scholarly journals Structural insights into ligand recognition and activation of the melanocortin-4 receptor

2021 ◽  
Author(s):  
Yan Zhang ◽  
Huibing Zhang ◽  
Li-Nan Chen ◽  
Dehua Yang ◽  
Chunyou Mao ◽  
...  
Keyword(s):  
Small Molecule ◽  
Energy Homeostasis ◽  
Structural Information ◽  
Sequence Similarity ◽  
Receptor Activation ◽  
Activation Mechanism ◽  
Receptor Subtype ◽  
High Sequence Similarity ◽  
Subtype Selectivity ◽  
Melanocortin 4 Receptor

Melanocortin-4 receptor (MC4R) plays a central role in the regulation of energy homeostasis. Its high sequence similarity to other MC receptor family members, low agonist selectivity and the lack of structural information concerning receptor activation have hampered the development of MC4R-seletive therapeutics to treat obesity. Here, we report four high-resolution structures of full-length MC4R in complex with the heterotrimeric Gs protein stimulated by the endogenous peptide α-MSH, FDA-approved drugs afamelanotide (Scenesse™) and bremelanotide (Vyleesi™), and selective small-molecule ligand THIQ, respectively. Together with pharmacological studies, our results reveal the conserved binding mode of peptidic agonists, the distinctive molecular details of small-molecule agonist recognition underlying receptor subtype selectivity, and distinct activation mechanism for MC4R, thereby offering new insights into G protein coupling. Our work may facilitate the discovery of selective therapeutic agents targeting MC4R.

scholarly journals YopT domain of the PfhB2 toxin from Pasteurella multocida: protein expression, characterization, crystallization and crystallographic analysis

2018 ◽  
Vol 74 (3) ◽  
pp. 128-134
Author(s):  
Sanjeev Kumar ◽  
Victoria Hedrick ◽  
Seema Mattoo
Keyword(s):  
Pasteurella Multocida ◽  
Opportunistic Infections ◽  
Structural Information ◽  
Sequence Similarity ◽  
Catalytic Triad ◽  
Crystallographic Analysis ◽  
High Sequence Similarity ◽  
Data Set ◽  
Cell Parameters ◽  
Tract Infections

Pasteurella multocida causes respiratory-tract infections in a broad range of animals, as well as opportunistic infections in humans. P. multocida secretes a multidomain toxin called PfhB2, which contains a YopT-like cysteine protease domain at its C-terminus. The YopT domain of PfhB2 contains a well conserved Cys–His–Asp catalytic triad that defines YopT family members, and shares high sequence similarity with the prototype YopT from Yersinia sp. To date, only one crystal structure of a YopT family member has been reported; however, additional structural information is needed to help characterize the varied substrate specificity and enzymatic action of this large protease family. Here, a catalytically inactive C3733S mutant of PfhB2 YopT that provides enhanced protein stability was used with the aim of gaining structural insight into the diversity within the YopT protein family. To this end, the C3733S mutant of PfhB2 YopT has been successfully cloned, overexpressed, purified and crystallized. Diffraction data sets were collected from native crystals to 3.5 Å resolution and a single-wavelength anomalous data set was collected from an iodide-derivative crystal to 3.2 Å resolution. Data pertaining to crystals belonging to space group P31, with unit-cell parameters a = 136.9, b = 136.9, c = 74.7 Å for the native crystals and a = 139.2, b = 139.2, c = 74.7 Å for the iodide-derivative crystals, are discussed.

scholarly journals 20 YEARS OF LEPTIN: Insights into signaling assemblies of the leptin receptor

2014 ◽  
Vol 223 (1) ◽  
pp. T9-T23 ◽  
Author(s):  
Frank Peelman ◽  
Lennart Zabeau ◽  
Kedar Moharana ◽  
Savvas N Savvides ◽  
Jan Tavernier
Keyword(s):  
Electron Microscopy ◽  
Energy Homeostasis ◽  
Leptin Receptor ◽  
Structural Information ◽  
Cell Types ◽  
Activation Mechanism ◽  
Peripheral Cell ◽  
X Ray ◽  
X Ray Crystallography ◽  
X Ray Scattering

Leptin plays a central role in the control of body weight and energy homeostasis, but is a pleiotropic cytokine with activities on many peripheral cell types. In this review, we discuss the interaction of leptin with its receptor, and focus on the structural and mechanistic aspects of the extracellular aspects of leptin receptor (LR) activation. We provide an extensive overview of all structural information that has been obtained for leptin and its receptor via X-ray crystallography, electron microscopy, small-angle X-ray scattering, homology modeling, and mutagenesis studies. The available knowledge is integrated into putative models toward a recapitulation of the LR activation mechanism.

scholarly journals Structures of active melanocortin-4 receptor–Gs-protein complexes with NDP-α-MSH and setmelanotide

Cell Research ◽  
2021 ◽  
Author(s):  
Nicolas A. Heyder ◽  
Gunnar Kleinau ◽  
David Speck ◽  
Andrea Schmidt ◽  
Sarah Paisdzior ◽  
...  
Keyword(s):  
G Protein ◽  
Energy Homeostasis ◽  
Protein Complexes ◽  
Transmembrane Helix ◽  
Receptor Activation ◽  
Binding Modes ◽  
Intracellular Loop ◽  
Melanocortin 4 Receptor ◽  
Biased Signaling ◽  
Gs Protein

AbstractThe melanocortin-4 receptor (MC4R), a hypothalamic master regulator of energy homeostasis and appetite, is a class A G-protein-coupled receptor and a prime target for the pharmacological treatment of obesity. Here, we present cryo-electron microscopy structures of MC4R–Gs-protein complexes with two drugs recently approved by the FDA, the peptide agonists NDP-α-MSH and setmelanotide, with 2.9 Å and 2.6 Å resolution. Together with signaling data from structure-derived MC4R mutants, the complex structures reveal the agonist-induced origin of transmembrane helix (TM) 6-regulated receptor activation. The ligand-binding modes of NDP-α-MSH, a high-affinity linear variant of the endogenous agonist α-MSH, and setmelanotide, a cyclic anti-obesity drug with biased signaling toward Gq/11, underline the key role of TM3 in ligand-specific interactions and of calcium ion as a ligand-adaptable cofactor. The agonist-specific TM3 interplay subsequently impacts receptor–Gs-protein interfaces at intracellular loop 2, which also regulates the G-protein coupling profile of this promiscuous receptor. Finally, our structures reveal mechanistic details of MC4R activation/inhibition, and provide important insights into the regulation of the receptor signaling profile which will facilitate the development of tailored anti-obesity drugs.

Assessment of a small molecule melanocortin-4 receptor-specific agonist on energy homeostasis

2004 ◽  
Vol 1000 (1-2) ◽  
pp. 64-71 ◽  
Author(s):  
David Cepoi ◽  
Teresa Phillips ◽  
Mary Cismowski ◽  
Val S Goodfellow ◽  
Nick Ling ◽  
...  
Keyword(s):  
Small Molecule ◽  
Energy Homeostasis ◽  
Melanocortin 4 Receptor

scholarly journals Structures of active melanocortin-4 receptor—Gs-protein complexes with NDP-α-MSH and setmelanotide

2021 ◽  
Author(s):  
Nicolas A. Heyder ◽  
Gunnar Kleinau ◽  
David Speck ◽  
Andrea Schmidt ◽  
Sarah Paisdzior ◽  
...  
Keyword(s):  
Energy Homeostasis ◽  
Protein Complexes ◽  
Transmembrane Helix ◽  
Receptor Activation ◽  
Binding Modes ◽  
Intracellular Loop ◽  
Melanocortin 4 Receptor ◽  
Biased Signaling ◽  
Gs Protein ◽  
Approved Drugs

The melanocortin-4 receptor (MC4R), a hypothalamic master regulator of energy homeostasis and appetite, is a G-protein coupled receptor and a prime target for the treatment of obesity. Here, we present cryo-electron microscopy structures of MC4R—Gs-protein complexes with two recently FDA-approved drugs, the peptide agonists NDP-α-MSH and setmelanotide, with 2.9 Å and 2.6 Å resolution. Together with signaling data, the complex structures reveal the agonist-induced origin of transmembrane helix (TM) 6 regulated receptor activation. In both structures, different ligand binding modes of NDP-α-MSH, a high-affinity variant of the endogenous agonist, and setmelanotide, an anti-obesity drug with biased signaling, underline the key role of TM3 for ligand-specific interactions and of calcium ion as a ligand-adaptable cofactor. The agonist-TM3 interplay subsequently impacts the receptor—Gs-protein interfaces, mainly at intracellular loop 2. These structures reveal mechanistic details of MC4R activation or inhibition and provide important insights into receptor selectivity that will facilitate the development of tailored anti-obesity drugs.

scholarly journals Structure guided protein engineering increases enzymatic activities of the SGNH family esterases

2020 ◽  
Author(s):  
Zhengyang Li ◽  
Long Li ◽  
Yingyi Huo ◽  
Zijun Chen ◽  
Yu Zhao ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Metal Ion ◽  
Catalytic Mechanism ◽  
Structural Information ◽  
Sequence Similarity ◽  
Catalytic Triad ◽  
Enzymatic Activities ◽  
Solvent Tolerance ◽  
Globular Domain ◽  
High Sequence Similarity

Abstract Background Esterases and lipases hydrolyze short-chain esters and long-chain triglycerides, respectively, and therefore play key roles in the synthesis and decomposition of ester bonds in the pharmaceutical and food industries. Many SGNH family esterases share high similarity in sequences, however, they have distinct enzymatic activities toward the same substrates. Due to lack of structural information, the detailed catalytic mechanisms of these esterases remain barely investigated.Results In this study, we identified two SGNH family esterases, CrmE10 and AlinE4, from marine bacteria with significantly different preferences for pH, temperature, metal ion and organic solvent tolerance despite of high sequence similarity. The crystal structures of these two esterases, including wild type and mutants, were determined to high resolutions ranging from 1.18 Å to 2.24 Å. Both CrmE10 and AlinE4 were composed of five β-strands and nine α-helices, which formed one compact N-terminal α/β globular domain and one extended C-terminal domain. The aspartic residues (D178 in CrmE10/ D162 in AlinE4) stabilized the conformations of the catalytic triad (Ser-Asp-His) in both esterases, and the metal ion Cd 2+ reduced enzymatic activity by blocking proton transfer and substrate binding. CrmE10 and AlinE4 showed distinctly different electrostatic surface potentials, despite of the similar atomic architectures and a similar swap catalytic mechanism. When five negative charged residues (Asp or Glu) were mutated to residue Lys, CrmE10 obtained elevated alkaline-adaptability and significantly increased the enzymatic activity from 0% to 20% at pH 10.5. In addition, CrmE10 mutants exhibited dramatically change for enzymatic properties when compared with wide-type enzyme.Conclusions These findings offer a perspective for understanding the catalytic mechanism of different esterases and might facilitate the industrial biocatalytic applications.

scholarly journals Structure guided protein engineering increases enzymatic activities of the SGNH family esterases

2020 ◽  
Author(s):  
Zhengyang Li ◽  
Long Li ◽  
Yingyi Huo ◽  
Zijun Chen ◽  
Yu Zhao ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Metal Ion ◽  
Catalytic Mechanism ◽  
Structural Information ◽  
Sequence Similarity ◽  
Catalytic Triad ◽  
Enzymatic Activities ◽  
Solvent Tolerance ◽  
Globular Domain ◽  
High Sequence Similarity

Abstract Background Esterases and lipases hydrolyze short-chain esters and long-chain triglycerides, respectively, and therefore play essential roles in the synthesis and decomposition of ester bonds in the pharmaceutical and food industries. Many SGNH family esterases share high similarity in sequences. However, they have distinct enzymatic activities toward the same substrates. Due to a lack of structural information, the detailed catalytic mechanisms of these esterases remain barely investigated. Results In this study, we identified two SGNH family esterases, CrmE10 and AlinE4, from marine bacteria with significantly different preferences for pH, temperature, metal ion, and organic solvent tolerance despite high sequence similarity. The crystal structures of these two esterases, including wild type and mutants, were determined to high resolutions ranging from 1.18 Å to 2.24 Å. Both CrmE10 and AlinE4 were composed of five β-strands and nine α-helices, which formed one compact N-terminal α/β globular domain and one extended C-terminal domain. The aspartic residues (D178 in CrmE10/ D162 in AlinE4) stabilized the conformations of the catalytic triad (Ser-Asp-His) in both esterases, and the metal ion Cd2+ might reduce enzymatic activity by blocking proton transfer or substrate binding. CrmE10 and AlinE4 showed distinctly different electrostatic surface potentials, despite the similar atomic architectures and a similar swap catalytic mechanism. When five negative charged residues (Asp or Glu) were mutated to residue Lys, CrmE10 obtained elevated alkaline-adaptability and significantly increased the enzymatic activity from 0% to 20% at pH 10.5. Also, CrmE10 mutants exhibited dramatic change for enzymatic properties when compared with the wide-type enzyme. Conclusions These findings offer a perspective for understanding the catalytic mechanism of different esterases and might facilitate the industrial biocatalytic applications.

scholarly journals Current Mechanistic and Pharmacodynamic Understanding of Melanocortin-4 Receptor Activation

Molecules ◽  
2019 ◽  
Vol 24 (10) ◽  
pp. 1892 ◽  
Author(s):  
Shubh Sharma ◽  
Alastair S. Garfield ◽  
Bhavik Shah ◽  
Patrick Kleyn ◽  
Ilia Ichetovkin ◽  
...  
Keyword(s):  
Small Molecule ◽  
Receptor Activation ◽  
Mechanistic Studies ◽  
Therapeutic Targeting ◽  
Activation Kinetics ◽  
Melanocortin 4 Receptor ◽  
Pathway Activation ◽  
Signaling Events ◽  
Biased Signaling ◽  
Targeting Strategy

In this work we summarize our understanding of melanocortin 4 receptor (MC4R) pathway activation, aiming to define a safe and effective therapeutic targeting strategy for the MC4R. Delineation of cellular MC4R pathways has provided evidence for distinct MC4R signaling events characterized by unique receptor activation kinetics. While these studies remain narrow in scope, and have largely been explored with peptidic agonists, the results provide a possible correlation between distinct ligand groups and differential MC4R activation kinetics. In addition, when a set of small-molecule and peptide MC4R agonists are compared, evidence of biased signaling has been reported. The results of such mechanistic studies are discussed.

scholarly journals Structure guided protein engineering increases enzymatic activities of the SGNH family esterases

2020 ◽  
Author(s):  
Zhengyang Li ◽  
Long Li ◽  
Yingyi Huo ◽  
Zijun Chen ◽  
Yu Zhao ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Metal Ion ◽  
Catalytic Mechanism ◽  
Structural Information ◽  
Sequence Similarity ◽  
Catalytic Triad ◽  
Enzymatic Activities ◽  
Solvent Tolerance ◽  
Globular Domain ◽  
High Sequence Similarity

Abstract Background: Esterases and lipases hydrolyze short-chain esters and long-chain triglycerides, respectively, and therefore play essential roles in the synthesis and decomposition of ester bonds in the pharmaceutical and food industries. Many SGNH family esterases share high similarity in sequences. However, they have distinct enzymatic activities toward the same substrates. Due to a lack of structural information, the detailed catalytic mechanisms of these esterases remain barely investigated. Results: In this study, we identified two SGNH family esterases, CrmE10 and AlinE4, from marine bacteria with significantly different preferences for pH, temperature, metal ion, and organic solvent tolerance despite high sequence similarity. The crystal structures of these two esterases, including wild type and mutants, were determined to high resolutions ranging from 1.18 Å to 2.24 Å. Both CrmE10 and AlinE4 were composed of five β-strands and nine α-helices, which formed one compact N-terminal α/β globular domain and one extended C-terminal domain. The aspartic residues (D178 in CrmE10/ D162 in AlinE4) destabilized the conformations of the catalytic triad (Ser-Asp-His) in both esterases, and the metal ion Cd2+ might reduce enzymatic activity by blocking proton transfer or substrate binding. CrmE10 and AlinE4 showed distinctly different electrostatic surface potentials, despite the similar atomic architectures and a similar swap catalytic mechanism. When five negative charged residues (Asp or Glu) were mutated to residue Lys, CrmE10 obtained elevated alkaline-adaptability and significantly increased the enzymatic activity from 0% to 20% at pH 10.5. Also, CrmE10 mutants exhibited dramatic change for enzymatic properties when compared with the wide-type enzyme. Conclusions: These findings offer a perspective for understanding the catalytic mechanism of different esterases and might facilitate the industrial biocatalytic applications.

scholarly journals Structure guided protein engineering increases enzymatic activities of the SGNH family esterases

2020 ◽  
Author(s):  
Zhengyang Li ◽  
Long Li ◽  
Yingyi Huo ◽  
Zijun Chen ◽  
Yu Zhao ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Metal Ion ◽  
Catalytic Mechanism ◽  
Structural Information ◽  
Sequence Similarity ◽  
Catalytic Triad ◽  
Enzymatic Activities ◽  
Solvent Tolerance ◽  
Globular Domain ◽  
High Sequence Similarity

Abstract Background Esterases and lipases hydrolyze short-chain esters and long-chain triglycerides, respectively, and therefore play essential roles in the synthesis and decomposition of ester bonds in the pharmaceutical and food industries. Many SGNH family esterases share high similarity in sequences. However, they have distinct enzymatic activities toward the same substrates. Due to a lack of structural information, the detailed catalytic mechanisms of these esterases remain barely investigated. Results In this study, we identified two SGNH family esterases, CrmE10 and AlinE4, from marine bacteria with significantly different preferences for pH, temperature, metal ion, and organic solvent tolerance despite high sequence similarity. The crystal structures of these two esterases, including wild type and mutants, were determined to high resolutions ranging from 1.18 Å to 2.24 Å. Both CrmE10 and AlinE4 were composed of five β-strands and nine α-helices, which formed one compact N-terminal α/β globular domain and one extended C-terminal domain. The aspartic residues (D178 in CrmE10/ D162 in AlinE4) destabilized the conformations of the catalytic triad (Ser-Asp-His) in both esterases, and the metal ion Cd2+ might reduce enzymatic activity by blocking proton transfer or substrate binding. CrmE10 and AlinE4 showed distinctly different electrostatic surface potentials, despite the similar atomic architectures and a similar swap catalytic mechanism. When five negative charged residues (Asp or Glu) were mutated to residue Lys, CrmE10 obtained elevated alkaline-adaptability and significantly increased the enzymatic activity from 0% to 20% at pH 10.5. Also, CrmE10 mutants exhibited dramatic changes for enzymatic properties when compared with the wide-type enzyme. Conclusions These findings offer a perspective for understanding the catalytic mechanism of different esterases and might facilitate the industrial biocatalytic applications.

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