ABSTRACT
Certain motile bacteria employ rotating flagella for propulsion. The relative flexibility of two key components of the flagellum, filament and hook, is partially responsible for the mechanistic workings of this motor. A new computational method, the quantized elastic deformational model, was employed in this article to calculate the dimensionless twist/bend ratio (EI/GJ) of the filament and hook, providing a quantitative means to compare their relative stiffness. Both ratios were much <1.0, an average of 0.0440 for the filament and 0.0512 for the hook, indicating that within each structure bending is favored over twisting. These two ratios, along with previous experimental measurements, allowed us to propose a theoretical Young's modulus (E) between 10(6) and 10(7) dyn/cm(2) for the hook. This value is orders of magnitude smaller than experimentally determined Young's moduli of the filament, hence in agreement with empirical evidence linking compliance in the flagellum mainly to the hook.
Subject(s)
Biomechanical Phenomena , Biophysics/methods , Flagella/chemistry , Flagella/ultrastructure , Animals , Bacteria/ultrastructure , Bacterial Physiological Phenomena , Bacterial Proteins/chemistry , Microscopy, Electron, Scanning , Models, Biological , Models, Theoretical , Movement , Salmonella typhimurium/physiologyABSTRACT
p97/VCP is a member of the AAA ATPase family and has roles in both membrane fusion and ubiquitin dependent protein degradation. Here, we present a 3.6A crystal structure of murine p97 in which D2 domain has been modelled as poly-alanine and the remaining approximately 100 residues are absent. The resulting structure illustrates a head-to-tail packing arrangement of the two p97 AAA domains in a natural hexameric state with D1 ADP bound and D2 nucleotide free. The head-to-tail packing arrangement observed in this structure is in contrast to our previously predicted tail-to-tail packing model. The linker between the D1 and D2 domains is partially disordered, suggesting a flexible nature. Normal mode analysis of the crystal structure suggests anti-correlated motions and distinct conformational states of the two AAA domains.
Subject(s)
Cell Cycle Proteins/chemistry , Adenosine Triphosphatases/chemistry , Animals , Anisotropy , Cloning, Molecular , Crystallography, X-Ray , Electrons , Endopeptidase Clp , Mice , Models, Molecular , Models, Statistical , Protein Conformation , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Serine Endopeptidases/chemistry , Valosin Containing ProteinABSTRACT
The crystal structures of lactose repressor protein (LacI) provide static endpoint views of the allosteric transition between DNA- and IPTG-bound states. To obtain an atom-by-atom description of the pathway between these two conformations, motions were simulated with targeted molecular dynamics (TMD). Strikingly, this homodimer exhibited asymmetric dynamics. All asymmetries observed in this simulation are reproducible and can begin on either of the two monomers. Asymmetry in the simulation originates around D149 and was traced back to the pre-TMD equilibrations of both conformations. In particular, hydrogen bonds between D149 and S193 adopt a variety of configurations during repetitions of this process. Changes in this region propagate through the structure via noncovalent interactions of three interconnected pathways. The changes of pathway 1 occur first on one monomer. Alterations move from the inducer-binding pocket, through the N-subdomain beta-sheet, to a hydrophobic cluster at the top of this region and then to the same cluster on the second monomer. These motions result in changes at (1) side chains that form an interface with the DNA-binding domains and (2) K84 and K84', which participate in the monomer-monomer interface. Pathway 2 reflects consequent reorganization across this subunit interface, most notably formation of a H74-H74rsquo; pi-stacking intermediate. Pathway 3 extends from the rear of the inducer-binding pocket, across a hydrogen-bond network at the bottom of the pocket, and transverses the monomer-monomer interface via changes in H74 and H74rsquo;. In general, intermediates detected in this study are not apparent in the crystal structures. Observations from the simulations are in good agreement with biochemical data and provide a spatial and sequential framework for interpreting existing genetic data.
Subject(s)
Bacterial Proteins/chemistry , Repressor Proteins/chemistry , Allosteric Regulation , Bacterial Proteins/metabolism , Binding Sites , DNA/chemistry , DNA/metabolism , Dimerization , Galactosides/chemistry , Galactosides/metabolism , Hydrogen Bonding , Lac Repressors , Ligands , Models, Molecular , Protein Binding , Protein Conformation , Protein Structure, Secondary , Protein Structure, Tertiary , Repressor Proteins/metabolismABSTRACT
p97, a Mg-ATPase belonging to the AAA (ATPase associated with various cellular activities) super family of proteins, has been proposed to function in two distinct cellular pathways, namely homotypic membrane fusion and ubiquitin protein degradation by utilizing differing adaptor complexes. We present the cryo-electron microscopy three-dimensional reconstruction of endogenous p97 in an AMP-PNP bound state at 24 A resolution. It reveals clear nucleotide-dependent differences when compared to our previously published "p97-ADP" reconstruction, including a striking rearrangement of N domains and a positional change of the two ATPase domains, D1 and D2, with respect to each other. The docking of the X-ray structure of N-D1 domains in an ADP bound state indicates that an upward repositioning of N domain is necessary to accommodate the cryo-EM map of "p97-AMP-PNP", suggesting a change in the orientation of N domains upon nucleotide hydrolysis. Furthermore, computational analysis of the deformational motions of p97, performed on the cryo-EM density map and the atomic structure of the N-D1 domains independently, shows the existence of a negative cooperativity between the D1 and D2 rings and the flexibility of the N domains. Together these results allow the identification of functionally important features that offer molecular insights into the dynamics of the proposed p97 chaperone function.
Subject(s)
Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/metabolism , Nuclear Proteins/chemistry , Nuclear Proteins/metabolism , Animals , Anisotropy , Biophysical Phenomena , Biophysics , Cryoelectron Microscopy , Crystallography, X-Ray , Electrons , Image Processing, Computer-Assisted , Liver/metabolism , Models, Molecular , Models, Statistical , Nucleotides/chemistry , Protein Binding , Protein Conformation , Protein Folding , Protein Structure, Secondary , Protein Structure, Tertiary , Rats , Recombinant Proteins/chemistryABSTRACT
Molecular dynamics trajectories for the bovine mitochondrial F(1)-ATPase are used to demonstrate the motions and interactions that take place during the elementary (120 degrees rotation) step of the gamma subunit. The results show how rotation of the gamma subunit induces the observed structural changes in the catalytic beta subunits. Both steric and electrostatic interactions contribute. An "ionic track" of Arg and Lys residues on the protruding portion of the gamma subunit plays a role in guiding the motions of the beta subunits. Experimental data for mutants of the DELSEED motif and the hinge region are interpreted on the basis of the molecular dynamics results. The trajectory provides a unified dynamic description of the coupled subunit motions involved in the 120 degrees rotation cycles of F(1)-ATPase.
