C-Mannosylation Enhances the Structural Stability of Human RNase 2

Abstract

C-Mannosylation is a relatively rare form of protein glycosylation involving the attachment of an α-mannopyranosyl residue to C-2 of the indole moiety of the amino acid tryptophan. This type of linkage was initially discovered in RNase 2 from human urine but later confirmed to be present in many other important proteins. Based on NMR experiments and extensive molecular dynamics simulations on the hundred microsecond timescale we demonstrate that, for isolated glycopeptides and denatured RNase 2, the C-linked mannopyranosyl residue exists as an ensemble of conformations, among which ¹C₄ is the most abundant. However, for native RNase 2, molecular dynamics and NMR studies revealed that the mannopyranosyl residue favors a specific conformation, which optimally stabilizes the protein fold through a network of hydrogen bonds and which leads to a significant reduction of the protein dynamics on the microsecond timescale. Our findings contribute to the understanding of the biological role of C-mannosylation.

Keywords: Biochemistry; Protein Structure Aspects; Structural Biology.

Graphical abstract

Figure 1

Examples of X-ray Crystal Structures Containing the α-D-Manp-Trp Moiety Well-resolved electron density supporting a ¹C₄ conformation of mannose is present in PDB entry 6s08 and 5m5e. Please note that in general the modeled ring shapes of carbohydrates in PDB structures should be taken with care (Agirre et al., 2017). As an example, PDB entry 5m5e is shown with a re-modeled mannose (shown in green) in ¹C₄ conformation, which fits at least equally well into the electron density than the distorted ring shape modeled in the original X-ray structure published. In most X-ray structures listed in Table S1 the electron density around the C-linked mannoses is not well resolved or the conformation of the mannose is ambiguous (indicated with a “?”). PDB entry 3ojy has β-D-Manp covalently attached to Trp, which is most likely not correct. The electron density in PDB entry 4nzd may support the existence of twisted ring shapes (skew, boat) of C-linked mannose. See also Table S1.

Figure 2

C²-α-D-mannopyranosyl-L-tryptophan Moiety 3D representations of two chair conformations: ¹C₄ (left), ⁴C₁ (right). The peptide backbone is indicated as ribbon. Selected atom and torsion labels are shown. For definitions of torsion angles see Supplemental Information, Molecular Modeling section.

Figure 3

Conformational Preferences of C²-α-D-mannopyranosyl-3-methyl-indole Glycosidic torsion ϕ_H as a function of ring pucker coordinate z. Values from 2 μs gas phase MD simulation using TINKER/MM3 (ε = 4) at 400 K. See also Figures S1–S4

Figure 4

Conformational Ring Transition Energy Profiles of (C²-α-D-Man-)Trp Calculated in explicit solvent at various temperatures (AMBER, NPT/NVT ensemble). See also Figure S5

Figure 5

Stability Check of “Skew/Boat” Conformations for Glycopeptide FTW^ManAQW Based on MD simulations in explicit solvent at 310 K (20 × 0.2 μs, AMBER, NPT ensemble). Left: Simplified representation of ring conformation trajectories (orange: skew/boat, red: ¹C₄, blue: ⁴C₁). Right: population of ring conformational states. Insert: Iso-contour plot indicating the location of the minima in ring pucker space.

Figure 6

Crystal Structure of Eosinophil-Derived Neurotoxin (EDN, RNase 2) Based on pdb code 1gqv (Swaminathan et al., 2002). Top left: Protein flexibility (beta factor) is indicated by the thickness and color of the backbone representation (PyMol “putty” representation). Top right: Crystallographic beta factors mapped to the atoms as a color code. Red color indicates a high beta factor value. TRP7 is shown as CPK. The arrow denotes the attachment point of α-mannose (C-glycosylation site). Bottom: Structure-based sequence alignment (Jung and Lee, 2000) of RNase 1 (sequence taken from PDB entry 1z7x) and RNase 2 (sequence taken from PDB entry 1gqv).

Figure 7

Dynamics of RNase 2 as Shown from MD Simulations on the Microsecond Timescale Left: “RMSD per residue” trajectory plot. Major conformational change (shown by a change to black coloring) of the insertion loop (residue 115–123, indicated by the boxed area) occurs at 1.1 (reversible) and 1.8 μs (irreversible). Right: RNase 2 shown as a cartoon representation. The starting structure of the MD is shown in green. The last snapshot of the trajectory is superimposed and colored by RMSD (blue-white-red, interval 0–7 Å). The change of the insertion loop orientation and the location of Trp7 are indicated. See also Figure S6.

Figure 8

Surface Representation Showing the Cleft Formed between Trp7 and the “Insertion Loop” (Asp112–Arg118) Left: Amino acids are colored by type: negatively charged (red), positively charged (blue), polar (green), lipophilic (white). Right: α-Mannose attached to Trp7 in various conformational states (see text for details). Atoms are colored by element: carbon (cyan), oxygen (red), nitrogen (blue).

Figure 9

Differences in Average RMSD (C-Alpha Atoms) between ¹C₄-Mannosylated RNase and “Apo” RNase 2 A slight reduction in RMSD occurs for Trp7 (highlighted with gray background), but a significant decrease in RMSD is evident for the insertion loop (residues 114–124, highlighted with gray background) when α-mannose is linked in a ¹C₄-synH conformation. The X-ray beta factors of the residues are indicated as a grayscale annotation bar above the plot (darker gray means higher value). See also Figure S7.

Figure 10

C-Linked α-mannose (¹C₄) Forms an Extensive Network of Hydrogen Bonds with RNase 2 See also Tables S5 and S6.