Decoding elegant interplay among different stereo-electronic effects due to the ancient prolyl-4-hydroxylation stabilizing collagenous helicity

Abstract

Prolyl-4-hydroxylation is an ancient evolutionarily conserved post-translational modification (PTM) critical for both structural and regulatory functions in multicellular life forms. This PTM plays a pivotal role in stabilizing collagen's triple helix by influencing the puckering of the pyrrolidine ring. The elegant interplay between ring pucker, torsional angles, peptide bond isomerization, and charge-transfer interactions (O···C=O n→π∗ and σ→σ∗) attaining the helical stability remains underappreciated. Using density functional theory calibrated against gold standard ab initio methods, we analyzed a physiologically relevant collagenous peptide proline-4-hydroxyproline-glycine (PO⁴G) to establish the correlation between stereo-electronic effects due to prolyl-4-hydroxylation. Our results show that 4(R)-hydroxylation promotes an exo ring pucker, optimizing main-chain torsional angles for a stable trans peptide bond and maximizing the n→π∗ interaction (E _n→π∗ = 0.9 kcal/mol) by tuning Bürgi-Dunitz trajectory, and maximizes σ→σ∗ interactions between axial C-H σ-electrons and C-OH∗ orbitals of the pyrrolidine ring. This study reveals the intricate stereo-electronic effects driving collagen's structural stability.

Keywords: Physical chemistry; Quantum chemical calculations; Quantum chemistry.

Graphical abstract

Figure 1

Stereoelectronic effects in collagen due to 4-HyP and quantum chemical studies of stabilization energies (A) The triple helical structure of collagen, a single strand of collagen, and the abundant triplet (–Xaa–4-HyP–Gly–). The sections in pink represent the –Xaa–4-HyP–Gly– triplet motif in a single strand in the triple helical domain. (B) The stereoelectronic parameters associated with 4-HyP responsible for collagenous helical stability; pyrrolidine ring pucker, key geometrical parameters (ring (χ) and main-chain (ϕ, ψ, and ω) torsional angles and Bürgi-Dunitz trajectory), peptide bond conformations, and charge-transfer (n→π∗ and σ→σ∗) interactions. (C) An illustration of the reported ΔE_endo–exo (kcal/mol) and E_n→π∗ (kcal/mol) values for single amino acid conformers using DFT methods.

Figure 2

Calibration of DFT methods against ab initio methods (A) An illustration of the category of DFT methods that are calibrated against ab initio MP2 and the DLPNO-CCSD(T) methods using the relative energy ΔE_endo–exo = E[4(R)-endo] – E[4(R)-exo] (kcal/mol) for 4-HyP conformers as the parameter. (B) Calibration of twenty-four DFT functionals including, pure GGA, hybrid GGA, pure meta-GGA, hybrid meta-GGA, and double hybrid functionals against the ab initio MP2 method. See also Figure S2 and Table S1. (C) Estimation of the interaction energy due to the n→π∗ charge-transfer (E_n→π∗) for the natural conformer of 4-HyP, i.e., 4(R)-HyP-exo at different DFT levels. See also Figure S5 and Table S4.

Figure 3

Geometry and natural bond orbital analysis of 4-HyP conformers (A) Natural bond orbitals and E_n→π∗ values in kcal/mol for four different conformers of 4-HyP. (B) Gas- and solvent-phase geometries of the single amino acid model of the natural 4(R)-HyP-exo conformer along with the natural bond orbitals and E_n→π∗ values. See also Figure S6.

Figure 4

Occurence of PPG triplet in collagen chain and building the physiologically-relevant PO⁴G peptide model (A) Collagen triple-helix structure highlighting a tripeptide unit in a single strand with H-bonding interactions with the other strands. The occupancy of the PPG triplet in the COL1A1 chain is shown. (B) Evaluation of the main-chain torsional angle and Bürgi-Dunitz trajectory of the DFT-M062X-optimized geometry and the crystal structure. The corresponding E_n→π∗ value and natural bond orbitals are also shown. See also Figures S7 and S8, and Tables S5 and S6.

Figure 5

Correlation between geometrical parameters and n→π∗ charge-transfer interaction in PO⁴G triplets (A) Main-chain torsional angles (ϕ and ψ) of triplets along with the crystal structure. (B) Correlations between E_n→π∗ and Bürgi-Dunitz trajectory and E_n→π∗ and main-chain torsional angles. (C) Natural bond orbitals and E_n→π∗ values for PO⁴G triplets bearing the four different conformers of 4-HyP.

Figure 6

Comparative analysis between PPG and PO⁴G triplets Comparison of geometrical parameters and charge-transfer (n→π∗ and σ→σ∗) interactions between the PPG-exo and PO⁴G-(R)-exo tripeptides showcasing the effect of 4-hydroxylation at the Yaa position. See also Figure S9.

Figure 7

Relative energy and NBO analyses of conformers from two-dimensional energy landscape of 4(R)-HyP-exo (A) A two-dimensional energy landscape of the relaxed torsional angle scans of the preceding (ω_prec) and succeeding (ω_succ) peptide bonds of the 4(R)-HyP-exo conformer at the M062X/6-31+G(d,p) level of theory. (B) The highest, lowest, and a few intermediate energy conformers are presented along with their relative energies (ΔE, kcal/mol). (C) Calculated E_n→π∗ and E_σ→σ∗ values (in kcal/mol) associated with the n→π∗ and σ→σ∗ interactions, respectively for five selected conformers. See also Tables S7 and S8.