NMR Studies of the Free α Subunit of Human Chorionic Gonadotropin

Beer, Tonny de; Zuylen, Carol W. E. M. Van; Leeflang, Bas R.; Hård, Karl; Boelens, Rolf; Kaptein, Robert; Kamerling, Johannis P.; Vliegenthart, Johannes F.G.

doi:10.1111/j.1432-1033.1996.0229t.x

Cited by 83 publications

(64 citation statements)

References 77 publications

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“…Here, the R-helix changes partly to a 3-helix or turn motif (Figure 10). The main effect that can be seen from the data is that removal of the first R-helix (amino acids [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] destabilizes the second R-helix (amino acids [23][24][25][26][27][28][29][30][31][32] in its relative position and also in its fold, as revealed from the analysis of the secondary structure ( Figure 10). This finding is not an artifact of superimposing residues 40-120 for the rmsd analysis: using all backbone atoms for superimposition gives the same result.…”

Section: Bs In Solventmentioning

confidence: 99%

The Structural Basis of the Difference in Sensitivity for PNGase F in the De-N-glycosylation of the Native Bovine Pancreatic Ribonucleases B and BS

et al. 2008

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In glycoanalysis protocols, N-glycans from glycoproteins are most frequently released with peptide-N 4 -(N-acetyl--glucosaminyl)asparagine amidase F (PNGase F). As the enzyme is an amidase, it cleaves the NH-CO linkage between the Asn side chain and the Asn-bound GlcNAc residue. Usually, the enzyme has a low activity, or is not active at all, on native glycoproteins. A typical example is native bovine pancreatic ribonuclease B (RNase B) with oligomannose-type N-glycans at Asn-34. However, native RNase BS, generated by subtilisin digestion of native RNase B, which comprises amino acid residues 21-124 of RNase B, is sensitive to PNGase F digestion. The same holds for carboxymethylated RNase B (RNase B cm ). In this study, NMR spectroscopy and molecular modeling have been used to explain the differences in PNGase F activity for native RNase B, native RNase BS, and RNase B cm . NMR analysis combined with literature data clearly indicated that the N-glycan at Asn-34 is more mobile in RNase BS than in RNase B. MD simulations showed that the region around Asn-34 in RNase B is not very flexible, whereby the R-helix of the amino acid residues 1-20 has a stabilizing effect. In RNase BS, the R-helix formed by amino acid residues 23-32 is significantly more flexible. Using these data, the possibilities for complex formation of both RNase B and RNase BS with PNGase F were studied, and a model for the RNase BS-PNGase F complex is proposed.Ribonucleases catalyze the breakdown of 3′,5′-phosphodiester linkages of single-stranded RNA, when a pyrimidine ring is at the 3′-side of the broken bond (1). Bovine pancreatic ribonuclease (RNase) 1 is intensively used as a model in protein chemistry, when studying folding, structure, and stability, as well as enzyme catalysis. This enzyme is present in large quantities in the forestomach of cows and digests the RNA produced by microorganisms. Bovine pancreatic RNase was one of the first proteins that could be synthesized chemically (2), and its three-dimensional structure is well-known from early X-ray (3) and NMR (4-6) studies.Bovine pancreatic RNase is composed of 124 amino acids; it has four disulfide bridges and a molecular mass of 13.7 kDa. RNase denoted RNase A is not glycosylated, whereas RNase B bears an oligomannose-type Man 5-9 GlcNAc 2 moiety at Asn-34. The N-glycan stabilizes the glycoprotein toward proteolytic and temperature degradation (7). Digestion of RNase B with subtilisin results in so-called S peptide (residues 1-20) and RNase BS (residues 21-124).

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Section: Bs In Solventmentioning

confidence: 99%

The Structural Basis of the Difference in Sensitivity for PNGase F in the De-N-glycosylation of the Native Bovine Pancreatic Ribonucleases B and BS

et al. 2008

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“…This showed that the location of the added cysteines made a difference in their ability to inhibit binding of the latch disulfide to either cysteine 26 or ␣-subunit S43C, revealing that the location of ␣-subunit loop 2, adjacent to the ␤-subunit, is most likely to be in the position occupied in Fig. 3 (D-F) as opposed to being disordered as observed during NMR analysis of hCG ␣-subunit loop 2 (28). The reason for this is that one ␤-subunit cysteine substitution (F64C) had a roughly equal ability to latch its seatbelt to ␤-subunit cysteine 26.…”

Section: Discussionmentioning

confidence: 93%

“…Based on the data in Table 4, it is clear that the ␣-subunit is already attached to the ␤-subunit in a position where both the ␣-and ␤-subunit cysteine knots are proximal to one another. This would permit ␣-subunit loop 2, which has been shown to be disordered in the free subunit (28), to be in the vicinity of these ␤-subunit residues as well as the ␤-subunit tail. In our model, the position of the ␣-subunit hydrophobic residues in loop 2 would allow for the migration of the hydrophobic ␤-subunit tail away from the seatbelt, enabling it to become latched to the cysteine at position 26.…”

Section: Discussionmentioning

confidence: 99%

Human Lutropin (hLH) and Choriogonadotropin (CG) Are Assembled by Different Pathways

Bernard¹,

Lin²,

Kholodovych

et al. 2014

Journal of Biological Chemistry

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“…substitution of cysteines for ␣Pro38 and ␣Pro40). Because the conformation of ␣-subunit loop 2 in the free ␣-subunit is known to be highly disordered (28), this observation suggested that docking of the subunits may stabilize the conformation of ␣-subunit loop 2 to one that is roughly similar to the conformation seen in the native heterodimer.…”

Section: Figmentioning

confidence: 88%

Alternatively Folded Choriogonadotropin Analogs

Xing

Lin

Jiang

et al. 2001

Journal of Biological Chemistry

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Most heterodimeric proteins are stabilized by intersubunit contacts or disulfide bonds. In contrast, human chorionic gonadotropin (hCG) and other glycoprotein hormones are secured by a strand of their ␤-subunits that is wrapped around ␣-subunit loop 2 "like a seatbelt." During studies of hCG synthesis in COS-7 cells, we found that, when the seatbelt was prevented from forming the disulfide that normally "latches" it to the ␤-subunit, its carboxyl-terminal end can "scan" the surface of the heterodimer and become latched by a disulfide to cysteines substituted for residues in the ␣-subunit. Analogs in which the seatbelt was latched to residues 35, 37, 41-43, and 56 of ␣-subunit loop 2 had similar lutropin activities to those of hCG; that in which it was latched to residue 92 at the carboxyl terminus had 10 -20% the activity of hCG. Attachment of the seatbelt to ␣-subunit residues 45-51, 86, 88, 90, and 91 reduced lutropin activity substantially. These findings show that the heterodimer can form before the ␤-subunit has folded completely and support the notions that the carboxylterminal end of the seatbelt, portions of ␣-subunit loop 2, and the end of the ␣-subunit carboxyl terminus do not participate in lutropin receptor interactions. They suggest also that several different architectures could have been sampled without disrupting hormone activity as the glycoprotein hormones diverged from other cysteine knot proteins.The heterodimeric placental glycoprotein hormone hCG 1 binds LHR and stimulates ovarian steroid synthesis during early pregnancy (1). The structure of hCG is known (2, 3), but the structures of the LHR and the hormone receptor complex remain to be elucidated. Each hCG subunit is divided into three elongated loops by cystine knots (2, 3), and the heterodimer is stabilized by a part of the ␤-subunit termed the "seatbelt" (2) that is wrapped around ␣-subunit loop 2. The composition of the seatbelt determines the receptor binding specificity of hCG (4 -6), but it is not known if this portion of the hormone contacts the receptor. The LHR is a G protein-coupled receptor that contains a large extracellular domain that binds hCG with high affinity and specificity (7). Based on its leucine-rich repeat motif (7), the LHR extracellular domain is usually presumed to be horseshoe-shaped, similar to ribonuclease inhibitor (8).The surfaces of hCG most likely to contact the LHR remain debated. The carboxyl-terminal end of the ␣-subunit, which is adjacent to a portion of the seatbelt in the heterodimer (2, 3), has been found to influence the affinity of all the glycoprotein hormones for their receptors (1, 9) and was proposed to be a receptor contact more than 25 years ago (1). Based partially on this observation, Jiang et al. (10) suggested that the long axis of hCG docks with the concave surface of a horseshoe-shaped extracellular domain. In their view, the hormone is perpendicular to the extracellular domain of the receptor such that the ␣-subunit carboxyl-terminal end, parts of ␤-subunit loop 2, and the seatbelt...

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NMR Studies of the Free α Subunit of Human Chorionic Gonadotropin

Cited by 83 publications

References 77 publications

The Structural Basis of the Difference in Sensitivity for PNGase F in the De-N-glycosylation of the Native Bovine Pancreatic Ribonucleases B and BS

The Structural Basis of the Difference in Sensitivity for PNGase F in the De-N-glycosylation of the Native Bovine Pancreatic Ribonucleases B and BS

Human Lutropin (hLH) and Choriogonadotropin (CG) Are Assembled by Different Pathways

Alternatively Folded Choriogonadotropin Analogs

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