UV-induced ligand exchange in MHC class I protein crystals.
High-throughput structure determination of protein-ligand complexes is central in drug development and structural proteomics. To facilitate such high-throughput structure determination we designed an induced replacement strategy. Crystals of a protein complex bound to a photosensitive ligand are exposed to UV light, inducing the departure of the bound ligand, allowing a new ligand to soak in. We exemplify the approach for a class of protein complexes that is especially recalcitrant to high-throughput strategies: the MHC class I proteins. We developed a UV-sensitive, "conditional", peptide ligand whose UV-induced cleavage in the crystals leads to the exchange of the low-affinity lytic fragments for full-length peptides introduced in the crystallant solution. This "in crystallo" exchange is monitored by the loss of seleno-methionine anomalous diffraction signal of the conditional peptide compared to the signal of labeled MHC beta2m subunit. This method has the potential to facilitate high-throughput crystallography in various protein families.
Celie
Patrick H N
PH
Division of Biochemistry, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands.
Toebes
Mireille
M
Rodenko
Boris
B
Ovaa
Huib
H
Perrakis
Anastassis
A
Schumacher
Ton N M
TN
D000595:Amino Acid Sequence; D003460:Crystallization; D018360:Crystallography, X-Ray; D015789:HLA-A2 Antigen / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D008958:Models, Molecular; D009842:Oligopeptides / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding / Q000528:radiation effects; D011487:Protein Conformation / Q000528:radiation effects; D012643:Selenium / Q000737:chemistry; D014466:Ultraviolet Rays
D015789:HLA-A2 Antigen; D008024:Ligands; D009842:Oligopeptides; D012643:Selenium