scholarly journals Identifying key residues and key interactions for the binding of LEAP2 to receptor GHSR1a

2020 ◽  
Vol 477 (17) ◽  
pp. 3199-3217
Author(s):  
Hao-Zheng Li ◽  
Li-Li Shou ◽  
Xiao-Xia Shao ◽  
Ya-Li Liu ◽  
Zeng-Guang Xu ◽  
...  
Keyword(s):  
Structural Model ◽  
Binding Pocket ◽  
Site Directed Mutagenesis ◽  
Alanine Scanning Mutagenesis ◽  
Competitive Antagonist ◽  
Future Studies ◽  
Cognate Receptor ◽  
Key Residues ◽  
G Protein Coupled ◽  
Positively Charged

Liver-expressed antimicrobial peptide 2 (LEAP2) was recently identified as a competitive antagonist for the G protein-coupled receptor GHSR1a, the cognate receptor for the gastric peptide ghrelin. LEAP2 plays important functions in energy metabolism by tuning the ghrelin–GHSR1a system. However, the molecular mechanism by which LEAP2 binds to GHSR1a is largely unknown. In the present study, we first conducted alanine-scanning mutagenesis on the N-terminal fragment of human LEAP2 and demonstrated that the positively charged Arg6 and the aromatic Phe4 are essential for LEAP2 binding to GHSR1a. To identify the receptor residues interacting with the essential Arg6 and Phe4 of LEAP2, we conducted extensive site-directed mutagenesis on GHSR1a. After all conserved negatively charged residues in the extracellular regions of human GHSR1a were mutated, only mutation of Asp99 caused much more detriments to GHSR1a binding to LEAP2 than binding to ghrelin, suggesting that the absolutely conserved Asp99 of GHSR1a probably interacts with the essential Arg6 of LEAP2. After five conserved Phe residues in the predicted ligand-binding pocket of human GHSR1a were mutated, three of them were identified as important for GHSR1a binding to LEAP2. According to a structural model of GHSR1a, we deduced that the adjacent Phe279 and Phe312 might interact with the essential Phe4 of LEAP2, while Phe119 might interact with the aromatic Trp5 of LEAP2. The present study provided new insights into the interaction of LEAP2 with its receptor, and would facilitate the design of novel ligands for GHSR1a in future studies.

scholarly journals Molecular sampling of the allosteric binding pocket of the TSH receptor provides discriminative pharmacophores for antagonist and agonists

2013 ◽  
Vol 41 (1) ◽  
pp. 213-217 ◽  
Author(s):  
Inna Hoyer ◽  
Ann-Karin Haas ◽  
Annika Kreuchwig ◽  
Ralf Schülein ◽  
Gerd Krause
Keyword(s):  
Amino Acids ◽  
Structural Model ◽  
Transmembrane Domain ◽  
Binding Pocket ◽  
Trigger Points ◽  
Antagonistic Effect ◽  
Site Directed Mutagenesis ◽  
Detailed Knowledge ◽  
Contact Points ◽  
Allosteric Binding Pocket

The TSHR (thyrotropin receptor) is activated endogenously by the large hormone thyrotropin and activated pathologically by auto-antibodies. Both activate and bind at the extracellular domain. Recently, SMLs (small-molecule ligands) have been identified, which bind in an allosteric binding pocket within the transmembrane domain. Modelling driven site-directed mutagenesis of amino acids lining this pocket led to the delineation of activation and inactivation sensitive residues. Modified residues showing CAMs (constitutively activating mutations) indicate signalling-sensitive positions and mark potential trigger points for agonists. Silencing mutations lead to an impairment of basal activity and mark contact points for antagonists. Mapping these residues on to a structural model of TSHR indicates locations where an SML may switch the receptor to an inactive or active conformation. In the present article, we report the effects of SMLs on these signalling-sensitive amino acids at the TSHR. Surprisingly, the antagonistic effect of SML compound 52 was reversed to an agonistic effect, when tested at the CAM Y667A. Switching agonism to antagonism and the reverse by changing either SMLs or residues covering the binding pocket provides detailed knowledge about discriminative pharmacophores. It prepares the basis for rational optimization of new high-affinity antagonists to interfere with the pathogenic activation of the TSHR.

scholarly journals Structural model of a putrescine-cadaverine permease from Trypanosoma cruzi predicts residues vital for transport and ligand binding

2013 ◽  
Vol 452 (3) ◽  
pp. 423-432 ◽  
Author(s):  
Radika Soysa ◽  
Hanka Venselaar ◽  
Jacqueline Poston ◽  
Buddy Ullman ◽  
Marie-Pierre Hasne
Keyword(s):  
Trypanosoma Cruzi ◽  
Structural Model ◽  
Three Dimensional ◽  
Salt Bridge ◽  
Homology Model ◽  
Binding Pocket ◽  
Dimensional Structure ◽  
Helical Structures ◽  
Structural Determinants ◽  
Key Residues

The TcPOT1.1 gene from Trypanosoma cruzi encodes a high affinity putrescine-cadaverine transporter belonging to the APC (amino acid/polyamine/organocation) transporter superfamily. No experimental three-dimensional structure exists for any eukaryotic member of the APC family, and thus the structural determinants critical for function of these permeases are unknown. To elucidate the key residues involved in putrescine translocation and recognition by this APC family member, a homology model of TcPOT1.1 was constructed on the basis of the atomic co-ordinates of the Escherichia coli AdiC arginine/agmatine antiporter crystal structure. The TcPOT1.1 homology model consisted of 12 transmembrane helices with the first ten helices organized in two V-shaped antiparallel domains with discontinuities in the helical structures of transmembrane spans 1 and 6. The model suggests that Trp241 and a Glu247–Arg403 salt bridge participate in a gating system and that Asn245, Tyr148 and Tyr400 contribute to the putrescine-binding pocket. To test the validity of the model, 26 site-directed mutants were created and tested for their ability to transport putrescine and to localize to the parasite cell surface. These results support the robustness of the TcPOT1.1 homology model and reveal the importance of specific aromatic residues in the TcPOT1.1 putrescine-binding pocket.

Probing the structural basis of oxygen binding in a cofactor-independent dioxygenase

2017 ◽  
Vol 73 (7) ◽  
pp. 573-580 ◽  
Author(s):  
Kunhua Li ◽  
Elisha N. Fielding ◽  
Heather L. Condurso ◽  
Steven D. Bruner
Keyword(s):  
Catalytic Mechanism ◽  
Binding Pocket ◽  
Binding Mode ◽  
Site Directed Mutagenesis ◽  
Structural Basis ◽  
General Mechanism ◽  
Oxygen Binding ◽  
Migration Pathway ◽  
And Migration ◽  
Key Residues

The enzyme DpgC is included in the small family of cofactor-independent dioxygenases. The chemistry of DpgC is uncommon as the protein binds and utilizes dioxygen without the aid of a metal or organic cofactor. Previous structural and biochemical studies identified the substrate-binding mode and the components of the active site that are important in the catalytic mechanism. In addition, the results delineated a putative binding pocket and migration pathway for the co-substrate dioxygen. Here, structural biology is utilized, along with site-directed mutagenesis, to probe the assigned dioxygen-binding pocket. The key residues implicated in dioxygen trafficking were studied to probe the process of binding, activation and chemistry. The results support the proposed chemistry and provide insight into the general mechanism of dioxygen binding and activation.

scholarly journals Functions of olfactory receptors are decoded from their sequence

2020 ◽  
Author(s):  
Xiaojing Cong ◽  
Wenwen Ren ◽  
Jody Pacalon ◽  
Claire A. de March ◽  
Lun Xu ◽  
...  
Keyword(s):  
Machine Learning ◽  
Olfactory Receptors ◽  
Binding Pocket ◽  
Site Directed Mutagenesis ◽  
Functional Assay ◽  
Machine Learning Approach ◽  
Activation Mechanisms ◽  
Odorant Binding ◽  
G Protein Coupled

AbstractG protein-coupled receptors (GPCRs) conserve common structural folds and activation mechanisms, yet their ligand spectra and functions are highly diversified. This work investigated how the functional variations in olfactory GPCRs (ORs)-the largest GPCR family-are encoded in the primary sequence. With the aid of site-directed mutagenesis and molecular simulations, we built machine learning models to predict OR-ligand pairs as well as basal activity of ORs. In vitro functional assay confirmed 20 new OR-odorant pairs, including 9 orphan ORs. Residues around the odorant-binding pocket dictate the odorant selectivity/specificity of the ORs. Residues that encode the varied basal activities of the ORs were found to mostly surround the conserved motifs as well as the binding pocket. The machine learning approach, which is readily applicable to mammalian OR families, will accelerate OR-odorant mapping and the decoding of combinatorial OR codes for odors.

scholarly journals Crystal structure of CotA laccase complexed with 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonate) at a novel binding site

2016 ◽  
Vol 72 (4) ◽  
pp. 328-335 ◽  
Author(s):  
Zhongchuan Liu ◽  
Tian Xie ◽  
Qiuping Zhong ◽  
Ganggang Wang
Keyword(s):  
Crystal Structure ◽  
Binding Site ◽  
Binding Pocket ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
The Novel ◽  
Wild Type ◽  
Outer Coat ◽  
Cota Laccase ◽  
Key Residues

The CotA laccase fromBacillus subtilisis an abundant component of the spore outer coat and has been characterized as a typical laccase. The crystal structure of CotA complexed with 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS) in a hole motif has been solved. The novel binding site was about 26 Å away from the T1 binding pocket. Comparison with known structures of other laccases revealed that the hole is a specific feature of CotA. The key residues Arg476 and Ser360 were directly bound to ABTS. Site-directed mutagenesis studies revealed that the residues Arg146, Arg429 and Arg476, which are located at the bottom of the novel binding site, are essential for the oxidation of ABTS and syringaldazine. Specially, a Thr480Phe variant was identified to be almost 3.5 times more specific for ABTS than for syringaldazine compared with the wild type. These results suggest this novel binding site for ABTS could be a potential target for protein engineering of CotA laccases.

scholarly journals Key Residues Defining the μ-Opioid Receptor Binding Pocket: A Site-Directed Mutagenesis Study

2002 ◽  
Vol 68 (1) ◽  
pp. 344-353 ◽  
Author(s):  
Alfred Mansour ◽  
Larry P. Taylor ◽  
Jeffrey L. Fine ◽  
Robert C. Thompson ◽  
Mary T. Hoversten ◽  
...  
Keyword(s):  
Opioid Receptor ◽  
Receptor Binding ◽  
Binding Pocket ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Μ Opioid Receptor ◽  
A Site ◽  
Key Residues ◽  
Mutagenesis Study ◽  
Opioid Receptor Binding

scholarly journals Improving the Activity and Stability of GL-7-ACA Acylase CA130 by Site-Directed Mutagenesis

2005 ◽  
Vol 71 (9) ◽  
pp. 5290-5296 ◽  
Author(s):  
Wei Zhang ◽  
Yuan Liu ◽  
Huabao Zheng ◽  
Sheng Yang ◽  
Weihong Jiang
Keyword(s):  
Specific Activity ◽  
Catalytic Efficiency ◽  
Binding Pocket ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Catalytic Function ◽  
Alkaline Shift ◽  
Key Residues

ABSTRACT In the present study, glutaryl-7-amino cephalosporanic acid acylase from Pseudomonas sp. strain 130 (CA130) was mutated to improve its enzymatic activity and stability. Based on the crystal structure of CA130, two series of amino acid residues, one from those directly involved in catalytic function and another from those putatively involved in surface charge, were selected as targets for site-directed mutagenesis. In the first series of experiments, several key residues in the substrate-binding pocket were substituted, and the genes were expressed in Escherichia coli for activity screening. Two of the mutants constructed, Y151αF and Q50βN, showed two- to threefold-increased catalytic efficiency (k cat/Km ) compared to wild-type CA130. Their Km values were decreased by ca. 50%, and the k cat values increased to 14.4 and 16.9 s−1, respectively. The ability of these mutants to hydrolyze adipoyl 6-amino penicillinic acid was also improved. In the second series of mutagenesis, several mutants with enhanced stabilities were identified. Among them, R121βA and K198βA had a 30 to 58% longer half-life than wild-type CA130, and K198βA and D286βA showed an alkaline shift of optimal pH by about 1.0 to 2.0 pH units. To construct an engineered enzyme with the properties of both increased activity and stability, the double mutant Q50βN/K198βA was expressed. This enzyme was purified and immobilized for catalytic analysis. The immobilized mutant enzyme showed a 34.2% increase in specific activity compared to the immobilized wild-type CA130.

scholarly journals Extracellular loop 2 of G protein-coupled olfactory receptors is critical for odorant recognition

2021 ◽  
Author(s):  
Yiqun Yu ◽  
Jody Pacalon ◽  
Zhenjie Ma ◽  
Lun Xu ◽  
Christine Belloir ◽  
...  
Keyword(s):  
G Protein ◽  
Drug Targets ◽  
Olfactory Receptors ◽  
3D Models ◽  
Binding Pocket ◽  
Site Directed Mutagenesis ◽  
Extracellular Loop ◽  
Odorant Binding ◽  
Loop 2 ◽  
G Protein Coupled

G protein-coupled olfactory receptors (ORs) enable us to detect innumerous odorants. They are also ectopically expressed, emerging as attractive drug targets. ORs can be promiscuous or highly specific, which is part of Nature′s strategy for odor discrimination. This work demonstrates that the extracellular loop 2 (ECL2) plays critical roles in OR promiscuity and specificity. Using site-directed mutagenesis and molecular modeling, we constructed 3D OR models in which ECL2 forms a lid of the orthosteric pocket. ECL2 controls the shape and the volume of the odorant-binding pocket, maintains the pocket hydrophobicity and acts as a gatekeeper of odorant binding. The interplay between the specific orthosteric pocket and the variable, less specific ECL2 controls OR specificity and promiscuity. The 3D models enabled virtual screening of new OR agonists and antagonists, exhibiting 78% hit rate in cell assays. This approach can be generalized to structure-based ligand screening for other GPCRs that lack high-resolution 3D structures.

Characterization of Recombinant Human Coagulation Factor XFriuli

1996 ◽  
Vol 75 (02) ◽  
pp. 313-317 ◽  
Author(s):  
D J Kim ◽  
A Girolami ◽  
H L James
Keyword(s):  
Catalytic Domain ◽  
Coagulation Factor ◽  
Molecular Size ◽  
Binding Pocket ◽  
Site Directed Mutagenesis ◽  
Bleeding Tendency ◽  
Factor X ◽  
Amino Terminal ◽  
Normal Plasma ◽  
Plasma Factor

SummaryNaturally occurring plasma factor XFriuli (pFXFr) is marginally activated by both the extrinsic and intrinsic coagulation pathways and has impaired catalytic potential. These studies were initiated to obtain confirmation that this molecule is multi-functionally defective due to the substitution of Ser for Pro at position 343 in the catalytic domain. By the Nelson-Long site-directed mutagenesis procedure a construct of cDNA in pRc/CMV was derived for recombinant factor XFriuli (rFXFr) produced in human embryonic (293) kidney cells. The rFXFr was purified and shown to have a molecular size identical to that of normal plasma factor X (pFX) by gel electrophoretic, and amino-terminal sequencing revealed normal processing cleavages. Using recombinant normal plasma factor X (rFXN) as a reference, the post-translational y-carboxy-glutamic acid (Gla) and (β-hydroxy aspartic acid (β-OH-Asp) content of rFXFr was over 85% and close to 100%, respectively, of expected levels. The specific activities of rFXFr in activation and catalytic assays were the same as those of pFXFr. Molecular modeling suggested the involvement of a new H-bond between the side-chains of Ser-343 and Thr-318 as they occur in anti-parallel (3-pleated sheets near the substrate-binding pocket of pFXFr. These results support the conclusion that the observed mutation in pFXFr is responsible for its dysfunctional activation and catalytic potentials, and that it accounts for the moderate bleeding tendency in the homozygous individuals who possess this variant procoagulant.

scholarly journals Cryo-EM structure of the human histamine H1 receptor/Gq complex

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Ruixue Xia ◽  
Na Wang ◽  
Zhenmei Xu ◽  
Yang Lu ◽  
Jing Song ◽  
...  
Keyword(s):  
Transmembrane Domain ◽  
Binding Pocket ◽  
Receptor Activation ◽  
Intracellular Loop ◽  
Cryo Electron Microscopy ◽  
Allergy Treatment ◽  
Domain 3 ◽  
Extracellular Side ◽  
Key Residues ◽  
Loop 2

AbstractHistamine receptors play important roles in various pathophysiological conditions and are effective targets for anti-allergy treatment, however the mechanism of receptor activation remain elusive. Here, we present the cryo-electron microscopy (cryo-EM) structure of the human H1R in complex with a Gq protein in an active conformation via a NanoBiT tethering strategy. The structure reveals that histamine activates receptor via interacting with the key residues of both transmembrane domain 3 (TM3) and TM6 to squash the binding pocket on the extracellular side and to open the cavity on the intracellular side for Gq engagement in a model of “squash to activate and expand to deactivate”. The structure also reveals features for Gq coupling, including the interaction between intracellular loop 2 (ICL2) and the αN-β junction of Gq/11 protein. The detailed analysis of our structure will provide a framework for understanding G-protein coupling selectivity and clues for designing novel antihistamines.

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