Elucidating the molecular determinants in the process of gastrin C-terminal pentapeptide amide end activating cholecystokinin 2 receptor by Gaussian accelerated molecular dynamics simulations

Abstract

Gastrin plays important role in stimulating the initiation and development of many gastrointestinal diseases through interacting with the cholecystokinin 2 receptor (CCK2R). The smallest bioactive unit of gastrin activating CCK2R is the C-terminal tetrapeptide capped with an indispensable amide end. Understanding the mechanism of this smallest bioactive unit interacting with CCK2R on a molecular basis could provide significant insights for designing CCK2R antagonists, which can be used to treat gastrin-related diseases. To this end, we performed extensive Gaussian accelerated molecular dynamics simulations to investigate the interaction between gastrin C-terminal pentapeptide capped with/without amide end and CCK2R. The amide cap influences the binding modes of the pentapeptide with CCK2R by weakening the electrostatic attractions between the C-terminus of the pentapeptide and basic residues near the extracellular domain in CCK2R. The C-terminus with the amide cap penetrates into the transmembrane domain of CCK2R while floating at the extracellular domain without the amide cap. Different binding modes induced different conformational dynamics of CCK2R. Residue pairs in CCK2R had stronger correlated motions when binding with the amidated pentapeptide. Key residues and interactions important for CCK2R binding with the amidated pentagastrin were also identified. Our results provide molecular insights into the determinants of the bioactive unit of gastrin activating CCK2R, which would be of great help for the design of CCK2R antagonists.

Keywords: GAMD; MM-PBSA analysis; PMF; cholecystokinin 2 receptor; gastrin; interaction mechanism.

FIGURE 1

2D PMF profiles of CCK2R-G5 (A) and CCK2R-G5NH₂ (B) along the reaction coordinates of distances between the N/C-terminus of G5/G5NH₂ (Cα atoms of G13 and F17, respectively) and the geometrical center of TM domain of CCK2R; The initial site from the cryo-EM structure was marked as symbol X (coordinate: 22.1, 10.8). The PMF profile of each system was calculated by reweighting the combined two 1 μs GaMD simulations.

FIGURE 2

(A) Projections of CCK2R conformations in the complexes of CCK2R-G5 (cyan dots) and CCK2R-G5NH₂ (orange triangles) with PMF equal to 0 kcal/mol onto the common subspace defined by the first two PCs. The representative conformations in B are marked as symbol X, and the cryo-EM structure is marked as symbol Y. (B) Comparison of representative conformations of CCK2R-G5 (cyan) and CCK2R-G5NH₂ (orange) complexes with PMF equal to 0 kcal/mol. CCK2R is shown as cartoons. G5 and G5NH₂ are shown as cartoons, and their C-termini are shown as sticks. The directions of TM movement of CCK2R along with the PC1 from lowest to highest are indicated by arrows. (C) Linear mutual information (LMI) correlations between Cα atom pairs within CCK2R-G5 (upper triangle) and CCK2R-G5NH₂ (lower triangle) complexes. Residues from 55 to 405 belong to CCK2R, and the rest 5 (at the end of the coordinate axis) belong to G5/G5NH₂. (D) Differences in correlation coefficients between CCK2R-G5NH₂ and CCK2R-G5. The correlation coefficient is color-coded (see color bars).

FIGURE 3

(A–D) Free energy (in unit of kcal/mol) contributions of residues in CCK2R for binding with G5 (A,B) and G5NH₂ (C,D). Charged residues (Asp, Glu, Arg and Lys) in CCK2R (A,C) located in the binding sites were labeled. Uncharged residues in CCK2R (B,D) with absolute values of binding free energy contributions larger than 1 kcal/mol were labeled, in which residues located in and outside binding sites were labeled in black and red, respectively. Dashed lines in A and C, B, and D are the reference lines with values of ±5 and ±1, respectively. Detailed binding poses of CCK2R-G5 (E) and CCK2R-G5NH₂ (F). Residues of G5 and G5NH₂ are shown in stick representation. Residues of CCK2R are shown in line representation, Salt bridges (R208^ECL2-F17 in E, R356^6.58-D16 and R215^5.36-D16 in F) and hydrogen bonds (N115^2.65-M15, and Q204^ECL2-G13 in E, Y189^4.60-D16 and H207^ECL2-D16 in F) between CCK2R and G5/G5NH₂ are depicted by yellow and red dotted lines, respectively.