NMR h3 JNC’ Couplings Provide Comprehensive Geometrical Constraints for Protein H-bonds in Solution

Protein backbone H-bonds (>N–H···O–C<) show relationships between NMR h3JNC\u27 couplings measured in solution and H-bond geometry parameters seen in X-ray crystal structures. Assuming that the solution and crystal structure of protein backbone is the same, the h3JNC\u27 couplings can be calibrated to provide good estimates of both angular and radial H-bond parameters in a solution. The crucial premise of equality between the solution and the crystal structure of protein backbone we validated on the level of the NH-bonds orientation, by comparing the orientations inferred from X-ray crystal structures with the solution ones determined from NMR residual dipolar couplings

Negative Regulation of TLR Signaling by BCAP Requires Dimerization of Its DBB Domain.

The B cell adaptor protein (BCAP) is a multimodular regulator of inflammatory signaling in diverse immune system cells. BCAP couples TLR signaling to phosphoinositide metabolism and inhibits MyD88-directed signal transduction. BCAP is recruited to the TLR signalosome forming multitypic interactions with the MAL and MyD88 signaling adaptors. In this study, we show that indirect dimerization of BCAP TIR is required for negative regulation of TLR signaling. This regulation is mediated by a transcription factor Ig (TIG/IPT) domain, a fold found in the NF-κB family of transcription factors. We have solved the crystal structure of the BCAP TIG and find that it is most similar to that of early B cell factor 1 (EBF1). In both cases, the dimer is stabilized by a helix-loop-helix motif at the C terminus and interactions between the β-sheets of the Ig domains. BCAP is exclusively localized in the cytosol and is unable to bind DNA. Thus, the TIG domain is a promiscuous dimerization module that has been appropriated for a range of regulatory functions in gene expression and signal transduction

The dominantly expressed class II molecule from a resistant MHC haplotype presents only a few Marek's disease virus peptides by using an unprecedented binding motif.

Funder: Deutschen Konsortium für Translationale Krebsforschung; funder-id: http://dx.doi.org/10.13039/501100012353Funder: Natural and Medical Sciences Institute (D)Viral diseases pose major threats to humans and other animals, including the billions of chickens that are an important food source as well as a public health concern due to zoonotic pathogens. Unlike humans and other typical mammals, the major histocompatibility complex (MHC) of chickens can confer decisive resistance or susceptibility to many viral diseases. An iconic example is Marek's disease, caused by an oncogenic herpesvirus with over 100 genes. Classical MHC class I and class II molecules present antigenic peptides to T lymphocytes, and it has been hard to understand how such MHC molecules could be involved in susceptibility to Marek's disease, given the potential number of peptides from over 100 genes. We used a new in vitro infection system and immunopeptidomics to determine peptide motifs for the 2 class II molecules expressed by the MHC haplotype B2, which is known to confer resistance to Marek's disease. Surprisingly, we found that the vast majority of viral peptide epitopes presented by chicken class II molecules arise from only 4 viral genes, nearly all having the peptide motif for BL2*02, the dominantly expressed class II molecule in chickens. We expressed BL2*02 linked to several Marek's disease virus (MDV) peptides and determined one X-ray crystal structure, showing how a single small amino acid in the binding site causes a crinkle in the peptide, leading to a core binding peptide of 10 amino acids, compared to the 9 amino acids in all other reported class II molecules. The limited number of potential T cell epitopes from such a complex virus can explain the differential MHC-determined resistance to MDV, but raises questions of mechanism and opportunities for vaccine targets in this important food species, as well as providing a basis for understanding class II molecules in other species including humans

Negative Regulation of TLR Signaling by BCAP Requires Dimerization of Its DBB Domain.

The B cell adaptor protein (BCAP) is a multimodular regulator of inflammatory signaling in diverse immune system cells. BCAP couples TLR signaling to phosphoinositide metabolism and inhibits MyD88-directed signal transduction. BCAP is recruited to the TLR signalosome forming multitypic interactions with the MAL and MyD88 signaling adaptors. In this study, we show that indirect dimerization of BCAP TIR is required for negative regulation of TLR signaling. This regulation is mediated by a transcription factor Ig (TIG/IPT) domain, a fold found in the NF-κB family of transcription factors. We have solved the crystal structure of the BCAP TIG and find that it is most similar to that of early B cell factor 1 (EBF1). In both cases, the dimer is stabilized by a helix-loop-helix motif at the C terminus and interactions between the β-sheets of the Ig domains. BCAP is exclusively localized in the cytosol and is unable to bind DNA. Thus, the TIG domain is a promiscuous dimerization module that has been appropriated for a range of regulatory functions in gene expression and signal transduction

NMR h3 JNC’ Couplings Provide Comprehensive Geometrical Constraints for Protein H-bonds in Solution

Protein backbone H-bonds (>N–H···O–C<) show relationships between NMR h3JNC\u27 couplings measured in solution and H-bond geometry parameters seen in X-ray crystal structures. Assuming that the solution and crystal structure of protein backbone is the same, the h3JNC\u27 couplings can be calibrated to provide good estimates of both angular and radial H-bond parameters in a solution. The crucial premise of equality between the solution and the crystal structure of protein backbone we validated on the level of the NH-bonds orientation, by comparing the orientations inferred from X-ray crystal structures with the solution ones determined from NMR residual dipolar couplings

Rotational and Translational Motion of Troponin C

Time resolved fluorescence anisotropy and sedimentation velocity has been used to study the rotational and translational hydrodynamic behavior of two mutants of chicken skeletal troponin C bearing a single tryptophan residue at position 78 or 154 in the metal-free-, metal-bound-, and troponin I peptide (residues 96–116 of troponin I)-ligated states. The fluorescence anisotropy data of both mutants were adequately described by two rotational correlation times, and these are compared with the theoretically expected values based on the rotational diffusion of an idealized dumbbell. These data imply that the motion of the N- and C-terminal domains of troponin C are independent. They also suggest that in the metal-free, calcium-saturated and calcium-saturated troponin I peptide-bound states, troponin C is elongated, having an axial ratio of 4–5. Calcium or magnesium binding to the high affinity sites alone reduces the axial ratio to approximately 3. However, with calcium bound to sites III and IV and in the presence of a 1:1 molar ratio of the troponin I peptide, troponin C is approximately spherical. The metal ion and troponin I peptide-induced length changes in troponin C may play a role in the mechanism by which the regulatory function of troponin C is effected

Anterograde trafficking of Toll-like receptors requires the cargo sorting adaptors TMED-2 and 7.

Toll-Like Receptors (TLRs) play a pivotal role in immunity by recognising conserved structural features of pathogens and initiating the innate immune response. TLR signalling is subject to complex regulation that remains poorly understood. Here we show that two small type I transmembrane receptors, TMED2 and 7, that function as cargo sorting adaptors in the early secretory pathway are required for transport of TLRs from the ER to Golgi. Protein interaction studies reveal that TMED7 interacts with TLR2, TLR4 and TLR5 but not with TLR3 and TLR9. On the other hand, TMED2 interacts with TLR2, TLR4 and TLR3. Dominant negative forms of TMED7 suppress the export of cell surface TLRs from the ER to the Golgi. By contrast TMED2 is required for the ER-export of both plasma membrane and endosomal TLRs. Together, these findings suggest that association of TMED2 and TMED7 with TLRs facilitates anterograde transport from the ER to the Golgi

Structure of the Glycosyltransferase EryCIII in Complex with its Activating P450 Homologue EryCII

In the biosynthesis of the clinically important antibiotic erythromycin D, the glycosyltransferase (GT) EryCIII, in concert with its partner EryCII, attaches a nucleotide-activated sugar to the macrolide scaffold with high specificity. To understand the role of EryCII, we have determined the crystal structure of the EryCIII·EryCII complex at 3.1 Å resolution. The structure reveals a heterotetramer with a distinctive, elongated quaternary organization. The EryCIII subunits form an extensive self-complementary dimer interface at the center of the complex, and the EryCII subunits lie on the periphery. EryCII binds in the vicinity of the putative macrolide binding site of EryCIII but does not make direct interactions with this site. Our biophysical and enzymatic data support a model in which EryCII stabilizes EryCIII and also functions as an allosteric activator of the GT