Mechanisms of Deamidation of Asparagine Residues and Effects of Main-Chain Conformation on Activation Energy

Abstract

Deamidation of asparagine (Asn) residues is a nonenzymatic post-translational modification of proteins. Asn deamidation is associated with pathogenesis of age-related diseases and hypofunction of monoclonal antibodies. Deamidation rate is known to be affected by the residue following Asn on the carboxyl side and by secondary structure. Information about main-chain conformation of Asn residues is necessary to accurately predict deamidation rate. In this study, the effect of main-chain conformation of Asn residues on deamidation rate was computationally investigated using molecular dynamics (MD) simulations and quantum chemical calculations. The results of MD simulations for γS-crystallin suggested that frequently deamidated Asn residues have common main-chain conformations on the N-terminal side. Based on the simulated structure, initial structures for the quantum chemical calculations were constructed and optimized geometries were obtained using the B3LYP density functional method. Structures that were frequently deamidated had a lower activation energy barrier than that of the little deamidated structure. We also showed that dihydrogen phosphate and bicarbonate ions are important catalysts for deamidation of Asn residues.

Keywords: age-related diseases; deamidation; molecular dynamics simulation; post-translational modification; quantum chemical calculation.

Figure 1

Succinimide-mediated deamidation pathways of asparagine (Asn) residues.

Figure 2

Proposed two-step succinimide formation pathway of Asn residues.

Figure 3

Conformation of Asn residues in γS-crystallin. (A) Experimentally determined structure of γS-crystallin. (B–F) Conformation of Asn residues in γS-crystallin.

Figure 4

Dihedral angles used in this study to analyze the main-chain conformationof Asn residues (A for φ, ψ, and χ, B for φ_H).

Figure 5

Root mean square deviation (RMSD) plots for the main-chain atoms of γS-crystallin.

Figure 6

Flexibility of the backbone of γS-crystallin. (A) The root mean square fluctuation (RMSF) plot of γS-crystallin. (B) The final structure of γS-crystallin obtained by MD simulations. The segments of backbone where RMSF values are >1.0 Å are shown in red.

Figure 7

The structure of the model compound.

Figure 8

Optimized geometries of the cyclization step for the syn conformation in the phosphate-catalyzed reaction. The carbon, nitrogen, oxygen, and phosphorus atoms are illustrated in gray, blue, red, and orange, respectively. Selected interatomic distances are in units of Å.

Figure 9

Optimized geometries of the deammoniation step for the syn conformation in phosphate-catalyzed reaction. The carbon, nitrogen, oxygen, and phosphorus atoms are illustrated in gray, blue, red, and orange, respectively. Selected interatomic distances are in units of Å.

Figure 10

Optimized geometries of the cyclization step for the anti conformation in the phosphate-catalyzed reaction. The carbon, nitrogen, oxygen, and phosphorus atoms are illustrated in gray, blue, red, and orange, respectively. Selected interatomic distances are in units of Å.

Figure 11

Optimized geometries of the deammoniation step for the anti conformation in the phosphate-catalyzed reaction. The carbon, nitrogen, oxygen, and phosphorus atoms are illustrated in gray, blue, red, and orange, respectively. Selected interatomic distances are in units of Å.

Figure 12

Relative energy profiles of the phosphate- and carbonate-catalyzed reaction calculated using the MP2/6-311+G(2d,2p)//B3LYP/6-31+G(d,p) level. Entire energy profiles of (A) syn conformation in the phosphate-catalyzed reaction, (B) anti conformation in the phosphate-catalyzed reaction, (C) syn conformation in the carbonate-catalyzed reaction, and (D) anti conformation in the carbonate-catalyzed reaction are shown.