Effects of substitutions in the CXXC active-site motif of the extracytoplasmic thioredoxin ResA

2008 ◽  
Vol 414 (1) ◽  
pp. 81-91 ◽  
Author(s):  
Allison Lewin ◽  
Allister Crow ◽  
Christopher T. C. Hodson ◽  
Lars Hederstedt ◽  
Nick E. Le Brun
Keyword(s):  
Active Site ◽  
Protein Unfolding ◽  
Cysteine Residues ◽  
Wild Type ◽  
Pka Values ◽  
Active Site Cysteine ◽  
Reduction Potentials ◽  
Type Protein ◽  
Wild Type Protein

The thiol–disulfide oxidoreductase ResA from Bacillus subtilis fulfils a reductive role in cytochrome c maturation. The pKa values for the CEPC (one-letter code) active-site cysteine residues of ResA are unusual for thioredoxin-like proteins in that they are both high (>8) and within 0.5 unit of each other. To determine the contribution of the inter-cysteine dipeptide of ResA to its redox and acid–base properties, three variants (CPPC, CEHC and CPHC) were generated representing a stepwise conversion into the active-site sequence of the high-potential DsbA protein from Escherichia coli. The substitutions resulted in large decreases in the pKa values of both the active-site cysteine residues: in CPHC (DsbA-type) ResA, ΔpKa values of −2.5 were measured for both cysteine residues. Increases in midpoint reduction potentials were also observed, although these were comparatively small: CPHC (DsbA-type) ResA exhibited an increase of +40 mV compared with the wild-type protein. Unfolding studies revealed that, despite the observed differences in the properties of the reduced proteins, changes in stability were largely confined to the oxidized state. High-resolution structures of two of the variants (CEHC and CPHC ResA) in their reduced states were determined and are discussed in terms of the observed changes in properties. Finally, the in vivo functional properties of CEHC ResA are shown to be significantly affected compared with those of the wild-type protein.

scholarly journals Identification of an autoinhibitory region in the activation loop of the Mos protein kinase.

1996 ◽  
Vol 16 (7) ◽  
pp. 3472-3479 ◽  
Author(s):  
S C Robertson ◽  
D J Donoghue
Keyword(s):  
Protein Kinase ◽  
Active Site ◽  
Kinase Activity ◽  
Molecular Model ◽  
Wild Type ◽  
Three Dimensional Model ◽  
Loop Region ◽  
Type Protein ◽  
Wild Type Protein ◽  
Ability To Act

The Mos protein is a serine/threonine protein kinase which acts to regulate progression through meiosis in vertebrate oocytes. Although Mos function is dependent on its ability to act as a protein kinase, little is known about the factors which regulate Mos kinase activity. To understand the mechanism by which Mos kinase activity is regulated, we have used molecular modeling to construct a three-dimensional model of Mos based on the crystallographic coordinates of cyclic AMP-dependent kinase (PKA). This model identified a loop in Mos which is positioned near the active site and appears capable of blocking substrate access to the active site. Mutagenesis was used to construct altered forms of the Mos protein with deletions of parts or all of the loop. In vitro kinase assays showed that Mos proteins with the loop removed had up to a fourfold increase in kinase activity compared with the wild-type protein, indicating that the loop acts in an autoinhibitory manner for Mos kinase activity. Point mutations were also made on individual residues of the loop which were determined from the molecular model to be capable of reaching the active site. Determination of the kinase activities of these mutants showed that individual mutations in the loop region are capable of either increasing or decreasing kinase activity with regard to the wild-type protein. These data suggest that the loop identified in Mos acts as an autoinhibitor of kinase activity.

scholarly journals Binding of Pyridine Nucleotide Coenzymes to the β-Subunit of the Voltage-sensitive K+Channel

2001 ◽  
Vol 276 (15) ◽  
pp. 11812-11820 ◽  
Author(s):  
Si-Qi Liu ◽  
Hongjun Jin ◽  
Albert Zacarias ◽  
Sanjay Srivastava ◽  
Aruni Bhatnagar
Keyword(s):  
Pyridine Nucleotide ◽  
K Channel ◽  
Β Subunit ◽  
Wild Type ◽  
Ionic Strength Dependence ◽  
Coenzyme Binding ◽  
Type Protein ◽  
Wild Type Protein ◽  
Tetrameric Structure

The β-subunit of the voltage-sensitive K+(Kv) channels belongs to the aldo-keto reductase superfamily, and the crystal structure of Kvβ2 shows NADP bound in its active site. Here we report that Kvβ2 displays a high affinity for NADPH (Kd= 0.1 μm) and NADP+(Kd= 0.3 μm), as determined by fluorometric titrations of the recombinant protein. The Kvβ2 also bound NAD(H) but with 10-fold lower affinity. The site-directed mutants R264E and N333W did not bind NADPH, whereas, theKdNADPHof Q214R was 10-fold greater than the wild-type protein. TheKdNADPHwas unaffected by the R189M, W243Y, W243A, or Y255F mutation. The tetrameric structure of the wild-type protein was retained by the R264E mutant, indicating that NADPH binding is not a prerequisite for multimer formation. A C248S mutation caused a 5-fold decrease inKdNADPH, shifted the pKaofKdNADPHfrom 6.9 to 7.4, and decreased the ionic strength dependence of NADPH binding. These results indicate that Arg-264 and Asn-333 are critical for coenzyme binding, which is regulated in part by Cys-248. The binding of both NADP(H) and NAD(H) to the protein suggests that several types of Kvβ2-nucleotide complexes may be formedin vivo.

scholarly journals Identification and Analysis of a Hyperactive Mutant Form of Drosophila P-Element Transposase

Genetics ◽  
2002 ◽  
Vol 162 (1) ◽  
pp. 217-227 ◽  
Author(s):  
Eileen L Beall ◽  
Matthew B Mahoney ◽  
Donald C Rio
Keyword(s):  
Dna Damage ◽  
Dna Cleavage ◽  
P Element ◽  
In Vitro Assays ◽  
Mutant Form ◽  
Wild Type ◽  
Type Protein ◽  
Wild Type Protein

Abstract Transposition in many organisms is regulated to control the frequency of DNA damage caused by the DNA breakage and joining reactions. However, genetic studies in prokaryotic systems have led to the isolation of mutant transposase proteins with higher or novel activities compared to those of the wild-type protein. In the course of our study of the effects of mutating potential ATM-family DNA damage checkpoint protein kinase sites in the Drosophila P-element transposase protein, we found one mutation, S129A, that resulted in an elevated level of transposase activity using in vivo recombination assays, including P-element-mediated germline transformation. In vitro assays for P-element transposase activity indicate that the S129A mutant exhibits elevated donor DNA cleavage activity when compared to the wild-type protein, whereas the strand-transfer activity is similar to that of wild type. This difference may reflect the nature of the in vitro assays and that normally in vivo the two reactions may proceed in concert. The P-element transposase protein contains 10 potential consensus phosphorylation sites for the ATM family of PI3-related protein kinases. Of these 10 sites, 8 affect transposase activity either positively or negatively when substituted individually with alanine and tested in vivo. A mutant transposase protein that contains all eight N-terminal serine and threonine residues substituted with alanine is inactive and can be restored to full activity by substitution of wild-type amino acids back at only 3 of the 8 positions. These data suggest that the activity of P-element transposase may be regulated by phosphorylation and demonstrate that one mutation, S129A, results in hyperactive transposition.

scholarly journals Redox Sensitive Cysteine Residues as Crucial Regulators of Wild-Type and Mutant p53 Isoforms

Cells ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 3149
Author(s):  
Elena Butturini ◽  
Giovanna Butera ◽  
Raffaella Pacchiana ◽  
Alessandra Carcereri de Prati ◽  
Sofia Mariotto ◽  
...  
Keyword(s):  
Redox State ◽  
P53 Protein ◽  
Tp53 Gene ◽  
Single Amino Acid ◽  
Cysteine Residues ◽  
Missense Mutations ◽  
Wild Type ◽  
Functional Changes ◽  
Type Protein ◽  
Wild Type Protein

The wild-type protein p53 plays a key role in preventing the formation of neoplasms by controlling cell growth. However, in more than a half of all cancers, the TP53 gene has missense mutations that appear during tumorigenesis. In most cases, the mutated gene encodes a full-length protein with the substitution of a single amino acid, resulting in structural and functional changes and acquiring an oncogenic role. This dual role of the wild-type protein and the mutated isoforms is also evident in the regulation of the redox state of the cell, with antioxidant and prooxidant functions, respectively. In this review, we introduce a new concept of the p53 protein by discussing its sensitivity to the cellular redox state. In particular, we focus on the discussion of structural and functional changes following post-translational modifications of redox-sensitive cysteine residues, which are also responsible for interacting with zinc ions for proper structural folding. We will also discuss therapeutic opportunities using small molecules targeting cysteines capable of modifying the structure and function of the p53 mutant isoforms in view of possible anticancer therapies for patients possessing the mutation in the TP53 gene.

scholarly journals Mutagenesis of the Active-Site Cysteine in the Ubiquitin-Specific Protease Contained in Large Tegument Protein pUL36 of Pseudorabies Virus Impairs Viral Replication In Vitro and Neuroinvasion In Vivo

2008 ◽  
Vol 82 (12) ◽  
pp. 6009-6016 ◽  
Author(s):  
Sindy Böttcher ◽  
Christina Maresch ◽  
Harald Granzow ◽  
Barbara G. Klupp ◽  
Jens P. Teifke ◽  
...  
Keyword(s):  
Active Site ◽  
Pseudorabies Virus ◽  
Wild Type Virus ◽  
Tegument Protein ◽  
Wild Type ◽  
Active Site Cysteine ◽  
Specific Protease ◽  
Ubiquitin Specific Protease

ABSTRACT Herpesviruses specify a ubiquitin-specific protease activity located within their largest tegument protein. Although its biological role is still largely unclear, mutation within the active site abolished deubiquitinating (DUB) activity and decreased virus replication in vitro and in vivo. To further elucidate the role of DUB activity for herpesvirus replication, the conserved active-site cysteine at amino acid position 26 within pUL36 of Pseudorabies virus (PrV) (Suid herpesvirus 1), a neurotropic alphaherpesvirus, was mutated to serine. Whereas one-step growth kinetics of the resulting mutant virus PrV-UL36(C26S) were moderately reduced, plaque size was decreased to 62% of that of the wild-type virus. Ultrastructural analysis revealed large accumulations of unenveloped nucleocapsids in the cytoplasm, but incorporation of the tegument protein pUL37 was not abolished. After intranasal infection with PrV-UL36(C26S) mice showed survival times two times longer than those of mice infected with wild-type or rescued virus. Thus, the DUB activity is important for PrV replication in vitro and for neuroinvasion in mice.

Generation of Novel, High Titre, Multi-Cistronic Retroviral Vectors Which Simultaneously Express Individual Proteins in Different Subcellular Locations of Hematopoietic Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 5255-5255
Author(s):  
Michael D. Milsom ◽  
Catherine Gavin ◽  
Rebecca Baldwin ◽  
Geoff Margison ◽  
Leslie J. Fairbairn
Keyword(s):  
Gene Therapy ◽  
Dna Methyltransferase ◽  
Growth Factor Receptor ◽  
Retroviral Vectors ◽  
High Titre ◽  
Target Cells ◽  
Wild Type ◽  
Type Protein ◽  
Wild Type Protein

Abstract As gene therapy strategies become ever more sophisticated, there is a need for vectors which efficiently deliver and express more than one gene product to target cells e.g. to enable in vivo selection; to correct multiple genetic loci; to provide a suicide mechanism for safety purposes. Employing alternate codon usage, we have synthesised a novel version of the FMDV 2A self-processing moiety which encodes the same primary amino-acid sequence as the wild type protein (and which possesses equivalent cleavage activity to the wild type protein). By including this synthetic FMDV 2A sequence in retroviral vectors which also contain the wild type sequence, we are able to facilitate the efficient co-translational separation of 3 different proteins encoded by a single transcript. This vector configuration also permits the inclusion of an IRES sequence and thus expression of a fourth protein. Using this technology we have created two tetracistronic retroviral vectors which express eGFP; O6-methylguanine DNA-methyltransferase and truncated human nerve growth factor receptor courtesy of 2A mediated cleavage, and a mitochondrially targeted dsRED via an IRES. Stable amphotropic producer lines containing these vectors generated supernatant which contained a high titre of retroviral particles (>106 IU/ml) which was sufficient for the transduction of K562 human erythroleukemic cells. Analysis of transduced cells confirmed that all four proteins were expressed individually and segragated to their correct subcellular locations. Furthermore, limiting dilution assay revealed that proteins expressed courtesy of the 2A sequences were still generated at high levels, even when cells were infected with a low MOI of virus. We propose that this technology will allow the creation of more sophisticated vectors for use in gene therapy of hematopoietic and other cells.

scholarly journals Substitutions in an Active Site Loop ofEscherichia coliIscS Result in Specific Defects in Fe-S Cluster and Thionucleoside Biosynthesisin Vivo

2004 ◽  
Vol 279 (19) ◽  
pp. 19551-19558 ◽  
Author(s):  
Charles T. Lauhon ◽  
Elizabeth Skovran ◽  
Hugo D. Urbina ◽  
Diana M. Downs ◽  
Larry E. Vickery
Keyword(s):  
Active Site ◽  
Cluster Formation ◽  
Wild Type ◽  
Cysteine Desulfurase ◽  
Mutant Proteins ◽  
Active Site Cysteine ◽  
Sulfur Transfer ◽  
Amino Acid Region

IscS catalyzes the fragmentation ofl-cysteine tol-alanine and sulfane sulfur in the form of a cysteine persulfide in the active site of the enzyme. InEscherichia coliIscS, the active site cysteine Cys328resides in a flexible loop that potentially influences both the formation and stability of the cysteine persulfide as well as the specificity of sulfur transfer to protein substrates. Alanine-scanning substitution of this 14 amino acid region surrounding Cys328identified additional residues important for IscS functionin vivo. Two mutations, S326A and L333A, resulted in strains that were severely impaired in Fe-S cluster synthesisin vivo. The mutant strains were deficient in Fe-S cluster-dependent tRNA thionucleosides (s2C and ms2i6A) yet showed wild type levels of Fe-S-independent thionucleosides (s4U and mnm5s2U) that require persulfide formation and transfer.In vitro, the mutant proteins were similar to wild type in both cysteine desulfurase activity and sulfur transfer to IscU. These results indicate that residues in the active site loop can selectively affect Fe-S cluster biosynthesisin vivowithout detectably affecting persulfide delivery and suggest that additional assays may be necessary to fully represent the functions of IscS in Fe-S cluster formation.

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