Studies on the biotin-binding site of avidin. Minimized fragments that bind biotin

Y Hiller; E A Bayer; M Wilchek

doi:10.1042/bj2780573

Studies on the biotin-binding site of avidin. Minimized fragments that bind biotin

Biochemical Journal ◽

10.1042/bj2780573 ◽

1991 ◽

Vol 278 (2) ◽

pp. 573-585 ◽

Cited By ~ 23

Author(s):

Y Hiller ◽

E A Bayer ◽

M Wilchek

Keyword(s):

Binding Site ◽

Binding Capacity ◽

Binding Activity ◽

Cleavage Sites ◽

Structural Elements ◽

Peptide Bonds ◽

Binding Function ◽

Biotin Binding ◽

Hydrolysis Of ◽

Extreme Stability

The object of this study was to define minimized biotin-binding fragments, or ‘prorecognition sites’, of either the egg-white glycoprotein avidin or its bacterial analogue streptavidin. Because of the extreme stability to enzymic hydrolysis, fragments of avidin were prepared by chemical means and examined for their individual biotin-binding capacity. Treatment of avidin with hydroxylamine was shown to result in new cleavage sites in addition to the known Asn-Gly cleavage site (position 88-89 in avidin). Notably, the Asn-Glu and Asp-Lys peptide bonds (positions 42-43 and 57-58 respectively) were readily cleaved; in addition, lesser levels of hydrolysis of the Gln-Pro (61-62) and Asn-Asp (12-13 and 104-105) bonds could be detected. The smallest biotin-binding peptide fragment, derived from hydroxylamine cleavage of either native or non-glycosylated avidin, was identified to comprise residues 1-42. CNBr cleavage resulted in a 78-amino acid-residue fragment (residues 19-96) that still retained activity. The data ascribe an important biotin-binding function to the overlapping region (residues 19-42) of avidin, which bears the single tyrosine moiety. This contention was corroborated by synthesizing a tridecapeptide corresponding to residues 26-38 of avidin; this peptide was shown to recognize biotin. Streptavidin was not susceptible to either enzymic or chemical cleavage methods used in this work. The approach taken in this study enabled the experimental distinction between the chemical and structural elements of the binding site. The capacity to assign biotin-binding activity to the tyrosine-containing domain of avidin underscores its primary chemical contribution to the binding of biotin by avidin.

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The vitronectin-binding function of PAI-1 exacerbates lung fibrosis in mice

Blood ◽

10.1182/blood-2010-12-324574 ◽

2011 ◽

Vol 118 (8) ◽

pp. 2313-2321 ◽

Cited By ~ 32

Author(s):

Anthony J. Courey ◽

Jeffrey C. Horowitz ◽

Kevin K. Kim ◽

Timothy J. Koh ◽

Margaret L. Novak ◽

...

Keyword(s):

Animal Studies ◽

Matrix Protein ◽

Binding Capacity ◽

Critical Role ◽

Plasminogen Activator Inhibitor ◽

Binding Activity ◽

Extracellular Matrix Protein ◽

Plasminogen Activator Inhibitor 1 ◽

Binding Function ◽

Pai 1

Abstract Plasminogen activator inhibitor-1 (PAI-1) is increased in the lungs of patients with pulmonary fibrosis, and animal studies have shown that experimental manipulations of PAI-1 levels directly influence the extent of scarring that follows lung injury. PAI-1 has 2 known properties that could potentiate fibrosis, namely an antiprotease activity that inhibits the generation of plasmin, and a vitronectin-binding function that interferes with cell adhesion to this extracellular matrix protein. To determine the relative importance of each PAI-1 function in lung fibrogenesis, we administered mutant PAI-1 proteins that possessed either intact antiprotease or vitronectin-binding activity to bleomycin-injured mice genetically deficient in PAI-1. We found that the vitronectin-binding capacity of PAI-1 was the primary determinant required for its ability to exacerbate lung scarring induced by intratracheal bleomycin administration. The critical role of the vitronectin-binding function of PAI-1 in fibrosis was confirmed in the bleomycin model using mice genetically modified to express the mutant PAI-1 proteins. We conclude that the vitronectin-binding function of PAI-1 is necessary and sufficient in its ability to exacerbate fibrotic processes in the lung.

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Studies on the biotin-binding site of streptavidin. Tryptophan residues involved in the active site

Biochemical Journal ◽

10.1042/bj2560279 ◽

1988 ◽

Vol 256 (1) ◽

pp. 279-282 ◽

Cited By ~ 22

Author(s):

G Gitlin ◽

E A Bayer ◽

M Wilchek

Keyword(s):

Binding Site ◽

Reversed Phase ◽

Binding Activity ◽

Complete Loss ◽

Tryptophan Residue ◽

Tryptophan Residues ◽

Lysine Residues ◽

Biotin Binding ◽

Peptide Fractions ◽

Specific Reagent

Streptavidin, the non-glycosylated bacterial analogue of the egg-white glycoprotein avidin, was modified with the tryptophan-specific reagent 2-hydroxy-5-nitrobenzyl (Hnb) bromide. As with avidin, complete loss of biotin-binding activity was achieved upon modification of an average of one tryptophan residue per streptavidin subunit. Tryptic peptides obtained from an Hnb-modified streptavidin preparation were fractionated by reversed-phase h.p.l.c., and three major Hnb-containing peptide fractions were isolated. Amino acid and N-terminal sequence analysis revealed that tryptophan residues 92, 108 and 120 are modified and probably comprise part of the biotin-binding site of the streptavidin molecule. Unlike avidin, the modification of lysine residues in streptavidin failed to result in complete loss of biotin-binding activity. The data imply subtle differences in the fine structure of the respective biotin-binding sites of the two proteins.

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Molecular dissection of a LIM domain.

Molecular Biology of the Cell ◽

10.1091/mbc.8.2.219 ◽

1997 ◽

Vol 8 (2) ◽

pp. 219-230 ◽

Cited By ~ 62

Author(s):

K L Schmeichel ◽

M C Beckerle

Keyword(s):

Protein Binding ◽

Protein Interactions ◽

Binding Capacity ◽

Specific Protein ◽

Binding Activity ◽

Zinc Binding ◽

Nucleic Acid Binding ◽

Lim Domain ◽

Binding Function ◽

Sequence Elements

LIM domains are novel sequence elements that are found in more than 60 gene products, many of which function as key regulators of developmental pathways. The LIM domain, characterized by the cysteine-rich consensus CX2CX16-23HX2CX2CX2CX16-21 CX2-3(C/H/ D), is a specific mental-binding structure that consists of two distinct zinc-binding subdomains. We and others have recently demonstrated that the LIM domain mediates protein-protein interactions. However, the sequences that define the protein-binding specificity of the LIM domain had not yet been identified. Because structural studies have revealed that the C-terminal zinc-binding module of a LIM domain displays a tertiary fold compatible with nucleic acid binding, it was of interest to determine whether the specific protein-binding activity of a LIM domain could be ascribed to one of its two zinc-binding subdomains. To address this question, we have analyzed the protein-binding capacity of a model LIM peptide, called zLIM1, that is derived from the cytoskeletal protein zyxin. These studies demonstrate that the protein-binding function of zLIM1 can be mapped to sequences contained within its N-terminal zinc-binding module. The C-terminal zinc-binding module of zLIM1 may thus remain accessible to additional interactive partners. Our results raise the possibility that the two structural subdomains of a LIM domain are capable of performing distinct biochemical functions.

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LIM domains of cysteine-rich protein 1 (CRP1) are essential for its zyxin-binding function

Biochemical Journal ◽

10.1042/bj3310885 ◽

1998 ◽

Vol 331 (3) ◽

pp. 885-892 ◽

Cited By ~ 20

Author(s):

Karen L. SCHMEICHEL ◽

Mary C. BECKERLE

Keyword(s):

Binding Site ◽

Protein Interactions ◽

Specific Protein ◽

Binding Activity ◽

Structural Motif ◽

Lim Domain ◽

Lim Domains ◽

Cysteine Rich Protein ◽

Binding Function ◽

Domain Mapping

Previous studies have demonstrated that the adhesion-plaque protein, zyxin, interacts specifically with a 23 kDa protein, called the cysteine-rich protein 1 (CRP1), which has been implicated in myogenesis. Primary sequence analyses have revealed that both zyxin and CRP1 exhibit multiple copies of a structural motif called the LIM domain. LIM domains, which are defined by the consensus CX2CX16–23HX2CX2CX2CX16–23CX2–3(C,H,D), are found in a variety of proteins that are involved in cell growth and differentiation. Recent studies have established that LIM domains are zinc-binding structures that mediate specific protein–protein interactions. For example, in the case of the zyxin–CRP1 interaction, one of zyxin's three LIM domains is necessary and sufficient for binding to CRP1. Because the CRP1 molecule is comprised primarily of two LIM domains, we were interested in the possibility that the binding site for zyxin on CRP1 might also be contained within a single LIM domain. Consistent with the hypothesis that the LIM domains of CRP1 are critical for the protein's zyxin-binding function, zinc-depleted CRP1 displays a reduced zyxin-binding activity. However, domain mapping analyses have revealed that neither of the two individual LIM domains of CRP1 can support a wild-type interaction with zyxin. Collectively, our results suggest that the binding site for zyxin on CRP1 is not contained within a single contiguous sequence of amino acids. Instead, the interaction appears to rely on the co-ordinate action of a number of residues that are displayed in both of CRP1's LIM domains.

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Erythropoietin binding sites in human foetal tissues

Acta Endocrinologica ◽

10.1530/acta.0.1160561 ◽

1987 ◽

Vol 116 (4) ◽

pp. 561-567 ◽

Cited By ~ 11

Author(s):

F. Pekonen ◽

K. Rosenlöf ◽

E.-M. Rutanen ◽

F. Fyhrquist

Keyword(s):

Binding Site ◽

Binding Sites ◽

Recombinant Dna ◽

Binding Capacity ◽

Specific Binding ◽

First Trimester ◽

Binding Activity ◽

Renin Substrate ◽

Human Erythropoietin ◽

Foetal Liver

Abstract. Using 125I labelled recombinant DNA human erythropoietin (EP), we have explored the presence and properties of EP binding sites in foetal human tissues. The EP binding site is present in the foetal liver already during the first trimester of pregnancy. The binding site has an equilibrium association constant of 4.1–6.2 × 109 1/mol and is specific for EP. The cross-reactivities of FSH, TSH, hCG, insulin and renin substrate were less than 0.01%. The EP binding capacity of foetal liver was 5.4–16 fmol/mg membrane protein. In foetal lung tissue, a slight EP binding activity was observed, whereas foetal spleen, muscle, brain, thyroid and placental tissues were virtually devoid of EP binding capacity. The same level of binding was reached at 37°C in 1 h and at 4°C in 24 h. The binding was pH-dependent with maximal specific binding at pH 7.7. SDS-PAGE gel electrophoresis analysis of covalently cross-linked 125I-EP to foetal liver membranes suggested that the EP binding site was composed of two subunits with an apparent mol wt of 41000 and 86000 dalton, respectively.

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Studies on the biotin-binding sites of avidin and streptavidin. Tyrosine residues are involved in the binding site

Biochemical Journal ◽

10.1042/bj2690527 ◽

1990 ◽

Vol 269 (2) ◽

pp. 527-530 ◽

Cited By ~ 45

Author(s):

G Gitlin ◽

E A Bayer ◽

M Wilchek

Keyword(s):

Binding Site ◽

Binding Sites ◽

Egg White ◽

Binding Activity ◽

Tyrosine Residue ◽

Tyrosine Residues ◽

Egg White Protein ◽

Biotin Binding ◽

The Difference ◽

Specific Reagent

The involvement of tyrosine in the biotin-binding sites of the egg-white glycoprotein avidin and the bacterial protein streptavidin was examined by using the tyrosine-specific reagent p-nitrobenzenesulphonyl fluoride (Nbs-F). Modification of an average of about 0.5 mol of tyrosine residue/mol of avidin subunit caused the complete loss of biotin binding. This indicates that the single tyrosine residue (Tyr-33) in the avidin subunit is directly involved in the biotin-binding site and that its modification by Nbs also abolishes the binding properties of a neighbouring subunit. This suggests that the tyrosine residues of the egg-white protein may also contribute to the stabilization of the native protein structure. In streptavidin, however, the modification of an average of 3 mol of tyrosine residue/mol of subunit was required to inactivate completely the biotin-binding activity of the protein, but only 1 mol (average) of tyrosine residue/mol of subunit was protected in the presence of biotin. The difference between the h.p.l.c. elution profiles of the enzymic digests of Nbs-modified streptavidin and the Nbs-modified streptavidin-biotin complex revealed two additional fractions in the unprotected protein that contain Nbs-modified tyrosine residues. These residues, Tyr-43 (major fraction) and Tyr-54 (minor fraction), appear to contribute to the biotin-binding site in streptavidin.

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Studies on the biotin-binding site of avidin. Tryptophan residues involved in the active site

Biochemical Journal ◽

10.1042/bj2500291 ◽

1988 ◽

Vol 250 (1) ◽

pp. 291-294 ◽

Cited By ~ 30

Author(s):

G Gitlin ◽

E A Bayer ◽

M Wilchek

Keyword(s):

Binding Site ◽

Active Site ◽

Egg White ◽

Binding Activity ◽

Complete Loss ◽

Tryptophan Residue ◽

Tryptophan Residues ◽

Biotin Binding ◽

Specific Reagent

Egg-white avidin was modified with the tryptophan-specific reagent 2-hydroxy-5-nitrobenzyl bromide. The complete loss of biotin-binding activity was achieved upon modification of an average of one tryptophan residue per avidin subunit. The identity of the modified residues was determined by isolating the relevant tryptic and chymotryptic peptides from CNBr-cleaved avidin fragments. The results demonstrate that Trp-70 and Trp-110 are modified in approximately equivalent proportions. It is believed that these residues are located in the active site of avidin and take part in the binding of biotin.

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Plasma Derived von Willebrand Factor Preparations: Collagen Binding and Ristocetin Cofactor Activities

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657654 ◽

1997 ◽

Vol 78 (02) ◽

pp. 930-933 ◽

Cited By ~ 11

Author(s):

Ping Chang ◽

D L Aronson

Keyword(s):

Von Willebrand Factor ◽

Functional Activity ◽

Binding Activity ◽

Collagen Binding ◽

Binding Function ◽

Cofactor Activity ◽

Von Willebrand ◽

Willebrand Factor ◽

Ristocetin Cofactor ◽

Ristocetin Cofactor Activity

SummaryFive plasma preparations (11 lots) used in the treatment of von Willebrand’s disease (vWD) were evaluated. The collagen binding function of von Willebrand factor (vWF) containing preparations was compared with the ristocetin cofactor activity and the vWF antigen. Some preparations have higher ratio of functional activity (ristocetin cofactor and collagen binding) relative to the antigen than is found in normal plasma. The ristocetin cofactor activity and the collagen binding activity are tightly correlated (r = .95). Ultracentrifugal (UCF) analysis was used to compare the size distribution of vWf antigen, ristocetin cofactor and collagen binding activity. The sedimentation of all of the vWF parameters in the plasma products was slower than in plasma. In plasma products the ristocetin cofactor activity sediments the most rapidly, the collagen binding activity is slower and the antigen the slowest. The collagen/antigen ratio decreases with decreasing vWF size. Assignment of potency to vWF containing preparations utilizing the collagen binding activity may be more precise and as accurate as with the traditional ristocetin cofactor assay.

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