Expression, purification and molecular dynamics simulation of extracellular domain of glucagon-like peptide-2 receptor linked to teduglutide.

Teduglutide is the only drug approved for long-term treatment of short bowel syndrome (SBS). This drug exerts its pharmacological effects via binding to the GLP-2 receptors (ECD-GLP2R) located in intestinal tissue. The three dimensional (3D) structure of ECD-GLP2R hasn't been determined yet and hence its mode of interaction with agonists/antagonists is not clear. Therefore, it would be of great importance to develop a structural scaffold for investigation of ECD-GLP2R interactions with its binders. For this, the current study aimed to produce fusion protein of ECD-GLP2R-teduglutide. The ECD-GLP2R-teduglutide protein was expressed in bacterial expression system and purified using affinity and size exclusion chromatography techniques. Using circular dichroism the secondary structure content of purified protein was determined which was comparable to that of theoretical calculations. The low structural stability of purified protein (ΔG = 3.64 kJ.mol-1) was elucidated by monitoring its fluorescence emission at the presence of various concentrations of GdnHCl as a denaturant. Finally, a 3D model for ECD-GLP2R-teduglutide protein was generated and validated using molecular dynamics simulation whose information alongside the experimental studies can be useful for providing new insight into the mode of interaction of ECD-GLP2R with its specific ligands in order to design potent and specific GLP2R agonists.

A unique hormonal recognition feature of the human glucagon-like peptide-2 receptor.

Glucagon-like peptides (GLP-1 and GLP-2) are two proglucagon-derived intestinal hormones that mediate distinct physiological functions through two related receptors (GLP-1R and GLP-2R) which are important drug targets for metabolic disorders and Crohn's disease, respectively. Despite great progress in GLP-1R structure determination, our understanding on the differences of peptide binding and signal transduction between these two receptors remains elusive. Here we report the electron microscopy structure of the human GLP-2R in complex with GLP-2 and a Gs heterotrimer. To accommodate GLP-2 rather than GLP-1, GLP-2R fine-tunes the conformations of the extracellular parts of transmembrane helices (TMs) 1, 5, 7 and extracellular loop 1 (ECL1). In contrast to GLP-1, the N-terminal histidine of GLP-2 penetrates into the receptor core with a unique orientation. The middle region of GLP-2 engages with TM1 and TM7 more extensively than with ECL2, and the GLP-2 C-terminus closely attaches to ECL1, which is the most protruded among 9 class B G protein-coupled receptors (GPCRs). Functional studies revealed that the above three segments of GLP-2 are essential for GLP-2 recognition and receptor activation, especially the middle region. These results provide new insights into the molecular basis of ligand specificity in class B GPCRs and may facilitate the development of more specific therapeutics.

Novel Descriptors and Digital Signal Processing- Based Method for Protein Sequence Activity Relationship Study.

The work aiming to unravel the correlation between protein sequence and function in the absence of structural information can be highly rewarding. We present a new way of considering descriptors from the amino acids index database for modeling and predicting the fitness value of a polypeptide chain. This approach includes the following steps: (i) Calculating Q elementary numerical sequences (Ele_SEQ) depending on the encoding of the amino acid residues, (ii) determining an extended numerical sequence (Ext_SEQ) by concatenating the Q elementary numerical sequences, wherein at least one elementary numerical sequence is a protein spectrum obtained by applying fast Fourier transformation (FFT), and (iii) predicting a value of fitness for polypeptide variants (train and/or validation set). These new descriptors were tested on four sets of proteins of different lengths (GLP-2, TNF alpha, cytochrome P450, and epoxide hydrolase) and activities (cAMP activation, binding affinity, thermostability and enantioselectivity). We show that the use of multiple physicochemical descriptors coupled with the implementation of the FFT, taking into account the interactions between residues of amino amides within the protein sequence, could lead to very significant improvement in the quality of models and predictions. The choice of the descriptor or of the combination of descriptors and/or FFT is dependent on the couple protein/fitness. This approach can provide potential users with value added to existing mutant libraries where screening efforts have so far been unsuccessful in finding improved polypeptide mutants for useful applications.