The crystal structure of affinity-matured human growth hormone at 2 A resolution.
A variant of human growth hormone (hGH), in which 15 mutations were introduced with phage display mutagenesis to improve receptor binding affinity by 400-fold, yielded two related crystal forms diffracting to high resolution. The structure of this variant was determined in both crystal forms, one at 2.0 A resolution and one at 2.4 A resolution, using molecular replacement with wild-type hGH taken from the receptor complex structure as a search model. Crystallographic refinement of the 2 A structure gave an R-value R-value of 18.5% for data in the resolution range 8 to 2 A. The final model consists of residues 1 to 128 and 155 to 191, with three side-chains modeled in alternative conformations, together with 77 water molecules. Comparison of the structure with wild-type hGH shows that most of the secondary structural elements are unchanged. The exception is the first turn of the third helix in the four-helix bundle core, which is unraveled in the present variant. Analysis of the two related packing environments suggests that this change is caused by crystal packing forces. A large change in the orientation of a short segment of helix found in the connection between the first two core helices is interpreted as evidence for rigid-body variability of this helical segment. Analysis of the mutations in light of the structure of the wild-type hGH/receptor complex shows that six of the mutations are buried in the hormone, whereas the remaining nine involve residues that interact with the receptor in the complex.
Ultsch
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Department of Protein Engineering Genetech, Inc, South San Francisco, CA 94080.
Somers
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Kossiakoff
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de Vos
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D000595:Amino Acid Sequence; D003196:Computer Graphics; D018360:Crystallography, X-Ray / Q000379:methods; D014644:Genetic Variation; D013006:Growth Hormone / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism
D011994:Recombinant Proteins; D013006:Growth Hormone