Structural requirements of the extracellular to transmembrane domain junction for erythropoietin receptor function.
The erythropoietin receptor (EpoR) is crucial for erythrocyte formation. The x-ray crystal structures of the EpoR extracellular domain lack the juxtamembrane (JM) region and the junction to the transmembrane (TM) domain. Yet the JM-TM regions are important for transmitting the conformational change imposed on the receptor dimer by Epo binding. Cysteine-scanning mutagenesis of the JM-TM regions identified three novel constitutively active mutants, demonstrating close disulfide-bonded juxtapositioning of these residues in the JM (L223C) and N-terminal TM domain (L226C, I227C). Chemical cross-linking defined the interface of the active helical TM dimer and revealed that the JM-TM segment encompassing Leu(226)-Leu(230) is non-helical. Molecular dynamics and NMR studies indicated that the TM-JM junction forms an N-terminal helix cap. This structure is important for EpoR function because replacement of this motif by consecutive leucines rendered the receptor constitutively active.
Kubatzky
Katharina F
KF
Ludwig Institute for Cancer Research, Bruxelles 1200, Belgium.
Liu
Wei
W
Goldgraben
Kerri
K
Simmerling
Carlos
C
Smith
Steven O
SO
Constantinescu
Stefan N
SN
D020816:Amino Acid Motifs; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D049109:Cell Proliferation; D002469:Cell Separation; D003432:Cross-Linking Reagents / Q000494:pharmacology; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry; D004252:DNA Mutational Analysis; D018076:DNA, Complementary / Q000378:metabolism; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry; D005434:Flow Cytometry; D005998:Glycine / Q000031:analogs & derivatives* / Q000737:chemistry; D006133:Growth Substances / Q000378:metabolism; D006801:Humans; D007930:Leucine / Q000737:chemistry; D008156:Luciferases / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017467:Receptors, Erythropoietin / Q000737:chemistry* / Q000378:metabolism; D014162:Transfection
D003432:Cross-Linking Reagents; D018076:DNA, Complementary; D004220:Disulfides; D006133:Growth Substances; D010455:Peptides; D017467:Receptors, Erythropoietin; D008156:Luciferases; D007930:Leucine; D003545:Cysteine; D005998:Glycine; C100184:tricine