Structural Biology Study Of Human Somatostatin Receptor 2

Somatostatin receptor 2（SSTR2）belongs to G protein-coupled receptors（GPCRs）,and its endogenous ligand is somatostatin（SST）.SSTR2 has a wide distribution in human body.Moreover,it is also highly expressed in many neuroendocrine tumors（NETs）.SSTR2 has diverse biologic actions including the regulation of the release of many hormones,the inhibition of proliferation of both normal and tumor cells,and the induction of apotosis.These make it an attractive drug target for the theurapy of acromegaly and NETs.Our goal is to solve the high-resolution crystal structures of SSTR2 in complex with its ligands by using the methods of molecular biology,biochemistry,biophysics and computational biology.Then we will analyze the ligand-binding pockets and conformational changes so that provide structural basis for SSTR2-targeted drug development.After solving the expression and purification problems,we successfully purified SSTR2 protein with high yield and quality.In addition,we screened many high affinity ligands and found two of them can dramatically improve SSTR2’s thermostability.One is a peptide antagonist CYN-154806,and the other is a small molecule agonist L-054,522.Then we co-crystallized the two ligands with SSTR2 protein respectively and screened many conditions.Finally,we got high-quality crystals.In the end,we successfully determined the crystal structure of SSTR2 recombinant protein 3862 in cpmplex with CYN-154806 at 2.5?,and the crystal structures of SSTR2 recombinant protein 3344 and 6097 in cpmplex with L-054,522 at 2.8 and 2.7?respectively.After that,we analyzed the atom details of the two ligand-binding pockets and the conformational changes of the structures.We elucidated the molecular mechanism of D-Trp⁸-Lys⁹ pair for its impact on ligand binding and receptor activation.Meanwhile,we also found that N125^3.35 played a key role in receptor activation.In addition,D89^2.50N mutation had an adverse impact on agonist binding and receptor activation.These findings deepen our understanding about the interaction mechanism between SSTR2 and its ligands,and the activation mechanism of SSTR2.We believe that these will facilitate SSTR2-targeted drug discovery.Despite the structural biology study of SSTR2,part of NK1R’s structural biology study is also included in this thesis.