Role of Ring6 in the Function of the E. coli MCE Protein LetB.

LetB is a tunnel-forming protein found in the cell envelope of some double-membraned bacteria, and is thought to be important for the transport of lipids between the inner and outer membranes. In Escherichia coli the LetB tunnel is formed from a stack of seven rings (Ring1 - Ring7), in which each ring is composed of a homo-hexameric assembly of MCE domains. The primary sequence of each MCE domain of the LetB protein is substantially divergent from the others, making each MCE ring unique in nature. The role of each MCE domain and how it contributes to the function of LetB is not well understood. Here we probed the importance of each MCE ring for the function of LetB, using a combination of bacterial growth assays and cryo-EM. Surprisingly, we find that ΔRing3 and ΔRing6 mutants, in which Ring3 and Ring6 have been deleted, confer increased resistance to membrane perturbing agents. Specific mutations in the pore-lining loops of Ring6 similarly confer increased resistance. A cryo-EM structure of the ΔRing6 mutant shows that despite the absence of Ring6, which leads to a shorter assembly, the overall architecture is maintained, highlighting the modular nature of MCE proteins. Previous work has shown that Ring6 is dynamic and in its closed state, may restrict the passage of substrate through the tunnel. Our work suggests that removal of Ring6 may relieve this restriction. The deletion of Ring6 combined with mutations in the pore-lining loops leads to a model for the tunnel gating mechanism of LetB. Together, these results provide insight into the functional roles of individual MCE domains and pore-lining loops in the LetB protein.

Affinity maturation is required for pathogenic monovalent IgG4 autoantibody development in myasthenia gravis.

Pathogenic muscle-specific tyrosine kinase (MuSK)-specific IgG4 autoantibodies in autoimmune myasthenia gravis (MG) are functionally monovalent as a result of Fab-arm exchange. The development of these unique autoantibodies is not well understood. We examined MG patient-derived monoclonal autoantibodies (mAbs), their corresponding germline-encoded unmutated common ancestors (UCAs), and monovalent antigen-binding fragments (Fabs) to investigate how affinity maturation contributes to binding and immunopathology. Mature mAbs, UCA mAbs, and mature monovalent Fabs bound to MuSK and demonstrated pathogenic capacity. However, monovalent UCA Fabs bound to MuSK but did not have measurable pathogenic capacity. Affinity of the UCA Fabs for MuSK was 100-fold lower than the subnanomolar affinity of the mature Fabs. Crystal structures of two Fabs revealed how mutations acquired during affinity maturation may contribute to increased MuSK-binding affinity. These findings indicate that the autoantigen drives autoimmunity in MuSK MG through the accumulation of somatic mutations such that monovalent IgG4 Fab-arm-exchanged autoantibodies reach a high-affinity threshold required for pathogenic capacity.

Effect of intravoxel incoherent motion on diffusion parameters in normal brain.

At very low diffusion weighting the diffusion MRI signal is affected by intravoxel incoherent motion (IVIM) caused by dephasing of magnetization due to incoherent blood flow in capillaries or other sources of microcirculation. While IVIM measurements at low diffusion weightings have been frequently used to investigate perfusion in the body as well as in malignant tissue, the effect and origin of IVIM in normal brain tissue is not completely established. We investigated the IVIM effect on the brain diffusion MRI signal in a cohort of 137 radiologically-normal patients (62 male; mean age = 50.2 ±â€¯17.8, range = 18 to 94). We compared the diffusion tensor parameters estimated from a mono-exponential fit at b = 0 and 1000 s/mm2 versus at b = 250 and 1000 s/mm2. The asymptotic fitting method allowed for quantitative assessment of the IVIM signal fraction f* in specific brain tissue and regions. Our results show a mean (median) percent difference in the mean diffusivity of about 4.5 (4.9)% in white matter (WM), about 7.8 (8.7)% in cortical gray matter (GM), and 4.3 (4.2)% in thalamus. Corresponding perfusion fraction f* was estimated to be 0.033 (0.032) in WM, 0.066 (0.065) in cortical GM, and 0.033 (0.030) in the thalamus. The effect of f* with respect to age was found to be significant in cortical GM (Pearson correlation ρ â€‹= â€‹0.35, p â€‹= â€‹3*10-5) and the thalamus (Pearson correlation ρ = 0.20, p = 0.022) with an average increase in f* of 5.17*10-4/year and 3.61*10-4/year, respectively. Significant correlations between f* and age were not observed for WM, and corollary analysis revealed no effect of gender on f*. Possible origins of the IVIM effect in normal brain tissue are discussed.

ß-Hydroxyacyl-acyl Carrier Protein Dehydratase (FabZ) from Francisella tularensis and Yersinia pestis: Structure Determination, Enzymatic Characterization, and Cross-Inhibition Studies.

The bacterial system for fatty acid biosynthesis (FAS) contains several enzymes whose sequence and structure are highly conserved across a vast array of pathogens. This, coupled with their low homology and difference in organization compared to the equivalent system in humans, makes the FAS pathway an excellent target for antimicrobial drug development. To this end, we have cloned, expressed, and purified the ß-hydroxyacyl-acyl carrier protein dehydratase (FabZ) from both Francisella tularensis (FtFabZ) and Yersinia pestis (YpFabZ). We also solved the crystal structures and performed an enzymatic characterization of both enzymes and several mutant forms of YpFabZ. Additionally, we have discovered two novel inhibitors of FabZ, mangostin and stictic acid, which show similar potencies against both YpFabZ and FtFabZ. Lastly, we selected several compounds from the literature that have been shown to be active against single homologues of FabZ and tested them against both YpFabZ and FtFabZ. These results have revealed clues as to which scaffolds are likely to lead to broad-spectrum antimicrobials targeted against FabZ as well as modifications to existing FabZ inhibitors that may improve potency.