Molecular basis for the interaction between stress-inducible phosphoprotein 1 (STIP1) and S100A1

2017 ◽  
Vol 474 (11) ◽  
pp. 1853-1866 ◽  
Author(s):  
Andrzej Maciejewski ◽  
Vania F. Prado ◽  
Marco A.M. Prado ◽  
Wing-Yiu Choy
Keyword(s):  
Calcium Binding ◽  
Molecular Basis ◽  
Structural Basis ◽  
Titration Calorimetry ◽  
Dependent Manner ◽  
S100 Family ◽  
Calcium Dependent ◽  
Binding Interface ◽  
Large Conformational Change ◽  
Α Helix

Stress-inducible phosphoprotein 1 (STIP1) is a cellular co-chaperone, which regulates heat-shock protein 70 (Hsp70) and Hsp90 activity during client protein folding. Members of the S100 family of dimeric calcium-binding proteins have been found to inhibit Hsp association with STIP1 through binding of STIP1 tetratricopeptide repeat (TPR) domains, possibly regulating the chaperone cycle. Here, we investigated the molecular basis of S100A1 binding to STIP1. We show that three S100A1 dimers associate with one molecule of STIP1 in a calcium-dependent manner. Isothermal titration calorimetry revealed that individual STIP1 TPR domains, TPR1, TPR2A and TPR2B, bind a single S100A1 dimer with significantly different affinities and that the TPR2B domain possesses the highest affinity for S100A1. S100A1 bound each TPR domain through a common binding interface composed of α-helices III and IV of each S100A1 subunit, which is only accessible following a large conformational change in S100A1 upon calcium binding. The TPR2B-binding site for S100A1 was predominately mapped to the C-terminal α-helix of TPR2B, where it is inserted into the hydrophobic cleft of an S100A1 dimer, suggesting a novel binding mechanism. Our data present the structural basis behind STIP1 and S100A1 complex formation, and provide novel insights into TPR module-containing proteins and S100 family member complexes.

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

2018 ◽  
Vol 115 (28) ◽  
pp. E6457-E6466 ◽  
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.

scholarly journals Initiating protease with modular domains interacts with β-glucan recognition protein to trigger innate immune response in insects

2015 ◽  
Vol 112 (45) ◽  
pp. 13856-13861 ◽  
Author(s):  
Daisuke Takahashi ◽  
Brandon L. Garcia ◽  
Michael R. Kanost
Keyword(s):  
Immune Responses ◽  
Molecular Basis ◽  
Molecular Mechanisms ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Dependent Manner ◽  
Calcium Dependent ◽  
Amino Terminal ◽  
Density Lipoprotein Receptor ◽  
Recognition Protein

The autoactivation of an initiating serine protease upon binding of pattern recognition proteins to pathogen surfaces is a crucial step in eliciting insect immune responses such as the activation of Toll and prophenoloxidase pathways. However, the molecular mechanisms responsible for autoactivation of the initiating protease remains poorly understood. Here, we investigated the molecular basis for the autoactivation of hemolymph protease 14 (HP14), an initiating protease in hemolymph of Manduca sexta, upon the binding of β-1,3-glucan by its recognition protein, βGRP2. Biochemical analysis using HP14 zymogen (proHP14), βGRP2, and the recombinant proteins as truncated forms showed that the amino-terminal modular low-density lipoprotein receptor class A (LA) domains within HP14 are required for proHP14 autoactivation that is stimulated by its interaction with βGRP2. Consistent with this result, recombinant LA domains inhibit the activation of proHP14 and prophenoloxidase, likely by competing with the interaction between βGRP2 and LA domains within proHP14. Using surface plasmon resonance, we demonstrated that immobilized LA domains directly interact with βGRP2 in a calcium-dependent manner and that high-affinity interaction requires the C-terminal glucanase-like domain of βGRP2. Importantly, the affinity of LA domains for βGRP2 increases nearly 100-fold in the presence of β-1,3-glucan. Taken together, these results present the first experimental evidence to our knowledge that LA domains of an insect modular protease and glucanase-like domains of a βGRP mediate their interaction, and that this binding is essential for the protease autoactivation. Thus, our study provides important insight into the molecular basis underlying the initiation of protease cascade in insect immune responses.

scholarly journals UHM–ULM interactions in the RBM39–U2AF65 splicing-factor complex

2016 ◽  
Vol 72 (4) ◽  
pp. 497-511 ◽  
Author(s):  
Galina A. Stepanyuk ◽  
Pedro Serrano ◽  
Eigen Peralta ◽  
Carol L. Farr ◽  
Herbert L. Axelrod ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Rna Binding ◽  
Splicing Factor ◽  
Structural Basis ◽  
Titration Calorimetry ◽  
Protein Protein Interactions ◽  
Rrm Domain ◽  
Nmr Structures ◽  
Binding Interface ◽  
Cell Extracts

RNA-binding protein 39 (RBM39) is a splicing factor and a transcriptional co-activator of estrogen receptors and Jun/AP-1, and its function has been associated with malignant progression in a number of cancers. The C-terminal RRM domain of RBM39 belongs to the U2AF homology motif family (UHM), which mediate protein–protein interactions through a short tryptophan-containing peptide known as the UHM-ligand motif (ULM). Here, crystal and solution NMR structures of the RBM39-UHM domain, and the crystal structure of its complex with U2AF65-ULM, are reported. The RBM39–U2AF65 interaction was confirmed by co-immunoprecipitation from human cell extracts, by isothermal titration calorimetry and by NMR chemical shift perturbation experiments with the purified proteins. When compared with related complexes, such as U2AF35–U2AF65 and RBM39–SF3b155, the RBM39-UHM–U2AF65-ULM complex reveals both common and discriminating recognition elements in the UHM–ULM binding interface, providing a rationale for the known specificity of UHM–ULM interactions. This study therefore establishes a structural basis for specific UHM–ULM interactions by splicing factors such as U2AF35, U2AF65, RBM39 and SF3b155, and a platform for continued studies of intermolecular interactions governing disease-related alternative splicing in eukaryotic cells.

scholarly journals Identification, localization, and functional implications of an abundant nematode annexin.

1996 ◽  
Vol 132 (6) ◽  
pp. 1079-1092 ◽  
Author(s):  
C E Creutz ◽  
S L Snyder ◽  
S N Daigle ◽  
J Redick
Keyword(s):  
Membrane Trafficking ◽  
Calcium Binding ◽  
Peptide Sequencing ◽  
Uterine Wall ◽  
Dependent Manner ◽  
Membrane Systems ◽  
Expression Library ◽  
Sequencing Project ◽  
Calcium Dependent ◽  
Chromosome Iii

Cultures of the nematode C. elegans were examined for the presence of calcium-dependent, phospholipid-binding proteins of the annexin class. A single protein of apparent mass on SDS-polyacrylamide gels of 32 kD was isolated from soluble extracts of nematode cultures on the basis of its ability to bind to phospholipids in a calcium-dependent manner. After verification of the protein as an annexin by peptide sequencing, an antiserum to the protein was prepared and used to isolate a corresponding cDNA from an expression library in phage lambda gt11. The encoded protein, herein referred to as the nex-1 annexin, has a mass of 35 kD and is 36-42% identical in sequence to 10 known mammalian annexins. Several unique modifications were found in the portions of the sequence corresponding to calcium-binding sites. Possible phosphorylation sites in the NH2-terminal domain of the nematode annexin correspond to those of mammalian annexins. The gene for this annexin (nex-1) was physically mapped to chromosome III in the vicinity of the dpy-17 genetic marker. Two other annexin genes (nex-2 and nex-3) were also identified in chromosome III sequences reported by the nematode genomic sequencing project (Sulston, J., Z. Du, K. Thomas, R. Wilson, L. Hillier, R. Staden, N. Halloran, P. Green, J. Thierry-Mieg, L. Qiu, et al. 1992. Nature (Lond.). 356:37-41). The nex-1 annexin was localized in the nematode by immunofluorescence and by electron microscopy using immunogold labeling. The protein is associated with membrane systems of the secretory gland cells of the pharynx, with sites of cuticle formation in the grinder in the pharynx, with yolk granules in oocytes, with the uterine wall and vulva, and with membrane systems in the spermathecal valve. The presence of the annexin in association with the membranes of the spermathecal valve suggests a novel function of the protein in the folding and unfolding of these membranes as eggs pass through the valve. The localizations also indicate roles for the annexin corresponding to those proposed in mammalian systems in membrane trafficking, collagen deposition, and extracellular matrix formation.

scholarly journals Calcium-dependent properties of CIB binding to the integrin αIIb cytoplasmic domain and translocation to the platelet cytoskeleton

1999 ◽  
Vol 342 (3) ◽  
pp. 729-735 ◽  
Author(s):  
David D. SHOCK ◽  
Ulhas P. NAIK ◽  
Julia E. BRITTAIN ◽  
Suresh K. ALAHARI ◽  
John SONDEK ◽  
...  
Keyword(s):  
Cellular Localization ◽  
Molecular Mechanisms ◽  
Cytoplasmic Domain ◽  
Titration Calorimetry ◽  
Dependent Manner ◽  
Calcium Dependent ◽  
Fibrinogen Binding ◽  
Ef Hand ◽  
Domain Peptide ◽  
Triton X

The αIIbβ3 integrin receives signals in agonist-activated platelets, resulting in its conversion to an active conformation that binds fibrinogen, thereby mediating platelet aggregation. Fibrinogen binding to αIIbβ3 subsequently induces a cascade of intracellular signalling events. The molecular mechanisms of this bi-directional αIIbβ3-mediated signalling are unknown but may involve the binding of proteins to the integrin cytoplasmic domains. We reported previously the sequence of a novel 22-kDa, EF-hand-containing, protein termed CIB (calcium- and integrin-binding protein) that interacts specifically with the αIIb cytoplasmic domain in the yeast two-hybrid system. Further analysis of numerous tissues and cell lines indicated that CIB mRNA and protein are widely expressed. In addition, isothermal titration calorimetry indicated that CIB binds to an αIIb cytoplasmic-domain peptide in a Ca2+-dependent manner, with moderate affinity (Kd, 700 nM) and 1:1 stoichiometry. In aggregated platelets, endogenous CIB and αIIbβ3 translocate to the Triton X-100-insoluble cytoskeleton in a parallel manner, demonstrating that the cellular localization of CIB is regulated, potentially by αIIbβ3. Thus CIB may contribute to integrin-related functions by mechanisms involving Ca2+-modulated binding to the αIIb cytoplasmic domain and changes in intracellular distribution.

Downstream regulatory element antagonistic modulator regulates islet prodynorphin expression

2006 ◽  
Vol 291 (3) ◽  
pp. E587-E595 ◽  
Author(s):  
David A. Jacobson ◽  
Julie Cho ◽  
Luis R. Landa ◽  
Natalia A. Tamarina ◽  
Michael W. Roe ◽  
...  
Keyword(s):  
Calcium Binding ◽  
Regulatory Element ◽  
Glucagon Secretion ◽  
Dependent Manner ◽  
Β Cells ◽  
Dynorphin A ◽  
Calcium Dependent ◽  
Low Glucose ◽  
Stimulation Of

Calcium-binding proteins regulate transcription and secretion of pancreatic islet hormones. Here, we demonstrate neuroendocrine expression of the calcium-binding downstream regulatory element antagonistic modulator (DREAM) and its role in glucose-dependent regulation of prodynorphin (PDN) expression. DREAM is distributed throughout β- and α-cells in both the nucleus and cytoplasm. As DREAM regulates neuronal dynorphin expression, we determined whether this pathway is affected in DREAM−/− islets. Under low glucose conditions, with intracellular calcium concentrations of <100 nM, DREAM−/− islets had an 80% increase in PDN message compared with controls. Accordingly, DREAM interacts with the PDN promoter downstream regulatory element (DRE) under low calcium (<100 nM) conditions, inhibiting PDN transcription in β-cells. Furthermore, β-cells treated with high glucose (20 mM) show increased cytoplasmic calcium (∼200 nM), which eliminates DREAM's interaction with the DRE, causing increased PDN promoter activity. As PDN is cleaved into dynorphin peptides, which stimulate κ-opioid receptors expressed predominantly in α-cells of the islet, we determined the role of dynorphin A-(1–17) in glucagon secretion from the α-cell. Stimulation with dynorphin A-(1–17) caused α-cell calcium fluctuations and a significant increase in glucagon release. DREAM−/− islets also show elevated glucagon secretion in low glucose compared with controls. These results demonstrate that PDN transcription is regulated by DREAM in a calcium-dependent manner and suggest a role for dynorphin regulation of α-cell glucagon secretion. The data provide a molecular basis for opiate stimulation of glucagon secretion first observed over 25 years ago.

scholarly journals Interferon-γ-dependent tyrosine phosphorylation of MEKK4 via Pyk2 is regulated by annexin II and SHP2 in keratinocytes

2005 ◽  
Vol 388 (1) ◽  
pp. 17-28 ◽  
Author(s):  
Ursula M. HALFTER ◽  
Zachary E. DERBYSHIRE ◽  
Richard R. VAILLANCOURT
Keyword(s):  
Tyrosine Phosphorylation ◽  
Calcium Binding ◽  
Interferon Γ ◽  
Janus Kinase ◽  
Mitogen Activated Protein Kinase ◽  
Annexin Ii ◽  
Dependent Manner ◽  
Hacat Cells ◽  
Perinuclear Region ◽  
Calcium Dependent

IFNγ (interferon-γ) binding to its cognate receptor results, through JAK (Janus kinase), in direct activation of receptor-bound STAT1 (signal transducer and activator of transcription 1), although there is evidence for additional activation of a MAPK (mitogen-activated protein kinase) pathway. In the present paper, we report IFNγ-dependent activation of the MEKK4 (MAPK/extracellular-signal-regulated kinase kinase kinase 4) pathway in HaCaT human keratinocytes. MEKK4 is tyrosine­-phosphorylated and the IFNγ-dependent phosphorylation requires intracellular calcium. Calcium-dependent phosphorylation of MEKK4 is mediated by Pyk2. Moreover, MEKK4 and Pyk2 co-localize in an IFNγ-dependent manner in the perinuclear region. Furthermore, the calcium-binding protein, annexin II, and the calcium-regulated kinase, Pyk2, co-immunoprecipitate with MEKK4 after treatment with IFNγ. Immunofluorescence imaging of HaCaT cells shows an IFNγ-dependent co-localization of annexin II with Pyk2 in the perinuclear region, suggesting that annexin II mediates the calcium-dependent regulation of Pyk2. Tyrosine phosphorylation of MEKK4 correlates with its activity to phosphorylate MKK6 (MAPK kinase 6) in vitro and subsequent p38 MAPK activation in an IFNγ-dependent manner. Additional studies demonstrate that the SH2 (Src homology 2)-domain-containing tyrosine phosphatase SHP2 co-immunoprecipitates with MEKK4 in an IFNγ-dependent manner and co-localizes with MEKK4 after IFNγ stimulation in the perinuclear region in HaCaT cells. Furthermore, we provide evidence that SHP2 dephosphorylates MEKK4 and Pyk2, terminating the MEKK4-dependent branch of the IFNγ signalling pathway.

Immunochemical studies on the cercarial-specific calcium binding protein ofSchistosoma mansoni

Parasitology ◽  
1994 ◽  
Vol 108 (3) ◽  
pp. 289-300 ◽  
Author(s):  
D. Ram ◽  
B. Romano ◽  
I. Schechter
Keyword(s):  
Calcium Binding ◽  
Time Course ◽  
Intestinal Wall ◽  
The Body ◽  
Immunogold Electron Microscopy ◽  
Dependent Manner ◽  
Specific Expression ◽  
Western Blots ◽  
Calcium Dependent ◽  
Putative Target

SUMMARYStage-specific expression of the mRNA encoding the cercarial-specific 8 kDa CaBP has been described previously. To gain information on possible function(s) of this protein we raised antibodies to the CaBP in rabbits immunized with a CaBP-TrpE fusion protein synthesized in bacteria. Western blots showed high levels of CaBP in cercariae and 3 h schistosomula, trace amounts in 24 h schistosomula, and none in miracidia sporocyst and adult worm, as found for the mRNA. The CaBP molecule has a short half-life (≤4 h) similar to that of the mRNA. Other experiments demonstrate that the CaBP may interact with a putative target molecule in a calcium-dependent manner to form a complex of 45 kDa. Immunogold electron microscopy showed CaBP in selected regions of cercariae and 3 h schistosomula: tegument, head gland, subtegumental cells, flame cells, intestinal wall and the body-tail junction. Other investigators have shown that the head gland and subtegumental cells synthesize and translocate granules to the tegument during transformation from cercariae (living freely in water) to schistosomula (residing in vertebrate host). These observations and the time-course of CaBP detection suggest that the CaBP synthesized in the head gland and subtegumental cells is translocated to the tegument where it plays a role in tegument modifications required for adaptation to parasite life in the host. CaBP was not found in muscles and mitochondria, suggesting that it is not involved in the rapid motility and aerobic metabolism characteristic of cercariae.

scholarly journals Biochemical and structural studies of target lectin SapL1 from the emerging opportunistic microfungus Scedosporium apiospermum

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Dania Martínez-Alarcón ◽  
Viviane Balloy ◽  
Jean-Philippe Bouchara ◽  
Roland J. Pieters ◽  
Annabelle Varrot
Keyword(s):  
Structural Basis ◽  
Soluble Form ◽  
Titration Calorimetry ◽  
Scedosporium Apiospermum ◽  
Dependent Manner ◽  
Bronchial Epithelial ◽  
Life Threatening ◽  
Opportunistic Fungal Pathogen ◽  
Conidial Surface

AbstractScedosporium apiospermum is an emerging opportunistic fungal pathogen responsible for life-threatening infections in humans. Host–pathogen interactions often implicate lectins that have become therapeutic targets for the development of carbohydrate mimics for antiadhesive therapy. Here, we present the first report on the identification and characterization of a lectin from S. apiospermum named SapL1. SapL1 was found using bioinformatics as a homolog to the conidial surface lectin FleA from Aspergillus fumigatus known to play a role in the adhesion to host glycoconjugates present in human lung epithelium. In our strategy to obtain recombinant SapL1, we discovered the importance of osmolytes to achieve its expression in soluble form in bacteria. Analysis of glycan arrays indicates specificity for fucosylated oligosaccharides as expected. Submicromolar affinity was measured for fucose using isothermal titration calorimetry. We solved SapL1 crystal structure in complex with α-methyl-L-fucoside and analyzed its structural basis for fucose binding. We finally demonstrated that SapL1 binds to bronchial epithelial cells in a fucose-dependent manner. The information gathered here will contribute to the design and development of glycodrugs targeting SapL1.

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