Solution structure of the α‐subunit of human chorionic gonadotropin

Erbel, P.; Karimi-Nejad, Yasmin; Beer, Tonny de; Boelens, Rolf; Kamerling, Johannis P.; Vliegenthart, Johannes F.G.

doi:10.1046/j.1432-1327.1999.00188.x

Cited by 46 publications

(38 citation statements)

References 48 publications

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“…To enable the comparison of the 2D homonuclear NOESY spectra of RhCG[GlcNAc 52,78 ] and RhCG-[glycan 78 ], they were recorded under the same experimental conditions i.e., at 328 K and pH 5.1 on a Varian Unity 750 or a Bruker AMX 600 spectrometer. Details regarding the acquisition of the NMR spectra were as reported previously (8,11 (10). Distance restraints within the glycan chain were increased by 1 Å, to account for the line widths, being smaller than those of the protein signals.…”

Section: Methodsmentioning

confidence: 99%

“…The calculation strategy was based on the hybrid distance geometry simulated annealing (DG/SA) method (12)(13)(14). To overcome the problems associated with the NMR structure determination of proteins containing complex disulfide bonding topologies, the parameters of the protocol were extensively modified, and an additional refinement step of isothermal molecular dynamics was added (10).…”

Section: Methodsmentioning

confidence: 99%

“…In detail, the -turn comprising amino acid residues 20-23 shows increased NOE intensities in the case of RhCG[glycan 78 ] for the cross-peaks between residues Phe18/Ile25, Gln20/Ala23, and Ala23/ Pro24, and decreased intensities for the NOEs between residues Ser19/Gln20 and Gly22/Ala23. In the -hairpin loop comprising residues 70-74, increased cross-peak intensities are found between the residues Val70/Phe74 78 ], in total 666 distance restraints were derived from the NOESY spectra as described previously (10). The number of intraprotein NOE distances was 620.…”

Section: General a Comparison Of The Nmr Parameters Ofmentioning

confidence: 99%

“…In RhCG[GlcNAc 52,78 ], the corresponding values are similar, viz., 34.2, 63.9, and 1.9%. Previously, we determined the conformation of RhCG[GlcNAc 52,78 ] with an X-PLOR protocol unable to handle monosaccharide residues (10). In comparison to the Φ and Ψ dihedral angles of Asn78, calculated with this elementary X-PLOR protocol, the backbone conformation of Asn78 in the conformers, generated with a X-PLOR protocol able to handle monosaccharides, improved from a conformation in the disallowed region to the allowed region of the Φ/Ψ space.…”

Section: General a Comparison Of The Nmr Parameters Ofmentioning

confidence: 99%

“…A detailed investigation of the two -hairpins reveals that the first -hairpin consists of two extended segments including residues 10-14 and 24-28. They are connected by two interlocking turns, namely a 3 10 78 ], the degree of local definition in the backbone is higher than in RhCG[GlcNAc 52,78 ], whereas for Gly72 and Phe74, the opposite is true. We also computed global displacements for all residues with backbone angular order The parallhdg force constant parameter set of X-PLOR 3.851 was used with the quartic repel core repulsion potential.…”

Section: General a Comparison Of The Nmr Parameters Ofmentioning

confidence: 99%

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Effects of the N-Linked Glycans on the 3D Structure of the Free α-Subunit of Human Chorionic Gonadotropin

et al. 2000

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To gain insight into intramolecular carbohydrate-protein interactions at the molecular level, the solution structure of differently deglycosylated variants of the R-subunit of human chorionic gonadotropin have been studied by NMR spectroscopy. Significant differences in chemical shifts and NOE intensities were observed for amino acid residues close to the carbohydrate chain at Asn78 upon deglycosylation beyond Asn78-bound GlcNAc. As no straightforward strategy is available for the calculation of the NMR structure of intact glycoproteins, a suitable computational protocol had to be developed. To this end, the X-PLOR carbohydrate force field designed for structure refinement was extended and modified. Furthermore, a computational strategy was devised to facilitate successful protein folding in the presence of extended glycans during the simulation. The values for φ and ψ dihedral angles of the glycosidic linkages of the oligosaccharide core fragments GlcNAc2( 1-4)GlcNAc1 and Man3( 1-4)GlcNAc2 are restricted to a limited range of the broad conformational energy minima accessible for free glycans. This demonstrates that the protein core affects the dynamic behavior of the glycan at Asn78 by steric hindrance. Reciprocally, the NMR structures indicate that the glycan at Asn78 affects the stability of the protein core. The backbone angular order parameters and displacement data of the generated conformers display especially for the -turn 20-23 a decreased structural order upon splitting off the glycan beyond the Asn78-bound GlcNAc. In particular, the Asn-bound GlcNAc shields the protein surface from the hydrophilic environment through interaction with predominantly hydrophobic amino acid residues located in both twisted -hairpins consisting of residues 10-28 and 59-84.Human chorionic gonadotropin (hCG) 1 is a glycoprotein hormone involved in the maintenance of the corpus luteum during early pregnancy. It is a heterodimer consisting of noncovalently associated R-and -subunits. Both subunits are glycosylated and contain a cystine knot motif formed by three disulfide bridges as a central structural element (1,2). Besides the role of R-subunit (RhCG or RhCG[glycan 52,78 ]) in the R -dimer, it has been discovered that the noncombined R-subunit of hCG (hCG-R f ), which is produced in significant amounts by the placenta during pregnancy, has an independent biological activity in being able to stimulate prolactin secretion from human decidual cells (3, 4).Glycosylation of proteins has been shown to have many functions, including stabilization of the protein structure (5). In the R-subunit of hCG, the two glycosylation sites at Asn52 and Asn78, respectively, have remarkably different properties with respect to the stability of the subunit as shown by sitedirected mutagenesis (6). The absence of glycosylation at Asn52 does not affect folding and stability of the R-subunit. In contrast, mutant R-subunits lacking glycosylation at Asn78 are poorly secreted and rapidly degraded, although the precise molecular origin of thi...

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Section: Methodsmentioning

confidence: 99%