Intracellular folding pathway of the cystine knot-containing glycoprotein hormone alpha-subunit.
Three of the five disulfide bonds in the glycoprotein hormone alpha-subunit (GPH-alpha) form a cystine knot motif that stabilizes a three-loop antiparallel structure. Previously, we described a mutant (alpha(k)) that contained only the three knot disulfide bonds and demonstrated that the cystine knot was necessary and sufficient for efficient GPH-alpha folding and secretion. In this study, we used alpha(k) as a model to study the intracellular GPH-alpha folding pathway. Cystine knot formation proceeded through a 1-disulfide intermediate that contained the 28-82 disulfide bond. Formation of disulfide bond 10-60, then disulfide bond 32-84, followed the formation of 28-82. Whether the two non-cystine knot bonds 7-31 and 59-87 could form independent of the knot was also tested. Disulfide bond 7-31 formed rapidly, whereas 59-87 did not form when all cysteine residues of the cystine knot were converted to alanine, suggesting that 7-31 forms early in the folding pathway and that 59-87 forms during or after cystine knot formation. Finally, loop 2 of GPH-alpha has been shown to be very flexible, suggesting that loop 2 does not actively drive GPH-alpha folding. To test this, we replaced residues 36-55 in the flexible loop 2 with an artificially flexible glycine chain. Consistent with our hypothesis, folding and secretion were unaffected when loop 2 was replaced with the glycine chain. Based on these findings, we describe a model for the intracellular folding pathway of GPH-alpha and discuss how these findings may provide insight into the folding mechanisms of other cystine knot-containing proteins.
Darling
R J
RJ
Department of Pharmacology, Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, Nebraska 68198, USA.
Wilken
J A
JA
Ruddon
R W
RW
Bedows
E
E
D020816:Amino Acid Motifs / Q000235:genetics; D002460:Cell Line; D018997:Chorionic Gonadotropin, beta Subunit, Human / Q000737:chemistry / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003553:Cystine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D015292:Glycoprotein Hormones, alpha Subunit / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007424:Intracellular Fluid / Q000737:chemistry*; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017510:Protein Folding; D017433:Protein Structure, Secondary / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D019163:Reducing Agents / Q000494:pharmacology
D018997:Chorionic Gonadotropin, beta Subunit, Human; D015292:Glycoprotein Hormones, alpha Subunit; D010446:Peptide Fragments; D019163:Reducing Agents; D003553:Cystine; D003545:Cysteine; D004229:Dithiothreitol