Combining NMR docking data with EPR distances and in silico calculations for a more complete model of colicin protein-protein interactions Morgan Bye, A. Stratford, C. Teutloff, G. Moore, F. MacMillan Henry Wellcome Unit for Biological EPR, University of East Anglia, Norwich, UK. School of Chemistry, University of East Anglia, Norwich, UK.  (Currently) Fachbereich Physik, Freie Universitat Berlin, Berlin, Germany morgan.bye@uea.ac.uk morganbye.net Work supported by [1] Ferentz et al, Rev Biophys, 33, 29 (2000) [2] Palma et al, Proteins, 39, 372 (2000) [3] de Vries et al, Proteins, 69, 726 (2007) [4] MMM – http://www.epr.ethz.ch/software [5] HADDOCK – http://www.nmr.chem.uu.nl/haddock [6] Cascales et al, MMBR, 71, 1 (2007) Currently atomic resolution structures of protein-protein interactions are commonly limited to X-ray crystallography or NMR, developments in these fields have allowed for more accurate and biologically relevant structures to be developed [1] however ab initio information still remains a developing field [2,3]. Site-directed spin labelling (SDSL) in combination with electron paramagnetic resonance (EPR) pulsed electron double resonance (PELDOR) allows for the precise measurement of intra and intermolecular distances. New in silico methods allow for comparison to the experimental [4]. SDSL at several sites within a complex allows a three dimensional model to be built up using distance constraints, which when combined with NMR docking data gives a comprehensive model [5]. Using the well-characterised system, of colicin E9 and its cognate inhibitor, Im9 [6] a comprehensive solution is being developed to combine such NMR docking models, with PELDOR distance (and angle) constraints and in silico computations which are comparable to X-ray crystallographic models. The biology During times of stress it is evolutionarily beneficial to have an advantage over the competition, resulting in E. coli having developed colicins which act as bactericides to other species [1]. Here we focus upon the DNase colicin E9. To provide immunity to the host it is co- expressed with an immunity protein, Im9. A simple diagram of the E9 action. The E9 DNase domain (C domain) is in red, whilst the E9 T and R domains are in blue, Im9 in green Spin labelling Initial spin labelling efficiencies were < 1% using standard procedures. A change to buffer, aerobic conditions and the addition of EDTA, acceptable labelling efficiencies were obtained. E9 S3C 56% E9 S30C 25% E9 S80C 27% Im9 C23 33% X-band cw-EPR Here it can be seen (by lineshape, width and position of peaks) that the binding of the cognate protein slightly influences the cw spectra. All experiments here were run with the same parameters, with the same protein concentrations. References X – band PELDOR (above) Colicin E9 DNase domain in grey with residues S3C, S30C and S80C MTSL spin labelled. Colicin Immunity Protein, Im9, in cyan with residue C23 MTSL spin labelled (left) E9 S80C position showing all 95 spin label rotamers at 298K, electron location shown as a purple sphere. Rotamer opacity and sphere volume is proportional to rotamer population (above) E9 S30C PELDOR trace prediction at various temperatures, highlighted is the 80K prediction for comparison to below. E9 S30C cw spectra comparison at 80K (top) and 298K (bottom). In blue E9S30C*, green E9S30C*- Im9 red E9S30C*-Im9* cyan E9S30C-Im9* magenta Im9*. Where * indicates a spin label. 298K 80K Complete the PELDOR with other mutants Complete the data set at other frequencies Develop technique to accurately model Expanded to characterise other ill- defined non-cognate binding pairs of colicin E9 with Im2, Im7 and Im8. Pump Detect Field Swept Echo Fourier Transformation Distance Distribution Pulse Sequence PELDOR (using the 4 pulse technique shown left) has been conducted on the E9 S30C* - Im9 C23* complex at 50K. The result of this shows the main distance distribution to lie at 38.1 Å. This compares well with the 38.2 Å pre