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
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.
Edited by Michael R. BubbKeywords: NM-2B Non-muscle myosin-2B Myosin MYH9-related disease X-ray crystallography a b s t r a c tWe 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 50kDa 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).
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