scholarly journals Electrostatics cause the molecular chaperone BiP to preferentially bind oligomerized states of a client protein

2021 ◽  
Author(s):  
Judy L.M. Kotler ◽  
Wei-Shao Wei ◽  
Erin E Deans ◽  
Timothy O. Street
Keyword(s):  
Endoplasmic Reticulum ◽  
Molecular Chaperone ◽  
Binding Sites ◽  
Client Protein ◽  
High Affinity ◽  
Hsp70 Family ◽  
Differential Affinity ◽  
Peptide Region ◽  
Positively Charged ◽  
Micromolar Range

Hsp70-family chaperones bind short monomeric peptides with a weak characteristic affinity in the low micromolar range, but can also bind some aggregates, fibrils, and amyloids, with low nanomolar affinity. While this differential affinity enables Hsp70 to preferentially target potentially toxic aggregates, it is unknown how Hsp70s differentiate between monomeric and oligomeric states of a target protein. Here we examine the interaction of BiP (the Hsp70 paralog in the endoplasmic reticulum) with proIGF2, the pro-protein form of IGF2 that includes a long and mostly disordered E-peptide region that promotes proIGF2 oligomerization. We discover that electrostatic attraction enables the negatively charged BiP to bind positively charged E-peptide oligomers with low nanomolar affinity. We identify the specific BiP binding sites on proIGF2, and although some are positively charged, as monomers they bind BiP with characteristically low affinity in the micromolar range. We conclude that electrostatics enable BiP to preferentially recognize oligomeric states of proIGF2. Electrostatic targeting of Hsp70 to aggregates may be broadly applicable, as all the currently-documented cases in which Hsp70 binds aggregates with high-affinity involve clients that are expected to be positively charged.

scholarly journals Electron microscopy of cervical-mucus glycoproteins and fragments therefrom. The use of colloidal gold to make visible ‘naked’ protein regions

1990 ◽  
Vol 265 (1) ◽  
pp. 169-177 ◽  
Author(s):  
J K Sheehan ◽  
I Carlstedt
Keyword(s):  
Electron Microscopy ◽  
Binding Site ◽  
Binding Sites ◽  
Colloidal Gold ◽  
Length Distribution ◽  
Cervical Mucus ◽  
High Affinity ◽  
Protein Rich ◽  
Peptide Region ◽  
Mucus Glycoproteins

Subunits of human cervical-mucus glycoproteins obtained by reductive cleavage of whole mucins and high-Mr glycopeptides (T-domains) obtained after their trypsin digestion were studied with electron microscopy after spreading the macromolecules in a monolayer of benzyldimethylalkylammonium chloride. Subunits were observed as linear and apparently flexible particles, with number- and weight-average lengths of 390 nm and 460 nm respectively. T-domains randomly distributed on the grid have number- and weight-average lengths of 90 nm and 103 nm respectively, whereas when aligned (possibly stretched by flow) they were longer, with number-average and weight-average lengths of 150 nm and 170 nm respectively. Subunits complexed with gold appeared as segmented structures, with a distribution of inter-gold distances similar to the length distribution for the relaxed T-domains. The whole mucins had few binding sites for gold, suggesting that reduction exposes hydrophobic protein-rich regions with high affinity for gold. Most T-domains had a binding site at one end, indicating the presence of a residual protruding naked peptide region. We conclude that mucins are assembled from subunits joined end-to-end, and that each subunit consists of alternating oligosaccharide ‘clusters’ (approx. 100 nm) and naked peptide regions which have (after reduction) a high affinity for colloidal gold.

Structural insights intoAspergillus fumigatuslectin specificity: AFL binding sites are functionally non-equivalent

2015 ◽  
Vol 71 (3) ◽  
pp. 442-453 ◽  
Author(s):  
Josef Houser ◽  
Jan Komarek ◽  
Gianluca Cioci ◽  
Annabelle Varrot ◽  
Anne Imberty ◽  
...  
Keyword(s):  
Amino Acid ◽  
Binding Sites ◽  
Opportunistic Pathogen ◽  
Biotechnological Applications ◽  
Binding Properties ◽  
High Affinity ◽  
Lectin Family ◽  
Structural Insights ◽  
Specific Ligand ◽  
Micromolar Range

TheAspergillus fumigatuslectin AFL was recently described as a new member of the AAL lectin family. As a lectin from an opportunistic pathogen, it might play an important role in the interaction of the pathogen with the human host. A detailed study of structures of AFL complexed with several monosaccharides and oligosaccharides, including blood-group epitopes, was combined with affinity data from SPR and discussed in the context of previous findings. Its six binding sites are non-equivalent, and owing to minor differences in amino-acid composition they exhibit a marked difference in specific ligand recognition. AFL displays a high affinity in the micromolar range towards oligosaccharides which were detected in plants and also those bound on the human epithelia. All of these results indicate AFL to be a complex member of the lectin family and a challenging target for future medical research and, owing to its binding properties, a potentially useful tool in specific biotechnological applications.

scholarly journals A kinetic model for the Ca2+ + Mg2+-activated ATPase of sarcoplasmic reticulum

1986 ◽  
Vol 237 (1) ◽  
pp. 217-227 ◽  
Author(s):  
G W Gould ◽  
J M East ◽  
R J Froud ◽  
J M McWhirter ◽  
H I Stefanova ◽  
...  
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Kinetic Model ◽  
Binding Sites ◽  
Ph Dependence ◽  
High Affinity ◽  
Phosphorylated Form ◽  
Low Concentrations ◽  
Kinetics Of ◽  
Single Binding Site ◽  
Micromolar Range

The Ca2+ + Mg2+-activated ATPase of sarcoplasmic reticulum exhibits complex kinetics of activation with respect to ATP. ATPase activity is pH-dependent, with similar pH-activity profiles at high and low concentrations of ATP. Low concentrations of Ca2+ in the micromolar range activate the ATPase, whereas activity is inhibited by Ca2+ at millimolar concentrations. The pH-dependence of this Ca2+ inhibition and the effect of the detergent C12E8 (dodecyl octaethylene glycol monoether) on Ca2+ inhibition are similar to those observed on activation by low concentrations of Ca2+. On the basis of these and other studies we present a kinetic model for the ATPase. The ATPase is postulated to exist in one of two conformations: a conformation (E1) of high affinity for Ca2+ and MgATP and a conformation (E2) of low affinity for Ca2+ and MgATP. Ca2+ binding to E2 and to the phosphorylated form E2P are equal. Proton binding at the Ca2+-binding sites in the E1 and E2 conformations explains the pH-dependence of Ca2+ effects. Binding of MgATP to the phosphorylated intermediate E1′PCa2 and to E2 modulate the rates of the transport step E1′PCa-E2′PCa2 and the return of the empty Ca2+ sites to the outside surface of the sarcoplasmic reticulum, as well as the rate of dephosphorylation of E2P. Only a single binding site for MgATP is postulated.

Binding of FKBP23 to BiP in ER Shown by Gel Filtration Chromatography

2007 ◽  
Vol 62 (1-2) ◽  
pp. 133-137 ◽  
Author(s):  
Chen Chen ◽  
Hui Ma ◽  
Huaifeng Mi ◽  
Ying Wang
Keyword(s):  
Endoplasmic Reticulum ◽  
Molecular Chaperone ◽  
Binding Sites ◽  
Gel Filtration ◽  
Gel Filtration Chromatography ◽  
Terminal Domain ◽  
Ppiase Domain ◽  
Main Protein ◽  
Chaperone Hsp70

FKBP23 was found in mouse endoplasmic reticulum (ER) in 1998. It consists of an N-terminal peptidyl-prolyl cis/trans isomerase (PPIase) domain and a C-terminal domain with Ca2+ binding sites. Previously, we reported that FKBP23 specifically binds to BiP, the main protein of the molecular chaperone Hsp70 in ER lumen, and the binding is interrelated with the Ca2+ concentration. In this work we have found the existence of the complex FKBP23/BiP by separation of an ER extract using gel filtration chromatography (GFC), and that the existence of this complex is Ca2+-interrelated. This result further verified the Ca2+- interrelated binding of these two proteins in vivo.

scholarly journals Characterization of high-affinity ryanodine-binding sites of rat liver endoplasmic reticulum. Differences between liver and skeletal muscle

1991 ◽  
Vol 276 (1) ◽  
pp. 41-46 ◽  
Author(s):  
V Shoshan-Barmatz ◽  
T A Pressley ◽  
S Higham ◽  
N Kraus-Friedmann
Keyword(s):  
Skeletal Muscle ◽  
Endoplasmic Reticulum ◽  
Binding Sites ◽  
Specific Binding ◽  
Dna Amplification ◽  
Single Class ◽  
High Affinity ◽  
Sodium Dantrolene

In this study, the binding of [3H]ryanodine to liver microsomal subfractions was investigated. The specific binding of [3H]ryanodine, as determined both by vacuum filtration and by ultracentrifugation, is to a single class of high-affinity binding sites with a Kd of 10 +/- 2.5 nM and density of 500 +/- 100 and 1200 +/- 200 fmol/mg of protein by the filtration and centrifugation methods respectively. [3H]Ryanodine binding reached equilibrium in about 1 min and 2 min at 36 degrees C and 24 degrees C respectively, and the half-time of dissociation at 37 degrees C was approx. 15 s. The binding of [3H]ryanodine is Ca(2+)-independent: it is slightly stimulated by NaCl, Mg2+, ATP and InsP3 but strongly inhibited by caffeine, diltiazem and sodium dantrolene. Thus the binding of ryanodine to endoplasmic reticulum membranes shares some of the characteristics of its binding to the sarcoplasmic reticulum but also differs from it in several important properties, such as its Ca(2+)-independence, its rapid association and dissociation, and its inhibition by caffeine. The structural similarities between the skeletal muscle and liver binding sites were further explored by employing in vitro DNA amplification techniques, using the known sequence of the skeletal muscle receptor as reference point. The data obtained with this method indicate that the liver does not process mRNA for the skeletal muscle ryanodine receptor.

scholarly journals Ribosome Binding to and Dissociation from Translocation Sites of the Endoplasmic Reticulum Membrane

2006 ◽  
Vol 17 (9) ◽  
pp. 3860-3869 ◽  
Author(s):  
Julia Schaletzky ◽  
Tom A. Rapoport
Keyword(s):  
Endoplasmic Reticulum ◽  
Binding Sites ◽  
Signal Recognition Particle ◽  
Structural Data ◽  
Endoplasmic Reticulum Membrane ◽  
Signal Recognition ◽  
Ribosome Binding ◽  
High Affinity ◽  
Chain Complexes ◽  
Nascent Chain

We have addressed how ribosome-nascent chain complexes (RNCs), associated with the signal recognition particle (SRP), can be targeted to Sec61 translocation channels of the endoplasmic reticulum (ER) membrane when all binding sites are occupied by nontranslating ribosomes. These competing ribosomes are known to be bound with high affinity to tetramers of the Sec61 complex. We found that the membrane binding of RNC–SRP complexes does not require or cause the dissociation of prebound nontranslating ribosomes, a process that is extremely slow. SRP and its receptor target RNCs to a free population of Sec61 complex, which associates with nontranslating ribosomes only weakly and is conformationally different from the population of ribosome-bound Sec61 complex. Taking into account recent structural data, we propose a model in which SRP and its receptor target RNCs to a Sec61 subpopulation of monomeric or dimeric state. This could explain how RNC–SRP complexes can overcome the competition by nontranslating ribosomes.

scholarly journals Enhanced Binding to the Molecular Chaperone BiP Slows Thyroglobulin Export from the Endoplasmic Reticulum

1998 ◽  
Vol 12 (3) ◽  
pp. 458-467 ◽  
Author(s):  
Zoia Muresan ◽  
Peter Arvan
Keyword(s):  
Endoplasmic Reticulum ◽  
B Cells ◽  
Unfolded Protein Response ◽  
Molecular Chaperone ◽  
Disulfide Bonds ◽  
Chinese Hamster ◽  
Hormone Synthesis ◽  
Unfolded Protein ◽  
Hsp70 Family ◽  
Protein Response

Abstract To examine how binding of BiP (a molecular chaperone of the hsp70 family that resides in the endoplasmic reticulum) influences the conformational maturation of thyroglobulin (Tg, the precursor for thyroid hormone synthesis), we have developed a system of recombinant Tg stably expressed in wild-type Chinese hamster ovary (CHO) cells and CHO-B cells genetically manipulated for selectively increased BiP expression. The elevation of immunoreactive BiP in CHO-B cells is comparable to that seen during the unfolded protein response in the thyrocytes of certain human patients and animals suffering from congenital hypothyroid goiter with defective Tg. However, in CHO-B cells, we expressed Tg containing no mutations that induce misfolding (i.e. no unfolded protein response), so that levels of all other endoplasmic reticulum chaperones were normal. Increased availability of BiP did not accelerate Tg secretion; rather, the export of newly synthesized Tg was delayed. Tg detained intracellularly was concentrated in the endoplasmic reticulum. By coimmunoprecipitation, BiP exhibited enhanced binding to Tg in CHO-B cells. Moreover, two-dimensional gel analysis showed that BiP associated especially well with intracellular Tg containing mispaired disulfide bonds, thought to represent early Tg folding intermediates. An endoplasmic reticulum chaperone of the hsp90 family, GRP94, was also associated in Tg-chaperone complexes. The results suggest that increased binding of BiP to Tg leads to its delayed conformational maturation in the endoplasmic reticulum.

High Affinity Binding Sites for Activated Protein C and Protein C on Cultured Human Umbilical Vein Endothelial Cells

1994 ◽  
Vol 72 (03) ◽  
pp. 465-474 ◽  
Author(s):  
Neelesh Bangalore ◽  
William N Drohan ◽  
Carolyn L Orthner
Keyword(s):  
Binding Sites ◽  
Protein C ◽  
Umbilical Vein ◽  
Activated Protein C ◽  
Affinity Binding ◽  
Cell Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
Gla Domain ◽  
Umbilical Vein Endothelial Cells

SummaryActivated protein C (APC) is an antithrombotic serine proteinase having anticoagulant, profibrinolytic and anti-inflammatory activities. Despite its potential clinical utility, relatively little is known about its clearance mechanisms. In the present study we have characterized the interaction of APC and its active site blocked forms with human umbilical vein endothelial cells (HUVEC). At 4° C 125I-APC bound to HUVEC in a specific, time dependent, saturable and reversible manner. Scatchard analysis of the binding isotherm demonstrated a Kd value of 6.8 nM and total number of binding sites per cell of 359,000. Similar binding isotherms were obtained using radiolabeled protein C (PC) zymogen as well as D-phe-pro-arg-chloromethylketone (PPACK) inhibited APC indicating that a functional active site was not required. Competition studies showed that the binding of APC, PPACK-APC and PC were mutually exclusive suggesting that they bound to the same site(s). Proteolytic removal of the N-terminal γ-carboxyglutamic acid (gla) domain of PC abolished its ability to compete indicating that the gla-domain was essential for cell binding. Surprisingly, APC binding to these cells appeared to be independent of protein S, a cofactor of APC generally thought to be required for its high affinity binding to cell surfaces. The identity of the cell binding site(s), for the most part, appeared to be distinct from other known APC ligands which are associated with cell membranes or extracellular matrix including phospholipid, thrombomodulin, factor V, plasminogen activator inhibitor type 1 (PAI-1) and heparin. Pretreatment of HUVEC with antifactor VIII antibody caused partial inhibition of 125I-APC binding indicating that factor VIII or a homolog accounted for ∼30% of APC binding. Studies of the properties of surface bound 125I-APC or 125I-PC and their fate at 4°C compared to 37 °C were consistent with association of ∼25% of the initially bound radioligand with an endocytic receptor. However, most of the radioligand appeared not to be bound to an endocytic receptor and dissociated rapidly at 37° C in an intact and functional state. These data indicate the presence of specific, high affinity binding sites for APC and PC on the surface of HUVEC. While a minor proportion of binding sites may be involved in endocytosis, the identity and function of the major proportion is presently unknown. It is speculated that this putative receptor may be a further mechanisms of localizing the PC antithrombotic system to the vascular endothelium.

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