NMR spectroscopic investigation of micropolymorphism-dependent dynamics of human major histocompatibility antigens
Zusammenfassung der Projektergebnisse
The goal of the project was the investigation of the dynamics of HLA-B*27 complexes at atomic resolution using NMR spectroscopy with the focus on differences between the two subtypes HLA-B*27:09 and HLA-B*27:05. Both HLA molecules were studied with two different peptides (pVIPR (RRKWRRWHL) and TIS (RRLPIFSRL)). A prerequisite of such an investigation is the availability of sufficient amount of samples labeled with 15N, 13C and 2H. Given the size of the complexes (45 kDa), only this approach enables an assignment of the resonances and the performance of experiments that yield the desired information on the dynamics. An efficient production of labeled complexes was established during the project. The production of the protein components and the complexes was based on prior work. For expression of labeled peptides, however, a new system was established since the synthesis using solid phase methods is prohibitively expensive. The dynamics of all three components of the four HLA-B*27 complexes (heavy chain, β2-microglobulin (β2m), peptide) was investigated. In addition, free β2m was also studied. Free β2m showed dynamics on the micro-to milli-second timescale as indicated by exchange broadening in the NMR spectra in the region that contacts the heavy chain in the complex. Exactly that region, part of which is also responsible for the interaction of the HLA- B*27 molecule with CD8 in the active complex, showed subtle differences with β2m being part of the complexes in a peptide- and subtype-dependent manner. Investigation of dynamics on the nanosecond to picosecond time scale using 15N relaxation times did not reveal major differences. A similar observation was made when investigating the heavy chains. An analysis of the lines in an H-N correlation revealed line doubeling as well as missing resonances, indicating mobility on several time scales. Again differences between complexes could be found that were peptide- as well as subtype-dependent. This, however, did not include the α3-domain of the heavy chain which behaved similarly in all complexes, but differently compared to the peptide-binding α1/α2-domains. The peptides exhibited surprisingly broad lines, indicating an independent mobility on the millisecond timescale, in sharp contrast to the expectations resulting from the x-ray structures. This mobility has so far prevented an assignment and thus an in-depth analysis of the dynamics. Given the results obtained so far, further experiments will be necessary. In particular, the millisecond time scale needs to be explored with relaxation dispersion experiments. For this, not only 15N should be used but also 13C. The possibility of labeling and assigning methyl groups has already been successfully explored. Last but not least an assignment and a full analysis of the peptide dynamics need to be performed.
Projektbezogene Publikationen (Auswahl)
- (2012). HLA class I-associated diseases with a suspected autoimmune etiology: HLA-B27 subtypes as a model system. Eur. J. Cell. Biol. 91, 274-86
Uchanska-Ziegler, B., Loll, B., Fabian, H., Hee, C. S., Saenger, W. & Ziegler, A.
(Siehe online unter https://doi.org/10.1016/j.ejcb.2011.03.003) - (2013). Dynamics of free versus complexed beta2-microglobulin and the evolution of interfaces in MHC class I molecules. Immunogenetics 65, 157-72
Hee, C. S., Beerbaum, M., Loll, B., Ballaschk, M., Schmieder, P., Uchanska-Ziegler, B. & Ziegler, A.
(Siehe online unter https://doi.org/10.1007/s00251-012-0667-4) - (2013). IR spectroscopic analyses of amyloid fibril formation of beta2-microglobulin using a simplified procedure for its in vitro generation at neutral pH. Biophys. Chem. 179, 35-46
Fabian, H., Gast, K., Laue, M., Jetzschmann, K. J., Naumann, D., Ziegler, A. & Uchanska-Ziegler, B.
(Siehe online unter https://doi.org/10.1016/j.bpc.2013.05.001) - (2013). NMR spectroscopy reveals unexpected structural variation at the protein-protein interface in MHC class I molecules. J. Biomol. NMR 57, 167-78
Beerbaum, M., Ballaschk, M., Erdmann, N., Schnick, C., Diehl, A., Uchanska-Ziegler, B., Ziegler, A. & Schmieder, P.
(Siehe online unter https://doi.org/10.1007/s10858-013-9777-z) - (2013). Structural and dynamic features of HLA-B27 subtypes. Curr. Opin. Rheumatol. 25, 411-8
Uchanska-Ziegler, B., Ziegler, A. & Schmieder, P.
(Siehe online unter https://doi.org/10.1097/BOR.0b013e32836203ab) - (2016). Increased Conformational Flexibility of HLA-B*27 Subtypes Associated With Ankylosing Spondylitis. Arthritis Rheumatol. 68, 1172-82
Loll, B., Fabian, H., Huser, H., Hee, C. S., Ziegler, A., Uchanska-Ziegler, B. & Ziegler, A.
(Siehe online unter https://doi.org/10.1002/art.39567)
