Insight into Structural Characteristics of Protein-Substrate Interaction in Pimaricin Thioesterase

Abstract

As a polyene antibiotic of great pharmaceutical significance, pimaricin has been extensively studied to enhance its productivity and effectiveness. In our previous studies, pre-reaction state (PRS) has been validated as one of the significant conformational categories before macrocyclization, and is critical to mutual recognition and catalytic preparation in thioesterase (TE)-catalyzed systems. In our study, molecular dynamics (MD) simulations were conducted on pimaricin TE-polyketide complex and PRS, as well as pre-organization state (POS), a molecular conformation possessing a pivotal intra-molecular hydrogen bond, were detected. Conformational transition between POS and PRS was observed in one of the simulations, and POS was calculated to be energetically more stable than PRS by 4.58 kcal/mol. The structural characteristics of PRS and POS-based hydrogen-bonding, and hydrophobic interactions were uncovered, and additional simulations were carried out to rationalize the functions of several key residues (Q29, M210, and R186). Binding energies, obtained from MM/PBSA calculations, were further decomposed to residues, in order to reveal their roles in product release. Our study advanced a comprehensive understanding of pimaricin TE-catalyzed macrocyclization from the perspectives of conformational change, protein-polyketide recognition, and product release, and provided potential residues for rational modification of pimaricin TE.

Keywords: macrocyclization; molecular dynamics (MD) simulation; pimaricin thioesterase; pre-reaction state; protein-substrate interaction.

Figure 1

Structures of pre-organization state (POS), active state and the pre-reaction state (PRS) of 6-deoxyerythronolide B synthase (DEBS) thioesterase (TE) system.

Figure 2

Conformational change of pima-TE system during molecular dynamics (MD) simulations. (a) Structural variations between post (opaque) and pre-simulation (transparent) complexes, with lid region, polyketide chain, α-helix αL2, αL3 and loop l1, l2 & l3 colored in tv_blue, gray, yellow, cyan, green, red and orange. (b) Root-mean-squared fluctuation (RMSF) of five trajectories with key structural elements highlighted.

Figure 3

Classification of trajectory frames based on polyketide chain conformation. (a) Representative structures of PRS, active state and POS extracted from clustering analysis. (b) Proportion of PRS, active state, and POS in each trajectory.

Figure 4

Conformational transformation between PRS and POS. (a) Dihedral value representation of md1 along with intermediate conformation change. (b) Presentation of dihedral angle C_α-C_β-C_γ-O₇.

Figure 5

Hydrogen-bonding and hydrophobic interaction network variations in PRS and POS. (a) Proportion of top-ranked hydrogen bonding interactions. (b) Diagram of protein-substrate interaction produced by ligplot⁺ [28]. Backbone of the polyketide substrate was colored in yellow, and residues providing hydrogen bonding and hydrophobic interactions in slate_blue and brown.

Figure 6

Instability of polyketide conformation. (a) Distance O₆-C_Q/A29 in wild type md4 and Q29A md1–3 along with conformation transformation. (b) Diagram of distance O₆-C_Q/A29.

Figure 7

Conformational change of intermediate change upon M210 mutation. (a) Distance RMSD (dRMSD) value of substrate backbone in wild type md4 and M210G (lightpink for wild type, lightblue, slate and tv_blue for M210G md1–3). (b) Diagram of the dominant structure in each trajectory.

Figure 8

Mutational trials on R186. (a) Radar chart indicating the proportion of hydrogen bond O₇-Nε_H261 and O₇-N_R186 formation within 5 wild type simulations. (b) Larger entrance of pima-TE after R186F mutation. (c) Coupling and non-coupling states of three entrance residues (R/F186, E80 and R266) located on different structure elements. (d) A favorable PRS emerged in R186Y md3.

Figure 9

(a) Diagram of representative PRS conformations in wild type (left) and S138C (right) trajectories. (b) Density map and marginal histogram indicating the distribution of all frames on the basis of distances O₇-Nε_H261 and O₇-C₁ in wild type and S138C trajectories. The rectangles in light-coral, slate-blue, and thistle highlight points with distance (O₇-Nε_H261) ≤ 3.0 Å, distance (O₇-C₁) ≤ 4.5 Å and 4.5 Å ≤ distance (O₇-C₁) ≤ 6.0 Å, respectively.

Figure 10

MM/PBSA analysis on the outward trend of macro-lactone product. (a) Binding energy between pima-TE and product ring. (b) Distance between ring mass center and Cα_S138. (c) Residues with top-ranked van der Waals (VDW) and electrostatic contributions to binding free energy.

Figure 11

Diagram of product movement in substrate channel in md1. (a) Three hydrogen bonding modes between Q29 and MOL and their transformation. (b) Spatial location of H187 with respect to MOL. (c) Distance H187-MOL in md1.