Intramolecular cooperativity in a protein binding site assessed by combinatorial shotgun scanning mutagenesis.
Combinatorial shotgun alanine-scanning was used to assess intramolecular cooperativity in the high affinity site (site 1) of human growth hormone (hGH) for binding to its receptor. A total of 19 side-chains were analyzed and statistically significant data were obtained for 145 of the 171 side-chain pairs. The analysis revealed that 90% of the side-chain pairs exhibited no statistically significant pair interactions, and the remaining 10% of side-chain pairs exhibited only small interactions corresponding to cooperative interaction energies with magnitudes less than 0.4 kcal/mol. The statistical predictions were tested by measuring affinities for purified mutant proteins and were found to be accurate for five of six side-chain pairs tested. The results reveal that hGH site 1 behaves in a highly additive manner and suggest that shotgun scanning should be useful for assessing cooperative effects in other protein-protein interactions.
Pál
Gábor
G
Department of Biochemistry and Molecular Biology and Institute for Biophysical Dynamics, Cummings Life Sciences Center, University of Chicago, 920 East 58th Street, Chicago, IL 60637, USA.
Ultsch
Mark H
MH
Clark
Kevin P
KP
Currell
Bridget
B
Kossiakoff
Anthony A
AA
Sidhu
Sachdev S
SS
D001665:Binding Sites; D019382:Human Growth Hormone / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D016296:Mutagenesis; D011485:Protein Binding; D017434:Protein Structure, Tertiary
D008565:Membrane Proteins; C405010:delta-hGHR; D019382:Human Growth Hormone