Protein Engineering (Christian Freund)
Projects
1. Proline-rich sequence recognition domains
2. T cell adhesion molecules and their effectors
3. MHC:peptide interactions
4. Alternative scaffolds
5. Redox potential
1. Proline-rich sequence recognition domains
Interactions between proline-rich sequences (PRS) and the respective PRS recognition domains are characterized by low affinities and correspondingly high off-rates. Often times, recognition codes for individual members of the respective fold families overlap and a structural comparison of individual domains (Figure 2) shows the convergence of the binding site towards a pocket enriched in aromatic amino acids and fine-tuned to bind the proline-rich ligand (Kofler & Freund, FEBS J. 273, 245,2006).
- Figure 2: Structures and binding mechanisms of PRD (Kofler & Freund, FEBS J. 2006). The structures of SH3 and GYF domains in complex with ligands that utilized charge complementarity for selective binding. For WW domains, no complex structure of a domain, recognizing positively charged ligands is available. The structure of a WW domain in complex with a PPxY ligand is shown instead. Aromatic residues which form part of the hydrophobic binding site (aromatic cradle) are depicted in yellow. Proline residues of the ligands that directly interact with the hydrophobic pockets are coloured dark blue, positively charged arginines are depicted in red, complementarily charged residues in the domains are shown in green. The hydrogen bond between carbonyl oxygens of the ligands and aromatic residues of the domains are represented as dashed red lines. Protein Data Base (PDB) accession codes for presented structures are: 1prm (SH3), 1eg4 (WW) and 1l2z (GYF).
Our work has a focus on GYF domain mediated protein-protein interactions. The GYF domain is an ancient domain probably derived from bacterial nucelotide or RNA-binding proteins. However, in eukaryotic cells, the major conserved function of GYF domains is the binding of proline-rich sequences and they do so in the context of larger complexes, as for example the spliceosome. Our goal is to define the structural and biophysical requirements of PRS recognition within the context of these multiprotein complexes. We and others have identified several PRS hubs that attract GYF and WW domain containing proteins. One major question arising from these studies is that of the spatiotemporal control of PRS recognition. Post-translational modifications as well as submodular compartmentalization potentially regulate PRS hub interactions with GYF, WW and SH3 domains. Studying the dynamic assembly of complex formation by NMR spectroscopy and other methods allows a better understanding of the principles that govern the physiological processes mediated by this interesting class of adaptor domains.
2. T cell adhesion molecules and their effectors
The ability to dramatically change their adhesive properties is a hallmark of immune cells and allows them to patrol body fluids, to contact epithelial surfaces and to interact amongst each other by forming specialized cell:cell junctions. In T cells, adhesion molecules like CD2 contribute to this process by forming interaction pairs with corresponding counter-receptors on antigen-presenting cells. Furthermore, the engagement of adhesion molecules results in the formation of intracellular complexes at the cytoplasmic membrane interface that constitute the first step in signal transduction. Thereby, transcription of T cell cytokines as for example Interleukin-2 can be induced or the properties of key adhesion molecules, the integrins, are modulated. We are interested in the order of events that trigger the formation of T cell receptor proximal protein complexes modulating integrin activity, thereby coupling the recognition of specific antigen with a change in the cells adhesive properties. A focus is on scaffolding molecules of the so-called inside-out signalling complex, especially ADAP and SKAP55. Both molecules drive the membrane recruitment of small G proteins and their regulators to the plasma membrane, however the mechanism behind is poorly understood. Having solved the NMR structure of the folded domains of ADAP we are beginning to deconvolute the contribution of individual domains to affinity and avidity modulation of integrins (Figure 1). We found that phosphorylation and redox-modifications are potentially important for the function of the adaptor proteins and we are now attempting to frame the understanding of the mutual interplay of these events within the living cell.
- Figure 3: T cell receptor (TCR) recognition of MHC:peptide on antigen presenting cells leads to highly regulated changes in integrin affinity and avidity that result in the formation of a stable cell-to-cell contact. The rapid "transversal" communication between the TCR and the integrins is mediated by an intracellular protein complex, that fulfils the criteria of a functional module.
3. MHC:peptide interactions
The central event of T cell mediated immunity is the recognition of MHC:peptide complexes by the clonotypic T cell receptor on a T cell. Despite the vast knowledge on the structure and function of MHC molecules, essential mechanistic insights are lacking in regard to antigen loading and exchange. As part of a research network we are investigating the role of small molecules (so-called MHC-loading enhancers or MLE; Hopner et al, JBC 2006) on peptide exchange by NMR spectroscopy. Having established a refolding protocol for MHC class II a/b heterodimers from E. coli inclusion bodies we are now able to investigate the effect of peptide loading and exchange by biophysical methods.
- Figure 4: MHC Class II molecule in complex with Influenza derived hemaglutinine peptide as pocket-depth-view (kindly provided by Dr. Bernd Rupp)
4. Alternative scaffolds
The development of recombinant antibodies as diagnostic tools and promising therapeutics is a success story in biotechnology. However, antibodies display unfavourable biophysical and biochemical features in certain applications that relate to their size and immunogenicity. Therefore, the development of so-called “alternative scaffolds” is desirable and has witnessed a burst in basic and applied research. We have identified several intracellular domains that may be well suited for loop randomization and the application of phage display. We are currently investigating the potential of these small size proteins to bind and inhibit the function of a variety of target proteins of diverse origin.
5. Redox potential
The importance of reactive oxygen species in physiological signalling cascades of the cell is increasingly recognized. Biochemically, it is important to identify the moieties within proteins and other biomolecules that are prone to oxidative modification. We use NMR spectroscopy to determine the redox potential of cysteine oxidation within intracellular protein domains. Using a combination of glutathione titrations, NMR spectroscopical analysis and mutagenesis allows a more detailed understanding of the molecular events that trigger reversible disulfide bond formation or oxidative modification to occur. Figure 5 gives an example of structural changes that were observed upon reversible oxidation of the ADAP hSH3N domain (Zimmermann et al., Biochemistry 46, 6971, 2007).
- Figure 5: Surface representation of the oxidized and reduced N-terminal hSH3 domain of the scaffolding protein ADAP. Yellow areas indicate hydrophobic patches while green depicts the hydrophilic surface epitopes.
