The N-Terminal Domain of the Flo1 Flocculation Protein from Saccharomyces cerevisiae Binds Specifically to Mannose Carbohydrates

ABSTRACTSaccharomyces cerevisiaecells possess a remarkable capacity to adhere to other yeast cells, which is called flocculation. Flocculation is defined as the phenomenon wherein yeast cells adhere in clumps and sediment rapidly from the medium in which they are suspended. These cell-cell interactions are mediated by a class of specific cell wall proteins, called flocculins, that stick out of the cell walls of flocculent cells. The N-terminal part of the three-domain protein is responsible for carbohydrate binding. We studied the N-terminal domain of the Flo1 protein (N-Flo1p), which is the most important flocculin responsible for flocculation of yeast cells. It was shown that this domain is both O and N glycosylated and is structurally composed mainly of β-sheets. The binding of N-Flo1p tod-mannose, α-methyl-d-mannoside, various dimannoses, and mannan confirmed that the N-terminal domain of Flo1p is indeed responsible for the sugar-binding activity of the protein. Moreover, fluorescence spectroscopy data suggest that N-Flo1p contains two mannose carbohydrate binding sites with different affinities. The carbohydrate dissociation constants show that the affinity of N-Flo1p for mono- and dimannoses is in the millimolar range for the binding site with low affinity and in the micromolar range for the binding site with high affinity. The high-affinity binding site has a higher affinity for low-molecular-weight (low-MW) mannose carbohydrates and no affinity for mannan. However, mannan as well as low-MW mannose carbohydrates can bind to the low-affinity binding site. These results extend the cellular flocculation model on the molecular level.

Download Full-text

Identification of a binding site on human FGF-2 for fibrinogen

Blood ◽

10.1182/blood-2003-08-2638 ◽

2004 ◽

Vol 103 (6) ◽

pp. 2114-2120 ◽

Cited By ~ 22

Author(s):

Hu Peng ◽

Abha Sahni ◽

Philip Fay ◽

Stephen Bellum ◽

Igor Prudovsky ◽

...

Keyword(s):

Binding Site ◽

Structural Changes ◽

Dissociation Constants ◽

Site Directed Mutagenesis ◽

Affinity Binding ◽

High Affinity Binding ◽

High Affinity ◽

The Third ◽

Fibrinogen Binding ◽

Adhesive Interactions

Abstract Endothelial cell adhesive interactions are mediated by both fibrinogen and fibrin, and growth is stimulated by fibroblast growth factor 2 (FGF-2). We have shown previously that FGF-2 binds specifically and with high affinity to fibrinogen and fibrin and that fibrinogen potentiates the proliferative capacity of FGF-2 and also protects it from proteolytic degradation. To further characterize this interaction we have performed FGF-2 mutagenesis to identify the interactive site. Because FGF-1 has a similar structure to FGF-2 but does not bind to fibrinogen, we used a strategy of cassette and site-directed mutagenesis, exchanging residues from FGF-1 and FGF-2 and correlating structural changes with fibrinogen binding. Two cassette interchange mutants, 2212 and 2211, contained either the third cassette or both the third and fourth cassettes from FGF-1, and neither exhibited any affinity for fibrinogen. Exchange of 5 residues (Phe95, Ser100, Asn102, Arg107, and Arg109) from FGF-2 into the corresponding sites in the third cassette of FGF-1 imparted high-affinity binding with apparent dissociation constants (Kd) of 5.3 nM and 8.6 nM, respectively, compared with 1.3 nM for wild-type FGF-2. We conclude that these 5 residues define a high-affinity binding site in FGF-2 for fibrinogen.

Download Full-text

Cyanovirin-N Binds Viral Envelope Proteins at the Low-Affinity Carbohydrate Binding Site without Direct Virus Neutralization Ability

Molecules ◽

10.3390/molecules26123621 ◽

2021 ◽

Vol 26 (12) ◽

pp. 3621

Author(s):

Irene Maier ◽

Robert H. Schiestl ◽

Georg Kontaxis

Keyword(s):

Binding Site ◽

Binding Sites ◽

Affinity Maturation ◽

Viral Envelope ◽

Biologically Active ◽

Carbohydrate Binding ◽

Affinity Binding ◽

Protein Patterns ◽

High Affinity Binding ◽

High Affinity

Glycan-targeting antibodies and pseudo-antibodies have been extensively studied for their stoichiometry, avidity, and their interactions with the rapidly modifying glycan shield of influenza A. Broadly neutralizing antiviral agents bind in the same order when they neutralize enveloped viruses regardless of the location of epitopes to the host receptor binding site. Herein, we investigated the binding of cyanovirin-N (CV–N) to surface-expressed glycoproteins such as those of human immunodeficiency virus (HIV) gp120, hemagglutinin (HA), and Ebola (GP)1,2 and compared their binding affinities with the binding response to the trimer-folded gp140 using surface plasmon resonance (SPR). Binding-site knockout variants of an engineered dimeric CV–N molecule (CVN2) revealed a binding affinity that correlated with the number of (high-) affinity binding sites. Binding curves were specific for the interaction with N-linked glycans upon binding with two low-affinity carbohydrate binding sites. This biologically active assembly of a domain-swapped CVN2, or monomeric CV–N, bound to HA with a maximum KD of 2.7 nM. All three envelope spike proteins were recognized at a nanomolar KD, whereas binding to HIV neutralizing 2G12 by targeting HA and Ebola GP1,2 was measured in the µM range and specific for the bivalent binding scheme in SPR. In conclusion, invariant structural protein patterns provide a substrate for affinity maturation in the membrane-anchored HA regions, as well as the glycan shield on the membrane-distal HA top part. They can also induce high-affinity binding in antiviral CV–N to HA at two sites, and CVN2 binding is achieved at low-affinity binding sites.

Download Full-text

Characterization of a Mode of Specific Binding of Fibrin Monomer through its Amino-Terminal Domain by Macrophages and Macrophage Cell-Lines

Thrombosis and Haemostasis ◽

10.1055/s-0038-1645194 ◽

1990 ◽

Vol 63 (02) ◽

pp. 193-203 ◽

Cited By ~ 9

Author(s):

John R Shainoff ◽

Deborah J Stearns ◽

Patricia M DiBello ◽

Youko Hishikawa-Itoh

Keyword(s):

Peritoneal Macrophages ◽

Affinity Binding ◽

Macrophage Cell ◽

High Affinity Binding ◽

Amino Terminal ◽

High Affinity ◽

Terminal Domain ◽

Weak Binding ◽

Amino Terminal Domain

SummaryThe studies reported here probe the existence of a receptor-mediated mode of fibrin-binding by macrophages that is associated with the chemical change underlying the fibrinogen-fibrin conversion (the release of fibrinopeptides from the amino-terminal domain) without depending on fibrin-aggregation. The question is pursued by 1) characterization of binding in relation to fibrinopeptide content of both the intact protein and the CNBr-fragment comprising the amino-terminal domain known as the NDSK of the protein, 2) tests of competition for binding sites, and 3) photo-affinity labeling of macrophage surface proteins. The binding of intact monomers of types lacking either fibrinopeptide A alone (α-fibrin) or both fibrinopeptides A and B (αβ-fibrin) by peritoneal macrophages is characterized as proceeding through both a fibrin-specific low density/high affinity (BMAX ≃ 200–800 molecules/cell, KD ≃ 10−12 M) interaction that is not duplicated with fibrinogen, and a non-specific high density/low affinity (BMAX ≥ 105 molecules/cell, KD ≥ 10−6 M) interaction equivalent to the weak binding of fibrinogen. Similar binding characteristics are displayed by monocyte/macrophage cell lines (J774A.1 and U937) as well as peritoneal macrophages towards the NDSK preparations of these proteins, except for a slightly weaker (KD ≃ 10−10 M) high-affinity binding. The high affinity binding of intact monomer is inhibitable by fibrin-NDSK, but not fibrinogen-NDSK. This binding appears principally dependent on release of fibrinopeptide-A, because a species of fibrin (β-fibrin) lacking fibrinopeptide-B alone undergoes only weak binding similar to that of fibrinogen. Synthetic Gly-Pro-Arg and Gly-His-Arg-Pro corresponding to the N-termini of to the α- and the β-chains of fibrin both inhibit the high affinity binding of the fibrin-NDSKs, and the cell-adhesion peptide Arg-Gly-Asp does not. Photoaffinity-labeling experiments indicate that polypeptides with elec-trophoretically estimated masses of 124 and 187 kDa are the principal membrane components associated with specifically bound fibrin-NDSK. The binding could not be up-regulated with either phorbol myristyl acetate, interferon gamma or ADP, but was abolished by EDTA and by lipopolysaccharide. Because of the low BMAX, it is suggested that the high-affinity mode of binding characterized here would be too limited to function by itself in scavenging much fibrin, but may act cooperatively with other, less limited modes of fibrin binding.

Download Full-text

Faculty Opinions recommendation of Distribution of the high-affinity binding site and intracellular target of botulinum toxin type A in the human bladder.

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

10.3410/f.1649968.1152067 ◽

2010 ◽

Author(s):

Apostolos Apostolidis

Keyword(s):

Botulinum Toxin ◽

Binding Site ◽

Botulinum Toxin Type A ◽

Botulinum Toxin Type ◽

Affinity Binding ◽

Human Bladder ◽

High Affinity Binding ◽

High Affinity ◽

High Affinity Binding Site ◽

Intracellular Target

Download Full-text

Ontogeny of a specific high-affinity binding site for ovine placental lactogen in fetal and postnatal liver

Domestic Animal Endocrinology ◽

10.1016/0739-7240(95)00030-i ◽

1995 ◽

Vol 12 (4) ◽

pp. 337-347 ◽

Cited By ~ 6

Author(s):

S.L. Pratt ◽

S.M. Kappes ◽

R.V. Anthony

Keyword(s):

Binding Site ◽

Placental Lactogen ◽

Affinity Binding ◽

High Affinity Binding ◽

High Affinity ◽

High Affinity Binding Site

Download Full-text

The collagen binding domain of fibronectin contains a high affinity binding site for Candida albicans.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)31752-0 ◽

1994 ◽

Vol 269 (35) ◽

pp. 22039-22045 ◽

Cited By ~ 6

Author(s):

E. Nègre ◽

T. Vogel ◽

A. Levanon ◽

R. Guy ◽

T.J. Walsh ◽

...

Keyword(s):

Candida Albicans ◽

Binding Site ◽

Binding Domain ◽

Affinity Binding ◽

Collagen Binding ◽

High Affinity Binding ◽

High Affinity ◽

High Affinity Binding Site

Download Full-text

Cloning and characterization of the low-affinity cyclic AMP phosphodiesterase gene of Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.7.10.3629-3636.1987 ◽

1987 ◽

Vol 7 (10) ◽

pp. 3629-3636

Author(s):

J Nikawa ◽

P Sass ◽

M Wigler

Keyword(s):

Saccharomyces Cerevisiae ◽

Cyclic Amp ◽

Copy Number ◽

Growth Arrest ◽

Yeast Cells ◽

Phosphodiesterase Activity ◽

Camp Phosphodiesterase ◽

High Affinity ◽

Cyclic Amp Phosphodiesterase

Saccharomyces cerevisiae contains two genes which encode cyclic AMP (cAMP) phosphodiesterase. We previously isolated and characterized PDE2, which encodes a high-affinity cAMP phosphodiesterase. We have now isolated the PDE1 gene of S. cerevisiae, which encodes a low-affinity cAMP phosphodiesterase. These two genes represent highly divergent branches in the evolution of phosphodiesterases. High-copy-number plasmids containing either PDE1 or PDE2 can reverse the growth arrest defects of yeast cells carrying the RAS2(Val-19) mutation. PDE1 and PDE2 appear to account for the aggregate cAMP phosphodiesterase activity of S. cerevisiae. Disruption of both PDE genes results in a phenotype which resembles that induced by the RAS2(Val-19) mutation. pde1- pde2- ras1- ras2- cells are viable.

Download Full-text