scholarly journals Molecular dissection of a LIM domain.

1997 ◽  
Vol 8 (2) ◽  
pp. 219-230 ◽  
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.

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

1998 ◽  
Vol 331 (3) ◽  
pp. 885-892 ◽  
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.

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

Blood ◽  
2011 ◽  
Vol 118 (8) ◽  
pp. 2313-2321 ◽  
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.

scholarly journals An unusual arrangement of 13 zinc fingers in the vertebrate gene Z13

1995 ◽  
Vol 311 (1) ◽  
pp. 219-224 ◽  
Author(s):  
T C Schulz ◽  
B Hopwood ◽  
P D Rathjen ◽  
J R Wells
Keyword(s):  
Dna Binding ◽  
Zinc Finger ◽  
Protein Interactions ◽  
Structural Organization ◽  
Zinc Fingers ◽  
Binding Activity ◽  
Protein Domain ◽  
Nucleic Acid Binding ◽  
C Terminus ◽  
Wide Range

The zinc finger is a protein domain that imparts specific nucleic acid-binding activity on a wide range of functionally important proteins. In this paper we report the molecular cloning and characterization of a novel murine zinc-finger gene, mZ13. Analysis of mZ13 cDNAs revealed that the gene expresses a 794-amino-acid protein encoded by a 2.7 kb transcript. The protein has an unusual arrangement of 13 zinc fingers into a ‘hand’ of 12 tandem fingers and a single isolated finger near the C-terminus. This structural organization is conserved with the probable chicken homologue, cZ13. mZ13 also contained an additional domain at the N-terminus which has previously been implicated in the regulation of zinc-finger transcription factor DNA-binding, via protein-protein interactions. mZ13 expression was detected in a wide range of murine embryonic and adult tissues. The structural organization of mZ13 and its expression profile suggest that it may function as a housekeeping DNA-binding protein that regulates the expression of specific genes.

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

1991 ◽  
Vol 278 (2) ◽  
pp. 573-585 ◽  
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.

scholarly journals Cell-cycle-specific transcription termination within the human histone H3.3 gene is correlated with specific protein–DNA interactions

1997 ◽  
Vol 69 (2) ◽  
pp. 101-110 ◽  
Author(s):  
ALAN TAYLOR ◽  
LIQUN ZHANG ◽  
JOHN HERRMANN ◽  
BEI WU ◽  
LARRY KEDES ◽  
...  
Keyword(s):  
Protein Binding ◽  
Rna Polymerase Ii ◽  
Transcription Termination ◽  
Transcription Elongation ◽  
Specific Protein ◽  
Binding Activity ◽  
Mobility Shift ◽  
Nuclear Extracts

In vitro studies using highly purified calf thymus RNA polymerase II and a fragment spanning the first intron of H3.3 as template DNA have demonstrated the existence of a strong transcription termination site consisting of thymidine stretches. In this study, nuclear run-on experiments have been performed to assess the extent to which transcription elongation is blocked in vivo using DNA probes corresponding to regions 5′ and 3′ of the in vitro termination sites. These studies suggest that H3.3 expression is stimulated following the inhibition of DNA synthesis through the elimination of the transcription elongation block. Interestingly, both the in vivo and in vitro experiments have revealed that the transcriptional block/termination sites are positioned immediately downstream of a 73 bp region that has been over 90% conserved between the chicken and human H3.3 genes. The extreme conservation of this intronic region suggests a possible role in maintaining cis-acting function. Electrophoretic mobility shift experiments show that HeLa cell nuclear extracts contain protein factors that bind specifically to the region of transcription elongation block. Furthermore, we demonstrate a correlation between the protein binding activity and the transcriptional block in cells that have been either arrested at the initiation of S phase or were replication-interrupted by hydroxyurea. DNA footprinting experiments indicate that the region of protein binding is at the 3′ end of the conserved region and overlaps with one of the three in-vitro-mapped termination sites.

scholarly journals Systemweite Anreicherung von RNA-Protein-Komplexen

BIOspektrum ◽  
2020 ◽  
Vol 26 (6) ◽  
pp. 618-620
Author(s):  
Stefan Pfister ◽  
Benedikt M. Beckmann
Keyword(s):  
Physicochemical Properties ◽  
Protein Interactions ◽  
Protein Complexes ◽  
Rna Metabolism ◽  
Specific Protein ◽  
Cross Linking ◽  
Cell Physiology ◽  
Key Players ◽  
Sequence Elements ◽  
Rna Protein Complexes

Abstract RNA-protein complexes (RNPs) are key players in cell physiology, especially in the context of RNA metabolism. However, their analysis has been dependent on specific protein epitopes or RNA sequence elements, preventing unbiased and cell-wide studies. We developed a three-step protocol, termed Phenol Toluol extraction (PTex), to isolate the complete suite of RNPs in cells, solely based on their unique physicochemical properties after UV-cross-linking. PTex, along with other recently developed unbiased techniques, has the potential to guide the way to cell-wide analysis of RNA-protein interactions.

scholarly journals That zincing feeling: the effects of EDTA on the behaviour of zinc-binding transcriptional regulators

2004 ◽  
Vol 381 (3) ◽  
Author(s):  
Jennifer K. NYBORG ◽  
Olve B. PEERSEN
Keyword(s):  
Protein Interactions ◽  
Binding Proteins ◽  
Chelating Agent ◽  
Specific Protein ◽  
Regulatory Proteins ◽  
Zinc Binding ◽  
Creb Binding Protein ◽  
Protein Protein Interactions ◽  
Activator Proteins ◽  
Biochemical Journal

Zinc-binding proteins account for nearly half of the transcription regulatory proteins in the human genome and are the most abundant class of proteins in the human proteome. The zinc-binding transcriptional regulatory proteins utilize Zn2+ to fold structural domains that participate in intermolecular interactions. A study by Matt et al. in this issue of the Biochemical Journal has examined the transcription factor binding properties of the zinc-binding module C/H1 (cysteine/histidine-rich region 1) found in the transcriptional co-activator proteins CBP (CREB-binding protein) and p300. Their studies revealed that EDTA treatment of native C/H1 leads to irreversible denaturation and aggregation. Of particular concern is their finding that unfolded C/H1 participates in non-specific protein–protein interactions. The implications of these results are significant. EDTA is a very potent zinc-chelating agent that is used ubiquitously in protein interaction studies and in molecular biology in general. The potentially detrimental effects of EDTA on the structure and interactions of zinc-binding proteins should be taken into account in the interpretation of a sizeable number of published studies and must be considered in future experiments.

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