Ecosystem
Beta Site
Session V
Structural mechanisms of AGPCR signaling and function
Structural Determinants Of GAIN Domain Autoproteolysis And Cleavage Resistance Of Adhesion G Protein-Coupled Receptors
Fabian Pohl
Abstract
"The GPCR autoproteolysis-inducing (GAIN) domain is a hallmark feature of adhe-sion G-protein coupled receptors (ADGRs), as this extracellular domain contains an integral agonistic sequence (Stachel) for activation via binding to the 7-transmembrane (7TM) helical domain of the receptor. Many ADGRs are autoproteo-lytically cleaved at the GPCR proteolysis site (GPS), an HXS/T motif within the GAIN domain. However, several ADGRs can be activated without GPS cleavage. We de-termined the crystal structure of the human ADGRB2/BAI2 hormone receptor (HormR) and GAIN domains and found that this ADGR is resistant to autoproteolysis despite the presence of a canonical HLS sequence at the GPS. By structural com-parison and with the help of molecular dynamics (MD) simulations we identified several unique structural features that are important for autoproteolytic cleavage, beyond the canonical HXS/T motif. Disruption of these features reduced autoproteo-lytic activity in ADGRL1/LPHN1 and restored cleavage competence of AD-GRB3/BAI3. Furthermore, conservation analysis indicates that wild type ADGRB2 and ADGRB3 are GPS cleavage-incompetent receptors."
Authors & Affiliations
"Fabian Pohl1, Florian Seufert2, Yin Kwan Chung3, Daniela Volke1, Ralf Hoffmann1, Torsten Schöneberg4, Tobias Langenhan3, Peter W. Hildebrand2, Norbert Sträter1
1 Institute of Bioanalytical Chemistry, Leipzig University, Deutscher Platz 5, 04103 Leipzig, Germany
2 Institute of Medical Physics and Biophysics, Leipzig University, Härtelstr. 16-18, D-04107 Leipzig
3 Rudolf-Schönheimer-Institute of Biochemistry, Division of General Biochemistry, Leipzig University, Johannisallee 30, D-04103 Leipzig
4 Rudolf-Schönheimer-Institute of Biochemistry, Division of Molecular Biochemis-try, Leipzig University, Johannisallee 30, D-04103 Leipzig"
About Fabian Pohl
"Mar 2023 – Today Postdoc, University Leipzig, Group of Prof. Langenhan
Apr 2016 – Nov 2022 PhD candidate, University Leipzig, Group of Prof. Sträter
Oct 2011 – Mar 2016 Master of Science in chemistry, University Leipzig
Oct 2008 – Sep 2011 Bachelor of Science in chemistry, University Leipzig"
Fabian Pohl on the web
Structural studies of the CELSR1 extracellular region reveal a compact multidomain module of fourteen domains which regulates signaling
Sumit Bandekar
Abstract
"Cadherin EGF Laminin G seven-pass G-type receptors (CELSRs) are conserved adhesion G protein-coupled receptors; they are essential for embryogenesis and neural development. CELSRs have large and enigmatic extracellular regions (ECRs) with nine cadherin repeats and a variety of adhesion domains which couple cell adhesion to signaling. CELSRs regulate planar cell polarity, including the closure of the neural tube. Despite numerous cell and animal studies, molecular details on CELSR proteins are sparsely available, precluding an integrative understanding of CELSR biology. Here, we report the 3.8 Å cryo-EM reconstruction of the CELSR1 ECR which enables unambiguous assignment of the 14 domains within the structure. These domains form a compact module mediated by robust and evolutionarily conserved interdomain interactions. This compact module provides a plethora of potential ligand binding sites for the various adhesion domains within the structure and hints at a model where the compact module could be pulled apart by robust mechanical force. We present biophysical evidence that the CELSR1 ECR forms an extended dimer in the presence of Ca2+, which we propose represents the cadherin repeats dimerizing in a configuration similar to protocadherins. We employ cellular assays with full-length CELSR1 and truncation constructs to assess the adhesive and signaling functions of this protein. We assign the N-terminal CADH1-8 module as necessary for cell adhesion and we show the C-terminal CAHD9-GAIN module regulates signaling. Our work provides molecular context to the literature on CELSR function and lays the groundwork for further elucidation of structure/function relationships."
Authors & Affiliations
"Garbett, Krassimira, Kordon, Szymon P., Shearer, Tanner, Sando, Richard C.*, and Araç, Demet*
Department of Biochemistry and Molecular Biology, The University of Chicago Neuroscience Institute, Institute for Biophysical Dynamics, and the Center for Mechanical Excitability, The University of Chicago, Chicago, IL, 60637, USA. Department of Pharmacology, Vanderbilt Brain Institute, Vanderbilt University, Nashville, TN 37240, USA."
About Sumit Bandekar
"I am an NIH F32 postdoctoral fellow in the Araç Laboratory at the University of Chicago. I study adhesion GPCRs using structural biology perspective and I am interested in how the large multidomain extracellular region regulates receptor function. In my free time, I enjoy biking around Chicago and trying new breweries and restaurants."
Sumit Bandekar on the web
Unveiling the GPS Cleavage Mechanism in ADGRL1 with QM/MM
Florian Seufert
Abstract
"Adhesion G-protein coupled receptors (aGPCR) are a family of 32 mammalian proteins with a defining conserved GPCR autoproteolysis inducing (GAIN) domain that catalyzes receptor self-cleavage at a GPCR proteolysis site (GPS). The autoproteolytic mechanism has been previously proposed, but remains to be validated.
A previous computational study has uncovered variable flexible protein regions, whose dynamics mediate solvent-accessibility of the catalytically active GPS triad HL|S/T, however classical molecular dynamics approaches fall short of explaining the chemical reaction.
Using a multiscale QM/MM approach - combining computational quantum mechanics with classical molecular dynamics - to study the GAIN domain cleavage mechanism of ADGRL1 reveals the sequence of events at the electronic level, suggesting relative energies for the individual states during the reaction, and provides insight into the structural determinants for a successful GPS cleavage exceeding the catalytically active GPS triad.
By directly scanning and comparing energetic sequences of reaction steps, the most likely pathway and the individual contribution of surrounding protein residues can be elucidated. A stable π-edge contact with a conserved phenylalanine and a protonated glutamate side-chain catalyze the reactant conformation. MD simulations with the parameterized ester intermediate reveal a protonation-dependent dynamic desolvation of the GPS for subsequent ester hydrolysis by restricting water conformations.
Mutational experiments on residues of interest showed that restoring the Phe-His interaction in the uncleaving ADGRB3 GAIN domain partially re-instates cleavage, while its deletion reduces cleavage in the ADGRL1 GAIN domain. We present a two-step GPS cleavage model and respective determinants of the reaction."
Authors & Affiliations
"Chung, Yin Kwan2, Pohl, Fabian2, Batebi, Hossein1 Sträter, Norbert3 , Langenhan, Tobias2 & Hildebrand, Peter Werner1
1 Institute of Medical Physics and Biophysics, Medical Faculty, Leipzig University, Germany
2 Rudolf Schönheimer Institute of Biochemistry, Division of General Biochemistry, Medical Faculty, Leipzig University, Germany
3 Institute of Bioanalytical Chemistry, Leipzig University, Germany"
About Florian Seufert
"Florian Seufert has studied Biochemistry in Leipzig, before joining the Hildebrand Lab in Leipzig for his PhD."