The Biologic Action of Single-chain Choriogonadotropin Is Not Dependent on the Individual Disulfide Bonds of the β Subunit

Abstract

Disrupting disulfide loops in the human chorionic gonadotropin ␤ subunit (CG␤) inhibits combination with the ␣ subunit. Because the bioactivity requires a heterodimer, studies on the role of disulfide bonds on receptor binding/signal transduction have previously been precluded. To address this problem, we bypassed the assembly step and genetically fused CG␤ subunits bearing paired cysteine mutations to a wild-type ␣ (WT␣) subunit. The changes altered secretion of the single-chain mutants which parallel that seen for the CG␤ monomeric subunit. Despite conformational changes in CG disulfide bond mutants (assayed by gel electrophoresis and conformationally sensitive monoclonal antibodies), the variants bind to the lutropin/CG receptor and activated adenylate cyclase in vitro. The data show that the structural requirements for secretion and bioactivity are not the same. The results also suggest that the extensive native subunit interactions determined by the cystine bonds are not required for signal transduction.Moreover, these studies demonstrate that the singlechain model is an effective approach to structure-activity relationships of residues and structural domains associated with assembly of multisubunit ligands.Mutational and structural studies have revealed that small clusters of amino acids rather than large structural motifs often contribute to most of the energy involved in proteinprotein interactions such as hormone binding to its receptor (for review, see Refs. 1-3). However, it is not clear how the conformation of a peptide ligand contributes to the coupling of binding to signal transduction. This is especially an issue for the function of hormone-receptor complexes involving multisubunit ligands. A convenient model for studying the tertiary and quaternary determinants in signaling is the glycoprotein hormone family which include human chorionic gonadotropin (hCG), 1 lutropin (LH), follitropin (FSH), and thyrotropin. Each is a non-covalent heterodimer composed of a common ␣ and unique ␤ subunit which allows recognition of the corresponding G-protein-coupled receptor (for review, see Ref. 4).Recent crystallographic studies of hCG revealed a significant structural similarity to several growth factor families, e.g. transforming growth factor ␤, which contain a cystine knot motif composed of three pairs of bridged cysteine residues (5-8). Each hCG subunit contains a cystine knot configuring three disulfide-bonded loops which are the major structural motifs (7,8). Based on the crystallographic studies of hCG (7,8), the disulfide bonds in the CG␤ subunit are at positions 9 -57, 34 -88, 38 -90, 23-72, 93-100, and 26 -110 (Fig. 1A). The folding intermediates associated with the ordered formation of these bridges to acquire an assembly-competent form is well documented (9, 10). Current models of hCG action presume that the conformation of the dimer and the highly interactive contacts between the two subunits are critical for function (11)(12)(13)(14). One method to examine the functional role of the structural mo...

The Solution Structure of the N-terminal Domain of Human Vitronectin

“…In the large family of CSH domains, it is observed that a variety of disulfide-bonding patterns can be tolerated within the core of the domain, providing a structural scaffold that orients the ␣-helix on the surface (41). Thus, the curious retention of PAI-1 binding among this set of clearly different folds for the SMB domain is consistent with the demonstrated malleability of cystine knot structures to accommodate cysteine substitutions, yet retain activity (41)(42)(43)(44)(45). Even though the recombinant forms of the SMB domain do not have the correct cystine bonds found in circulating vitronectin, PAI-1 binding appears to be maintained because the various disulfide cross-linked frameworks support the formation of the sole ␣-helix in this domain.…”

Modulation of the steroidogenic related activity according to the design of single-chain bovine FSH analogs

“…The generation of variants in the presence or absence of a linker sequence to space the tethered subunit domains, as well as switching their relative position, enabled to generate SC gonadotropin analogs with differences in the conformation (Argos, 1990;Ben-Menahem et al, 2001Garcia-Campayo and Boime, 2001;Grinberg et al, 2008;Sugahara et al, 1995Sugahara et al, , 1996b. Interestingly, gonadotropin analogs, mostly SC variants but also mutated heterodimers and nonpeptide mimetics, with a conformation that is different from that of the wild-type heterodimer, bound to the cognate receptor and stimulated cAMP formation, which is a key signaling mediator for steroidogenesis (Ben-Menahem et al, 1997;Fralish et al, 2003;Garcia-Campayo and Boime, 2001;Grossmann et al, 1997;Heikoop et al, 1997;Jackson et al, 1999;Maclean et al, 2004;Moyle et al, 1987;Perlman et al, 2003;Sato et al, 1997;Sugahara et al, 1995;van Straten et al, 2002;Weenen et al, 2004;Xing et al, 2001). These and additional studies indicated that not all the interactions between the a and b subunits as seen in the crystal structure of the heterodimer are mandatory for receptor binding and activation (Bhowmick et al, 1996;Fan and Hendrickson, 2005;Fox et al, 2001;Lapthorn et al, 1994).…”

Alternatively Folded Choriogonadotropin Analogs

“…The curve for ␣E56C-␤C26A, which is between those for hCG and ␣L41C-␤C26A is not shown. chain gonadotropin analogs have also led to the suggestion that the region of the seatbelt near the latch disulfide is not needed for lutropin activity (29,30,32). Although this conclusion is consistent with the data described here, the finding that a single-chain hCG analog lacking this disulfide was recognized well by B111 (hCGsc␤C26A, C110A-␣, Table I) suggests that fusing the subunits together offsets the role of the seatbelt latch disulfide in stabilizing this region of the protein.…”

“…This result confirmed that the carboxyl-terminal end of the seatbelt in all the mutant analogs occupies a position different from that in hCG. Others have shown that the disulfide between ␤-subunit residues 26 and 110 that latches the ␤-subunit is not required for hormone folding or for receptor binding activities of single-chain analogs (29,30). To learn if the carboxyl-terminal end of the seatbelt has a similar location in a single-chain hCG analog lacking the seatbelt latch disulfide as it has in hCG, we compared hCGsc␤C26A, C110A-␣ (Fig.…”

“…As is apparent from the instability of heterodimers missing the seatbelt latch disulfide (18), the hCG subunit cores do not have high affinity for one another, at least once they have left the endoplasmic reticulum. Unless these proteins originated from dimers of subunits that had high affinities for one another, it seems likely that they could have evolved from intermediates lacking seatbelts only if their subunits were expressed in tandem (30) or if they were produced downstream of dimerization domains (23,27). The latter mechanism appears to be responsible for the assembly of inhibins and activins, other cystine knot proteins with key roles in reproduction and development (41).…”