Structural Basis of Ligand Interactions of the Large Extracellular Domain of Tetraspanin CD81

Hepatitis C virus (HCV) causes chronic liver disease, cirrhosis, and primary liver cancer. Despite 130 million people being at risk worldwide, no vaccine exists, and effective therapy is limited by drug resistance, toxicity, and high costs. The tetraspanin CD81 is an essential entry-level receptor required for HCV infection of hepatocytes and represents a critical target for intervention. In this study, we report the first structural characterization of the large extracellular loop of CD81, expressed in mammalian cells and studied in physiological solutions. The HCV E2 glycoprotein recognizes CD81 through a dynamic loop on the helical bundle, which was shown by nuclear magnetic resonance (NMR) spectroscopy to adopt a conformation distinct from that seen in crystals. A novel membrane binding interface was revealed adjacent to the exposed HCV interaction site in the extracellular loop of CD81. The binding pockets for two proposed inhibitors of the CD81-HCV interaction, namely, benzyl salicylate and fexofenadine, were shown to overlap the HCV and membrane interaction sites. Although the dynamic loop region targeted by these compounds presents challenges for structure-based design, the NMR assignments enable realistic screening and validation of ligands. Together, these data provide an improved avenue for developing potent agents that specifically block CD81-HCV interaction and also pave a way for elucidating the recognition mechanisms of diverse tetraspanins.

Download Full-text

Faculty Opinions recommendation of Residues SFQ (173-175) in the large extracellular loop of CD9 are required for gamete fusion.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1010335.150909 ◽

2002 ◽

Author(s):

Martin Hemler

Keyword(s):

Extracellular Loop ◽

Gamete Fusion ◽

Large Extracellular Loop

Download Full-text

Structural basis for proficient oxidized ribonucleotide insertion in double strand break repair

Nature Communications ◽

10.1038/s41467-021-24486-x ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Joonas A. Jamsen ◽

Akira Sassa ◽

Lalith Perera ◽

David D. Shock ◽

William A. Beard ◽

...

Keyword(s):

Mammalian Cells ◽

Time Lapse ◽

Strand Break ◽

Structural Basis ◽

End Joining ◽

Strand Breaks ◽

Nucleotide Pools ◽

Nucleotide Insertion ◽

Non Homologous End Joining

AbstractReactive oxygen species (ROS) oxidize cellular nucleotide pools and cause double strand breaks (DSBs). Non-homologous end-joining (NHEJ) attaches broken chromosomal ends together in mammalian cells. Ribonucleotide insertion by DNA polymerase (pol) μ prepares breaks for end-joining and this is required for successful NHEJ in vivo. We previously showed that pol μ lacks discrimination against oxidized dGTP (8-oxo-dGTP), that can lead to mutagenesis, cancer, aging and human disease. Here we reveal the structural basis for proficient oxidized ribonucleotide (8-oxo-rGTP) incorporation during DSB repair by pol μ. Time-lapse crystallography snapshots of structural intermediates during nucleotide insertion along with computational simulations reveal substrate, metal and side chain dynamics, that allow oxidized ribonucleotides to escape polymerase discrimination checkpoints. Abundant nucleotide pools, combined with inefficient sanitization and repair, implicate pol μ mediated oxidized ribonucleotide insertion as an emerging source of widespread persistent mutagenesis and genomic instability.

Download Full-text

Visualization of Head–Head Interactions in the Inhibited State of Smooth Muscle Myosin

The Journal of Cell Biology ◽

10.1083/jcb.147.7.1385 ◽

1999 ◽

Vol 147 (7) ◽

pp. 1385-1390 ◽

Cited By ~ 84

Author(s):

Thomas Wendt ◽

Dianne Taylor ◽

Terri Messier ◽

Kathleen M. Trybus ◽

Kenneth A. Taylor

Keyword(s):

Smooth Muscle ◽

Smooth Muscle Myosin ◽

Intramolecular Interactions ◽

Actin Binding ◽

Structural Basis ◽

Binding Interface ◽

Structural Differences ◽

Muscle Myosin ◽

Positively Charged ◽

Phosphoryla Tion

The structural basis for the phosphoryla- tion-dependent regulation of smooth muscle myosin ATPase activity was investigated by forming two- dimensional (2-D) crystalline arrays of expressed unphosphorylated and thiophosphorylated smooth muscle heavy meromyosin (HMM) on positively charged lipid monolayers. A comparison of averaged 2-D projections of both forms at 2.3-nm resolution reveals distinct structural differences. In the active, thiophosphorylated form, the two heads of HMM interact intermolecularly with adjacent molecules. In the unphosphorylated or inhibited state, intramolecular interactions position the actin-binding interface of one head onto the converter domain of the second head, thus providing a mechanism whereby the activity of both heads could be inhibited.

Download Full-text

Functional interaction between p21rap1A and components of the budding pathway in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.12.9.4084-4092.1992 ◽

1992 ◽

Vol 12 (9) ◽

pp. 4084-4092

Author(s):

P C McCabe ◽

H Haubruck ◽

P Polakis ◽

F McCormick ◽

M A Innis

Keyword(s):

Saccharomyces Cerevisiae ◽

Mammalian Cells ◽

Bound States ◽

Yeast Cells ◽

Temperature Sensitive ◽

Wild Type ◽

Amino Terminal ◽

Loop Region

The rap1A gene encodes a 21-kDa, ras-related GTP-binding protein (p21rap1A) of unknown function. A close structural homolog of p21rap1A (65% identity in the amino-terminal two-thirds) is the RSR1 gene product (Rsr1p) of Saccharomyces cerevisiae. Although Rsr1p is not essential for growth, its presence is required for nonrandom selection of bud sites. To assess the similarity of these proteins at the functional level, wild-type and mutant forms of p21rap1A were tested for complementation of activities known to be fulfilled by Rsr1p. Expression of p21rap1A, like multicopy expression of RSR1, suppressed the conditional lethality of a temperature-sensitive cdc24 mutation. Point mutations predicted to affect the localization of p21rap1A or its ability to cycle between GDP and GTP-bound states disrupted suppression of cdc24ts, while other mutations in the 61-65 loop region improved suppression. Expression of p21rap1A could not, however, suppress the random budding phenotype of rsr1 cells. p21rap1A also apparently interfered with the normal activity of Rsrlp, causing random budding in diploid wild-type cells, suggesting an inability of p21rap1A to interact appropriately with Rsr1p regulatory proteins. Consistent with this hypothesis, we found an Rsr1p-specific GTPase-activating protein (GAP) activity in yeast membranes which was not active toward p21rap1A, indicating that p21rap1A may be predominantly GTP bound in yeast cells. Coexpression of human Rap1-specific GAP suppressed the random budding due to expression of p21rap1A or its derivatives, including Rap1AVal-12. Although Rap1-specific GAP stimulated the GTPase of Rsr1p in vitro, it did not dominantly interfere with Rsr1p function in vivo. A chimera consisting of Rap1A1-165::Rsr1p166-272 did not exhibit normal Rsr1p function in the budding pathway. These results indicated that p21rap1A and Rsr1p share at least partial functional homology, which may have implications for p21rap1A function in mammalian cells.

Download Full-text

MLL1 minimal catalytic complex is a dynamic conformational ensemble susceptible to pharmacological allosteric disruption

10.1101/308676 ◽

2018 ◽

Author(s):

Lilia Kaustov ◽

Alexander Lemak ◽

Hong Wu ◽

Marco Faini ◽

Scott Houliston ◽

...

Keyword(s):

Hybrid Methods ◽

Structural Basis ◽

Epigenetic Mark ◽

Conformational Ensemble ◽

Interaction Sites ◽

Catalytic Function ◽

Individual Interaction ◽

Mixed Lineage Leukaemia ◽

Repeat Domain ◽

Histone H3k4

ABSTRACTHistone H3K4 methylation is an epigenetic mark associated with actively transcribed genes. This modification is catalyzed by the mixed lineage leukaemia (MLL) family of histone methyltransferases including MLL1, MLL2, MLL3, MLL4, SET1A and SET1B. Catalytic activity of MLL proteins is dependent on interactions with additional conserved proteins but the structural basis for subunit assembly and the mechanism of regulation is not well understood. We used a hybrid methods approach to study the assembly and biochemical function of the minimally active MLL1 complex (MLL1, WDR5 and RbBP5). A combination of small angle X-ray scattering (SAXS), cross-linking mass spectrometry (XL-MS), NMR spectroscopy, and computational modeling were used to generate a dynamic ensemble model in which subunits are assembled via multiple weak interaction sites. We identified a new interaction site between the MLL1 SET domain and the WD40 repeat domain of RbBP5, and demonstrate the susceptibility of the catalytic function of the complex to disruption of individual interaction sites.

Download Full-text

Large Extracellular Loop of Tetraspanin as a Potential Vaccine Candidate for Filariasis

PLoS ONE ◽

10.1371/journal.pone.0077394 ◽

2013 ◽

Vol 8 (10) ◽

pp. e77394 ◽

Cited By ~ 16

Author(s):

Gajalakshmi Dakshinamoorthy ◽

Gnanasekar Munirathinam ◽

Kristen Stoicescu ◽

Maryada Venkatarami Reddy ◽

Ramaswamy Kalyanasundaram

Keyword(s):

Vaccine Candidate ◽

Extracellular Loop ◽

Potential Vaccine Candidate ◽

Large Extracellular Loop ◽

Potential Vaccine

Download Full-text

Structural basis for recognition of human 7SK long noncoding RNA by the La-related protein Larp7

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1806276115 ◽

2018 ◽

Vol 115 (28) ◽

pp. E6457-E6466 ◽

Cited By ~ 20

Author(s):

Catherine D. Eichhorn ◽

Yuan Yang ◽

Lucas Repeta ◽

Juli Feigon

Keyword(s):

Noncoding Rna ◽

Rna Binding ◽

Rna Binding Proteins ◽

Transcription Elongation ◽

Structural Basis ◽

Titration Calorimetry ◽

Rna Recognition Motif ◽

Related Protein ◽

Binding Interface ◽

And Function

The La and the La-related protein (LARP) superfamily is a diverse class of RNA binding proteins involved in RNA processing, folding, and function. Larp7 binds to the abundant long noncoding 7SK RNA and is required for 7SK ribonucleoprotein (RNP) assembly and function. The 7SK RNP sequesters a pool of the positive transcription elongation factor b (P-TEFb) in an inactive state; on release, P-TEFb phosphorylates RNA Polymerase II to stimulate transcription elongation. Despite its essential role in transcription, limited structural information is available for the 7SK RNP, particularly for protein–RNA interactions. Larp7 contains an N-terminal La module that binds UUU-3′OH and a C-terminal atypical RNA recognition motif (xRRM) required for specific binding to 7SK and P-TEFb assembly. Deletion of the xRRM is linked to gastric cancer in humans. We report the 2.2-Å X-ray crystal structure of the human La-related protein group 7 (hLarp7) xRRM bound to the 7SK stem-loop 4, revealing a unique binding interface. Contributions of observed interactions to binding affinity were investigated by mutagenesis and isothermal titration calorimetry. NMR 13C spin relaxation data and comparison of free xRRM, RNA, and xRRM–RNA structures show that the xRRM is preordered to bind a flexible loop 4. Combining structures of the hLarp7 La module and the xRRM–7SK complex presented here, we propose a structural model for Larp7 binding to the 7SK 3′ end and mechanism for 7SK RNP assembly. This work provides insight into how this domain contributes to 7SK recognition and assembly of the core 7SK RNP.

Download Full-text

Structural basis for auxiliary subunit KCTD16 regulation of the GABABreceptor

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1903024116 ◽

2019 ◽

Vol 116 (17) ◽

pp. 8370-8379 ◽

Cited By ~ 11

Author(s):

Hao Zuo ◽

Ian Glaaser ◽

Yulin Zhao ◽

Igor Kurinov ◽

Lidia Mosyak ◽

...

Keyword(s):

Structural Basis ◽

Functional Coupling ◽

Girk Channels ◽

Receptor Complexes ◽

Binding Interface ◽

Tetramerization Domain ◽

High Resolution Structure ◽

Inwardly Rectifying ◽

The Brain ◽

Oligomerization Domain

Metabotropic GABABreceptors mediate a significant fraction of inhibitory neurotransmission in the brain. Native GABABreceptor complexes contain the principal subunits GABAB1and GABAB2, which form an obligate heterodimer, and auxiliary subunits, known as potassium channel tetramerization domain-containing proteins (KCTDs). KCTDs interact with GABABreceptors and modify the kinetics of GABABreceptor signaling. Little is known about the molecular mechanism governing the direct association and functional coupling of GABABreceptors with these auxiliary proteins. Here, we describe the high-resolution structure of the KCTD16 oligomerization domain in complex with part of the GABAB2receptor. A single GABAB2C-terminal peptide is bound to the interior of an open pentamer formed by the oligomerization domain of five KCTD16 subunits. Mutation of specific amino acids identified in the structure of the GABAB2–KCTD16 interface disrupted both the biochemical association and functional modulation of GABABreceptors and G protein-activated inwardly rectifying K+channel (GIRK) channels. These interfacial residues are conserved among KCTDs, suggesting a common mode of KCTD interaction with GABABreceptors. Defining the binding interface of GABABreceptor and KCTD reveals a potential regulatory site for modulating GABAB-receptor function in the brain.

Download Full-text

The Cruciality of Single Amino Acid Replacement for the Spectral Tuning of Biliverdin-Binding Cyanobacteriochromes

International Journal of Molecular Sciences ◽

10.3390/ijms21176278 ◽

2020 ◽

Vol 21 (17) ◽

pp. 6278

Author(s):

Keiji Fushimi ◽

Hiroki Hoshino ◽

Naeko Shinozaki-Narikawa ◽

Yuto Kuwasaki ◽

Keita Miyake ◽

...

Keyword(s):

Mammalian Cells ◽

Molecular Mechanisms ◽

Near Infrared ◽

Structural Characteristics ◽

Blue Shift ◽

Amino Acid Replacement ◽

Red Shift ◽

Single Amino Acid ◽

Structural Basis ◽

Small Unit

Cyanobacteriochromes (CBCRs), which are known as linear tetrapyrrole-binding photoreceptors, to date can only be detected from cyanobacteria. They can perceive light only in a small unit, which is categorized into various lineages in correlation with their spectral and structural characteristics. Recently, we have succeeded in identifying specific molecules, which can incorporate mammalian intrinsic biliverdin (BV), from the expanded red/green (XRG) CBCR lineage and in converting BV-rejective molecules into BV-acceptable ones with the elucidation of the structural basis. Among the BV-acceptable molecules, AM1_1870g3_BV4 shows a spectral red-shift in comparison with other molecules, while NpF2164g5_BV4 does not show photoconversion but stably shows a near-infrared (NIR) fluorescence. In this study, we found that AM1_1870g3_BV4 had a specific Tyr residue near the d-ring of the chromophore, while others had a highly conserved Leu residue. The replacement of this Tyr residue with Leu in AM1_1870g3_BV4 resulted in a blue-shift of absorption peak. In contrast, reverse replacement in NpF2164g5_BV4 resulted in a red-shift of absorption and fluorescence peaks, which applies to fluorescence bio-imaging in mammalian cells. Notably, the same Tyr/Leu-dependent color-tuning is also observed for the CBCRs belonging to the other lineage, which indicates common molecular mechanisms.

Download Full-text