Certain surfactants significantly enhance the activity of antibiotics in the mouse model of MTB and drug resistant MTB infection and effectively remove the bacteria from a pulmonary cavity in human ex-vivo study.

Surfactants have the potential to overcome natural resistance of MTB to antibiotics which is mediated by barriers that impede the penetration of drugs to their targets. A major component of this barrier is trehalose dimycolate (TDM) which surrounds the bacteria with a thick lipid shield. In this study dodecyl maltoside (DDM) was evaluated for this purpose. This surfactant is an excellent cellular permeabilizing agent with associated low toxicity. The administration of the surfactant as an aerosol into the lungs of the infected mice achieved a 5-10 times enhancement of the isoniazid (INH) treatment gauged by the reduction of the colony forming units. This study also established proof of principle that surfactants alone applied as an aerosol can reduce the bacteria count in lungs infected with MTB. The potential of the surfactant in the therapy of human cavitary TB was also investigated using a surgically removed lung from a patient with extreme drug resistant MTB (XDR-TB). A cavity in this lung was flushed with DDM solution ex-vivo. The procedure readily removed the bacteria, excessive amounts of TDM and necrotic tissue from the cavity. These studies demonstrate that DDM can disrupt the layers of TDM and free embedded MTB and, consequently, surfactants have promise as a proficient modality for the treatment of pulmonary MTB.

Certain surfactants show promise in the therapy of pulmonary tuberculosis.

BACKGROUND/AIM: The aim of the present study was to develop the basis for the use of surfactants in the treatment of pulmonary tuberculosis (TB). Bacteria are surrounded by a thick lipid coat primarily consisting of trehalose dimycolate (TDM) and, consequently, are well shielded from the immune system's response and antibiotics. This protective barrier was removed by exposing the bacteria to certain surfactants. MATERIALS AND METHODS: Dodecyl maltoside (DDM) and octyl glucoside (OG) were utilized as non-toxic surfactants. RESULTS: Electron microscopy (EM) studies revealed that aggregated bacteria were also covered with excessive TDM which exacerbate the treatment efforts. Light and EM studies demonstrated that DDM and OG disperse the aggregated bacteria and are bactericidal. CONCLUSION: The studies presented here establish that certain surfactants are proficient in removing MTB's shield and, because they are well known as cell permeabilizing agents, they may also enhance the effectiveness of antibiotics and the immune system's response in the treatment of pulmonary TB.

Structures of the human pyruvate dehydrogenase complex cores: a highly conserved catalytic center with flexible N-terminal domains.

Dihydrolipoyl acetyltransferase (E2) is the central component of pyruvate dehydrogenase complex (PDC), which converts pyruvate to acetyl-CoA. Structural comparison by cryo-electron microscopy (cryo-EM) of the human full-length and truncated E2 (tE2) cores revealed flexible linkers emanating from the edges of trimers of the internal catalytic domains. Using the secondary structure constraints revealed in our 8 A cryo-EM reconstruction and the prokaryotic tE2 atomic structure as a template, we derived a pseudo atomic model of human tE2. The active sites are conserved between prokaryotic tE2 and human tE2. However, marked structural differences are apparent in the hairpin domain and in the N-terminal helix connected to the flexible linker. These permutations away from the catalytic center likely impart structures needed to integrate a second component into the inner core and provide a sturdy base for the linker that holds the pyruvate dehydrogenase for access by the E2-bound regulatory kinase/phosphatase components in humans.

The endosome-associated protein Hrs is hexameric and controls cargo sorting as a "master molecule".

The structure of the endosomal-associated protein, Hrs, has been determined with cryo-electron microscopy. Hrs interacts with a number of proteins, including SNAP-25 and STAM1, forming a complex that binds ubiquitin moieties. Analytical ultracentrifugation studies revealed that Hrs exists as a hexamer. The symmetry and the structure of the hexameric form of Hrs were determined with the single-particle reconstruction method. Hrs comprises three antiparallel dimers with a central core and distinct caps on either end. Crystal structures of VHS and FYVE domains fit into the Hrs end caps in the EM density map. Thus, the location of domains that interact with the endosomal membrane, the VHS, FYVE, and C-terminal domains, facilitates the anchorage of Hrs to the membrane, initiating the functional processes of Hrs on the endosome. Based on our model, the Hrs hexamer interacts with the membrane and acts as a "master molecule" that presents multiple sites for protein binding.

Rearrangement of the 16S precursor subunits is essential for the formation of the active 20S proteasome.

Proteasome-dependent proteolysis is essential for a number of key cellular processes and requires a sophisticated biogenesis pathway to function. Here, we have arrested the assembly process in its dynamic progression at the short-lived 16S state. Structural analysis of the 16S proteasome precursor intermediates by electron microscopy, and single particle analysis reveals major conformational changes in the structure of the beta-ring in comparison with one-half of the 20S proteasome. The individual beta-subunits in the 16S precursor complex rotate with respect to their positions in the x-ray crystallographic structure of the fully assembled 20S. This rearrangement results in a movement of the catalytic residue threonine-1 from the protected location in 16S precursor complexes to a more exposed position in the 20S structure. Thereby, our findings provide a molecular explanation for the structural rearrangements necessary for the dimerization of two 16S precursor complexes and the subsequent final maturation to active 20S proteasomes.

Comparative analyses of the three-dimensional structures and enzymatic properties of alpha, beta, gamma and delta isoforms of Ca2+-calmodulin-dependent protein kinase II.

Ca(2+)-calmodulin-dependent protein kinase II (CaM-kinase II) is a ubiquitous Ser/Thr-directed protein kinase that is expressed from a family of four genes (alpha, beta, gamma, and delta) in mammalian cells. We have documented the three-dimensional structures and the biophysical and enzymatic properties of the four gene products. Biophysical analyses showed that each isoform assembles into oligomeric forms and their three-dimensional structures at 21-25 A revealed that all four isoforms were dodecamers with similar but highly unusual architecture. A gear-shaped core comprising the association domain has the catalytic domains tethered on appendages, six of which extend from both ends of the core. At this level of resolution, we can discern no isoform-dependent differences in ultrastructure of the holoenzymes. Enzymatic analyses showed that the isoforms were similar in their K(m) for ATP and the peptide substrate syntide, but showed significant differences in their interactions with Ca(2+)-calmodulin as assessed by binding, substrate phosphorylation, and autophosphorylation. Interestingly, the rank order of CaM binding affinity (gamma > beta > delta > alpha) does not directly correlate with the rank order of their CaM dependence for autophosphorylation (beta > gamma > delta > alpha). Simulations utilizing this data revealed that the measured differences in CaM binding affinities play a minor role in the autophosphorylation of the enzyme, which is largely dictated by the rate of autophosphorylation for each isoform.

Conformational flexibility of pyruvate dehydrogenase complexes: a computational analysis by quantized elastic deformational model.

Pyruvate dehydrogenase complex (PDC) is one of the largest multienzyme complexes known and consists of a dodecahedral E2 core to which other components are attached. We report the results of applying a new computational method, quantized elastic deformational model, to simulating the conformational fluctuations of the truncated E2 core, using low-resolution electron cryomicroscopy density maps. The motional features are well reproduced; especially, the symmetric breathing mode revealed in simulation is nearly identical with what was observed experimentally. Structural details of the motions of the trimeric building blocks, which are critical to facilitating the global expansion and contraction of the complex, were revealed. Using the low-resolution maps from electron cryomicroscopy reconstructions, the simulations showed a picture of the motional mechanism of the PDC core, which is an example without precedent of thermally activated global dynamics. Moreover, the current results support an earlier suggestion that, at low resolution and without the use of amino acid sequence and atomic coordinates, it is possible for computer simulations to provide an accurate description of protein dynamics.

3D electron microscopy reveals the variable deposition and protein dynamics of the peripheral pyruvate dehydrogenase component about the core.

Cryo-electron microscopy was exploited to reveal and study the influence of pyruvate dehydrogenase (E1) occupancy on the conformational states of the Saccharomyces cerevisiae pyruvate dehydrogenase complex (PDC). Structures representative of PDC preparations with approximately 40% and full E1 occupancy were determined after the electron microscopy images from each preparation were classified according to their sizes. The reconstructions derived from two size groups showed that the deposition of the E1 molecules associated with the larger complex is, unexpectedly, not icosahedrally arranged, whereas in the smaller complex the E1 molecules have an arrangement and architecture similar to their more ordered deposition in the WT bovine kidney PDC. This study also shows that the linker of dihydrolipamide acetyltransferase (E2) that tethers E1 to the E2 core increases in length from approximately 50 to 75 A, accounting largely for the size difference of the smaller and larger structures, respectively. Extensive E1 occupancy of its 60 E2 binding sites favors the extended conformation of the linker associated with the larger complex and appears to be related to the loss of icosahedral symmetry of the E1 molecules. However, the presence of a significant fraction of larger molecules also in the WT PDC preparation with low E1 occupancy indicates that the conformational variability of the linker contributes to the overall protein dynamics of the PDC and the variable deposition of E1. The flexibility of the complex may enhance the catalytic proficiency of this macromolecular machine by promoting the channeling of the intermediates of catalysis between the active sites.

The three-dimensional structure of the human alpha 2-macroglobulin dimer reveals its structural organization in the tetrameric native and chymotrypsin alpha 2-macroglobulin complexes.

Three-dimensional electron microscopy reconstructions of the human alpha(2)-macroglobulin (alpha(2)M) dimer and chymotrypsin-transformed alpha(2)M reveal the structural arrangement of the two dimers that comprise native and proteinase-transformed molecules. They consist of two side-by-side extended strands that have a clockwise and counterclockwise twist about their major axes in the native and transformed structures, respectively. This and other studies show that there are major contacts between the two strands at both ends of the molecule that evidently sequester the receptor binding domains. Upon proteinase cleavage of the bait domains and subsequent thiol ester cleavages, which occur near the central region of the molecule, the two strands separate by 40 A at both ends of the structure to expose the receptor binding domains and form the arm-like extensions of the transformed alpha(2)M. During the transformation of the structure, the strands untwist to expose the alpha(2)M central cavity to the proteinase. This extraordinary change in the architecture of alpha(2)M functions to completely engulf two molecules of chymotrypsin within its central cavity and to irreversibly encapsulate them.