scholarly journals Structural basis for heme-dependent NCoR binding to the transcriptional repressor REV-ERBβ

2021 ◽  
Vol 7 (5) ◽  
pp. eabc6479
Author(s):  
Sarah A. Mosure ◽  
Timothy S. Strutzenberg ◽  
Jinsai Shang ◽  
Paola Munoz-Tello ◽  
Laura A. Solt ◽  
...  
Keyword(s):  
Nuclear Receptors ◽  
Binding Mode ◽  
Structural Basis ◽  
Receptor Interaction ◽  
Heme Binding ◽  
Endogenous Ligand ◽  
Interaction Domain ◽  
Response Elements ◽  
Biochemical Assays ◽  
Chemical Cross Linking

Heme is the endogenous ligand for the constitutively repressive REV-ERB nuclear receptors, REV-ERBα (NR1D1) and REV-ERBβ (NR1D2), but how heme regulates REV-ERB activity remains unclear. Cellular studies indicate that heme is required for the REV-ERBs to bind the corepressor NCoR and repress transcription. However, fluorescence-based biochemical assays suggest that heme displaces NCoR; here, we show that this is due to a heme-dependent artifact. Using ITC and NMR spectroscopy, we show that heme binding remodels the thermodynamic interaction profile of NCoR receptor interaction domain (RID) binding to REV-ERBβ ligand-binding domain (LBD). We solved two crystal structures of REV-ERBβ LBD cobound to heme and NCoR peptides, revealing the heme-dependent NCoR binding mode. ITC and chemical cross-linking mass spectrometry reveals a 2:1 LBD:RID stoichiometry, consistent with cellular studies showing that NCoR-dependent repression of REV-ERB transcription occurs on dimeric DNA response elements. Our findings should facilitate renewed progress toward understanding heme-dependent REV-ERB activity.

scholarly journals Structural basis for heme-dependent NCoR binding to the transcriptional repressor REV-ERBβ

2020 ◽  
Author(s):  
Sarah A. Mosure ◽  
Jinsai Shang ◽  
Paola Munoz-Tello ◽  
Douglas J. Kojetin
Keyword(s):  
Nmr Spectroscopy ◽  
Ligand Binding ◽  
Crystal Structures ◽  
Binding Affinity ◽  
Isothermal Titration Calorimetry ◽  
Nuclear Receptors ◽  
Structural Basis ◽  
Titration Calorimetry ◽  
Endogenous Ligand ◽  
Biochemical Assays

ABSTRACTHeme is the endogenous ligand for the constitutively repressive REV-ERB nuclear receptors, REV-ERBα (NR1D1) and REV-ERBβ (NR1D2), but how heme regulates REV-ERB activity remains unclear. While cellular studies indicate heme is required for the REV-ERBs to bind the corepressor NCoR and repress transcription, fluorescence-based biochemical assays and crystal structures suggest that heme displaces NCoR. Here, we show that heme artifactually influences detection of NCoR interaction in fluorescence-based assays. However, using fluorescence-independent methods, isothermal titration calorimetry and NMR spectroscopy, we demonstrate that heme directly increases REV-ERBβ ligand-binding domain (LBD) binding affinity for NCoR. We further report two crystal structures of REV-ERBβ LBD cobound to heme and NCoR peptides, which reveal the structural basis for heme-dependent NCoR binding to REV-ERBβ. By resolving previous contradictory biochemical, structural, and cellular studies, our findings should facilitate renewed progress toward understanding heme-dependent REV-ERB activity.

scholarly journals Structure of cyanobacterial phycobilisome core revealed by structural modeling and chemical cross-linking

2021 ◽  
Vol 7 (2) ◽  
pp. eaba5743
Author(s):  
Haijun Liu ◽  
Mengru M. Zhang ◽  
Daniel A. Weisz ◽  
Ming Cheng ◽  
Himadri B. Pakrasi ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Excitation Energy Transfer ◽  
Energy Coupling ◽  
Regulatory Elements ◽  
Photochemical Activity ◽  
Structural Basis ◽  
Bottom Surface ◽  
Cross Linking ◽  
Orange Carotenoid Protein ◽  
Chemical Cross Linking

In cyanobacteria and red algae, the structural basis dictating efficient excitation energy transfer from the phycobilisome (PBS) antenna complex to the reaction centers remains unclear. The PBS has several peripheral rods and a central core that binds to the thylakoid membrane, allowing energy coupling with photosystem II (PSII) and PSI. Here, we have combined chemical cross-linking mass spectrometry with homology modeling to propose a tricylindrical cyanobacterial PBS core structure. Our model reveals a side-view crossover configuration of the two basal cylinders, consolidating the essential roles of the anchoring domains composed of the ApcE PB loop and ApcD, which facilitate the energy transfer to PSII and PSI, respectively. The uneven bottom surface of the PBS core contrasts with the flat reducing side of PSII. The extra space between two basal cylinders and PSII provides increased accessibility for regulatory elements, e.g., orange carotenoid protein, which are required for modulating photochemical activity.

scholarly journals Structural basis of subunit selectivity for competitive NMDA receptor antagonists with preference for GluN2A over GluN2B subunits

2017 ◽  
Vol 114 (33) ◽  
pp. E6942-E6951 ◽  
Author(s):  
Genevieve E. Lind ◽  
Tung-Chung Mou ◽  
Lucia Tamborini ◽  
Martin G. Pomper ◽  
Carlo De Micheli ◽  
...  
Keyword(s):  
Nmda Receptor ◽  
Binding Affinity ◽  
Binding Mode ◽  
Structural Basis ◽  
Nmda Receptor Antagonists ◽  
Receptor Antagonists ◽  
Glutamate Binding ◽  
Subunit Interface ◽  
The Central Nervous System ◽  
Contact Residues

NMDA-type glutamate receptors are ligand-gated ion channels that contribute to excitatory neurotransmission in the central nervous system (CNS). Most NMDA receptors comprise two glycine-binding GluN1 and two glutamate-binding GluN2 subunits (GluN2A–D). We describe highly potent (S)-5-[(R)-2-amino-2-carboxyethyl]-4,5-dihydro-1H-pyrazole-3-carboxylic acid (ACEPC) competitive GluN2 antagonists, of which ST3 has a binding affinity of 52 nM at GluN1/2A and 782 nM at GluN1/2B receptors. This 15-fold preference of ST3 for GluN1/2A over GluN1/2B is improved compared with NVP-AAM077, a widely used GluN2A-selective antagonist, which we show has 11-fold preference for GluN1/2A over GluN1/2B. Crystal structures of the GluN1/2A agonist binding domain (ABD) heterodimer with bound ACEPC antagonists reveal a binding mode in which the ligands occupy a cavity that extends toward the subunit interface between GluN1 and GluN2A ABDs. Mutational analyses show that the GluN2A preference of ST3 is primarily mediated by four nonconserved residues that are not directly contacting the ligand, but positioned within 12 Å of the glutamate binding site. Two of these residues influence the cavity occupied by ST3 in a manner that results in favorable binding to GluN2A, but occludes binding to GluN2B. Thus, we reveal opportunities for the design of subunit-selective competitive NMDA receptor antagonists by identifying a cavity for ligand binding in which variations exist between GluN2A and GluN2B subunits. This structural insight suggests that subunit selectivity of glutamate-site antagonists can be mediated by mechanisms in addition to direct contributions of contact residues to binding affinity.

scholarly journals Characterization of Receptor Interaction and Transcriptional Repression by the Corepressor SMRT

1997 ◽  
Vol 11 (13) ◽  
pp. 2025-2037 ◽  
Author(s):  
Hui Li ◽  
Christopher Leo ◽  
Daniel J. Schroen ◽  
J. Don Chen
Keyword(s):  
Nuclear Receptors ◽  
Nuclear Receptor ◽  
Transcriptional Repression ◽  
Thyroid Hormone Receptor ◽  
Receptor Interaction ◽  
Basal Transcription ◽  
Protein Protein Interaction ◽  
Stable Association ◽  
Interacting Surfaces ◽  
Transcriptional Corepressors

Abstract SMRT (silencing mediator of retinoic acid and thyroid hormone receptor) and N-CoR (nuclear receptor corepressor) are two related transcriptional corepressors that contain separable domains capable of interacting with unliganded nuclear receptors and repressing basal transcription. To decipher the mechanisms of receptor interaction and transcriptional repression by SMRT/N-CoR, we have characterized protein-protein interacting surfaces between SMRT and nuclear receptors and defined transcriptional repression domains of both SMRT and N-CoR. Deletional analysis reveals two individual nuclear receptor domains necessary for stable association with SMRT and a C-terminal helix essential for corepressor dissociation. Coordinately, two SMRT domains are found to interact independently with the receptors. Functional analysis reveals that SMRT contains two distinct repression domains, and the corresponding regions in N-CoR also repress basal transcription. Both repression domains in SMRT and N-CoR interact weakly with mSin3A, which in turn associates with a histone deacetylase HDAC1 in a mammalian two-hybrid assay. Far-Western analysis demonstrates a direct protein-protein interaction between two N-CoR repression domains with mSin3A. Finally we demonstrate that overexpression of full-length SMRT further represses basal transcription from natural promoters. Together, these results support a role of SMRT/N-CoR in corepression through the utilization of multiple mechanisms for receptor interactions and transcriptional repression.

scholarly journals Human Group C Rotavirus VP8*s Recognize Type A Histo-Blood Group Antigens as Ligands

2018 ◽  
Vol 92 (11) ◽  
Author(s):  
Xiaoman Sun ◽  
Lihong Wang ◽  
Jianxun Qi ◽  
Dandi Li ◽  
Mengxuan Wang ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Binding Site ◽  
Blood Group ◽  
Specific Interaction ◽  
Type A ◽  
Host Susceptibility ◽  
Structural Basis ◽  
Receptor Interaction ◽  
Blood Group Antigens ◽  
Group Species

ABSTRACTGroup/species C rotaviruses (RVCs) have been identified as important pathogens of acute gastroenteritis (AGE) in children, family-based outbreaks, as well as animal infections. However, little is known regarding their host-specific interaction, infection, and pathogenesis. In this study, we performed serial studies to characterize the function and structural features of a human G4P[2] RVC VP8* that is responsible for the host receptor interaction. Glycan microarrays demonstrated that the human RVC VP8* recognizes type A histo-blood group antigens (HBGAs), which was confirmed by synthetic glycan-/saliva-based binding assays and hemagglutination of red blood cells, establishing a paradigm of RVC VP8*-glycan interactions. Furthermore, the high-resolution crystal structure of the human RVC VP8* was solved, showing a typical galectin-like structure consisting of two β-sheets but with significant differences from cogent proteins of group A rotaviruses (RVAs). The VP8* in complex with a type A trisaccharide displays a novel ligand binding site that consists of a particular set of amino acid residues of the C-D, G-H, and K-L loops. RVC VP8* interacts with type A HBGAs through a unique mechanism compared with that used by RVAs. Our findings shed light on the host-virus interaction and the coevolution of RVCs and will facilitate the development of specific antivirals and vaccines.IMPORTANCEGroup/species C rotaviruses (RVCs), members ofReoviridaefamily, infect both humans and animals, but our knowledge about the host factors that control host susceptibility and specificity is rudimentary. In this work, we characterized the glycan binding specificity and structural basis of a human RVC that recognizes type A HBGAs. We found that human RVC VP8*, the rotavirus host ligand binding domain that shares only ∼15% homology with the VP8* domains of RVAs, recognizes type A HBGA at an as-yet-unknown glycan binding site through a mechanism distinct from that used by RVAs. Our new advancements provide insights into RVC-cell attachment, the critical step of virus infection, which will in turn help the development of control and prevention strategies against RVs.

scholarly journals Structural basis of the activation of the CC chemokine receptor 5 by a chemokine agonist

2020 ◽  
Author(s):  
Polina Isaikina ◽  
Ching-Ju Tsai ◽  
Nikolaus Dietz ◽  
Filip Pamula ◽  
Anne Grahl ◽  
...  
Keyword(s):  
Chemokine Receptor ◽  
Chemokine Receptors ◽  
Binding Mode ◽  
Activation Mechanism ◽  
Structural Basis ◽  
Cc Chemokine ◽  
Gi Protein ◽  
Agonist And Antagonist ◽  
Cc Chemokine Receptor 5 ◽  
Chemokine Receptor 5

AbstractThe human CC chemokine receptor 5 (CCR5) is a G protein-coupled receptor (GPCR) that plays a major role in inflammation and is involved in the pathology of cancer, HIV, and COVID-19. Despite its significance as a drug target, the activation mechanism of CCR5, i.e. how chemokine agonists transduce the activation signal through the receptor, is yet unknown. Here, we report the cryo-EM structure of wild-type CCR5 in an active conformation bound to the chemokine super-agonist [6P4]CCL5 and the heterotrimeric Gi protein. The structure provides the rationale for the sequence-activity relation of agonist and antagonist chemokines. The N-terminus of agonist chemokines pushes onto an aromatic connector that transmits activation to the canonical GPCR microswitch network. This activation mechanism differs significantly from other CC chemokine receptors that bind shorter chemokines in a shallow binding mode and have unique sequence signatures and a specialized activation mechanism.One-sentence summaryThe structure of CCR5 in complex with the chemokine agonist [6P4]CCL5 and the heterotrimeric Gi protein reveals its activation mechanism

scholarly journals Structural basis for VPg-induced formation of RNA-dependent RNA polymerase multimeric complexes

2014 ◽  
Vol 70 (a1) ◽  
pp. C1601-C1601
Author(s):  
Ji-Hye Lee ◽  
Yeon Bin Chung ◽  
Jong Hyeon Seok ◽  
Kang Rok Han ◽  
Sella Kim ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
De Novo ◽  
Helical Structure ◽  
Science Research ◽  
Binding Mode ◽  
Structural Basis ◽  
Murine Norovirus ◽  
Virion Protein ◽  
Electron Microscopic ◽  
Rna Dependent Rna Polymerase

Norovirus is the leading cause of epidemic acute, nonbacterial gastroenteritis, and adopts de novo and VPg (Virion protein genome linked)-primed RNA synthesis by RNA-dependent RNA polymerase (RdRp). To understand the interaction between RdRp and VPg in replication of murine norovirus-1 (MNV-1), we determined the crystal structure of MNV-1 RdRp-VPg(1-73)-RNA complex. VPg was bound to the base of the palm domain and the tip of the fingers domain of RdRp simultaneously, but RNA template could not be modeled. The binding affinity constants (Kd) for RdRp-VPg was 3.7411.57 nM and VPg(1-73) showed approximately 90-fold less affinity than that of full-length VPg. In addition to this multiple binding mode, VPg enhanced the interactions of RdRp hexamers, leading to the formation of high-order multimers or tubular fibrils with significantly increased polymerase activity, confirmed by electron microscopic and biochemical studies. Our data indicated that MNV-1 VPg with helical structure was bound to RdRp at multiple sites and induces RdRp multimerization in viral replication. The multimers of RdRp-VPg-RNA can provide a mechanistic understanding of viral polymerase multimeric arrays and a new tool for development of antivirals to control norovirus outbreaks. This work was supported by a grant of the Korea Healthcare Technology R&D Project, Ministry of Health, Welfare and Family Affairs (A085119 K.H.K), Basic Science Research Program through the National Research Foundation (NRF-2013R1A1A2064940, L.J-H), Korea University Grant (L.J-H), and the BK21 plus program of the Ministry of Education, Korea.

scholarly journals Structural Basis for the Inhibition of RNase H Activity of HIV-1 Reverse Transcriptase by RNase H Active Site-Directed Inhibitors

2010 ◽  
Vol 84 (15) ◽  
pp. 7625-7633 ◽  
Author(s):  
Hua-Poo Su ◽  
Youwei Yan ◽  
G. Sridhar Prasad ◽  
Robert F. Smith ◽  
Christopher L. Daniels ◽  
...  
Keyword(s):  
Active Site ◽  
Thermal Denaturation ◽  
Binding Mode ◽  
Rnase H ◽  
New Drugs ◽  
Structural Basis ◽  
Independent Manner ◽  
Approved Drugs ◽  
Hiv 1 ◽  
Rapid Emergence

ABSTRACT HIV/AIDS continues to be a menace to public health. Several drugs currently on the market have successfully improved the ability to manage the viral burden in infected patients. However, new drugs are needed to combat the rapid emergence of mutated forms of the virus that are resistant to existing therapies. Currently, approved drugs target three of the four major enzyme activities encoded by the virus that are critical to the HIV life cycle. Although a number of inhibitors of HIV RNase H activity have been reported, few inhibit by directly engaging the RNase H active site. Here, we describe structures of naphthyridinone-containing inhibitors bound to the RNase H active site. This class of compounds binds to the active site via two metal ions that are coordinated by catalytic site residues, D443, E478, D498, and D549. The directionality of the naphthyridinone pharmacophore is restricted by the ordering of D549 and H539 in the RNase H domain. In addition, one of the naphthyridinone-based compounds was found to bind at a second site close to the polymerase active site and non-nucleoside/nucleotide inhibitor sites in a metal-independent manner. Further characterization, using fluorescence-based thermal denaturation and a crystal structure of the isolated RNase H domain reveals that this compound can also bind the RNase H site and retains the metal-dependent binding mode of this class of molecules. These structures provide a means for structurally guided design of novel RNase H inhibitors.

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