Project 1 – (Germany) – Research Group Prof. Andreas Ziegler
The human major histocompatibility complex is a genetic region that is of crucial importance for the immune system, the body’s defence line against foreign invaders such as bacteria or viruses. It encodes several membrane proteins (HLA molecules) whose function it is to bind components (so-called peptides) derived from proteins of infectious agents that are present within cells. The resulting complexes can then be recognized by specialized white blood cells, so-called cytotoxic lymphocytes (CTL), and the cells carrying the complexes are destroyed as a consequence. In this way, the immune system can get rid of dangerous intruders. Unfortunately, this sophisticated system is not entirely perfect and may turn against the host, resulting in autoimmunity.
Here, some CTL recognize not only antigenic fragments derived from a foreign protein, but react also with HLA molecules that have bound a peptide which has been cleaved from a normal component of the body, a so-called self-peptide. There are many autoimmune disorders for which such a mechanism of disease initiation is suspected, as in the case of the association between HLA-B*27 and ankylosing spondylitis (AS). We use biophysical techniques in an attempt to understand the differential association of two HLA-B*27 variants (also called subtypes) with AS. The following description of our projects is very technical and directed more to a specialist. It is still largely enigmatic how the deeply buried polymorphic heavy chain (HC) residue 116, which distinguishes the HLA-B*27 subtypes B*27:05 and B*27:09, exerts its effects such that these two proteins are either associated (B*27:05) or not associated (B*27:09) with AS.
Despite more than 15 years of research to solve the structures of the two HLA-B*27 subtypes in complex with more than ten distinct peptides, X-ray crystallography has not been able to shed light on this issue. We also investigate two other subtypes that are differentially associated with AS, B*27:04 and B*27:06, that are distinguished by two amino acids. Two experimental techniques to assess also the dynamic properties of HLA-B*27 subtypes, infrared (IR) spectroscopy (in cooperation with Dr. Heinz Fabian, Robert Koch-Institut, Berlin) and nuclear magnetic resonance (NMR) spectroscopy (in cooperation with Dr. Peter Schmieder, Leibniz-Institut für Molekulare Pharmakologie, Berlin) are in the center of our research. In contrast to IR spectroscopy, NMR spectroscopy allows investigating the flexibility of individual residues at various time-scales, but requires 10-20x more highly purified protein that has to be labeled with certain non-radioactive isotopes (13C, 15N, 2H).
The introduction of these isotopes into the individual components of HLA-B*27 complexes (HC, ?2m, peptide) is cumbersome, time-consuming and, due in particular to the low yields of labeled proteins, very expensive. Using IR spectroscopy, we want to verify that AS-associated subtypes (B*27:04 and B*27:05) are indeed more flexible than those that lack the disease association (B*27:06 and B*27:09). A challenging further series of experiments will assess the dynamics of Cys67, an amino acid residue within the peptide binding groove of the molecule that is thought by some researchers to be involved in AS pathogenesis. The NMR spectroscopic experiments are aimed at analyzing NMR spectra that have already been obtained from HC and b2m of four complexes.
These comprise the analysis of B*27:05 and B*27:09, each in complex with the peptides TIS and pVIPR. The results will provide us with information on the flexibility of all components of the complexes at atomic resolution.