Identification of functioning regulatory sites and a new myosin binding site in the C-terminal 288 amino acids of caldesmon expressed from a human clone

1993 ◽  
Vol 14 (4) ◽  
pp. 385-391 ◽  
Author(s):  
P. A. J. Huber ◽  
C. S. Redwood ◽  
N. D. Avent ◽  
M. J. A. Tanner ◽  
S. B. Marston
Keyword(s):  
Amino Acids ◽  
Binding Site ◽  
Human Clone ◽  
Myosin Binding ◽  
Regulatory Sites

scholarly journals Peptides presenting the binding site of human CD4 for the HIV-1 envelope glycoprotein gp120

2012 ◽  
Vol 8 ◽  
pp. 1858-1866 ◽  
Author(s):  
Julia Meier ◽  
Kristin Kassler ◽  
Heinrich Sticht ◽  
Jutta Eichler
Keyword(s):  
Amino Acids ◽  
Binding Site ◽  
Envelope Glycoprotein ◽  
Conformational Stability ◽  
Cellular Receptor ◽  
Binding Assays ◽  
Glycoprotein Gp120 ◽  
Dynamics Simulations ◽  
Key Residues ◽  
Hiv 1

Based on the structure of the HIV-1 glycoprotein gp120 in complex with its cellular receptor CD4, we have designed and synthesized peptides that mimic the binding site of CD4 for gp120. The ability of these peptides to bind to gp120 can be strongly enhanced by increasing their conformational stability through cyclization, as evidenced by binding assays, as well as through molecular-dynamics simulations of peptide–gp120 complexes. The specificity of the peptide–gp120 interaction was demonstrated by using peptide variants, in which key residues for the interaction with gp120 were replaced by alanine or D-amino acids.

scholarly journals Finding the Binding Site of Peloruside A and its Secondary Effects in Saccharomyces Cerevisiae using a  Chemical Genetics Approach

2021 ◽  
Author(s):  
◽  
Reem Hanna
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Amino Acids ◽  
Flow Cytometry ◽  
Binding Site ◽  
Mammalian Cells ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Peloruside A ◽  
Microtubule Function

<p>Peloruside A, a natural product isolated from the marine sponge Mycale hentscheli, is a microtubule-stabilising agent that has a similar mechanism of action to the anticancer drug paclitaxel and is cytotoxic to cultured mammalian cells. Peloruside appears to bind to a distinct site on mammalian tubulin that is different from that of the taxoid-site drugs. Because of the high sequence homology between yeast and mammalian tubulin, Saccharomyces cerevisiae (S. cerevisiae) was used as a model organism to characterise the peloruside-binding site with the aim of advancing our understanding about this site on mammalian tubulin. Wild type S. cerevisiae (BY4741) was sensitive to peloruside at uM concentrations; however, a strain that lacks the mad2 (Mitotic Arrest Deficient 2) gene showed increased sensitivity to the drug at much lower uM concentrations. This gene is a component of the spindle-assembly checkpoint complex that delays the onset of anaphase in cells with defects in mitotic spindle assembly. The main aims of this project were to define the binding site of peloruside A using yeast tubulin to see if microtubule function and/or morphology is altered in yeast by peloruside, and to identify any secondary drug targets "friends of the target" through chemical genetic interactions profiling (Homozygous deletion profiling microarray). Site-directed mutagenesis was used to mutate two conserved amino acids (A296T; R306H) known to confer resistance to peloruside in mammalian cells. Based on a published computer model of the peloruside binding site on mammalian tubulin, we also mutated three other amino acids, two that were predicted to affect peloruside binding (Q291M and N337L), and one that was predicted to affect laulimalide binding but have little affect on peloruside binding (V333W). We also included a negative control that was predicted to have no effect on peloruside binding (R282Q) and would affect epothilone binding. We found that of the six point mutations, only Q291M failed to confer resistance in yeast and instead it increased the inhibition to the drug. Using a bud index assay, confocal microscopy, and flow cytometry, 40-50 uM peloruside was shown to block cells in G2/M of the cell cycle, confirming a direct action of the drug on microtubule function. Homozygous profiling (HOP) microarray analysis of a deletion mutant set of yeast genes was also carried out to identify gene products that interact with peloruside in order to link the drug to specific networks or biochemical pathways in the cells. From site-directed mutagenesis, we concluded that peloruside binds to yeast B-tubulin in the region predicted by the published model of the binding site, and therefore mapping the site on yeast tubulin could provide useful information about the mammalian binding site for peloruside. The bud index, flow cytometry, and confocal microscopy experiments provided further evidence that peloruside interacts with yeast tubulin. From HOP we found that peloruside has roles in the cell cycle, as expected, and has effects on protein transport, secretion, cell wall synthesis, and steroid biosynthesis pathways.</p>

scholarly journals In-silico Investigation of the Interaction between Beta-class Glutathione S-Transferase and Five Antibiotics, namely; Ampicillin, Tetracycline, Chloramphenicol, Ciprofloxacin and Cephalexin

2021 ◽  
Vol 25 (4) ◽  
pp. 497-502
Author(s):  
D. Shehu ◽  
S Danlami ◽  
M. Ya’u ◽  
A. Babandi ◽  
H.M. Yakasai ◽  
...  
Keyword(s):  
Amino Acids ◽  
Antibiotic Resistance ◽  
Molecular Docking ◽  
Binding Site ◽  
Substrate Binding ◽  
Docking Study ◽  
Glutathione S Transferase ◽  
Molecular Docking Study ◽  
Substrate Binding Site ◽  
Glutathione S Transferases

Glutathione s-transferases(GSTs) are enzymes involved in the conjugation and deactivation of various xenobiotics including drugs. Thisin-silico study was undertaken in order to investigate the interaction between beta-class glutathione s-transferase and five selected antibiotics, namely; ampicillin, tetracycline, chloramphenicol, ciprofloxacin and cephalexin using molecular docking study. RaptorX server was used to predict the amino acids involved at the binding sitewhile molecular docking study was employed in order to investigate the binding interactions.RaptorX predicted several amino acids which were different from the ones observed in molecular docking because of the variability in the substrate binding site of GSTs however, all the amino acids predicted by RaptorX were also found to be involved in the GSH binding.Lys107, Phe109, Ser110, Leu113, Trp114, His115 and Arg123, Leu168 were the amino acids involved in the binding of various antibiotics to the substrate binding site of the protein while Ala9, Cys10, Leu32, Tyr51, Val52, Pro53, Glu65 and Ala66were involved in the binding of the co-substrate GSH to the binding site of the protein. The results indicated that all the antibiotics showed a good binding affinity with the beta class GST and are therefore capable of deactivating the drugs. With these, finding a beta class GST inhibitors alongside antibiotics during a treatment of diseases will be of beneficial in the current fight against antibiotic resistance.

A crosslinked and ribosylated actin trimer does not interact productively with myosin

2019 ◽  
Vol 97 (2) ◽  
pp. 140-147 ◽  
Author(s):  
Navneet Sidhu ◽  
John F. Dawson
Keyword(s):  
Binding Site ◽  
Protein Interactions ◽  
Binding Proteins ◽  
Binding Protein ◽  
Dnase I ◽  
Protein Protein Interactions ◽  
Adp Ribosylation ◽  
Myosin Binding ◽  
Pointed End

A purified F-actin-derived actin trimer that interacts with end-binding proteins did not activate or bind the side-binding protein myosin under rigor conditions. Remodeling of the actin trimer by the binding of gelsolin did not rescue myosin binding, nor did the use of different means of inhibiting the polymerization of the trimer. Our results demonstrate that ADP-ribosylation on all actin subunits of an F-actin-derived trimer inhibits myosin binding and that the binding of DNase-I to the pointed end subunits of a crosslinked trimer also remodels the myosin binding site. Taken together, this work highlights the need for a careful balance between modification of actin subunits and maintaining protein–protein interactions to produce a physiologically relevant short F-actin complex.

Identification of the A-band localization domain of myosin binding proteins C and H (MyBP-C, MyBP-H) in skeletal muscle

1999 ◽  
Vol 112 (1) ◽  
pp. 69-79 ◽  
Author(s):  
R. Gilbert ◽  
J.A. Cohen ◽  
S. Pardo ◽  
A. Basu ◽  
D.A. Fischman
Keyword(s):  
Skeletal Muscle ◽  
Amino Acids ◽  
Binding Proteins ◽  
Thick Filament ◽  
Terminal Segment ◽  
Binding Domain ◽  
Striated Muscles ◽  
Fibronectin Type Iii ◽  
Chimeric Construct ◽  
Myosin Binding

Although major constituents of the thick filaments of vertebrate striated muscles, the myosin binding proteins (MyBP-C and MyBP-H) are still of uncertain function. Distributed in the cross-bridge bearing zone of the A-bands of myofibrils, in a series of transverse 43 nm stripes, the proteins are constructed of a tandem series of small globular domains, each composed of approximately 90–100 amino acids, which have sequence similarities to either the C2-set of immunoglobulins (IgC2) and the fibronectin type III (FnIII) motifs. MyBP-C is composed of ten globular domains (approximately 130 kDa) whereas MyBP-H is smaller (approximately 58 kDa) and consists of a unique N-terminal segment followed by four globular domains, the order of which is identical to that of MyBP-C (FnIII-IgC2-FnIII-IgC2). To improve our understanding of this protein family we have characterized the domains in each of these two proteins which are required for targeting the proteins to their native site(s) in the sarcomere during myogenesis. Cultures of skeletal muscle myoblasts were transfected with expression plasmids encoding mutant constructs of the MyBPs bearing an N-terminal myc epitope, and their localization to the A-band examined by immunofluorescence microscopy. Based on the clarity and intensity of the myc A-band signals we concluded that constructs encoding the four C-terminal motifs of MyBP-C and MyBP-H (approximately 360 amino acids) were all that was necessary to efficiently localize each of these peptides to the A-band. Truncation mutants lacking one of these 4 domains were less efficiently targeted to the C-zone of the sarcomere. Deletion of the last C-terminal motif of MyBP-H, its myosin binding domain, abolished all localization to the A-band. A chimeric construct, HU-3C10, in which the C-terminal motif of MyBP-H was replaced by the myosin binding domain of MyBP-C, efficiently localized to the A-band. Taken together, these observations indicate that MyBP-C and MyBP-H are localized to the A-band by the same C-terminal domain, composed of two IgC2 and two FnIII motifs. A model has been proposed for the interaction and positioning of the MyBPs in the thick filament through a ternary complex of the four C-terminal motifs with the myosin rods and titin.

The carboxyl terminus of myosin binding protein C (MyBP-C, C-protein) specifies incorporation into the A-band of striated muscle

1996 ◽  
Vol 109 (1) ◽  
pp. 101-111 ◽  
Author(s):  
R. Gilbert ◽  
M.G. Kelly ◽  
T. Mikawa ◽  
D.A. Fischman
Keyword(s):  
Amino Acids ◽  
Protein C ◽  
Binding Protein ◽  
Thick Filament ◽  
Binding Domain ◽  
C Protein ◽  
Amino Terminal ◽  
Myosin Binding Protein ◽  
Myosin Binding Protein C ◽  
Myosin Binding

Myosin binding protein-C (MyBP-C), also known as C-protein, is a major constituent of the thick filaments of vertebrate striated muscles. The protein, approximately 130 kDa, consists of a series of 10 globular motifs (numbered I to X) each of approximately 90–100 amino acids, bearing resemblance to the C2-set of immunoglobins (Ig C2) and to the fibronectin type III (FnIII) motifs. Using pure preparations of myosin and MyBP-C, it has been demonstrated that the major myosin binding domain of MyBP-C resides within the C-terminal Ig C2 motif (motif X). However, in the context of the in vivo thick filament, it is uncertain if the latter domain is sufficient to target MyBP-C correctly to the A-band or if other regions of the molecule are required for this process. To answer this question, cultures of skeletal muscle myoblasts were transfected with expression plasmids encoding seven truncation mutants of MyBP-C, and their targeting to the A-band investigated by immunofluorescence microscopy. To distinguish the recombinant proteins from endogenous MyBP-C, a myc epitope was inserted at each amino terminus. Recombinant MyBP-C exhibited an identical distribution in the sarcomere to that of native MyBP-C; i.e. it was found exclusively in the C-zone of the A-band. A mutant encoding the C-terminal 372 amino acids, but lacking motifs I-VI (termed delta 1–6), also targeted correctly to the A-band. This fragment, which is composed of two Ig C2 and two FnIII motifs, was the minimal protein fragment required for correct A-band incorporation. Larger amino-terminal deletions or deletion of motif X, the myosin binding domain, abolished all localization to the A-band. One construct (delta 10) lacking only motif X strongly inhibited myofibril assembly. We conclude that the myosin binding domain of MyBP-C, although essential, is not sufficient for correct incorporation into the A-band and that motifs VII to IX are required for this process. The data suggest a topological model in which MyBP-C is associated with the thick filament through its C terminus.

An essential Saccharomyces cerevisiae gene homologous to SNF2 encodes a helicase-related protein in a new family

1992 ◽  
Vol 12 (4) ◽  
pp. 1893-1902
Author(s):  
B C Laurent ◽  
X Yang ◽  
M Carlson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Binding Site ◽  
Transcriptional Activation ◽  
Nucleoside Triphosphate ◽  
Related Protein ◽  
New Family ◽  
Eukaryotic Proteins ◽  
Beta Galactosidase

The Saccharomyces cerevisiae SNF2 gene affects the expression of many diversely regulated genes and has been implicated in transcriptional activation. We report here the cloning and characterization of STH1, a gene that is homologous to SNF2. STH1 is essential for mitotic growth and is functionally distinct from SNF2. A bifunctional STH1-beta-galactosidase protein is located in the nucleus. The predicted 155,914-Da STH1 protein is 72% identical to SNF2 over 661 amino acids and 46% identical over another stretch of 66 amino acids. Both STH1 and SNF2 contain a putative nucleoside triphosphate-binding site and sequences resembling the consensus helicase motifs. The large region of homology shared by STH1 and SNF2 is conserved among other eukaryotic proteins, and STH1 and SNF2 appear to define a novel family of proteins related to helicases.

scholarly journals Effects of Substrate-Binding Site Residues on the Biochemical Properties of a Tau Class Glutathione S-Transferase from Oryza sativa

Genes ◽  
2019 ◽  
Vol 11 (1) ◽  
pp. 25 ◽  
Author(s):  
Xue Yang ◽  
Jinchi Wei ◽  
Zhihai Wu ◽  
Jie Gao
Keyword(s):  
Amino Acids ◽  
Enzyme Activity ◽  
Abiotic Stress ◽  
Binding Site ◽  
Stress Responses ◽  
Stress Resistance ◽  
Substrate Binding ◽  
Biochemical Properties ◽  
Biotic And Abiotic Stress ◽  
Substrate Binding Site

Glutathione S-transferases (GSTs)—an especially plant-specific tau class of GSTs—are key enzymes involved in biotic and abiotic stress responses. To improve the stress resistance of crops via the genetic modification of GSTs, we predicted the amino acids present in the GSH binding site (G-site) and hydrophobic substrate-binding site (H-site) of OsGSTU17, a tau class GST in rice. We then examined the enzyme activity, substrate specificity, enzyme kinetics and thermodynamic stability of the mutant enzymes. Our results showed that the hydrogen bonds between Lys42, Val56, Glu68, and Ser69 of the G-site and glutathione were essential for enzyme activity and thermal stability. The hydrophobic side chains of amino acids of the H-site contributed to enzyme activity toward 4-nitrobenzyl chloride but had an inhibitory effect on enzyme activity toward 1-chloro-2,4-dinitrobenzene and cumene hydroperoxide. Different amino acids of the H-site had different effects on enzyme activity toward a different substrate, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. Moreover, Leu112 and Phe162 were found to inhibit the catalytic efficiency of OsGSTU17 to 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, while Pro16, Leu112, and Trp165 contributed to structural stability. The results of this research enhance the understanding of the relationship between the structure and function of tau class GSTs to improve the abiotic stress resistance of crops.

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