Protein design: reengineering cellular retinoic acid binding protein II into a rhodopsin protein mimic.
Rational redesign of the binding pocket of Cellular Retinoic Acid Binding Protein II (CRABPII) has provided a mutant that can bind retinal as a protonated Schiff base, mimicking the binding observed in rhodopsin. The reengineering was accomplished through a series of choreographed manipulations to ultimately orient the reactive species (the epsilon-amino group of Lys132 and the carbonyl of retinal) in the proper geometry for imine formation. The guiding principle was to achieve the appropriate Bürgi-Dunitz trajectory for the reaction to ensue. Through crystallographic analysis of protein mutants incapable of forming the requisite Schiff base, a highly ordered water molecule was identified as a key culprit in orienting retinal in a nonconstructive manner. Removal of the ordered water, along with placing reinforcing mutations to favor the desired orientation of retinal, led to a triple mutant CRABPII protein capable of nanomolar binding of retinal as a protonated Schiff base. The high-resolution crystal structure of all-trans-retinal bound to the CRABPII triple mutant (1.2 A resolution) unequivocally illustrates the imine formed between retinal and the protein.
Vasileiou
Chrysoula
C
Department of Chemistry, Michigan State University, East Lansing, Michigan 48824, USA.
Vaezeslami
Soheila
S
Crist
Rachael M
RM
Rabago-Smith
Montserrat
M
Geiger
James H
JH
Borhan
Babak
B
D001665:Binding Sites; D018360:Crystallography, X-Ray; D008956:Models, Chemical; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D015202:Protein Engineering; D017510:Protein Folding; D017433:Protein Structure, Secondary; D018168:Receptors, Retinoic Acid / Q000737:chemistry* / Q000235:genetics*; D012172:Retinaldehyde / Q000737:chemistry; D012243:Rhodopsin / Q000737:chemistry*; D012545:Schiff Bases
D018168:Receptors, Retinoic Acid; D012545:Schiff Bases; C082928:retinoic acid binding protein II, cellular; D012243:Rhodopsin; D012172:Retinaldehyde