NDLI: Mapping leptin-interacting sites in recombinant leptin-binding domain (LBD) subcloned from chicken leptin receptor

Content Provider	PubMed Central
Author	Niv-spector, L. Raver, N. Friedman-einat, M. Grosclaude, J. Gussakovsky, E. E. Livnah, O. Gertler, A.
Copyright Year	2005
Abstract	The binding domain of the chicken leptin receptor [chLBD (chicken leptin-binding domain)], subcloned from the full-size chicken leptin receptor and prepared in an Escherichia coli system, was subjected to site-directed mutagenesis to identify the amino acids involved in leptin binding. A total of 22 electrophoretically pure, >90% monomer-containing mutants were expressed, refolded and purified. The effects of the mutations were tested by the ability to form complexes with ovine leptin, and the kinetic parameters of interaction were determined by surface plasmon resonance. Six mutants were used to determine whether mutations of several amino acids that differ between chLBD and mammalian LBDs will affect affinity: none showed any such effect, except the mutant A105D (Ala105→Asp), which exhibited some decrease in affinity. Surface plasmon resonance analysis identified six mutants in which binding activity was totally abolished (F73A, Y14A/F73A, V76A/F77A, L78A/L79A, V76A/F77A/L78A/L79A and A105D/D106V) and six mutants (Y14A, R41A, R41A/S42A/K43A, V103A, V135A/F136A and F136A) in which affinity for the hormone was reduced, mainly by increased dissociation rates. Gel-filtration experiments indicated the formation of a 1:1 ovine or human leptin–chLBD complex with a molecular mass of approx. 41 kDa. Gel-filtration experiments yielded 1:1 complexes with those mutants in which affinity had decreased, but not with the six mutants, which had totally lost their binding capacity. Modelling the leptin–chLBD complex indicated that the binding domain of the latter is located mainly in the L3 loop, which contributes nine amino acid residues interacting with leptin. Contact-surface analysis identified the residues having the highest contribution to the recognition site to be Phe73, Phe77 and Leu79.
Related Links	http://dx.doi.org/10.1042/bj20050233
Ending Page	484
Page Count	10
Starting Page	475
File Format	PDF
ISSN	02646021
e-ISSN	14708728
Journal	Biochemical Journal
Issue Number	Pt 2
Volume Number	390
Language	English
Publisher	Portland Press Ltd.
Publisher Date	2005-09-01
Access Restriction	Open
Rights Holder	Portland Press Ltd.
Subject Keyword	Cell Biology Biochemistry Molecular Biology Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Cell Biology Molecular Biology Biochemistry

Sl.	Authority	Responsibilities	Communication Details
1	Ministry of Education (GoI), Department of Higher Education	Sanctioning Authority	https://www.education.gov.in/ict-initiatives
2	Indian Institute of Technology Kharagpur	Host Institute of the Project: The host institute of the project is responsible for providing infrastructure support and hosting the project	https://www.iitkgp.ac.in
3	National Digital Library of India Office, Indian Institute of Technology Kharagpur	The administrative and infrastructural headquarters of the project	Dr. B. Sutradhar bsutra@ndl.gov.in
4	Project PI / Joint PI	Principal Investigator and Joint Principal Investigators of the project	Dr. B. Sutradhar bsutra@ndl.gov.in Prof. Saswat Chakrabarti will be added soon
5	Website/Portal (Helpdesk)	Queries regarding NDLI and its services	support@ndl.gov.in
6	Contents and Copyright Issues	Queries related to content curation and copyright issues	content@ndl.gov.in
7	National Digital Library of India Club (NDLI Club)	Queries related to NDLI Club formation, support, user awareness program, seminar/symposium, collaboration, social media, promotion, and outreach	clubsupport@ndl.gov.in
8	Digital Preservation Centre (DPC)	Assistance with digitizing and archiving copyright-free printed books	dpc@ndl.gov.in
9	IDR Setup or Support	Queries related to establishment and support of Institutional Digital Repository (IDR) and IDR workshops	idr@ndl.gov.in

Subcloning, Expression, Purification, and Characterization of Recombinant Human Leptin-binding Domain

Ligand Displaces Heat Shock Protein 90 from Overlapping Binding Sites within the Aryl Hydrocarbon Receptor Ligand-binding Domain*

Analysis of two CBP (cAMP-response-element-binding protein-binding protein) interacting sites in GRIP1 (glucocorticoid-receptor-interacting protein), and their importance for the function of GRIP1

Mapping Targetable Sites on Human Telomerase RNA Pseudoknot/Template Domain Using 2′-OMe RNA-interacting Polynucleotide (RIPtide) Microarrays*

Mapping of the Leptin Binding Sites and Design of a Leptin Antagonist

Identification and Dissection of Ca2+-binding Sites in the Extracellular Domain of Ca2+-sensing Receptor*

Mapping the Interacting Regions between Troponins T and C BINDING OF TnT AND TnI PEPTIDES TO TnC AND NMR MAPPING OF THE TnT-BINDING SITE ON TnC

Mapping of the interface between leptin and the leptin receptor CRH2 domain

Functional Probing of the Human Glucocorticoid Receptor Steroid-interacting Surface by Site-directed Mutagenesis Gln-642 PLAYS AN IMPORTANT ROLE IN STEROID RECOGNITION AND BINDING

Mapping leptin-interacting sites in recombinant leptin-binding domain (LBD) subcloned from chicken leptin receptor

Similar Documents

Subcloning, Expression, Purification, and Characterization of Recombinant Human Leptin-binding Domain

Ligand Displaces Heat Shock Protein 90 from Overlapping Binding Sites within the Aryl Hydrocarbon Receptor Ligand-binding Domain*

Analysis of two CBP (cAMP-response-element-binding protein-binding protein) interacting sites in GRIP1 (glucocorticoid-receptor-interacting protein), and their importance for the function of GRIP1

Mapping Targetable Sites on Human Telomerase RNA Pseudoknot/Template Domain Using 2′-OMe RNA-interacting Polynucleotide (RIPtide) Microarrays*

Mapping of the Leptin Binding Sites and Design of a Leptin Antagonist

Identification and Dissection of Ca2+-binding Sites in the Extracellular Domain of Ca2+-sensing Receptor*

Mapping the Interacting Regions between Troponins T and C BINDING OF TnT AND TnI PEPTIDES TO TnC AND NMR MAPPING OF THE TnT-BINDING SITE ON TnC

Mapping of the interface between leptin and the leptin receptor CRH2 domain

Functional Probing of the Human Glucocorticoid Receptor Steroid-interacting Surface by Site-directed Mutagenesis Gln-642 PLAYS AN IMPORTANT ROLE IN STEROID RECOGNITION AND BINDING

Mapping leptin-interacting sites in recombinant leptin-binding domain (LBD) subcloned from chicken leptin receptor