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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2024 Oct 3;121(41):e2400298121. doi: 10.1073/pnas.2400298121

Selective ligand recognition and activation of somatostatin receptors SSTR1 and SSTR3

Yujue Wang a,b,c,1, Youwei Xu d,1, Yue Wang d, Jie Zhang a,b,c, Lan Chen a,b,c, Xinheng He d,e, Wenjia Fan d,f, Kai Wu g, Wen Hu g, Xi Cheng d, Guizhu Yang a,b,c, H Eric Xu d,2, Youwen Zhuang d,h,2, Shuyang Sun a,b,c,2
PMCID: PMC11474030  PMID: 39361640

Significance

Somatostatin receptors (SSTRs) are important targets for treating endocrine disorders such as neuroendocrine tumors. The clinical use of SSTR drugs shows limited therapeutic efficacy and considerable side effects, potentially arising from the poor selectivity to distinct SSTR subtypes. We present the cryoelectron microscopy structures of SSTR1 and SSTR3 bound to the panagonist pasireotide and subtype-selective agonists L-797591 and L-796778. Key findings include the unique feature of pasireotide binding, the identification of determinants controlling ligand selectivity across the orthosteric binding pockets of SSTRs, and diversity and conservation of SSTRs activation triggered by distinct agonists. This study provides a framework for rational design of SSTR drugs with enhanced selectivity and potency, which may facilitate the development of more effective therapies targeting SSTRs.

Keywords: GPCR, somatostatin receptors, ligand selectivity, neuroendocrine tumor, cryo-EM

Abstract

Somatostatin receptors (SSTRs) exert critical biological functions such as negatively regulating hormone release and cell proliferation, making them popular targets for developing therapeutics to treat endocrine disorders, especially neuroendocrine tumors. Although several panagonists mimicking the endogenous ligand somatostatin are available, the development of more effective and safer somatostatinergic therapies is limited due to a lack of molecular understanding of the ligand recognition and regulation of divergent SSTR subtypes. Here, we report four cryoelectron microscopy structures of Gi-coupled SSTR1 and SSTR3 activated by distinct agonists, including the FDA-approved panagonist pasireotide as well as their selective small molecule agonists L-797591 and L-796778. Our structures reveal a conserved recognition pattern of pasireotide in SSTRs attributed to the binding with a conserved extended binding pocket, distinct from SST14, octreotide, and lanreotide. Together with mutagenesis analyses, our structures further reveal the dynamic feature of ligand binding pockets in SSTR1 and SSTR3 to accommodate divergent agonists, the key determinants of ligand selectivity lying across the orthosteric pocket of different SSTR subtypes, as well as the molecular mechanism underlying diversity and conservation of receptor activation. Our work provides a framework for rational design of subtype-selective SSTR ligands and may facilitate drug development efforts targeting SSTRs with improved therapeutic efficacy and reduced side effects.

Somatostatin (SST) is a hormone peptide known for its broad antisecretory effects. There are two active isoforms of the endogenous SST peptide with different amino acid lengths, namely SST14 and SST28 (1), both of which are widely distributed in the human central nervous system, such as the cerebral cortex, pituitary, and hypothalamus, as well as peripheral tissues including the pancreas and gastrointestinal tract (2, 3). SST negatively regulates the secretion of various hormones, including growth hormone, insulin, glucagon, adrenocorticotropic hormone, and cholecystokinin (1, 4–6). The biological functions of SST are mediated by activating human SST receptors (SSTRs), a group of Class A G protein-coupled receptors comprising five members, SSTR1-SSTR5 (7), which transmit signals into cells through coupling with inhibitory G protein (Gi) (7, 8). The five SSTRs are further divided into two classes based on sequence homologies and pharmacological properties, SRIF1 (SSTR2, 3, 5) and SRIF2 (SSTR1, 4) (9).

Each SSTR subtype regulates the secretion of distinct hormones. For instance, secretion of growth hormone is dually regulated by SSTR2, SSTR3, and SSTR5 (1, 5, 10). Insulin secretion is inhibited by SSTR1, SSTR2, and SSTR5, while glucagon secretion is predominantly down-regulated by SSTR2 (6). The extensive hormonal regulatory effects make SSTRs attractive therapeutic targets for diseases characterized by abnormal hormonal hypersecretion, such as acromegaly and Cushing’s disease. However, the ultrashort half-life (<3 min) of SST limits its clinical usage and has promoted the development of longer-acting SST analogs. Several long-acting SST analogs have been developed, including the first-generation SST analogues octreotide and lanreotide for acromegaly (11, 12), and the second-generation SST analogue pasireotide for Cushing’s disease (13). These SST analogs retain the two essential pharmacophoric residues (Trp-Lys), and display greatly extended half-life and stability compared to SST. Octreotide and lanreotide show high affinity for SSTR2 and lower affinities for SSTR3 and SSTR5, whereas pasireotide shows high affinity for all subtypes except SSTR4 (11). However, the clinical application of current SST analogs was accompanied by limited effectiveness and several side effects such as hyperglycemia, hypothyroidism, and gallbladder stones (14–18). These side effects are possibly due to the poor selectivity of SST analogs for distinct SSTR subtypes, leading to inhibition of nonpathological hormone secretion. Drug discovery targeting SSTRs has been in pursuit of small molecule agonists with extended half-life, oral availability, and high selectivity toward SSTR subtypes (11, 17). L-797591 and L-796778 are two small-molecule SSTR agonists identified by combinatorial chemistry and high-throughput screening that showed selective activity toward SSTR1 and SSTR3, respectively (19). Despite extensive efforts made recently to elucidate the structural mechanisms of ligand binding and pharmacology of SSTR2 and SSTR4 (20–25), little is known about how other SSTR subtypes are recognized and selectively activated by their specific ligands.

Here, we determined cryoelectron microscopy (cryo-EM) structures of active SSTR1-Gi and SSTR3-Gi complexes with the panagonist pasireotide and their selective small molecules, L-797591 and L-796778. Together with functional analyses, these structures reveal the conserved binding mode of pasireotide in SSTR1 and SSTR3 and provide key insights into the selective ligand recognition and receptor activation profiles of SSTR subtypes.

Results

Structures of SSTR1-Gi and SSTR3-Gi Activated by Pasireotide.

Pasireotide (Hyp-Phg-Trp-Lys-(Bzl)Tyr-Phe) is a cyclohexapeptide SST analog that lacks the intramolecular disulfide bond present in most SSTR peptide ligands, but retains the common “Trp-Lys” motif (Fig. 1A). We first characterized pasireotide activity toward SSTRs using cAMP accumulation assay. In our assay, pasireotide showed pan-activating effects on SSTRs and activated SSTR4 with 10,100 times lower potency versus other SSTR subtypes, consistent with the previous studies (26) (Fig. 1B). To elucidate the ligand binding properties of SSTR1 and SSTR3, we thought to determine the cryo-EM structures of pasireotide-bound SSTR1-Gi and SSTR3-Gi signaling complexes. To obtain stably assembled protein complexes for structure determination, an engineered form of Gαi1 was designed based on the reported mini-Gαs sequence and replaced the αN and α5 helices with the corresponding amino acids from Gαi1 (27). The V6.40Y mutation, which was shown to enhance SSTR2/4 thermal stability (21), was induced to SSTR1 and SSTR3, respectively. We coexpressed SSTR1 or SSTR3 with the dominant-negative heterotrimeric Gi and purified the protein complexes to homogeneity in detergent buffer. The single-chain antibody, Nb35, was added to further stabilize the nucleotide-free SSTR1-Gi or SSTR3-Gi complexes. The structures of pasireotide-bound SSTR1-Gi and SSTR3-Gi complexes were determined to a global resolution of 3.29 Å and 2.52 Å, respectively (Fig. 1 C and D and SI Appendix, Fig. S1 and Table S1).

Fig. 1.

Fig. 1.

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Overall structures of pasireotide-SSTR1-Gi and pasireotide-SSTR3-Gi signaling complexes. (A) Sequence alignment of SST14, octreotide, lanreotide, and pasireotide. (B) Activities of SSTRs-induced Gi signaling by pasireotide, measured using GloSensor cAMP assays. Data represent means ± SEM from three independent experiments. (C and D) Orthogonal views of the cryo-EM density map (Left panel) and model (Right panel) of (C) pasireotide-SSTR1-Gi complex and (D) pasireotide-SSTR3- Gi complex. SSTR1, pasireotide, Gαi, Gβ, Gγ, and Nb35 are colored in dodger blue, yellow green, tan, dark cyan, magenta, and gray, respectively, while SSTR3 and pasireotide are colored in medium spring green and orange. (E and F) The side view of pasireotide binding pose in (E) SSTR1 and (F) SSTR3.

For both pasireotide-bound SSTR-Gi complexes, the high-quality density maps allowed the clear modeling of most residues of SSTR1 from S52 to R336, SSTR3 from L40 to L328, the Gi heterotrimer, and Nb35, as well as the precise definition of the peptide agonist pasireotide (Fig. 1 C and D and SI Appendix, Fig. S1 E and F).

No clear density could be observed for the N terminus and C terminus of SSTR1 and SSTR3 due to conformational flexibility, similar to the reported structures of the SSTR family (21, 22). In our structures, both SSTR1 and SSTR3 showed typical seven αhelical transmembrane configuration, with the ligand binding pocket laid on the extracellular portion of the transmembrane domain (TMD) and the G protein insert into the intracellular cavity (Fig. 1 C and D).

In our structures, pasireotide occupied a large binding pocket in both SSTR1 and SSTR3 to accommodate bulky side chains, with the top region widely open to the extracellular portion (Fig. 1 C and D). The whole pasireotide was nearly embedded into the receptors, forming close hydrophobic and polar contacts with surrounding residues from TM2-TM3, TM5-TM7, and ECL1-ECL3 of SSTR1 and SSTR3. In SSTR1 and SSTR3, the orthosteric binding pocket (OBP) of pasireotide could be divided into three parts (Fig. 1 E and F). The conserved Trp-Lys motif, located at the tip of the cyclic peptide, vertically inserts into the TMD core, adopting the amphiphilic bottom subpocket of receptors. It has been reported that the Trp-Lys motif of SST analogues is crucial for their biological activity (21, 28). The side chains of Hyp, Phg, and Phe extend toward the extracellular milieu, and occupy the top OBP region composed of residues from ECL2-ECL3 and extracellular ends of TM5-TM7. In addition to the interactions with the bottom and top regions of OBP, the side chain of (Bzl)Tyr of pasireotide extends toward the cleft of TM2 and TM3 and occupy the extended binding pocket (EBP) (Fig. 1 E and F).

A Conserved Binding Pattern of Pasireotide in SSTR1 and SSTR3.

In SSTR1, the side chain of lysine residue of pasireotide makes close ionic contact with D1373.32 [superscript based on Ballesteros-Weinstein numbering rules (29), hereafter] and hydrogen bond interaction with Y3137.43 by the positively charged head group, and formed hydrophobic interactions with nearby residues M1413.36, F2876.51, and V3097.39 by the alkyl chain (Fig. 2A). The tryptophan residue in Trp-Lys motif faces toward TM4, with the indole ring engaging in hydrophobic contacts with M1413.36, M210ECL2, T2275.42, and F2876.51 (Fig. 2A). Consistent with the binding pose, mutations of D1373.32 and Y3137.43 into alanine showed attenuated Gi activities of SSTR1 induced by pasireotide (Fig. 2C and SI Appendix, Table S2). It should be noted that neither the D1373.32A nor the Y3137.43A mutation reduced the cell surface expression of SSTR1, indicating that the decreased pasireotide-induced SSTR1 activation can be directly attributed to the mutational effects on these specific residues, rather than to a reduction of cell surface expression level (SI Appendix, Table S2). The conformation of Hyp is stabilized via a direct salt bridge between its aminoethylcarbamoyloxy moiety and D300ECL3 (SI Appendix, Fig. S2A). The Phg inserts into a hydrophobic cavity formed by M210ECL2, M212ECL2, and L2205.35, while the side chain of Phe stretches to ECL3 and the extracellular end of TM7 (SI Appendix, Fig. S2A). In addition to the interactions with the bottom and top regions of OBP, the side chain of (Bzl)Tyr of pasireotide extends toward the cleft of TM2 and TM3, forming a hydrophobic network with surrounding residues F1132.59, L1142.60, W12323.50, V1333.28, and L1343.29, as well as hydrogen bond interaction with S1172.63 (Fig. 2B). Mutations of L1142.60, W12323.50, and L1343.29 into alanine all largely diminished the pasireotide-induced SSTR1 activation, indicating the indispensable role of the interaction with the EBP in the activity of pasireotide to SSTR1 (Fig. 2C and SI Appendix, Table S2).

Fig. 2.

Fig. 2.

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Binding modes of pasireotide in SSTR1 and SSTR3. (A and B) Detailed interaction of pasireotide with (A) the bottom OBP and (B) EBP of SSTR1. The hydrogen bonds are shown in orange dashed lines. (C) The effects of SSTR1 mutations within the pasireotide binding pocket. Responses were normalized to 100% for the wild-type (WT). (D and E) Detailed interaction of pasireotide with the (D) bottom OBP and (E) EBP of SSTR3. (F) The effects of SSTR3 mutations within the pasireotide binding pocket. (G) Structural superposition of pasireotide-bound SSTR1 and SSTR3. (H) Comparison of the binding poses of pasireotide in SSTR1 with those of SST14 in SSTR2 (PDB:7XAT) and SSTR4 (PDB:7XMS) when aligning the structures on the receptor part. SST14 and SSTR2 in SST14-bound SSTR2 structure are colored in dark green and light tan, respectively. SST14 and SSTR4 in SST14-bound SSTR4 structure are colored in light pink and powder blue, respectively. (I) Comparison of the binding poses of pasireotide in SSTR1 with those of octreotide (tomato) in SSTR2 (pale green) (PDB:7XAU), and lanreotide (violet) in SSTR2 (wheat) (PDB:7XAV) when aligning the structures on the receptor part. The EBP is circled in oval frames.

SSTR3 and SSTR1 belong to different SSTR classes and share only 40% sequence similarity, in contrast to the 56 to 66% identity among SRIF1 members. Nevertheless, structural alignment of pasireotide-bound SSTR1 and SSTR3 suggested that only subtle conformational changes in the side chains of pasireotide are observed, mainly attributed to different interactions of pasireotide with nonconserved residues between receptors (Fig. 2G). In addition to the ionic interaction with the conserved D3.32 residue, the protonated nitrogen atom of lysine of pasireotide forms a hydrogen bond with Q1273.36 instead of Y3037.43 in the bottom OBP of SSTR3 (Fig. 2D). In the top region of OBP of SSTR3, the benzyl side chain of Phg in pasireotide adopts an upward shift and forms hydrophobic interaction with the aliphatic portion of R2035.35, while the Phe residue of the ligand inserts into a hydrophobic pocket near ECL3 and extracellular end of TM7 (SI Appendix, Fig. S2B). Unlike in SSTR1, there are no polar interactions between SSTR3 and the Hyp or (Bzl)Tyr residue of pasireotide (Fig. 2E). Consistent with the binding pose of pasireotide in SSTR3, alanine mutation on the critical interaction residues, such as D1233.32 and Q1273.36 in the bottom OBP, and W10923.50 in the EBP, largely decreased the pasireotide-induced Gi activation of SSTR3 (Fig. 2F and SI Appendix, Table S3). Interestingly, pasireotide adopted a highly similar pose in both receptors albeit with minor differences observed in specific interactions (Fig. 2G), in contrast to the distinct binding conformations of SST14 observed between SSTR2 and SSTR4 (21, 22) (Fig. 2H). The total surface area of pasireotide binding on either receptor is almost identical, with a size of 833.6 Å2 and 884.5 Å2, respectively, in SSTR1 and SSTR3. The conserved binding mode adopted by pasireotide between SSTR1 and SSTR3 may be attributed to the additional anchoring point provided by the EBP, which is absent in SST14 (Fig. 2H). The additional anchor enables pasireotide to maintain a rigid binding conformation across different SSTR subtypes, in contrast to SST14 which exhibits a certain level of flexibility when binding to SSTR2 and SSTR4 (21). It is worth noting that interaction with such hydrophobic EBP is also absent in structures of SSTRs bound to first-generation SST analogues octreotide and lanreotide (Fig. 2I), presenting the possibility of rationally designing more potent peptide drugs selectively targeting the EBP across SSTRs.

Selectivity of Pasireotide to SSTR4.

Pasireotide shows high potency to all SSTR subtypes except for SSTR4 (Fig. 1B). The ECL regions of SSTRs have been reported to participate in subtype selectivity of SSTR peptide ligands (28). Structural analysis revealed high diversities in residue compositions and topologies of the extracellular vestibules near ECL3 among SSTRs, which mainly interacts with the Phe moiety of pasireotide in the pasireotide-bound SSTR1 and SSTR3 structures (21) (SI Appendix, Fig. S2 C and D). To investigate whether these differences account for the poor activity of pasireotide toward SSTR4, we generated chimeric constructs by swapping the nonconserved extracellular vestibules around ECL3 from SSTR1 or SSTR2 with the corresponding sequence of SSTR4, or the other way around (SI Appendix, Fig. S2E), and tested their effects on pasireotide-induced cAMP accumulation. Remarkably, substitutions of SSTR1 and SSTR2 with those of SSTR4 impaired the potency of pasireotide (SI Appendix, Fig. S2F and Table S5). Conversely, swapped mutation of SSTR4 with that from SSTR2 increased the potency of pasireotide to SSTR4 (SI Appendix, Fig. S2F and Table S5). These findings suggest that the differences in the extracellular region near ECL3 among SSTRs may partially explain the reduced specificity of pasireotide toward SSTR4.

Binding Mode of L-797591 in SSTR1.

SSTRs differ in their pharmacological roles and tissue expression distributions (2, 3). The selective SSTR agonists were suggested to provide advantages over panagonists in treating endocrine tumors with fewer side effects (14–16). To understand the mechanism of ligand selectivity of SSTR1, we also determined the structure of SSTR1 bound to L-797591. L-797591 was synthesized through combinatorial chemistry based on cyclic hexapeptide SST agonist, showing high selectivity to SSTR1(Fig. 3 A and C). In our structure, the density of L-797591 is clearly defined (Fig. 3B and SI Appendix, Fig. S3 A–D and Table S1). The overall L-797591- and pasireotide-bound SSTR1 structures display high conformational similarity, with the RMSD at 0.57 Å when aligned in the receptor part. However, noticeable divergencies could be observed in the topologies of TM7 and ECL3 of SSTR1 when bound to L-797591 and pasireotide, mainly attributed to the distinct binding modes between these two ligands (SI Appendix, Fig. S3 E and F).

Fig. 3.

Fig. 3.

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The binding pocket of L-797591 in SSTR1. (A) Dose-dependent response curves of SSTRs activated by L-797591 measured by cAMP accumulation assays. (B) Orthogonal view of the cryo-EM model (Left panel) of L-797591-SSTR1-Gi complex. The chemical structure of small molecule ligand L-797591 is shown (Right panel). SSTR1 and L-797591 are colored in plum and medium slate blue, respectively. (C) Comparison of the binding modes of pasireotide and L-797591 in SSTR1. The crucial pharmacophore group Trp-Lys of pasireotide and the corresponding region in L-797591 are circled in oval frames. (D) Cross-section of the SSTR1 binding pockets occupied by pasireotide and L-797591. (E and F) Detailed interactions of L-797591 at the bottom pocket of (E) SSTR1 and (F) differential effects of the SSTR1 mutants on cAMP accumulation assay induced by L-797591. (G and H) (G) Detailed interactions of L-797591 at the EBP of SSTR1 and (H) differential effects of corresponding amino acid residues on cAMP inhibition induced by L-797591.

L-797591 lacks the classical Trp-Lys motif of SSTR peptide ligands. Instead, in our structure, the naphthalene and phenylethyl moieties of L-797591, which mimics the role of Trp-Lys motif of SSTR peptide (Fig. 3 C and D). The naphthalene group of L-797591 engages an identical hydrophobic pocket in SSTR1 as occupied by Trp in pasireotide (Fig. 3 D and E). Compared to the binding pose of Lys residue in pasireotide-bound SSTR1, the side chain of phenylethyl moiety of L-797591 extends downward by an additional 2.3 Å when measured at the Cα atoms, and forms extensive hydrophobic packing with surrounding residues, including M1413.36, W2846.48, F2876.51, and Y3137.43 (Fig. 3 D and E). Consistent with the structure observation, mutation of F2235.38A, Y3137.43A, and D1373.32A largely impair the activity of L-797591-induced SSTR1 activation (Fig. 3F and SI Appendix, Table S2). In the top OBP of SSTR1, the amino-trimethylhexyl group of L-797591 engages a hydrophobic subpocket around TM5 and ECL2, including M21045.52 and L2205.35, similar to that occupied by Phg residue of pasireotide (SI Appendix, Fig. S3G). However, L-797591 lacks interactions with the extracellular milieu near ECL3 of SSTR1, as occupied by Hyp and Phe of pasireotide. Consequently, the ECL3 and extracellular end of TM7 of SSTR1 undergo an inward movement to accommodate the binding of L-797591, suggesting the plasticity of the SSTR1 OBP in recognition of diverse ligands as SSTR2 and SSTR4 (21, 28) (SI Appendix, Fig. S3F). Several water molecules are observed in the binding pocket of L-797591 rather than that of pasireotide (SI Appendix, Fig. S3H). These water molecules participate in the stable binding of L-797591 in the SSTR1 OBP through forming a polar network with Q2916.55, N2946.58, S3057.35, and L-797591(SI Appendix, Fig. S3H). Coordinately, mutation of Q2916.55A showed much pronounced effect on the agonistic activity of L-797591 on SSTR1 when compared to pasireotide (SI Appendix, Fig. S3 I and J). In addition, individual mutation of the residues of the polar network, such as Q2916.55A, N2946.58A, and S3057.35A, decreased the L-797591-induced Gi activation of SSTR1 by 3.5- to 6-folds, while combined mutations of these residues reduced it by 3- to 14.7-folds (SI Appendix, Fig. S3J). L-797591 also possesses a hydrophobic EBP around TM2 and TM3 of SSTR1 by the pyridine ring, which is overlapped with that occupied by the (Bzl)Tyr moiety of pasireotide (Fig. 3G). Mutations of the nearby residues of EBP to alanine, such as L1343.29A, W12323.50A, and L1142.60A, significantly reduced the potency of L-797591-induced SSTR1 activation (Fig. 3H and SI Appendix, Table S2).

Selective Recognition of L-797591 to SSTR1.

L-797591 showed highly selectivity to SSTR1 versus the other SSTR subtypes (19) (Fig. 3A). To reveal the potential ligand selective determinants of SSTR1, we aligned the structure of L-797591-bound SSTR1 with those of the other SSTR subtypes in their activated states. An active SSTR5 structure generated by Alphafold2 method was used for structural analysis (30). Structural comparisons indicated that the residue composition and topological conformation of SSTRs in the extracellular vestibule is much less conserved relative to the bottom OBP (Fig. 4A). Several nonconserved residues, especially those in the bottom OBP, played a critical role in determining the ligand selectivity of L-797591 across the SSTR family. The deeply inserted phenylethyl moiety of L-797591, which is well accommodated in the bottom OBP of SSTR1, would form steric clash with the bulky side chains of F/Y2.53 in SSTR2-SSTR5 (Fig. 4 B and C). In addition, the polar head group of Q3.36 in SSTR2, SSTR3, and SSTR5 would be incompatible for the hydrophobic profile of phenylethyl moiety in L-797591 (Fig. 4C). Consistently, the M1413.36Q and L1072.53F mutations in SSTR1 reduced the potency of L-797591 toward SSTR1 by 10- to 100-fold (Fig. 4D and SI Appendix, Table S4). Substituting phenylalanine or tyrosine with leucine at position 2.53 rescued the agonist activity of L-797591 toward SSTR2 and SSTR5, while simultaneously increasing its potency toward SSTR4 by ~10-fold (Fig. 4D and SI Appendix, Table S4). Moreover, the F/Y2.53L and Q3.36M double mutation restored the activation of L-797591 to SSTR3, while further enhanced the potency of L-797591 to SSTR2 and SSTR5 compared to single mutation (Fig. 4D and SI Appendix, Table S4). Additionally, the nonconserved residues in the extracellular region may also contribute to the ligand selectivity of L797591 among different SSTRs. For instance, the elongated side chain of the positively charged residue R2035.35 in SSTR3 would sterically hinder the aminotrimethylhexyl group of L-797591. Indeed, mutation of L2205.35R decreased the potency of L-797591-induced activation of SSTR1 by ~10-fold (SI Appendix, Fig. S4B and Table S4). However, swapped mutation of residue at position 5.35 into leucine failed to improve the L-797591-induced activation of SSTR2-5(SI Appendix, Fig. S4 C–F and Table S4). In addition, mutation of residue at position 45.52 into methionine only increased the potency of L-797591 toward SSTR2 (SI Appendix, Fig. S4 C–F and Table S4). These results unveil a complex role of the extracellular ECL2-TM5 region in determining the selectivity of L-797591, which may vary across different SSTR subtypes.

Fig. 4.

Fig. 4.

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Characterization of the ligand selectivity of L-797591 to SSTRs. (A) Sequence alignment of SSTRs in the OBP region. (B and C) Detailed comparison of the residues of (B) SRIF1 and (C) SRIF2 at the bottom pocket of L-797591 in SSTR1. SSTR2 (PDB: 7XN9), SSTR4 (PDB: 7XMT), and SSTR5 (Alphafold2) are colored in light tan, powder blue, and green, respectively. (D) The effects of residues at positions 2.53 and 3.36, as well as double mutations, on cAMP inhibition in SSTRs.

Interaction and Selectivity of L-796778 to SSTR3.

L-796778 is a selective small-molecule agonist of SSTR3 that shows at least ten times more potency than other SSTR subtypes (19) (Fig. 5A). To gain insights into the ligand binding properties of SSTR3, we determined the cryo-EM structure of the SSTR3-Gi signaling complex bound to L-796778 (SI Appendix, Fig. S5 A–D and Table S1). The high-resolution density map of the protein complex enabled us to unambiguously define the binding pose of L-796778 within SSTR3 (Fig. 5B). Comparison of the structures of SSTR3 bound to L-796778 and pasireotide revealed distinct binding modes of the two ligands in SSTR3 due to different chemical scaffolds, resulting in considerable conformational changes in the receptor topology, with an RMSD of 0.9 Å when aligned on the Cα atoms of the receptor (Fig. 5C and SI Appendix, Fig. S6 B and C).

Fig. 5.

Fig. 5.

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The binding pocket of L-796778 in SSTR3. (A) Dose-dependent response curves of SSTRs activated by L-796778 measured by cAMP accumulation assays. (B) Orthogonal view of the cryo-EM model of L-796778-SSTR3-Gi complex (Left panel). The chemical structure of small molecule ligand L-796778 is shown (Right panel). SSTR3 and L-796778 are colored in salmon and khaki, respectively. (C) Comparison of the binding modes of pasireotide and L-796778 in SSTR3. The crucial pharmacophore group Trp-Lys of pasireotide and the corresponding region in L-796778 are circled in oval frames. (D) Cross-section of the SSTR3 binding pockets occupied by L-796778. (E) Interaction of L-796778 at the bottom pocket of SSTR3. (F) Interaction of L-796778 at the top binding pocket of SSTR3. H192ECL2 participates in the stable binding of L-796778 in SSTR3 via a polar network mediated by water molecules. (G) The effect of H192ECL2A mutation of SSTR3 on cAMP inhibition induced by L-796778. (H) Interaction between the lysine group of L-796778 and SSTR3. (I) The nitroanilino moiety of L-796778 forms hydrogen bond with Q1032.63 of SSTR3. (J and K) Detailed comparison of the amino acid residues of (J) SRIF1 and (K) SRIF2 at the bottom pocket of L-796778 in SSTR3. SSTR2 (7XN9), SSTR4 (7XMT), and SSTR5 (Alphafold2) are colored in light tan, powder blue, and green, respectively. (L) The effects of SSTR1 and SSTR3 mutants on the cAMP inhibition analysis induced by L-796778. The pEC50 values of the mutants relative to their respective. The significance was determined with two-side, one-way ANOVA followed by Fisher’s LSD multiple comparisons test compared with WT. *P < 0.05; **P < 0.01, and ***P < 0.001 were considered statistically significant.

In the bottom OBP, the ethylbenzene group of L-796778 extends toward the cleft of transmembrane helices TM4 and TM5 and inserts into a hydrophobic pocket composed of residues from TM3-TM5, including F1283.37, V1784.60, V1794.61, and F2065.38, which are conserved among SSTRs (Fig. 5 D and E). Of note, such an extended hydrophobic pocket is not occupied by pasireotide in SSTR3. Consequently, compared to pasireotide-bound SSTR3, the extracellular TMD of TM4 in L-796778-bound SSTR3 adopts a dramatic outward movement at 4.6 Å when measured at the Cα atoms of the conserved P1774.59 (SI Appendix, Fig. S6C). In the top OBP of SSTR3, L-796778 has no interactions with ECL3-TM7 side of the receptor, where only the hydrocarbyl moiety extends upwardly and makes hydrophobic contacts with R2035.35, I2075.39, and V2816.59 (Fig. 5F). Furthermore, the H19245.51 forms a water-mediated polar interaction with L-796778 but not pasireotide (Fig. 5F). Mutation of H19245.51A reduced the Gi activation of SSTR1 induced by L-796778, rather than pasireotide (Fig. 5G and SI Appendix, Table S3). Despite the above differences, several similarities were observed in the recognition of L-796778 and pasireotide with SSTR3. First, the lysine group of L-796778, mimicking the role of lysine residue in Trp-Lys motif of pasireotide, forms salt bridge interaction with D1233.32 and hydrogen bond interaction with Q1273.36 (Fig. 5H). Next, the nitroanilino moiety of L-796778 engages in a similar EBP around TM2 and TM3 of SSTR3, as occupied by the (Bzl)Tyr moiety of pasireotide, with the nitro group forming a hydrogen bond with Q1032.63 (Fig. 5I). Consistently, mutations of F1283.37A, V1784.60I, D1233.32A, Q1273.36A, and Q1032.63A compromised the Gi activation of SSTR3 by L-796778, supporting the binding mode of L-796778 (SI Appendix, Fig. S6D and Tables S3 and S4).

Superposition of the structures of L-796778-bound SSTR3 with the other SSTR subtypes enables the discovery of several key determinants in the selective recognition of L-796778 to SSTR3. The amino acid residues Q3.36 and F2.53 are identical in SSTR2 and SSTR3, while they differ from those in SSTR1, SSTR4, and SSTR5 (Fig. 5 J and K). Mutation of M1413.36Q or L1072.53F in SSTR1 both increased the potency of L-796778 to SSTR1 by over 10-fold (Fig. 5L and SI Appendix, Fig. S6E and Table S4). In addition, mutation of L2205.35R rescued the potency of L-796778 to SSTR1 (SI Appendix, Fig. S6F and Table S4). However, individually replacing the residues at positions 2.53 and 3.36 in the bottom OBP (SI Appendix, Fig. S6E and Table S4), and 5.35 and 6.55 in the top OBP of SSTR2, SSTR4, and SSTR5 with the corresponding residues from SSTR3, displayed little to no effect on the potency of L-796778 to the receptors (SI Appendix, Fig. S6F and Table S4). These results reveal that the selectivity of L-796778 may be caused by a comprehensive effect of multiple nonconserved residues around the L-796778 binding pocket. Moreover, the selective determinants of L-796778 may differ across SSTR subtypes, similar to L-797591.

Mechanisms of SSTR1 and SSTR3 Activation.

To date, no inactive structures of SSTR1 and SSTR3 were reported. However, the inactive structure of their homology receptor CYN-154806-bound SSTR2 was available and was used to investigate the potential activation mechanism of SSTR1 and SSTR3 (21). Structural alignment elucidated that the conformational changes upon SSTR1 and SSTR3 activation are quite similar to those observed in SSTR2 activation (Fig. 6 A and B). Nevertheless, it should be noted that the detailed mechanisms underlying these conformational changes in SSTR1 and SSTR3 are not identical.

Fig. 6.

Fig. 6.

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Activation of SSTR1 and SSTR3. (A and B) Structural comparison of the topology of (A) pasireotide- or L-797591-bound SSTR1 structures and (B) pasireotide- or L-796778- bound SSTR3 structures with inactive SSTR2 (PDB:7XNA) in the extracellular region and the cytoplasmic region. The inactive SSTR2 structure is colored in light gray. (C and D) Conformational changes of the toggle switch residue and polar network beneath the OBP in (C) pasireotide-bound SSTR1 and (D) L-797591-bound SSTR1 that are involved in receptor activation. (E and F) Conformational changes of the toggle switch residue and polar network beneath the OBP in (E) pasireotide-bound SSTR3 and (F) L-796778-bound SSTR3 that are involved in receptor activation.

In SSTR1, the agonist pasireotide induces an outward kick and an anticlockwise rotation of TM3 through the deeply inserted Trp residue and the (Bzl)Tyr group. As a result, the side chain of M3.36 in TM3 makes direct contact with the toggle switch residue W2846.48, resulting in a downward shift of W2846.48 toward F2806.44 of the P5.50I3.40F6.44 core triad (Fig. 6C). The twist of TM3 and down-swing of W6.48 further induce a conformational rearrangement of the P5.50I3.40F6.44 motif (SI Appendix, Fig. S7A), the reconstruction of a polar network containing residues D1042.50, N1403.35, S1443.39, W2846.48, N3157.45, and S3167.46 (Fig. 6C), the rearrangement of the NP7.50xxY motif (SI Appendix, Fig. S7B), and the breaking of the ionic lock in DR3.50Y motif (SI Appendix, Fig. S7C). L-797591-bound SSTR1 share conserved activation mechanism with pasireotide-bound SSTR1(Fig. 6D and SI Appendix, Fig. S7 A–C). In L-797591-bound SSTR1, the naphthalene moiety of L-797591 induces the rotation of TM3 (Fig. 6D). In SSTR3, the binding of pasireotide induced similar conformational changes of TM3 as in SSTR1, which then causes the inward movement of the extracellular half of TM2 and the rearrangement of “F2.53-Y7.43” motif (Fig. 6E). As a result, the middle portion of TM7 adopts an inner translation toward TM6, leading to the downward shift of toggle switch residue W2706.48 partially attributed to the steric clash between P3027.42 and W2706.48, as well as the direct contact with Q1273.36 (Fig. 6E). Consistent with the structural observation, mutation of P3027.42G attenuated the Gi activation of SSTR3 by pasireotide (SI Appendix, Table S4). Compared to the pasireotide-bound SSTR3, the TM3 in L-796778-bound SSTR3 adopts a more outward movement owing to the long-stretched ethylbenzene moiety. Consequently, the Q1273.36 in TM3 adopts a conformation far away from the toggle-switch residue W2706.48 (Fig. 6F). In both active SSTR1 and SSTR3 structures, the side chain of R3.50 in DR3.50Y motif forms a hydrogen bond interaction with Y5.58 (SI Appendix, Fig. S7 C and F), facilitating the stabilization of the active states of the receptors. Of note, this polar interaction is a common feature in most active GPCR structures (31).

Discussion

SSTRs represent an essential class of GPCR drug targets for the treatment of neuroendocrine tumors owing to their important role in broadly regulating the secretion of hormones and suppressing cell proliferation. While the five SSTR subtypes (SSTR1-5) share high sequence homology and downstream Gi signaling pathways, they exhibit different expression patterns and physiological functions in normal tissues and tumor types. Understanding ligand binding and regulation profiles, especially the selective recognition of specific SSTR subtypes, is critical for SSTR-targeted drug discovery with reduced side effects. SSTR1 and SSTR3 belong to two distinct SSTR subfamilies, SRIF1 and SRIF2, respectively. In this study, we report four structures of SSTR1 and SSTR3 bound to either the panagonist drug pasireotide or a subtype-selective agonist. Structural comparisons reveal that, in contrast to the flexible binding modes of SST14, pasireotide exhibits a conserved binding pose in SSTR1 and SSTR3, possibly due to additional contacts with the EBP near TM2, TM3, and ECL1, occupied by pasireotide but not SST14. Mutagenesis analysis of key EBP residues suggests the binding of pasireotide with the EBP is essential for its high potency at SSTR1 and SSTR3. It is worth noting that such EBP is absent in the binding pocket of octreotide and lanreotide in SSTR2 and SSTR4, presenting the possibility to rationally design more potent peptide drugs selectively targeting the highly conserved EBP across SSTRs.

It was suggested that the nonconserved residues in the top OBP, such as residues in positions 2.63, 5.35, 6.54, 6.55, and 7.35 of SSTR2 and SSTR4, played an important role in determining the peptide ligands selectivity of SSTRs (21, 22, 24, 25, 28). Our structures demonstrate extensive contacts between the pasireotide and ECLs of SSTRs, and SSTRs have low conservation in this region. In particular, the substitution of extracellular vestibules around ECL3 in SSTR4 with corresponding residues in SSTR2 increased the Gi activity induced by pasireotide. We profile that the subtype selectivity of pasireotide may partially attributed to the nonconvergency of the extracellular regions of SSTRs near ECL3. In addition to the residue divergency in the top OBP, several nonconserved residues in the bottom OBP also contributed to the small molecule agonists selectivity of SSTRs, such as Q/M3.36 and F/Y/L2.53. We found that both residues in position 2.53 and 3.36 are critical for the selectivity of L-797591 and L-796778 to SSTRs, while the other potential determinants, such as residue in position 5.35 and 45.52, differ from different ligands and SSTR subtypes. This observation revealed a complex landscape of selective determinants for SSTR ligands, which heavily depends on both the ligands and receptor subtypes.

Structural comparison of active SSTR1/3 structures with the inactive SSTR2 revealed a cascade of consensus activation mechanism among SSTRs, although differences arise in triggering the conformational change of toggle switch residue W6.48. Notably, activation of SSTRs relies considerably on reconstructing a polar network beneath the OBP, in a way similar to opioid receptors (32, 33) and various other GPCRs such as the adenosine A2A receptor (A2AR) (34) and complement C5a receptor 1 (C5aR1) (35).

Overall, our work expands molecular understanding of ligand recognition and activation mechanisms for SSTRs, offering opportunities to develop more effective and safer therapies for cancers and other diseases involving these important receptor targets.

Materials and Methods

Cell Lines.

Trichoplusia ni (Hi5) cells were grown in ESF 921 medium at 27 °C and 120 rpm. HEK293 cells were grown in a humidified 37 °C incubator with 5% CO2 using media supplemented with 100 I.U./mL penicillin and 100 mg/mL streptomycin (Invitrogen). The human cell lines HEK293 were maintained in DMEM (VWR) containing 10% fetal bovine serum (FBS, VWR).

Constructs.

Both full-length SSTR1 and SSTR3 were subcloned into pFastBac vector (ThermoFisher) with the prolactin precursor sequence as a signaling peptide followed by a FLAG tag, along with a fragment of β2AR N-terminal tail region (BN) to facilitate the protein expression. One mutation, V6.40Y was introduced into the SSTR1 and SSTR3 to assist in the formation of pasireotide-SSTR1-Gi, L-797591-SSTR1-Gi, pasireotide-SSTR3-Gi complexes. NanoBiT tethering strategy was introduced to facilitate the assembly of the L-796778-SSTR3-Gi complex, the large part of NanoBiT (LgBiT) was fused to the C-terminal of SSTR3, while the small part of NanoBiT (HiBiT) was fused to the C terminus of Gβ1. The engineered Gαi was designed based on the mini-Gs/i43 scaffold, in which the N-terminal 1–18 amino acids and the α-helical domain were replaced by the corresponding sequences from Gi. This provided possible binding sites for Nb35 and scFv16 (27). The engineered Gαi, Gβ1 with C-terminal HiBiT and Gγ2 were cloned into pFastBac1 vector, respectively.

GloSensor cAMP Assay.

WT or with various mutations of SSTR1 and SSTR3 were cloned into pcDNA6.0 vector (Invitrogen) with a FLAG tag at its N-terminal. HEK293 cells were seeded in 6-well plates at 300,000 cells per well. After 12 h, the cells were transfected with SSTR1 or SSTR3 or mutant plasmids and the cAMP biosensor GloSensor-22F (Promega) at a 1.5:1 ratio. At least 24 h later, the cells were detached and transferred to 96-well plates at 1,500 cells per well. After 16 h, the medium was removed and the cells were starved in 50 μL of Hank’s balanced salt solution for 30 min. The cells were then incubated with 50 μL of CO2-independent media containing 2% GloSensor cAMP Reagent (Promega) at 37 °C for 1 h. After that, the cells were incubated with varying concentrations of test ligands at room temperature for 10 min. Forskolin (Sigma-Aldrich) was then added to each well in the final concentration of 3 μM and incubates for 10 min at room temperature prior to measurement for luminescence. All luminescence signals are tested by EnVision multiplate reader according to the manufacturer’s instructions. All data were analyzed using the sigmoidal log(agonist) versus dose–response in GraphPad Prism.

Surface Expression Analysis.

Cell preparation and transfection were performed as described for the GloSensor cAMP assay. After 24 h of transfection, cells were washed once with PBS and digested with 0.2% (w/v) EDTA in PBS. Cells were blocked with 5% (w/v) BSA at room temperature for 30 min, followed by incubation with primary anti-FLAG antibody (diluted with PBS containing 5% BSA at a ratio of 1:150, ABclonal) at room temperature for 1 h. After that, cells were washed three times with PBS before incubating with antimouse Alexa-488-conjugated secondary antibody (diluted with PBS containing 5% BSA at a ratio of 1:1,000, ABclonal) at 4 °C in the dark for 1 h. Finally, after washing three times with PBS, the cells were resuspended in PBS. The surface expression of SSTR1 and SSTR3 was monitored by detecting the fluorescent intensity of FITC at excitation 488 nm and emission 519 nm with Guava easyCyte 8HT (Merck Millipore). The FACS data were analyzed by Guava software 2.1.

Data Processing.

Surface expression levels of WT SSTRs and SSTR mutants were determined by chemiluminescence assays. The values were normalized to the WT receptor and reported as a percentage of WT using Prism 8 (GraphPad) software. All functional study data were analyzed using Prism 8 (GraphPad) and presented as means ± SE of measurement (SEM) from at least three independent experiments.

Nonlinear curve fit was performed using a three-parameter logistic equation [log (agonist vs. response)] for cAMP assay. The significance was determined with two-side, one-way ANOVA followed by Fisher’s LSD multiple comparisons test compared with WT.

Supplementary Material

Appendix 01 (PDF)

pnas.2400298121.sapp.pdf (16.8MB, pdf)

Acknowledgments

The cryo-EM data were collected at the Advanced Center for Electron Microscopy, Shanghai Institute of Materia Medica (SIMM). We sincerely thank all the staffs at the EM center for their kind assistance in cryo-EM grid checking and data collection. This work was partially supported by grants from the Key Program of National Natural Science Foundation of China (82030085 to S.S.); the National Key Research and Development Program of China (2023YFC2506403 and 2017YFC0908500 to S.S.); the CAS Strategic Priority Research Program (XDB37030103 to H.E.X.); Shanghai Municipal Science and Technology Major Project (2019SHZDZX02 to H.E.X.); Shanghai Municipal Science and Technology Major Project (H.E.X.); the National Natural Science Foundation of China (32130022,82121005); the Lingang Laboratory, Grant No.LG-GG-202204-01 (H.E.X.); the National Key R&D Program of China (2022YFC2703105 to H.E.X.); the Youth Talent Promotion Project of Chinese Association for Science and Technology (to Y.Z.); the Youth Innovation Promotion Association of Chinese Academy of Sciences (2023298 to Y.Z.); Natural Science Foundation of Shanghai, China (23ZR1475300 to Y.Z.); the Sailing Program of Shanghai Venus Project (23YF1456700 to Y.Z.); the Special Research Assistant Project of Chinese Academy of Sciences (to Y.W.); and Innovative Research Team of Highlevel Local Universities in Shanghai (SHSMU-ZLCX20212300 and SSMU-ZLCX20180500); Shanghai Clinical Research Center for Oral Diseases (19MC1910600); Shanghai Municipal Key Clinical Specialty (shslczdzk01601); Shanghai’s Top Priority Research Center (2022ZZ01017).

Author contributions

Y.Z., H.E.X., and S.S. designed research; Yujue Wang, Y.X., Yue Wang, J.Z., X.H., W.F., K.W., and W.H. performed research; X.H., K.W., W.H., and X.C. contributed new reagents/analytic tools; Yujue Wang, Y.X., Yue Wang, and Y.Z. analyzed data; and Yujue Wang, Y.X., J.Z., L.C., G.Y., Y.Z., H.E.X., and S.S. wrote the paper.

Competing interests

H.E.X is a founder of Cascade Pharmaceutics. All the other authors declare no competing interests.

Footnotes

This article is a PNAS Direct Submission.

Contributor Information

H. Eric Xu, Email: eric.xu@simm.ac.cn.

Youwen Zhuang, Email: youwen_zhuang@sjtu.edu.cn.

Shuyang Sun, Email: sunshuyang@sjtu.edu.cn.

Data, Materials, and Software Availability

The atomic coordinates and cryo-EM maps included in this study have been deposited in the Protein Data Bank (PDB) and Electron Microscopy Data Bank (EMDB), respectively. The cryo-EM density maps of pasireotide-SSTR1-Gi, L-797591-SSTR1-Gi, pasireotide-SSTR3Gi, and L-796778-SSTR3-Gi complexes have been deposited in the EMDB under the Accession Nos. EMD-38386 (36), EMD-38385 (37), EMD-38388 (38), and EMD-38387 (39), respectively. Atomic coordinates for the atomic models of pasireotide-SSTR1-Gi, L-797591-SSTR1-Gi, pasireotide-SSTR3-Gi, and L-796778-SSTR3-Gi complex structures have been deposited in the PDB under the Accession Nos. 8XIP (40), 8XIO (41), 8XIR (42), and 8XIQ (43), respectively. These codes or accession numbers are also listed in the key resources table. The software used in this study is available from the key resources table. All other data are included in the manuscript and/or SI Appendix.

Supporting Information

References

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Appendix 01 (PDF)

pnas.2400298121.sapp.pdf (16.8MB, pdf)

Data Availability Statement

The atomic coordinates and cryo-EM maps included in this study have been deposited in the Protein Data Bank (PDB) and Electron Microscopy Data Bank (EMDB), respectively. The cryo-EM density maps of pasireotide-SSTR1-Gi, L-797591-SSTR1-Gi, pasireotide-SSTR3Gi, and L-796778-SSTR3-Gi complexes have been deposited in the EMDB under the Accession Nos. EMD-38386 (36), EMD-38385 (37), EMD-38388 (38), and EMD-38387 (39), respectively. Atomic coordinates for the atomic models of pasireotide-SSTR1-Gi, L-797591-SSTR1-Gi, pasireotide-SSTR3-Gi, and L-796778-SSTR3-Gi complex structures have been deposited in the PDB under the Accession Nos. 8XIP (40), 8XIO (41), 8XIR (42), and 8XIQ (43), respectively. These codes or accession numbers are also listed in the key resources table. The software used in this study is available from the key resources table. All other data are included in the manuscript and/or SI Appendix.

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

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