Protein-Protein Interactions in Receptor Activation and Intracellular Signalling

Abstract

We review here signalling complexes that we have defined using X-ray analysis in our laboratory. They include growth factors and their receptors: nerve growth factor (NGF) and its hetero-hexameric 7S NGF storage complex, hepatocyte growth factor/scatter factor (HGF/SF) NK1 dimers and fibroblast growth factor (FGF1) in complex with its receptor (FGFR2) ectodomain and heparin. We also review our recent structural studies on intracellular signalling complexes, focusing on phosducin transducin GPry, CK2 protein kinase and its complexes, and the cyclin D-dependent kinase, Cdk6, bound to the cell cycle inhibitor p19INK4d. Comparing the structures of these complexes with others we show that the surface area buried in signalling interactions does not always give a good indication of the strength of the interactions. We show that conformational changes are often important in complexes with intermediate buried surface areas of 1500 to 2000 A2, such as Cdk6INK4 interactions. Some interactions involve recognition of continuous epitopes, where there is no necessity for a tertiary structure and very often the binding conformation is induced during the process of interaction, for example phosducin binding to the betagamma subunits (Gtbetagamma) of the heterotrimeric G protein transducin.

Understanding protein non-folding

“…As accessed from available crystal structures, the majority of small molecule effectors greater than 300 Da bind a contiguous epitope of five or more residues [350]. In contrast, many interfaces between two ordered proteins are much more complex, with epitopes consisting of discontinuous or a combination of multiple contiguous epitopes [351]. The divergent natures of these two binding systems can help to explain why attempts to find small molecule inhibitors of protein-protein interactions have so far met with limited success.…”

Synthetic Multivalent Ligands as Probes of Signal Transduction

“…[54][55][56] Ligand binding can transduce signals by directly stabilizing or destabilizing protein assemblies. [57][58][59] For example, data collected from diverse experiments suggest that attractant binding to a single MCP can impact signaling from nearby MCP partners. [46] [60][61][62][63][64] Similarly, crossphosphorylation of some growth factor receptors is facilitated by ligand-induced contacts.…”

Macromolecular complexes in crystals and solutions

“…it may vary depending on the external conditions. Protein-protein complexes, which may dissociate or associate depending on the biochemical environment, play an important role in many biological processes, such as signal transduction (Gomperts et al, 2002), electron transport (Brown et al, 1999;Doyle et al, 1986;Ren et al, 1993), transcriptional regulation (Huang et al, 1997;Sengchanthalangsy et al, 1999), growth factors (Lu et al, 1995;Hsu et al, 1997;Bianchet et al, 2000;Blundell et al, 2000), molecular switches (Darling et al, 2000;Pan & Heitman, 2002;Ma & Karplus, 1997), cell-cell recognition (Alattia et al, 1997) and many others (Waas & Dalby, 2002;Cho et al, 2006;Johannes, 2007;Bonet et al, 2006;Ansari & Helms, 2005;Nooren & Thornton, 2003;Schnarr & Khosla, 2006;Vaynberg & Qin, 2006;Fuentes et al, 2006Fuentes et al, , 2007Boelens et al, 1991;Ceres & Zlotnick, 2002;Buts et al, 2001;Nyfeler et al, 2005;Hamelryck et al, 2000). The dissociation constant K d of weak complexes may reach a few hundred mM (Nooren & Thornton, 2003), which corresponds to a ÁG diss of only a few kcal mol…”

“…If this happens then the most significant crystal interface does not correspond to the biologically related interaction or, in other words, the interaction is misrepresented by the crystal packing. Although it was found in a number of studies that weak interactions may manifest themselves in highly condensed pre-crystal solutions and crystalline states (examples are given in Ren et al, 1993;Bianchet et al, 2000;Blundell et al, 2000;Nooren & Thornton, 2003;Boelens et al, 1991;Ceres & Zlotnick, 2002;Buts et al, 2001;Hamelryck et al, 2000), the overall probability of observing a weak biologically related interaction as a crystal interface remains unclear.…”