Mutational Analysis of the Putative Anti-Müllerian Hormone (AMH) Binding Interface on its Type II Receptor, AMHR2

Abstract Anti-Müllerian hormone (AMH) or Müllerian inhibiting substance is a unique member of the TGF-β family responsible for development and differentiation of the reproductive system. AMH signals through its own dedicated type II receptor, anti-Müllerian hormone receptor type II (AMHR2), providing an exclusive ligand-receptor pair within the broader TGF-β family. In this study, we used previous structural information to derive a model of AMH bound to AMHR2 to guide mutagenesis studies to identify receptor residues important for AMH signaling. Nonconserved mutations were introduced in AMHR2 and characterized in an AMH-responsive cell-based luciferase assay and native PAGE. Collectively, our results identified several residues important for AMH signaling within the putative ligand binding interface of AMHR2. Our results show that AMH engages AMHR2 at a similar interface to how activin and BMP class ligands bind the type II receptor, ACVR2B; however, there are significant molecular differences at the ligand interface of these 2 receptors, where ACVR2B is mostly hydrophobic and AMHR2 is predominately charged. Overall, this study shows that although the location of ligand binding on the receptor is similar to ACVR2A, ACVR2B, and BMPR2; AMHR2 uses unique ligand-receptor interactions to impart specificity for AMH.

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Structural characterization of an activin class ternary receptor complex reveals a third paradigm for receptor specificity

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1906253116 ◽

2019 ◽

Vol 116 (31) ◽

pp. 15505-15513 ◽

Cited By ~ 11

Author(s):

Erich J. Goebel ◽

Richard A. Corpina ◽

Cynthia S. Hinck ◽

Magdalena Czepnik ◽

Roselyne Castonguay ◽

...

Keyword(s):

Mutational Analysis ◽

Structural Information ◽

Type I ◽

Receptor Complex ◽

Type Ii ◽

Dimer Interface ◽

Ligand Type ◽

Receptor Interactions ◽

Receptor Assembly

TGFβ family ligands, which include the TGFβs, BMPs, and activins, signal by forming a ternary complex with type I and type II receptors. For TGFβs and BMPs, structures of ternary complexes have revealed differences in receptor assembly. However, structural information for how activins assemble a ternary receptor complex is lacking. We report the structure of an activin class member, GDF11, in complex with the type II receptor ActRIIB and the type I receptor Alk5. The structure reveals that receptor positioning is similar to the BMP class, with no interreceptor contacts; however, the type I receptor interactions are shifted toward the ligand fingertips and away from the dimer interface. Mutational analysis shows that ligand type I specificity is derived from differences in the fingertips of the ligands that interact with an extended loop specific to Alk4 and Alk5. The study also reveals differences for how TGFβ and GDF11 bind to the same type I receptor, Alk5. For GDF11, additional contacts at the fingertip region substitute for the interreceptor interactions that are seen for TGFβ, indicating that Alk5 binding to GDF11 is more dependent on direct contacts. In support, we show that a single residue of Alk5 (Phe84), when mutated, abolishes GDF11 signaling, but has little impact on TGFβ signaling. The structure of GDF11/ActRIIB/Alk5 shows that, across the TGFβ family, different mechanisms regulate type I receptor binding and specificity, providing a molecular explanation for how the activin class accommodates low-affinity type I interactions without the requirement of cooperative receptor interactions.

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Mechanisms of Transforming Growth Factor-β Receptor Endocytosis and Intracellular Sorting Differ between Fibroblasts and Epithelial Cells

Molecular Biology of the Cell ◽

10.1091/mbc.12.3.675 ◽

2001 ◽

Vol 12 (3) ◽

pp. 675-684 ◽

Cited By ~ 38

Author(s):

Jules J.E. Doré ◽

Diying Yao ◽

Maryanne Edens ◽

Nandor Garamszegi ◽

Elizabeth L. Sholl ◽

...

Keyword(s):

Growth Factor ◽

Epithelial Cells ◽

Ligand Binding ◽

Transforming Growth Factor ◽

Point Mutations ◽

Type I ◽

Receptor Complex ◽

Type Ii ◽

Down Regulation ◽

Β Receptor

Transforming growth factor-βs (TGF-β) are multifunctional proteins capable of either stimulating or inhibiting mitosis, depending on the cell type. These diverse cellular responses are caused by stimulating a single receptor complex composed of type I and type II receptors. Using a chimeric receptor model where the granulocyte/monocyte colony-stimulating factor receptor ligand binding domains are fused to the transmembrane and cytoplasmic signaling domains of the TGF-β type I and II receptors, we wished to describe the role(s) of specific amino acid residues in regulating ligand-mediated endocytosis and signaling in fibroblasts and epithelial cells. Specific point mutations were introduced at Y182, T200, and Y249 of the type I receptor and K277 and P525 of the type II receptor. Mutation of either Y182 or Y249, residues within two putative consensus tyrosine-based internalization motifs, had no effect on endocytosis or signaling. This is in contrast to mutation of T200 to valine, which resulted in ablation of signaling in both cell types, while only abolishing receptor down-regulation in fibroblasts. Moreover, in the absence of ligand, both fibroblasts and epithelial cells constitutively internalize and recycle the TGF-β receptor complex back to the plasma membrane. The data indicate fundamental differences between mesenchymal and epithelial cells in endocytic sorting and suggest that ligand binding diverts heteromeric receptors from the default recycling pool to a pathway mediating receptor down-regulation and signaling.

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Structural models of SARS-CoV-2 Omicron variant in complex with ACE2 receptor or antibodies suggest altered binding interfaces

10.1101/2021.12.12.472313 ◽

2021 ◽

Author(s):

Joseph H. Lubin ◽

Christopher Markosian ◽

D. Balamurugan ◽

Renata Pasqualini ◽

Wadih Arap ◽

...

Keyword(s):

Structural Information ◽

Structural Models ◽

Interaction Network ◽

Neutralizing Antibody ◽

Data Bank ◽

Binding Interface ◽

Global Pandemic ◽

Residue Interaction ◽

Protein Interfaces ◽

Class 1

There is enormous ongoing interest in characterizing the binding properties of the SARS-CoV-2 Omicron Variant of Concern (VOC) (B.1.1.529), which continues to spread towards potential dominance worldwide. To aid these studies, based on the wealth of available structural information about several SARS-CoV-2 variants in the Protein Data Bank (PDB) and a modeling pipeline we have previously developed for tracking the ongoing global evolution of SARS-CoV-2 proteins, we provide a set of computed structural models (henceforth models) of the Omicron VOC receptor-binding domain (omRBD) bound to its corresponding receptor Angiotensin-Converting Enzyme (ACE2) and a variety of therapeutic entities, including neutralizing and therapeutic antibodies targeting previously-detected viral strains. We generated bound omRBD models using both experimentally-determined structures in the PDB as well as machine learning-based structure predictions as starting points. Examination of ACE2-bound omRBD models reveals an interdigitated multi-residue interaction network formed by omRBD-specific substituted residues (R493, S496, Y501, R498) and ACE2 residues at the interface, which was not present in the original Wuhan-Hu-1 RBD-ACE2 complex. Emergence of this interaction network suggests optimization of a key region of the binding interface, and positive cooperativity among various sites of residue substitutions in omRBD mediating ACE2 binding. Examination of neutralizing antibody complexes for Barnes Class 1 and Class 2 antibodies modeled with omRBD highlights an overall loss of interfacial interactions (with gain of new interactions in rare cases) mediated by substituted residues. Many of these substitutions have previously been found to independently dampen or even ablate antibody binding, and perhaps mediate antibody-mediated neutralization escape (e.g., K417N). We observe little compensation of corresponding interaction loss at interfaces when potential escape substitutions occur in combination. A few selected antibodies (e.g., Barnes Class 3 S309), however, feature largely unaltered or modestly affected protein-protein interfaces. While we stress that only qualitative insights can be obtained directly from our models at this time, we anticipate that they can provide starting points for more detailed and quantitative computational characterization, and, if needed, redesign of monoclonal antibodies for targeting the Omicron VOC Spike protein. In the broader context, the computational pipeline we developed provides a framework for rapidly and efficiently generating retrospective and prospective models for other novel variants of SARS-CoV-2 bound to entities of virological and therapeutic interest, in the setting of a global pandemic.

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gp330 on type II pneumocytes mediates endocytosis leading to degradation of pro-urokinase, plasminogen activator inhibitor-1 and urokinase-plasminogen activator inhibitor-1 complex

Journal of Cell Science ◽

10.1242/jcs.108.6.2361 ◽

1995 ◽

Vol 108 (6) ◽

pp. 2361-2368 ◽

Cited By ~ 1

Author(s):

S. Stefansson ◽

M.Z. Kounnas ◽

J. Henkin ◽

R.K. Mallampalli ◽

D.A. Chappell ◽

...

Keyword(s):

Ligand Binding ◽

Plasminogen Activator ◽

Solid Phase ◽

Polyclonal Antibodies ◽

Plasminogen Activator Inhibitor ◽

Type Ii ◽

Plasminogen Activator Inhibitor 1 ◽

Type Ii Pneumocytes ◽

Activator Inhibitor ◽

Pai 1

Glycoprotein 330 (gp330) is a member of a family of receptors related to the low density lipoprotein receptor (LDLR). Although several ligands have been shown to bind gp330 in solid-phase assays, the ability of gp330 to mediate ligand endocytosis has not been demonstrated. To develop a cellular model for gp330 function we screened a variety of cultured cell lines and identified several that expressed this protein, including immortalized rat type II pneumocytes and a human and two rodent tumor cell lines. Using type II pneumocytes, endocytosis of a previously described gp330 ligand, urokinase (uPA) complexed with plasminogen activator inhibitor-1 (uPA:PAI-1) and two new ligands, PAI-1 and pro-uPA, was demonstrated. RAP, the 39 kDa receptor-associated protein known to antagonize ligand binding to gp330 in solid-phase binding assays, completely inhibited both internalization and degradation of the radiolabeled ligands by type II pneumocytes. This suggested that the clearance of these ligands was dependent on either gp330 or the LDLR-related protein (LRP), which shares several ligand-binding characteristics with gp330. By using polyclonal antibodies to gp330, the cellular internalization and degradation of the ligands were inhibited by 30–50%; remaining ligand internalization and degradation activity could be partially inhibited by polyclonal antibodies against LRP. These findings indicate that gp330, like other LDLR family members, mediates endocytosis of its ligands. In addition, gp330 acts in concert with LRP in type II pneumocytes to mediate clearance of a variety of proteins involved in plasminogen activation, including uPA:PAI-1 complexes PAI-1 and pro-uPA.(ABSTRACT TRUNCATED AT 250 WORDS)

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SAT-718 Inhibition of Androgen Receptor Activity by DDE Is Affected by Mutations in the BF3 Site

Journal of the Endocrine Society ◽

10.1210/jendso/bvaa046.1648 ◽

2020 ◽

Vol 4 (Supplement_1) ◽

Author(s):

Andrea Lozano ◽

Evangelia Kotsikorou ◽

Frank B Dean

Keyword(s):

Androgen Receptor ◽

Ligand Binding ◽

Binding Domain ◽

Ligand Binding Domain ◽

Hek293 Cells ◽

Luciferase Assay ◽

Breakdown Product ◽

Reporter System ◽

Surface Binding ◽

Endocrine Disrupting

Abstract The androgen receptor (AR) plays an important role in the development of the male phenotype and traits. Some diphenyl compounds inhibit AR activity by binding to a hydrophobic surface binding site, BF3. A similar diphenyl structure is found in 4,4’ DDT and its breakdown product 4,4’ DDE. Previous results showed that DDT and DDE induced the release of bound dihydrotestosterone from the AR ligand binding domain, with IC50 values ranging from 54 to 82uM. This suggested that DDT and related compounds may act as endocrine disrupting chemicals by binding to the BF3 site and inducing allosteric changes in the AR structure, disrupting binding of the steroid to the ligand binding domain. Here, an AR reporter system was transiently transfected into HEK293 cells and AR activity was measured using a dual luciferase assay. The system was used to measure the response of the AR protein to varying concentrations of dihydrotestosterone in the presence and absence of DDE. DDE inhibited the activation of AR by dihydrotestosterone under these conditions. Five mutant AR genes with amino acid changes in the BF3 site were tested for alterations in the ability of DDE to disrupt AR activity. The five mutations tested were F673K, F673W, G724R, G724M, and L830D. The ability of DDE to inhibit AR activity was reduced by the mutations in the BF3 site. These results suggest that DDE acts as an endocrine disrupting chemical (EDC) by binding to the BF3 site and allosterically regulating AR activity.

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NMR Solution Structure of Type II Human Cellular Retinoic Acid Binding Protein: Implications for Ligand Binding†,‡

Biochemistry ◽

10.1021/bi9808924 ◽

1998 ◽

Vol 37 (37) ◽

pp. 12727-12736 ◽

Cited By ~ 19

Author(s):

Lincong Wang ◽

Yue Li ◽

Frits Abildgaard ◽

John L. Markley ◽

Honggao Yan

Keyword(s):

Retinoic Acid ◽

Ligand Binding ◽

Solution Structure ◽

Binding Protein ◽

Type Ii ◽

Nmr Solution Structure ◽

Acid Binding Protein

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Mutational analysis of the CD6 ligand binding domain

Protein Engineering Design and Selection ◽

10.1093/protein/10.8.943 ◽

1997 ◽

Vol 10 (8) ◽

pp. 943-947 ◽

Cited By ~ 12

Author(s):

J. E. Skonier ◽

D. L. Bodian ◽

J. Emswiler ◽

M. A. Bowen ◽

A. Aruffo ◽

...

Keyword(s):

Ligand Binding ◽

Mutational Analysis ◽

Binding Domain ◽

Ligand Binding Domain

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Structure, interactions and action of Mycobacterium tuberculosis 3-hydroxyisobutyric acid dehydrogenase

Biochemical Journal ◽

10.1042/bcj20180271 ◽

2018 ◽

Vol 475 (15) ◽

pp. 2457-2471 ◽

Cited By ~ 1

Author(s):

Rajapiramuthu Srikalaivani ◽

Amrita Singh ◽

Mamannamana Vijayan ◽

Avadhesha Surolia

Keyword(s):

Mycobacterium Tuberculosis ◽

Active Site ◽

Mutational Analysis ◽

Gel Filtration ◽

Ph Optimum ◽

Native Page ◽

Acid Dehydrogenase ◽

Binary Complexes ◽

Solution Studies ◽

Hydroxyisobutyric Acid

Biochemical and crystallographic studies on Mycobacterium tuberculosis 3-hydroxyisobutyric acid dehydrogenase (MtHIBADH), a member of the 3-hydroxyacid dehydrogenase superfamily, have been carried out. Gel filtration and blue native PAGE of MtHIBADH show that the enzyme is a dimer. The enzyme preferentially uses NAD+ as the cofactor and is specific to S-hydroxyisobutyric acid (HIBA). It can also use R-HIBA, l-serine and 3-hydroxypropanoic acid (3-HP) as substrates, but with much less efficiency. The pH optimum for activity is ∼11. Structures of the native enzyme, the holoenzyme, binary complexes with NAD+, S-HIBA, R-HIBA, l-serine and 3-HP and ternary complexes involving the substrates and NAD+ have been determined. None of the already known structures of HIBADH contain a substrate molecule at the binding site. The structures reported here provide for the first time, among other things, a clear indication of the location and interactions of the substrates at the active site. They also define the entrance of the substrates to the active site region. The structures provide information on the role of specific residues at the active site and the entrance. The results obtained from crystal structures are consistent with solution studies including mutational analysis. They lead to the proposal of a plausible mechanism of the action of the enzyme.

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