Fig 1.
FSH/FSHR quaternary and primary structures examined in this study.
(a). Structure showing the FSH-FSHR complex with the partial glycosylation of NAG (N-acetylglucosamine). Ribbons colored green and cyan identify FSHα and FSHβ subunits, respectively. The former is glycosylated by NAG at sequence positions 52 and 78, while the latter is glycosylated at positions 7 and 24. NAGs are shown as grey colored stick models. The pink ribbon shows a portion of the FSH receptor extracellular domain that possesses the high affinity FSH binding site. (b). The amino acid sequences for FSHα residues 3–92 (yellow), FSHβ 3–107 (green), and FSHR 1–241 (brown) are shown below. In subsequent figures a colored bar will indicate each protein, as the software numbers the residues 1–437.
Fig 2.
FSH glycoforms result from partial glycosylation of the hFSHβ subunit.
The variants are defined by the molecular weights of the FSHβ subunit based on Western blotting experiments. The primary structures are indicated as solid lines, 92 residues for FSHα and 111 for FSHβ. The tuning forks represent N-glycans, when present. The N is the single-letter code for asparagine and the superscript represents each residue’s position in the primary structure.
Fig 3.
Schematic diagram for the tetra-antennary glycan used in the simulations.
The α/β 1–6 linkage line is longer, which indicates the carbon atom is exocyclic. All other linkages involve ring carbons.
Fig 4.
Root mean-squared positional deviation among FSH protein backbone atom positions during molecular dynamics simulations computed relative to the original X-ray crystallographic coordinates.
A. Comparison of FSH glycoforms decorated with single NAG residues. B. Comparison of FSH glycoforms decorated with TAG oligosaccharides.
Fig 5.
Comparison of FSH dynamic structural features at the FSH/FSHR interface for FSH(TAG) glycoforms.
A. FSH15(TAG) and B. FSH24(TAG). The protein portion of FSH is rendered in ribbon form, while the spatial extent of TAG glycan residues is shown via transparent green features. FSH ribbons are colored as follows: blue = FSH residues with FSHR contact surfaces; red = FSH residues with backbone RMSD shifts of greater than 1.5 Å; purple = FSH residues with FSHR contact surfaces and backbone RMSD shifts of greater than 1.5 Å.
Fig 6.
(a) Plot showing RMSF values of Cα atoms from MD simulations of dgFSH (in green), FSH15(NAG) (in black), and FSH24(NAG) (in red). Residues associated with the RMSF, showing each subunit as a bar (α-subunit: yellow bar and β-subunit: green bar) and single-letter code sequences with residue numbers for the regions where RMSF changes reasonably. Oval dots over the bar are shown for the potential N-glycosylation sites (Black & Orange dots for Asn52 & Asn78 on FSHα and Green & Purple dots for Asn7 & Asn24 on FSHβ). Residue sequences with reasonable RMSF changes of at least >1.0 Å are labeled inside the bars in each subunit. Ribbon models: Color-coded mapping of the averaged protein flexibility profiles (RMSF values) from MD simulations of the dgFSH, FSH15(NAG) and FSH24(NAG) (from left to right). The color-coded sliding scheme corresponds to the following ranges of protein flexibility values: red (highly flexible with +5.00 Å values), brown (+4.00 Å values), yellow (+3.00 Å), green (+2.00 Å), cyan (+1.00 Å) and blue (the least flexible). The amino acid sequences for FSHα residues 3–92 (yellow), and FSHβ 3–107 (green) are shown in the lower panel of the figure. (b) Difference of RMSF values for FSH15(TAG) and FSH24(TAG) from dgFSH. The specific residues with absolute difference larger than 0.50 Å (dashed lines) are labeled.
Fig 7.
(a) Plot showing RMSF values of Cα atoms from MD simulations of dgFSH / FSH24(TAG) (in green), FSH15(TAG) (in black), and FSH24(TAG) (in red). Residues associated with the RMSF, showing each subunit as a bar (α-subunit: yellow bar, β-subunit: green bar and FSHR: light-orange bar) and single-letter code sequences with residue numbers for the regions where RMSF changes reasonably. Residue sequences with reasonable RMSF changes of at least >1.0 Å are labeled inside the bars in each subunit. Ribbon models: Color-coded mapping of the averaged protein flexibility profiles (RMSF values) from MD simulations of the dgFSH, FSH15(TAG) and FSH24(TAG) FSH-FSHR complexes (from left to right). The color-coded sliding scheme is the same as was adopted for Fig 6a. The amino acid sequences for FSHα residues 3–92 (yellow), FSHβ 3–107 (green), and FSHR 1–241 (brown) are shown below. (b) Difference of RMSF values for FSH15(TAG) and FSH24(TAG)from dgFSH. The residues with absolute differences greater than 0.50 Å (dashed lines) are labeled.
Fig 8.
(a) Plot showing RMSF values of Cα atoms from MD simulations of dgFSH / FSH24(TAG) (in green), FSH15(TAG) (in black), and FSH24(TAG) (in red). Residues associated with the RMSF, showing each subunit as a bar (α-subunit: yellow bar, β-subunit: green bar and FSHR: light-orange bar) and single-letter code sequences with residue numbers for the regions where RMSF changes reasonably. Residue sequences with reasonable RMSF changes of at least >1.0 Å are labeled inside the bars in each subunit. Ribbon models: Color-coded mapping of the averaged protein flexibility profiles (RMSF values) from MD simulations of the dgFSH, FSH15(TAG) and FSH24(TAG) FSH-FSHR complexes (from left to right). The color-coded sliding scheme is the same as was adopted for Fig 6a. The amino acid sequences for FSHα residues 3–92 (yellow), FSHβ 3–107 (green), and FSHR 1–241 (brown) are shown below. (b) Difference of RMSF values for FSH15(TAG) and FSH24(TAG) from dgFSH. The residues with absolute difference larger than 0.50 Å are labeled by two cutoff dashed black lines.
Fig 9.
(a) Plot showing RMSF values of Cα atoms from MD simulations of free/unbound FSH and FSHr-bound models of FSH. RMSF changes were noted in two regions in the FSHα subunit centered on residues Met45 and Met69 and one region in the FSHβ subunit centered on residues Lys128-Ala131. Residues with RMSF changes of at least >2.0 Å are labeled inside the bars in each subunit. (b) Difference of RMSF values for FSHR-bound FSH and free-FSH models. The residues with absolute difference larger than 1.0 Å are labeled by one cutoff dashed black line.
Fig 10.
Energy components (kcal/mol) for the binding of FSH/FSHR in different systems like the dgFSH, FSH15(NAG), FSH24(NAG), FSH15(TAG), and FSH24(TAG): INT: Internal energies arising from bond, angle, and dihedral terms, ELE: Electrostatic energy in the gas phase; VDW: van der Waals energy; GBSOL: sum of polar and non-polar solvation energies; GBTOT: Total binding free energies.
Error bars shown in black solid line specifies the difference in terms of standard deviations.
Table 1.
FSH/FSHR binding free energies as computed from GBSA molecular dynamics studies.
Values are in kcal/mol. Parenthetical values represent standard deviations in the free energies.
Fig 11.
Key FSH/FSHR interface salt bridges underlying the electrostatic distinctions between FSH15(TAG) and FSH24(TAG).
FSH is depicted as cyan ribbons with key residues shown as green/CPK-colored sticks, whereas FSHR is rendered via yellow ribbons and yellow/CPK-colored sticks.
Table 2.
Median O-N distances for selected inter-protein salt bridges.
Values are in Angstroms, averaged of molecular dynamics conformational sampling. Parenthetical values represent the fraction of time in which the O-N distance is less than 3.5 Å.
Fig 12.
Difference in Solvent Accessible Surface Area (SASA) for FSH15(NAG) and FSH24(NAG) relative to de-glycosylated FSH.
Fig 13.
Difference in Solvent Accessible Surface Area (SASA) for FSH15 (NAG)-FSHR and FSH24(NAG)-FSHR from dgFSH.
Fig 14.
Difference in Solvent Accessible Surface Area (SASA) for FSH15 (TAG)-FSHR and FSH24(TAG)-FSHR from dgFSH.