Probing Dictyostelium severin structure and function by cross linking to actin

2004 ◽  
Vol 82 (2) ◽  
pp. 343-350
Author(s):  
Joanna Summerscales ◽  
John F Dawson
Keyword(s):  
Structural Model ◽  
Sequence Similarity ◽  
Comparative Modeling ◽  
Actin Binding ◽  
Cross Linking ◽  
High Sequence Similarity ◽  
Calcium Dependent ◽  
A Minor ◽  
And Function ◽  
Chemical Cross Linking

DS151 is the first 151 amino acids of the Dictyostelium discoidium protein severin, which shares high sequence similarity with segment 1 of the actin-severing protein gelsolin. DS151 is able to mediate the depolymerization of F-actin in a calcium-dependent fashion, much like segment 1 of gelsolin. A structural model of DS151 was obtained by comparative modeling studies with segment 1 of gelsolin. This model was tested by studies of chemical cross linking between DS151 and bound actin, suggesting that Cys residues on DS151 are cross linked with Lys residues of actin. The model suggests that Cys125 of DS151 cross links with either Lys326 or Lys328 of actin. Mutagenesis of DS151 demonstrates that Cys125 of DS151 dominates the cross linking, whereas Cys25 of DS151 makes a minor con tribution through a longer-range cross link with Cys374 of actin, which likely involves flexibility of both proteins in that region.Key words: actin-binding proteins, homology modeling, chemical cross linking, actin-severing proteins.

scholarly journals Structure and Function of Filamin C in the Muscle Z-Disc

2020 ◽  
Vol 21 (8) ◽  
pp. 2696 ◽  
Author(s):  
Zhenfeng Mao ◽  
Fumihiko Nakamura
Keyword(s):  
Sequence Similarity ◽  
Actin Binding ◽  
Amino Acid Residues ◽  
Filamin A ◽  
Dimerization Domain ◽  
High Sequence Similarity ◽  
Actin Binding Domain ◽  
And Function ◽  
Filamin C

Filamin C (FLNC) is one of three filamin proteins (Filamin A (FLNA), Filamin B (FLNB), and FLNC) that cross-link actin filaments and interact with numerous binding partners. FLNC consists of a N-terminal actin-binding domain followed by 24 immunoglobulin-like repeats with two intervening calpain-sensitive hinges separating R15 and R16 (hinge 1) and R23 and R24 (hinge-2). The FLNC subunit is dimerized through R24 and calpain cleaves off the dimerization domain to regulate mobility of the FLNC subunit. FLNC is localized in the Z-disc due to the unique insertion of 82 amino acid residues in repeat 20 and necessary for normal Z-disc formation that connect sarcomeres. Since phosphorylation of FLNC by PKC diminishes the calpain sensitivity, assembly, and disassembly of the Z-disc may be regulated by phosphorylation of FLNC. Mutations of FLNC result in cardiomyopathy and muscle weakness. Although this review will focus on the current understanding of FLNC structure and functions in muscle, we will also discuss other filamins because they share high sequence similarity and are better characterized. We will also discuss a possible role of FLNC as a mechanosensor during muscle contraction.

scholarly journals Cross-linking of the electron-transfer flavoprotein to electron-transfer flavoprotein-ubiquinone oxidoreductase with heterobifunctional reagents

1988 ◽  
Vol 255 (3) ◽  
pp. 869-876 ◽  
Author(s):  
D J Steenkamp
Keyword(s):  
Electron Transfer ◽  
Small Subunit ◽  
Cross Linking ◽  
Minor Effect ◽  
Electron Transfer Flavoprotein ◽  
Ubiquinone Oxidoreductase ◽  
Lysine Residues ◽  
Cross Links ◽  
A Minor ◽  
Chemical Cross Linking

The mitochondrial electron-transfer flavoprotein (ETF) is a heterodimer containing only one FAD. In previous work on the structure-function relationships of ETF, its interaction with the general acyl-CoA dehydrogenase (GAD) was studied by chemical cross-linking with heterobifunctional reagents [D. J. Steenkamp (1987) Biochem. J. 243, 519-524]. GAD whose lysine residues were substituted with 3-(2-pyridyldithio)propionyl groups was preferentially cross-linked to the small subunit of ETF, the lysine residues of which had been substituted with 4-mercaptobutyramidine (MBA) groups. This work was extended to the interaction of ETF with ETF-ubiquinone oxidoreductase (ETF-Q ox). ETF-Q ox was partially inactivated by modification with N-succinimidyl 3-(2-pyridyldithio)propionate to introduce pyridyl disulphide structures. A similar modification of ETF caused a large increase in the apparent Michaelis constant of ETF-Q ox for modified ETF owing to the loss of positive charge on some critical lysines of ETF. When ETF-Q ox was modified with 2-iminothiolane to introduce 4-mercaptobutyramidine groups, only a minor effect on the activity of the enzyme was observed. To retain the positive charges on the lysine residues of ETF, pyridyl disulphide structures were introduced by treating ETF with 2-iminothiolane in the presence of 2,2′-dithiodipyridyl. The electron-transfer activity of the resultant ETF preparation containing 4-(2-pyridyldithio)butyramidine (PDBA) groups was only slightly affected. When ETF-Q ox substituted with MBA groups was mixed with ETF bearing PDBA groups, at least 70% of the cross-links formed between the two proteins were between the small subunit of ETF and ETF-Q ox. ETF-Q ox, therefore, interacts predominantly with the same subunit of ETF as GAD. Variables which affect the selectivity of ETF-Q ox cross-linking to the subunits of ETF are considered.

scholarly journals Involvement of caldesmon at the actin-myosin interface

1992 ◽  
Vol 287 (2) ◽  
pp. 633-637 ◽  
Author(s):  
M C Harricane ◽  
E Fabbrizio ◽  
C Arpin ◽  
D Mornet
Keyword(s):  
Ternary Complex ◽  
Actin Binding ◽  
Cross Linking ◽  
Binary Complex ◽  
Binding Region ◽  
Myosin Subfragment ◽  
Myosin Subfragment 1 ◽  
Spacer Arm ◽  
Protein Components ◽  
Chemical Cross Linking

Addition of myosin subfragment 1 (S-1) to the actin-caldesmon binary complex, which forms bundles of actin filaments resulted in the formation of actin/caldesmon-decorated filaments [Harricane, Bonet-Kerrache, Cavadore & Mornet (1991) Eur. J. Biochem. 196, 219-224]. The present data provide further evidence that caldesmon and S-1 compete for a common actin-binding region and demonstrate that a change occurs in the actin-myosin interface induced by caldesmon. S-1 digested by trypsin, which has an actin affinity 100-fold weaker than that of native S-1, was efficiently removed from actin by caldesmon, but not completely dissociated. This particular ternary complex was stabilized by chemical cross-linking with carbodi-imide, which does not have any spacer arm, and revealed contact interfaces between the different protein components. Cross-linking experiments showed that the presence of caldesmon had no effect on stabilization of actin-(20 kDa domain), whereas the actin-(50 kDa domain) covalent association was significantly decreased, to the point of being virtually abolished.

scholarly journals Identification of a novel receptor for an invertebrate oxytocin/vasopressin superfamily peptide: molecular and functional evolution of the oxytocin/vasopressin superfamily

2004 ◽  
Vol 382 (1) ◽  
pp. 231-237 ◽  
Author(s):  
Tsuyoshi KAWADA ◽  
Atsuhiro KANDA ◽  
Hiroyuki MINAKATA ◽  
Osamu MATSUSHIMA ◽  
Honoo SATAKE
Keyword(s):  
Sequence Similarity ◽  
Genomic Analysis ◽  
Egg Laying ◽  
Pcr Analysis ◽  
High Sequence Similarity ◽  
Calcium Dependent ◽  
Functional Correlation ◽  
Dependent Signal ◽  
Egg Laying Behaviour

Annetocin is structurally related to an OT (oxytocin)/VP (vasopressin) family peptide, which has been isolated from the earthworm Eisenia foetida and has been shown to induce OT-like egg-laying behaviour. We now report the identification of an endogenous AnR (annetocin receptor). The deduced AnR precursor displays high sequence similarity with OT/VP receptors. Genomic analysis of the AnR gene revealed that the intron-inserted position is conserved between the AnR gene and the mammalian OT/VP receptor genes. These results indicate that AnR and mammalian OT/VP receptors share a common ancestor gene. Administration of annetocin to the AnR expressed in Xenopus oocytes induced a calcium-dependent signal transduction. Reverse transcriptase–PCR analysis and in situ hybridization showed that the AnR gene is expressed specifically in the nephridia located in the clitellum region, although the nephridia are distributed throughout the worm body. This result suggests that annetocin induces egg-laying behaviour through its action on the nephridia. This is the first description concerning the functional correlation between an invertebrate OT/VP-related peptide and egg-laying behaviour.

scholarly journals The model structure of the copper-dependent ammonia monooxygenase

2020 ◽  
Vol 25 (7) ◽  
pp. 995-1007
Author(s):  
Francesco Musiani ◽  
Valquiria Broll ◽  
Elisa Evangelisti ◽  
Stefano Ciurli
Keyword(s):  
Crop Production ◽  
Rational Design ◽  
Structural Model ◽  
Sequence Similarity ◽  
Methane Monooxygenase ◽  
Ammonia Oxidizing Bacteria ◽  
Ammonia Monooxygenase ◽  
Particulate Methane Monooxygenase ◽  
High Sequence Similarity ◽  
Copper Site

Abstract Ammonia monooxygenase is a copper-dependent membrane-bound enzyme that catalyzes the first step of nitrification in ammonia-oxidizing bacteria to convert ammonia to hydroxylamine, through the reductive insertion of a dioxygen-derived O atom in an N–H bond. This reaction is analogous to that carried out by particulate methane monooxygenase, which catalyzes the conversion of methane to methanol. The enzymatic activity of ammonia monooxygenase must be modulated to reduce the release of nitrogen-based soil nutrients for crop production into the atmosphere or underground waters, a phenomenon known to significantly decrease the efficiency of primary production as well as increase air and water pollution. The structure of ammonia monooxygenase is not available, rendering the rational design of enzyme inhibitors impossible. This study describes a successful attempt to build a structural model of ammonia monooxygenase, and its accessory proteins AmoD and AmoE, from Nitrosomonas europaea, taking advantage of the high sequence similarity with particulate methane monooxygenase and the homologous PmoD protein, for which crystal structures are instead available. The results obtained not only provide the structural details of the proteins ternary and quaternary structures, but also suggest a location for the copper-containing active site for both ammonia and methane monooxygenases, as well as support a proposed structure of a CuA-analogue dinuclear copper site in AmoD and PmoD. Graphic abstract

scholarly journals Steric Regulation of Tandem Calponin Homology Domain Actin-Binding Affinity

2019 ◽  
Author(s):  
Andrew R Harris ◽  
Brian Belardi ◽  
Pamela Jreij ◽  
Kathy Wei ◽  
Hengameh Shams ◽  
...  
Keyword(s):  
Cellular Localization ◽  
Sequence Similarity ◽  
Point Mutations ◽  
Negative Regulator ◽  
Actin Binding ◽  
Binding Properties ◽  
High Sequence Similarity ◽  
Calponin Homology ◽  
Binding Domains

ABSTRACTTandem calponin homology (CH1-CH2) domains are common actin-binding domains in proteins that interact with and organize the actin cytoskeleton. Despite regions of high sequence similarity, CH1-CH2 domains can have remarkably different actin-binding properties, with disease-associated point mutants known to increase as well as decrease affinity for f-actin. To investigate features that affect CH1-CH2 affinity for f-actin in cells and in vitro, we perturbed the utrophin actin-binding domain by making point mutations at the CH1-CH2 interface, replacing the linker domain, and adding a PEG polymer to CH2. Consistent with a previous model describing CH2 as a steric negative regulator of actin binding, we find that utrophin CH1-CH2 affinity is both increased and decreased by modifications that change the effective ‘openness’ of CH1 and CH2 in solution. We also identified interface mutations that caused a large increase in affinity without changing solution ‘openness’, suggesting additional influences on affinity. Interestingly, we also observe non-uniform sub-cellular localization of utrophin CH1-CH2 that depends on the N-terminal flanking region but not on bulk affinity. These observations provide new insights into how small sequence changes, such as those found in diseases, can affect CH1-CH2 binding properties.

An analysis of splicing, actin-binding properties, heterodimerization and molecular interactions of the non-muscle α-actinins

2013 ◽  
Vol 452 (3) ◽  
pp. 477-488 ◽  
Author(s):  
Kate S. Foley ◽  
Paul W. Young
Keyword(s):  
Molecular Interactions ◽  
Molecular Basis ◽  
Sequence Similarity ◽  
Actin Binding ◽  
Interacting Proteins ◽  
Binding Properties ◽  
High Sequence Similarity ◽  
Glomerular Function ◽  
Proteomics Approach ◽  
First Time

The non-muscle α-actinin isoforms (actinin-1 and -4) are closely related dimeric actin filament cross-linking proteins. Despite high sequence similarity, unique properties have been ascribed to actinin-4 in particular. For example, actinin-4, but not actinin-1, is essential for normal glomerular function in the kidney, is overexpressed in several cancers and can translocate to the nucleus to regulate transcription. To understand the molecular basis for such isoform-specific functions we have, for the first time, comprehensively compared these proteins in terms of alternative splicing, actin-binding properties, heterodimer formation and molecular interactions. We find that the Ca2+-insensitive variant of actinin-4 is expressed only in the nervous system and thus cannot be regarded as a smooth muscle isoform, as is the case for the Ca2+-insensitive variant of actinin-1. The actin-binding properties of actinin-1 and -4 are similar and are unlikely to explain isoform-specific functions. Surprisingly, we reveal that actinin-1/-4 heterodimers, rather than homodimers, are the most abundant form of actinin in many cell lines. Finally, we use a proteomics approach to identify potential isoform-specific interactions. The results of the present study indicate that actinin-1 and -4 can readily form heterodimers composed of monomers that may have different properties and interacting proteins. This significantly alters our view of non-muscle actinin function.

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