ß-sheet and the HO helix, suggesting a mechanism for communicating changes in Ca(2+) binding in the neck region to the actin and nucleotide binding regions of the motor domain. The structural context and the chemical environment of Myo1c mutations that are involved in sensorineural hearing loss in humans are described and their impact on motor function is discussed. We show that a construct consisting of the motor domain of Myo1c and the first IQ motif is sufficient to establish a tight interaction with 14-3-3ß (KD=0.9 µM) and present the model of a double-headed Myo1c-14-3-3 complex. This complex has been implicated in the exocytosis of glucose transporter 4 storage vesicles during insulin-stimulated glucose uptake.
Subject(s)
14-3-3 Proteins/metabolism , Calcium/metabolism , Exocytosis , Glucose Transporter Type 4/metabolism , Myosin Type I/chemistry , Myosin Type I/metabolism , 14-3-3 Proteins/chemistry , Calmodulin/chemistry , Calmodulin/metabolism , Crystallography, X-Ray , Hearing Loss, Sensorineural/genetics , Humans , Models, Molecular , Mutation , Myosin Type I/genetics , Protein Binding , Protein Conformation , Protein TransportABSTRACT
Injectisomes are multi-protein transmembrane machines allowing pathogenic bacteria to inject effector proteins into eukaryotic host cells, a process called type III secretion. Here we present the first three-dimensional structure of Yersinia enterocolitica and Shigella flexneri injectisomes in situ and the first structural analysis of the Yersinia injectisome. Unexpectedly, basal bodies of injectisomes inside the bacterial cells showed length variations of 20%. The in situ structures of the Y. enterocolitica and S. flexneri injectisomes had similar dimensions and were significantly longer than the isolated structures of related injectisomes. The crystal structure of the inner membrane injectisome component YscD appeared elongated compared to a homologous protein, and molecular dynamics simulations documented its elongation elasticity. The ring-shaped secretin YscC at the outer membrane was stretched by 30-40% in situ, compared to its isolated liposome-embedded conformation. We suggest that elasticity is critical for some two-membrane spanning protein complexes to cope with variations in the intermembrane distance. DOI:http://dx.doi.org/10.7554/eLife.00792.001.
Subject(s)
Membrane Proteins/metabolism , Yersinia enterocolitica/metabolism , Cryoelectron Microscopy , Membrane Proteins/chemistry , Osmotic Pressure , Protein ConformationABSTRACT
Myosin 1c (Myo1c) is implicated in several cellular processes such as vesicle transport and the mediation of adaptation in the inner ear. Consequently, mutations impairing Myo1c motor activity lead to hearing loss in humans. To understand the role of Myo1c in this process on a molecular level, its crystal structure in complex with the light chain calmodulin was determined. A human Myo1c construct encompassing the motor domain and the first IQ motif was co-expressed with calmodulin in Sf9 cells and purified to homogeneity. The protein complex crystallized readily, and the crystals belonged to space group P2(1) and diffracted to 3â Å resolution. Attempts to determine the structure by molecular replacement are currently under way.
Subject(s)
Calmodulin/chemistry , Myosin Type I/chemistry , Animals , Binding Sites , Calmodulin/genetics , Calmodulin/isolation & purification , Crystallography, X-Ray , Escherichia coli/genetics , Gene Expression , Humans , Myosin Type I/genetics , Myosin Type I/isolation & purification , Protein Binding , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Sf9 Cells , SpodopteraABSTRACT
Methyltransferases form a major class of tRNA-modifying enzymes that are needed for the proper functioning of tRNA. Here, the expression, purification and crystallization of two related putative tRNA methyltransferases from two kingdoms of life are reported. The protein encoded by the gene pf1002 from the archaeon Pyrococcus furiosus was crystallized in the monoclinic space group P2(1). A complete data set was collected to 2.2 Å resolution. The protein encoded by the gene ttc1157 from the eubacterium Thermus thermophilus was crystallized in the trigonal space group P3(2)21. A complete data set was collected to 2.05 Å resolution.
