Crystallography-based methods of fragment screening often rely on growing many crystals of a protein and soaking these in fragment-containing buffers. But how do you get biologically relevant crystals in the first place? Many proteins adopt a variety of different conformations in solution, and their freedom of movement is constrained once they are forced into a crystal lattice. Crystallizing the protein in a state that is relevant for binding ligands often means co-crystallizing them in the presence of a known ligand. In fact, some proteins are so disordered on their own that the only way you can get them to crystallize at all is by adding a small molecule. In many cases, these “co-crystals” can then be soaked in a solution containing new ligands; the existing ligands will diffuse out of the crystal, making room for new ligands. Unfortunately, in some cases the original molecule binds so tightly that it can’t be forced out. Two recent papers in J. Am. Chem. Soc. provide a clever solution.
Both papers focus on the major histocompatibility complex (MHC) Class I proteins. These proteins bind 8-11 amino acid intracellular peptides and present them on the cell surface, allowing passing T cells to survey the contents of cells for viruses, bacteria, or other nasties and, when appropriate, eliminate the infected cells. As might be expected given their function, the MHC proteins are quite promiscuous in which peptides they bind to, frustrating a general understanding of the molecular recognition. Moreover, crystallography is complicated by the fact that MHC class I proteins do not crystallize in the absence of a bound ligand.
In the first paper, Anastassis Perrakis, Ton Schumacher, and colleagues at the Netherlands Cancer Institute designed a 9-amino acid "conditional" peptide ligand for MHC that contains two internal photosensitive nitrophenyl substituents. They were able to crystallize this in complex with MHC and solve the structure. When they exposed these crystals to UV-light, the nitrophenyl groups caused the peptide to break apart into into three pieces. Interestingly, structural characterization after this exposure revealed that while the central portion of the peptide was gone, the two end bits were still bound to MHC. However, the researchers were able to successfully replace these remnants with new, full length peptides derived from HIV and avian flu proteins by soaking the crystals for just a few hours in buffer containing the new peptides. The resulting structures were identical with previously determined structures, even revealing some side-chain movement. A second paper from Ton Schumacher, Huib Ovaa, and colleagues reports a similar strategy, this time using diol-containing peptides and mild chemical cleavage with sodium periodate rather than UV-light, although in this case the reaction is done in solution rather than in crystals.
This seems like an interesting approach for tackling peptide-binding proteins, and possibly even small-molecule binding proteins, though this would require more effort to design destructible ligands.