Flexibility and extracellular opening determine the interaction between ligands and insect sulfakinin receptors

Abstract

Despite their fundamental importance for growth, the mechanisms that regulate food intake are poorly understood. Our previous work demonstrated that insect sulfakinin (SK) signaling is involved in inhibiting feeding in an important model and pest insect, the red flour beetle Tribolium castaneum. Because the interaction of SK peptide and SK receptors (SKR) initiates the SK signaling, we have special interest on the structural factors that influence the SK-SKR interaction. First, the three-dimensional structures of the two T. castaneum SKRs (TcSKR1 and TcSKR2) were generated from molecular modeling and they displayed significance in terms of the outer opening of the cavity and protein flexibility. TcSKR1 contained a larger outer opening of the cavity than that in TcSKR2, which allows ligands a deep access into the cavity through cell membrane. Second, normal mode analysis revealed that TcSKR1 was more flexible than TcSKR2 during receptor-ligand interaction. Third, the sulfated SK (sSK) and sSK-related peptides were more potent than the nonsulfated SK, suggesting the importance of the sulfate moiety.

Figure 1. Cartoon diagram of Tribolium castaneum sulfakinin receptor 1 (TcSKR1) and T. castaneum sulfakinin receptor 2 (TcSKR2).

The seven transmembrane α-helices building the three-dimensional fold of the proteins are differently colored from blue (N-terminus) to red (C-terminus) (upper and lower on the left) by secondary structure (upper and lower in the middle) and with extracellular loops (ECL) colored differently (upper and lower on the right). The ECL 1 is colored in orange, ECL 2 in yellow and ECL 3 in green. The N-terminus is colored in blue while C-terminus red.

Figure 2

Surface, ribbon, flexibility and interaction diagrams generated by normal mode analysis of TcSKR1 (A,C,E,G) and TcSKR2 (B,D,F,H) with nonsulfated sulfakinin (nsSK) peptide docked (colored by atom type). Cavities are colored by depth from blue (shallow) to orange (deep) in TcSKR1 (A) and TcSKR2 (B). The flexibility for each amino in SKR1 and SKR2 was retrieved from normal model analysis and the root-mean-square fluctuation (RMSF) was plotted onto each structure and colored from dark blue (rigid) to red (highly flexible) (E,F). The picture demonstrates a deep cavity in both structures but with an extended opening out cavity in SKR1 (C). The red arrows (F) indicate the most significant region responsible for reduction in outer openings of SKR2 cavity (D). The top view of TcSKR2 (D,F) displays a rigid core that restricts a larger outer opening of the cavity, which to some extend hinders a deeper intrusion of peptides into cavity. The figure C shows the larger outer opening of SKR1 allows the peptides to go deeper into cavity (see lupe detail). Docking of nsSK to TcSKR1 (C) and TcSKR2 (F). The clips show the binding of nsSK in both receptors through several hydrogen bonds colored by pink dashed lines. The residues are indicated by names and colored as light cyan.

Figure 3

Flexibility demonstrated by a graph constructed using root-mean-square fluctuation (RMSF) for each amino acid in TcSKR1 model (A) and RMSF plotted onto TcSKR1 structure (B–D). The corresponding amino acids for TcSKR1 are indicated along with the x-axis. The calculations were performed with empty cavity of TcSKR1 in oxidized state (SKR1_oxy, black line), with docked nonsulfated sulfakinin (SKR1_oxy_lig_ns, blue line) and with docked sulfated sulfakinin (SKR1_oxy_lig_s, red line) and the retrieved flexibility (RMSF) was plotted onto structures (B–D), respectively. The transparent ellipses indicate regions with remarkable changes. The red ellipse corresponds to transmembrane region I and blue ellipse corresponds to extracellular loop 3. These regions are also indicated in the line graph by a red arrow or blue arrows.

Figure 4

Amino acid residues involved in binding of SK-related peptides in TcSKR1 (A) and TcSKR2 (B). The residues were collected for the binding of TcSKRs with sSK, nsSK, alanine-substituted and truncated SK peptides in Table 1 and Table supplement 1. The residues are represented with white single-letter codes in black filled circles. Figures were generated online with Protter v.1.0.