The mechanism of the converter domain rotation in the recovery stroke of myosin motor protein

Upon ATP binding, myosin motor protein is found in two alternative conformations, prerecovery state M* and postrecovery state M**. The transition from one state to the other, known as the recovery stroke, plays a key role in the myosin functional cycle. Despite much recent research, the microscopic details of this transition remain elusive. A critical step in the recovery stroke is the rotation of the converter domain from “up” position in prerecovery state to “down” position in postrecovery state that leads to the swing of the lever arm attached to it. In this work, we demonstrate that the two rotational states of the converter domain are determined by the interactions within a small structural motif in the force‐generating region of the protein that can be accurately modeled on computers using atomic representation and explicit solvent. Our simulations show that the transition between the two states is controlled by a small helix (SH1) located next to the relay helix and relay loop. A small translation in the position of SH1 away from the relay helix is seen to trigger the transition from “up” state to “down” state. The transition is driven by a cluster of hydrophobic residues I687, F487, and F506 that make significant contributions to the stability of both states. The proposed mechanism agrees well with the available structural and mutational studies. Proteins 2012; © 2012 Wiley Periodicals, Inc.     

The structure of the Alzheimer amyloid beta 10-35 peptide probed through replica-exchange molecular dynamics simulations in explicit solvent

The conformational states sampled by the Alzheimer amyloid beta (10-35) (Abeta 10-35) peptide were probed using replica-exchange molecular dynamics (REMD) simulations in explicit solvent. The Abeta 10-35 peptide is a fragment of the full-length Abeta 40/42 peptide that possesses many of the amyloidogenic properties of its full-length counterpart. Under physiological temperature and pressure, our simulations reveal that the Abeta 10-35 peptide does not possess a single unique folded state. Rather, this peptide exists as a mixture of collapsed globular states that remain in rapid dynamic equilibrium with each other. This conformational ensemble is dominated by random coil and bend structures with insignificant presence of an alpha-helical or beta-sheet structure. The 3D structure of Abeta 10-35 is seen to be defined by a salt bridge formed between the side-chains of K28 and D23. This salt bridge is also observed in Abeta fibrils and our simulations suggest that monomeric conformations of Abeta 10-35 contain pre-folded structural motifs that promote rapid aggregation of this peptide.     

Early stages of the recovery stroke in myosin II studied by molecular dynamics simulations

The recovery stroke is a key step in the functional cycle of muscle motor protein myosin, during which pre-recovery conformation of the protein is changed into the active post-recovery conformation, ready to exersice force. We study the microscopic details of this transition using molecular dynamics simulations of atomistic models in implicit and explicit solvent. In more than 2 μs of aggregate simulation time, we uncover evidence that the recovery stroke is a two-step process consisting of two stages separated by a time delay. In our simulations, we directly observe the first stage at which switch II loop closes in the presence of adenosine triphosphate at the nucleotide binding site. The resulting configuration of the nucleotide binding site is identical to that detected experimentally. Distribution of inter-residue distances measured in the force generating region of myosin is in good agreement with the experimental data. The second stage of the recovery stroke structural transition, rotation of the converter domain, was not observed in our simulations. Apparently it occurs on a longer time scale. We suggest that the two parts of the recovery stroke need to be studied using separate computational models.     

Effects of rottlerin on silica-exacerbated systemic autoimmune disease in New Zealand mixed mice

Environmental crystalline silica exposure has been associated with formation of autoantibodies and development of systemic autoimmune disease, but the mechanisms leading to these events are unknown. Silica exposure in autoimmune-prone New Zealand mixed (NZM) mice results in a significant exacerbation of systemic autoimmunity as measured by increases in autoantibodies and glomerulonephritis. Previous studies have suggested that silica-induced apoptosis of alveolar macrophages (AM) contributes to the generation of the autoantibodies and disease. Rottlerin has been reported to inhibit apoptosis in many cell types, possibly through direct or indirect effects on PKCdelta. In this study, rottlerin reduced silica-induced apoptosis in bone marrow-derived macrophages as measured by DNA fragmentation. In NZM mice, RNA and protein levels of PKCdelta were significantly elevated in AM 14 wk after silica exposure. Therefore, rottlerin was used to reduce apoptosis of AM and evaluate the progress of silica-exacerbated systemic autoimmune disease. Fourteen weeks after silica exposure, NZM mice had increased levels of anti-histone autoantibodies, high proteinuria, and glomerulonephritis. However, silica-instilled mice that also received weekly instillations of rottlerin had significantly lower levels of proteinuria, anti-histone autoantibodies, complement C3, and IgG deposition within the kidney. Weekly instillations of rottlerin in silica-instilled NZM mice also inhibited the upregulation of PKCdelta in AM. Together, these data demonstrate that in vivo treatment with rottlerin significantly decreased the exacerbation of autoimmunity by silica exposure.     

Effects of familial Alzheimer's disease mutations on the folding nucleation of the amyloid beta-protein

The effect of single amino acid substitutions associated with the Italian (E22K), Arctic (E22G), Dutch (E22Q) and Iowa (D23N) familial forms of Alzheimer's disease and cerebral amyloid angiopathy on the structure of the 21-30 fragment of the Alzheimer amyloid β-protein (Aβ) is investigated by replica-exchange molecular dynamics simulations. The 21-30 segment has been shown in our earlier work to adopt a bend structure in solution that may serve as the folding nucleation site for Aβ. Our simulations reveal that the 24-28 bend motif is retained in all E22 mutants, suggesting that mutations involving residue E22 may not affect the structure of the folding nucleation site of Aβ. Enhanced aggregation in Aβ with familial Alzheimer's disease substitutions may result from the depletion of the E22-K28 salt bridge, which destabilizes the bend structure. Alternately, the E22 mutations may affect longer-range interactions outside the 21-30 segment that can impact the aggregation of Aβ. Substituting at residue D23, on the other hand, leads to the formation of a turn rather than a bend motif, implying that in contrast to E22 mutants, the D23N mutant may affect monomer Aβ folding and subsequent aggregation. Our simulations suggest that the mechanisms by which E22 and D23 mutations affect the folding and aggregation of Aβ are fundamentally different.     

Scavenger receptor class A type I/II (CD204) null mice fail to develop fibrosis following silica exposure

Alveolar macrophages express the class A scavenger receptor (CD204) (Babaev VR, Gleaves LA, Carter KJ, Suzuki H, Kodama T, Fazio S, and Linton MF. Arterioscler Thromb Vasc Biol 20: 2593-2599, 2000); yet its role in vivo in lung defense against environmental particles has not been clearly defined. In the current study, CD204 null mice (129Sv background) were used to investigate the link between CD204 and downstream events of inflammation and fibrosis following silica exposure in vivo. CD204-/- macrophages were shown to recognize and uptake silica in vitro, although this response was attenuated compared with 129Sv wild-type mice. The production of tumor necrosis factor-alpha in lavage fluid was significantly enhanced in CD204 null mice compared with wild-type mice following silica exposure. Moreover, after exposure to environmental particles, CD204-/- macrophages exhibited improved cell viability in a dose-dependent manner compared with wild-type macrophages. Finally, histopathology from a murine model of chronic silicosis in 129Sv wild-type mice displayed typical focal lesions, interstitial thickening with increased connective tissue matrix, and cellular infiltrate into air space. In contrast, CD204-/- mice exhibited little to no deposition of collagen, yet they demonstrated enhanced accumulation of inflammatory cells largely composed of neutrophils. Our findings point to an important role of CD204 in mounting an efficient and appropriately regulated immune response against inhaled particles. Furthermore, these results indicate that the functions of CD204 are critical to the development of fibrosis and the resolution of inflammation.     

Free energy landscapes for amyloidogenic tetrapeptides dimerization

The oligomerization of four peptide sequences, KFFE, KVVE, KLLE, and KAAE is studied using replica-exchange molecular dynamics simulations with an atomically detailed peptide model. Previous experimental studies reported that of these four peptides, only those containing phenylalanine and valine residues form fibrils. We show that the fibrillogenic propensities of these peptides can be rationalized in terms of the equilibrium thermodynamics of their early oligomers. Thermodynamic stability of dimers, as measured by the temperature of monomer association, is seen to be higher for those peptides that are able to form fibrils. Although the relative high and low stabilities of the KFFE and KAAE dimers arise from their respective high and low interpeptide interaction energies, the higher stability of the KVVE dimer over the KLLE system results from the smaller loss of configurational entropy accompanying the dimerization of KVVE. Free energy landscapes for dimerization are found to be strongly sequence-dependent, with a high free energy barrier separating the monomeric and dimeric states for KVVE, KLLE, and KAAE sequences. In contrast, the most fibrillogenic peptide, KFFE, displayed downhill assembly, indicating enhanced kinetic accessibility of its dimeric states. The dimeric phase for all peptide sequences is found to be heterogeneous, containing both antiparallel beta-sheet structures that can grow into full fibrils as well as disordered dimers acting as on- or off-pathway intermediates for fibrillation.     

Silica binding and toxicity in alveolar macrophages

Inhalation of the crystalline form of silica is associated with a variety of pathologies, from acute lung inflammation to silicosis, in addition to autoimmune disorders and cancer. Basic science investigators looking at the mechanisms involved with the earliest initiators of disease are focused on how the alveolar macrophage interacts with the inhaled silica particle and the consequences of silica-induced toxicity on the cellular level. Based on experimental results, several rationales have been developed for exactly how crystalline silica particles are toxic to the macrophage cell that is functionally responsible for clearance of the foreign particle. For example, silica is capable of producing reactive oxygen species (ROS) either directly (on the particle surface) or indirectly (produced by the cell as a response to silica), triggering cell-signaling pathways initiating cytokine release and apoptosis. With murine macrophages, reactive nitrogen species are produced in the initial respiratory burst in addition to ROS. An alternative explanation for silica toxicity includes lysosomal permeability, by which silica disrupts the normal internalization process leading to cytokine release and cell death. Still other research has focused on the cell surface receptors (collectively known as scavenger receptors) involved in silica binding and internalization. The silica-induced cytokine release and apoptosis are described as the function of receptor-mediated signaling rather than free radical damage. Current research ideas on silica toxicity and binding in the alveolar macrophage are reviewed and discussed.