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Structure-Based Analysis of the Ligand-Binding Mechanism for DhelOBP21, a C-minus Odorant Binding Protein, from Dastarcus helophoroides (Fairmaire; Coleoptera: Bothrideridae).

Li DZ, Yu GQ, Yi SC, Zhang Y, Kong DX, Wang MQ - Int. J. Biol. Sci. (2015)

Bottom Line: Ligand-binding experiments using N-phenylnaphthylamine (1-NPN) as a fluorescent probe showed that DhelOBP21 exhibited better binding affinities against those ligands with a molecular volume between 100 and 125 Å(³) compared with ligands with a molecular volume between 160 and 185 Å(³).Ligand-binding experiments and cyber molecular docking assays indicated that hydrophobic interactions are more significant than hydrogen-bonding interactions.This study provides a basis to explore the ligand-binding mechanisms of Minus-C OBP.

View Article: PubMed Central - PubMed

Affiliation: 1. Hubei Insect Resources Utilization and Sustainable Pest Management Key Laboratory, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, P. R. China.

ABSTRACT
Odorant binding proteins (OBPs) transport hydrophobic odor molecules across the sensillar lymph to trigger a neuronal response. Herein, the Minus-C OBP (DhelOBP21) was characterized from Dastarcus helophoroides, the most important natural parasitic enemy insect that targets Monochamus alternatus. Homology modeling and molecular docking were conducted on the interaction between DhelOBP21 and 17 volatile molecules (including volatiles from pine bark, the larva of M. alternatus, and the faeces of the larva). The predicted three-dimensional structure showed only two disulfide bridges and a hydrophobic binding cavity with a short C-terminus. Ligand-binding experiments using N-phenylnaphthylamine (1-NPN) as a fluorescent probe showed that DhelOBP21 exhibited better binding affinities against those ligands with a molecular volume between 100 and 125 Å(³) compared with ligands with a molecular volume between 160 and 185 Å(³). Molecules that are too big or too small are not conducive for binding. We mutated the amino acid residues of the binding cavity to increase either hydrophobicity or hydrophilia. Ligand-binding experiments and cyber molecular docking assays indicated that hydrophobic interactions are more significant than hydrogen-bonding interactions. Although hydrogen-bond interactions could be predicted for some binding complexes, the hydrophobic interactions had more influence on binding following hydrophobic changes that affected the cavity. The orientation of ligands affects binding by influencing hydrophobic interactions. The binding process is controlled by multiple factors. This study provides a basis to explore the ligand-binding mechanisms of Minus-C OBP.

No MeSH data available.


Binding affinities (indicated by 1/Ki*1000) assays of DhelOBP21-WT. (A, B) Relationship between molecular volume and Ki at pH 7.4 and pH 5.0. (C, D) Relationship between LogD and Ki at pH 7.4 and pH 5.0. (E) Relationship between difference value and Ki at pH 7.4. (F) Relationship between difference value and LogD. (G) Relationship between difference value and molecular volume. The dashed lines express confidence interval.
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Figure 6: Binding affinities (indicated by 1/Ki*1000) assays of DhelOBP21-WT. (A, B) Relationship between molecular volume and Ki at pH 7.4 and pH 5.0. (C, D) Relationship between LogD and Ki at pH 7.4 and pH 5.0. (E) Relationship between difference value and Ki at pH 7.4. (F) Relationship between difference value and LogD. (G) Relationship between difference value and molecular volume. The dashed lines express confidence interval.

Mentions: After protein purification, the identity and integrity of the recombinant proteins were confirmed using SDS-PAGE (Fig. 3). Those proteins were used in the following fluorescence binding assays. The ligand-binding assays of 1-NPN of DhelOBP21 and the mutant are shown in Fig. 4. The binding affinities (indicated by 1/Ki*1000) of DhelOBP21 and the mutant are shown in Table 1. Some binding and structural studies have shown remarkable plasticity in the ligand-binding site of OBP 40. We must first consider the molecular volumes of the ligands. Based on the molecular volume and hydrophobicity of these ligands, they were divided into two major groups (Fig. 5): (1) ligands with a molecular volume between 100 and 125 ų; and (2) ligands with a molecular volume between 160 and 185 ų. By plotting these values in different pH and molecular volumes (Fig. 6A-B), one could find that the ligands with a molecular volume between 100 and 125 ų had a stronger binding ability than the ligands with a molecular volume between 160 and 185 ų at pH 7.4. Hydrophobic contacts with the cavity wall residues have been reported for AmelOBP14 bound to citralva. Considering the high hydrophobicity of the cavity, ligands with a molecular volume between 100 and 125 ų were selected to plot against the LogD of these ligands, which denotes hydrophobicity of ligands at different pH values. We found that ligands that are more hydrophobic have a stronger binding affinity at pH 7.4 (Fig. 6C). However, when the pH reduced to 5.0, this trend was not obvious. Furthermore, this difference was obtained by subtracting the binding ability at pH 7.4 from the corresponding value at pH 5.0 to study the influence of pH. Similarly, we plotted these values (Fig. 6D). Compared with the binding ability at pH 7.4, most ligands have a lower binding ability at pH5.0, except for (+)-α-longipinene and (-)-caryophyllene oxide. We also found that pH had a stronger influence on those ligands that had a stronger binding ability at pH 7.4 (Fig. 6E-F).


Structure-Based Analysis of the Ligand-Binding Mechanism for DhelOBP21, a C-minus Odorant Binding Protein, from Dastarcus helophoroides (Fairmaire; Coleoptera: Bothrideridae).

Li DZ, Yu GQ, Yi SC, Zhang Y, Kong DX, Wang MQ - Int. J. Biol. Sci. (2015)

Binding affinities (indicated by 1/Ki*1000) assays of DhelOBP21-WT. (A, B) Relationship between molecular volume and Ki at pH 7.4 and pH 5.0. (C, D) Relationship between LogD and Ki at pH 7.4 and pH 5.0. (E) Relationship between difference value and Ki at pH 7.4. (F) Relationship between difference value and LogD. (G) Relationship between difference value and molecular volume. The dashed lines express confidence interval.
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Figure 6: Binding affinities (indicated by 1/Ki*1000) assays of DhelOBP21-WT. (A, B) Relationship between molecular volume and Ki at pH 7.4 and pH 5.0. (C, D) Relationship between LogD and Ki at pH 7.4 and pH 5.0. (E) Relationship between difference value and Ki at pH 7.4. (F) Relationship between difference value and LogD. (G) Relationship between difference value and molecular volume. The dashed lines express confidence interval.
Mentions: After protein purification, the identity and integrity of the recombinant proteins were confirmed using SDS-PAGE (Fig. 3). Those proteins were used in the following fluorescence binding assays. The ligand-binding assays of 1-NPN of DhelOBP21 and the mutant are shown in Fig. 4. The binding affinities (indicated by 1/Ki*1000) of DhelOBP21 and the mutant are shown in Table 1. Some binding and structural studies have shown remarkable plasticity in the ligand-binding site of OBP 40. We must first consider the molecular volumes of the ligands. Based on the molecular volume and hydrophobicity of these ligands, they were divided into two major groups (Fig. 5): (1) ligands with a molecular volume between 100 and 125 ų; and (2) ligands with a molecular volume between 160 and 185 ų. By plotting these values in different pH and molecular volumes (Fig. 6A-B), one could find that the ligands with a molecular volume between 100 and 125 ų had a stronger binding ability than the ligands with a molecular volume between 160 and 185 ų at pH 7.4. Hydrophobic contacts with the cavity wall residues have been reported for AmelOBP14 bound to citralva. Considering the high hydrophobicity of the cavity, ligands with a molecular volume between 100 and 125 ų were selected to plot against the LogD of these ligands, which denotes hydrophobicity of ligands at different pH values. We found that ligands that are more hydrophobic have a stronger binding affinity at pH 7.4 (Fig. 6C). However, when the pH reduced to 5.0, this trend was not obvious. Furthermore, this difference was obtained by subtracting the binding ability at pH 7.4 from the corresponding value at pH 5.0 to study the influence of pH. Similarly, we plotted these values (Fig. 6D). Compared with the binding ability at pH 7.4, most ligands have a lower binding ability at pH5.0, except for (+)-α-longipinene and (-)-caryophyllene oxide. We also found that pH had a stronger influence on those ligands that had a stronger binding ability at pH 7.4 (Fig. 6E-F).

Bottom Line: Ligand-binding experiments using N-phenylnaphthylamine (1-NPN) as a fluorescent probe showed that DhelOBP21 exhibited better binding affinities against those ligands with a molecular volume between 100 and 125 Å(³) compared with ligands with a molecular volume between 160 and 185 Å(³).Ligand-binding experiments and cyber molecular docking assays indicated that hydrophobic interactions are more significant than hydrogen-bonding interactions.This study provides a basis to explore the ligand-binding mechanisms of Minus-C OBP.

View Article: PubMed Central - PubMed

Affiliation: 1. Hubei Insect Resources Utilization and Sustainable Pest Management Key Laboratory, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, P. R. China.

ABSTRACT
Odorant binding proteins (OBPs) transport hydrophobic odor molecules across the sensillar lymph to trigger a neuronal response. Herein, the Minus-C OBP (DhelOBP21) was characterized from Dastarcus helophoroides, the most important natural parasitic enemy insect that targets Monochamus alternatus. Homology modeling and molecular docking were conducted on the interaction between DhelOBP21 and 17 volatile molecules (including volatiles from pine bark, the larva of M. alternatus, and the faeces of the larva). The predicted three-dimensional structure showed only two disulfide bridges and a hydrophobic binding cavity with a short C-terminus. Ligand-binding experiments using N-phenylnaphthylamine (1-NPN) as a fluorescent probe showed that DhelOBP21 exhibited better binding affinities against those ligands with a molecular volume between 100 and 125 Å(³) compared with ligands with a molecular volume between 160 and 185 Å(³). Molecules that are too big or too small are not conducive for binding. We mutated the amino acid residues of the binding cavity to increase either hydrophobicity or hydrophilia. Ligand-binding experiments and cyber molecular docking assays indicated that hydrophobic interactions are more significant than hydrogen-bonding interactions. Although hydrogen-bond interactions could be predicted for some binding complexes, the hydrophobic interactions had more influence on binding following hydrophobic changes that affected the cavity. The orientation of ligands affects binding by influencing hydrophobic interactions. The binding process is controlled by multiple factors. This study provides a basis to explore the ligand-binding mechanisms of Minus-C OBP.

No MeSH data available.