n→π* Interactions Modulate the Properties of Cysteine Residues and Disulfide Bonds in Proteins

Abstract

Noncovalent interactions are ubiquitous in biology, taking on roles that include stabilizing the conformation of and assembling biomolecules, and providing an optimal environment for enzymatic catalysis. Here, we describe a noncovalent interaction that engages the sulfur atoms of cysteine residues and disulfide bonds in proteins-their donation of electron density into an antibonding orbital of proximal amide carbonyl groups. This n→ π* interaction tunes the reactivity of the CXXC motif, which is the critical feature of thioredoxin and other enzymes involved in redox homeostasis. In particular, an n→ π* interaction lowers the p K_a value of the N-terminal cysteine residue of the motif, which is the nucleophile that initiates catalysis. In addition, the interplay between disulfide n→ π* interactions and C5 hydrogen bonds leads to hyperstable β-strands. Finally, n→ π* interactions stabilize vicinal disulfide bonds, which are naturally diverse in function. These previously unappreciated n→ π* interactions are strong and underlie the ability of cysteine residues and disulfide bonds to engage in the structure and function of proteins.

Figure 1.

Images of the sulfur lone pairs in N-acetyl-cysteine methyl amide disulfide with surrounding carbonyl groups. (A) Sulfur lone pair in the n_s orbital. (B) Sulfur lone pair in the n_p orbital. (C) n_s^γ→π* interaction between S_i^γ and C_i=O_i. (D) n_p^γ→π* interaction between S_i^γ and C_i=O_i.

Figure 2.

Network of n→π* interactions within the CXXC motif. (A) Electron donation in the oxidized state. (B) S_i^γ···C_i=O_i n→π* interaction in the oxidized state. (C) S_i^γ···C_i=O_i n→π* interaction in the thiol state. (D) S_i^γ···C_i=O_i n→π* interaction in the thiolate state. (E) C_i=O_i···C_i+1=O_i+1 n→π* interaction in the thiolate state. (F) C_i+1=O_i+1···C_i+2=O_i+2 n→π* interaction in the thiolate state. Structures are from PDB entries 1ert and 1eru.

Figure 3.

Graph of the relationship between calculated E_n→π* values and measured E°′ values for CXXC motifs: Escherichia coli DsbA (black; PDB entry 1a2j) and three variants of Staphylococcus aureus thioredoxin (blue; PDB entries 2o7k, 2o85, and 2o87).

Figure 4.

Graph showing calculated E_n→π* values (in kcal/mol) within the CXXC motifs of Homo sapiens thioredoxin and thioredoxin-2, and Drosophila melanogaster thioredoxin. Data are listed in Table S2.

Figure 5.

Interplay between a disulfide n→π* interaction and C5 hydrogen bond in a β-strand. (A) Natural bond orbitals showing a disulfide n→π* interaction. (B) Network of natural bond orbitals in which the n→π* interaction from panel A enhances an n→σ* interaction (that is, a C5 hydrogen bond) within the half-cystine residue. (C) Image of a model disulfide bond. (D) Scan of the dihedral angle ξ (which is defined in the inset of panel E) in the presence of a disulfide n→π* interaction of E_n→π* = 1.65 kcal/mol; data are listed in Table S3. (E) Scan of the dihedral angle ξ in the absence of a disulfide n→π* interaction; data are listed in Table S4. The structure in panels A–C is from PDB entry 4gn2 (Table S5) and was used in the calculations of panels D and E.

Figure 6.

n→π* Interactions of vicinal disulfide bonds. (A) Image of a model vicinal disulfide bond (PDB entry 3cu9). (B) Histograms of n→π* interaction energies of vicinal disulfide bonds in protein crystal structures. Twenty-four vicinal disulfide bonds from the PDB were subjected to NBO analysis, and the resulting E_n→π* values were put into bins of 0.25 kcal/mol. (C–F) Natural bond orbitals for the strongest disulfide n→π* interaction in the four conformations of vicinal disulfide bonds: trans-up conformation (panel C; 3cu9), trans-down conformation (panel D; 4aah), cis-up conformation (panel E; 1wd3), and cis-down conformation (panel F; 4mge).