scholarly journals Functional domain organization of the potato α-glucan, water dikinase (GWD): evidence for separate site catalysis as revealed by limited proteolysis and deletion mutants

2005 ◽  
Vol 385 (2) ◽  
pp. 355-361 ◽  
Author(s):  
René MIKKELSEN ◽  
Andreas BLENNOW
Keyword(s):  
Structural Changes ◽  
Sequence Similarity ◽  
Limited Proteolysis ◽  
Phosphoryl Group ◽  
Deletion Mutants ◽  
Functional Domain ◽  
Partial Reaction ◽  
Atp Binding Site ◽  
Binding Domains ◽  
Terminal Domains

The potato tuber (Solanum tuberosum) GWD (α-glucan, water dikinase) catalyses the phosphorylation of starch by a dikinase-type reaction mechanism in which the β-phosphate of ATP is transferred to the glucosyl residue of amylopectin. GWD shows sequence similarity to bacterial pyruvate, water dikinase and PPDK (pyruvate, phosphate dikinase). In the present study, we examine the structure–function relationship of GWD. Analysis of proteolytic fragments of GWD, in conjunction with peptide microsequencing and the generation of deletion mutants, indicates that GWD is comprised of five discrete domains of 37, 24, 21, 36 and 38 kDa. The catalytic histidine, which mediates the phosphoryl group transfer from ATP to starch, is located on the 36 kDa fragment, whereas the 38 kDa C-terminal fragment contains the ATP-binding site. Binding of the glucan molecule appears to be confined to regions containing the three N-terminal domains. Deletion mutants were generated to investigate the functional interdependency of the putative ATP- and glucan-binding domains. A truncated form of GWD expressing the 36 and 38 kDa C-terminal domains was found to catalyse the E+ATP→E-P+AMP+Pi (where Pi stands for orthophosphate) partial reaction, but not the E-P+glucan→E+glucan-P partial reaction. CD experiments provided evidence for large structural changes on autophosphorylation of GWD, indicating that GWD employs a swivelling-domain mechanism for enzymic phosphotransfer similar to that seen for PPDK.

A Unique Chitinase with Dual Active Sites and Triple Substrate Binding Sites from the Hyperthermophilic Archaeon Pyrococcus kodakaraensis KOD1

1999 ◽  
Vol 65 (12) ◽  
pp. 5338-5344 ◽  
Author(s):  
Takeshi Tanaka ◽  
Shinsuke Fujiwara ◽  
Shingo Nishikori ◽  
Toshiaki Fukui ◽  
Masahiro Takagi ◽  
...  
Keyword(s):  
Active Sites ◽  
Sequence Similarity ◽  
Chitinase Activity ◽  
Deletion Mutants ◽  
Gene Encoding ◽  
Hyperthermophilic Archaeon ◽  
Chromatography Analysis ◽  
Binding Domains ◽  
Activity Measurements ◽  
Cellulose Binding

ABSTRACT We have found that the hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 produces an extracellular chitinase. The gene encoding the chitinase (chiA) was cloned and sequenced. ThechiA gene was found to be composed of 3,645 nucleotides, encoding a protein (1,215 amino acids) with a molecular mass of 134,259 Da, which is the largest among known chitinases. Sequence analysis indicates that ChiA is divided into two distinct regions with respective active sites. The N-terminal and C-terminal regions show sequence similarity with chitinase A1 from Bacillus circulans WL-12 and chitinase from Streptomyces erythraeus (ATCC 11635), respectively. Furthermore, ChiA possesses unique chitin binding domains (CBDs) (CBD1, CBD2, and CBD3) which show sequence similarity with cellulose binding domains of various cellulases. CBD1 was classified into the group of family V type cellulose binding domains. In contrast, CBD2 and CBD3 were classified into that of the family II type. chiA was expressed inEscherichia coli cells, and the recombinant protein was purified to homogeneity. The optimal temperature and pH for chitinase activity were found to be 85°C and 5.0, respectively. Results of thin-layer chromatography analysis and activity measurements with fluorescent substrates suggest that the enzyme is an endo-type enzyme which produces a chitobiose as a major end product. Various deletion mutants were constructed, and analyses of their enzyme characteristics revealed that both the N-terminal and C-terminal halves are independently functional as chitinases and that CBDs play an important role in insoluble chitin binding and hydrolysis. Deletion mutants which contain the C-terminal half showed higher thermostability than did N-terminal-half mutants and wild-type ChiA.

scholarly journals The Divergent Key Residues of Two Agrobacterium fabrum (tumefaciens) CheY Paralogs Play a Key Role in Distinguishing Their Functions

2021 ◽  
Vol 9 (6) ◽  
pp. 1134
Author(s):  
Dawei Gao ◽  
Renjie Zong ◽  
Zhiwei Huang ◽  
Jingyang Ye ◽  
Hao Wang ◽  
...  
Keyword(s):  
Cellular Localization ◽  
Chemotactic Response ◽  
Phosphoryl Group ◽  
Deletion Mutants ◽  
Functional Domain ◽  
Functional Difference ◽  
Flagellar Motor ◽  
Swimming Pattern ◽  
Binding Surface ◽  
Key Residues

The chemotactic response regulator CheY, when phosphorylated by the phosphoryl group from phosphorylated CheA, can bind to the motor switch complex to control the flagellar motor rotation. Agrobacterium fabrum (previous name: Agrobacterium tumefaciens), a phytopathogen, carries two paralogous cheY genes, cheY1 and cheY2. The functional difference of two paralogous CheYs remains unclear. Three cheY-deletion mutants were constructed to test the effects of two CheYs on the chemotaxis of A. fabrum. Phenotypes of three cheY-deletion mutants show that deletion of each cheY significantly affects the chemotactic response, but cheY2-deletion possesses more prominent effects on the chemotactic migration and swimming pattern of A. fabrum than does cheY1-deletion. CheA-dependent cellular localization of two CheY paralogs and in vitro pull-down of two CheY paralogs by FliM demonstrate that the distinct roles of two CheY paralogs arise mainly from the differentiation of their binding affinities for the motor switch component FliM, agreeing with the divergence of the key residues on the motor-binding surface involved in the interaction with FliM. The single respective replacements of key residues R93 and A109 on the motor-binding surface of CheY2 by alanine (A) and valine (V), the corresponding residues of CheY1, significantly enhanced the function of CheY2 in regulating the chemotactic response of A. fabrum CheY-deficient mutant Δy to nutrient substances and host attractants. These results conclude that the divergence of the key residues in the functional subdomain is the decisive factor of functional differentiation of these two CheY homologs and protein function may be improved by the substitution of the divergent key residues in the functional domain for the corresponding residues of its paralogs. This finding will help us to better understand how paralogous proteins sub-functionalize. In addition, the acquirement of two CheY2 variants, whose chemotactic response functions are significantly improved, will be very useful for us to further explore the mechanism of CheY to bind and regulate the flagellar motor and the role of chemotaxis in the pathogenicity of A. fabrum.

scholarly journals Within-Arctic horizontal gene transfer as a driver of convergent evolution in distantly related microalgae

2021 ◽  
Author(s):  
Richard G Dorrell ◽  
Alan Kuo ◽  
Zoltan Fussy ◽  
Elisabeth H Richardson ◽  
Asaf Salamov ◽  
...  
Keyword(s):  
Sequence Data ◽  
Sequence Similarity ◽  
Algal Species ◽  
Gene Families ◽  
The Arctic ◽  
Arctic Water ◽  
Diverse Range ◽  
Binding Domains ◽  
Ice Binding ◽  
Ice Conditions

The Arctic Ocean is being impacted by warming temperatures, increasing freshwater and highly variable ice conditions. The microalgal communities underpinning Arctic marine food webs, once thought to be dominated by diatoms, include a phylogenetically diverse range of small algal species, whose biology remains poorly understood. Here, we present genome sequences of a cryptomonad, a haptophyte, a chrysophyte, and a pelagophyte, isolated from the Arctic water column and ice. Comparing protein family distributions and sequence similarity across a densely-sampled set of algal genomes and transcriptomes, we note striking convergences in the biology of distantly related small Arctic algae, compared to non-Arctic relatives; although this convergence is largely exclusive of Arctic diatoms. Using high-throughput phylogenetic approaches, incorporating environmental sequence data from Tara Oceans, we demonstrate that this convergence was partly explained by horizontal gene transfers (HGT) between Arctic species, in over at least 30 other discrete gene families, and most notably in ice-binding domains (IBD). These Arctic-specific genes have been repeatedly transferred between Arctic algae, and are independent of equivalent HGTs in the Antarctic Southern Ocean. Our data provide insights into the specialised Arctic marine microbiome, and underlines the role of geographically-limited HGT as a driver of environmental adaptation in eukaryotic algae.

scholarly journals The primary structural photoresponse of phytochrome proteins captured by a femtosecond X-ray laser

2019 ◽  
Author(s):  
Elin Claesson ◽  
Weixiao Yuan Wahlgren ◽  
Heikki Takala ◽  
Suraj Pandey ◽  
Leticia Castillon ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Structural Changes ◽  
Red Light ◽  
Crystallographic Data ◽  
Water Dissociation ◽  
X Ray ◽  
Binding Domains ◽  
Delay Times

Phytochrome proteins control the growth, reproduction, and photosynthesis of plants, fungi, and bacteria. Light is detected by a bilin cofactor, but it remains elusive how this leads to activation of the protein through structural changes. We present serial femtosecond X-ray crystallographic data of the chromophore-binding domains of a bacterial phytochrome at delay times of 1 ps and 10 ps after photoexcitation. The structures reveal a twist of the D-ring, which lead to partial detachment of the chromophore from the protein. Unexpectedly, the conserved so-called pyrrole water is photodissociated from the chromophore, concomitant with movement of the A-ring and a key signalling aspartate. The changes are wired together by ultrafast backbone and water movements around the chromophore, channeling them into signal transduction towards the output domains. We suggest that the water dissociation is key to the phytochrome photoresponse, explaining the earliest steps of how plants, fungi and bacteria sense red light.

scholarly journals Proteolytic modification of the heparin-binding affinity of extracellular superoxide dismutase

1993 ◽  
Vol 290 (2) ◽  
pp. 623-626 ◽  
Author(s):  
K Karlsson ◽  
A Edlund ◽  
J Sandström ◽  
S L Marklund
Keyword(s):  
Superoxide Dismutase ◽  
Binding Affinity ◽  
Heparan Sulphate ◽  
Limited Proteolysis ◽  
Size Exclusion ◽  
Extracellular Superoxide Dismutase ◽  
Heparin Binding ◽  
Binding Domains ◽  
Heparin Affinity

The heparin-binding affinity of the tetrameric extracellular superoxide dismutase (EC-SOD) is a result of the cooperative effect of the heparin-binding domains of the subunits, located in the hydrophilic, strongly positively charged C-terminal ends. EC-SOD C, the high-heparin-affinity type, exposed to immobilized trypsin and plasmin was found to rapidly lose its affinity for heparin, without any loss of enzymic activity or major change in molecular mass as judged by size-exclusion chromatography. Heparin and dextran sulphate 5000 inhibited the proteolysis, suggesting that EC-SOD C sequestered by heparan sulphate proteoglycan in vivo is partially protected against proteolysis. The loss of heparin-affinity occurred with the stepwise formation of intermediates, and the pattern upon chromatography on heparin-Sepharose and subsequent immunoblotting was compatible with the notion that the changes are due to sequential truncations of heparin-binding domains from subunits composing the EC-SOD tetramers. A similar pattern with intermediates and apparent truncations has previously been found with EC-SOD of human plasma. The findings show that the unique design of the heparin-binding domain of EC-SOD allows easy modification of the heparin-affinity by means of limited proteolysis, and suggest that such proteolysis is a major contributor to the heterogeneity in heparin-affinity of EC-SOD in mammalian plasma.

Differences in the exposure of C- and N-terminal tubulin domains in cytoplasmic microtubules detected with domain-specific monoclonal antibodies

1989 ◽  
Vol 92 (3) ◽  
pp. 519-528 ◽  
Author(s):  
P. Draber ◽  
E. Draberova ◽  
I. Linhartova ◽  
V. Viklicky
Keyword(s):  
Monoclonal Antibodies ◽  
Polyclonal Antibodies ◽  
Limited Proteolysis ◽  
Antigenic Determinants ◽  
Alpha Tubulin ◽  
Domain Specific ◽  
Double Label ◽  
Cytoplasmic Microtubules ◽  
C And N ◽  
Terminal Domains

A panel of 11 monoclonal antibodies specific to alpha- or beta-tubulin subunits was used to study the location of tubulin molecules in cytoplasmic microtubules. Specificity of antibodies was confirmed by immunoblotting and immunofluorescence experiments on fixed cells. The limited proteolysis of tubulin with trypsin and chymotrypsin followed by immunoblotting demonstrated that the antibodies discriminated between structural domains of both subunits. Epitope mapping of isolated alpha-tubulin revealed that a set of antibodies against the N-terminal domain of the alpha-subunit (TU-01, TU-02, TU-03, TU-09, 6–11B-1) recognized at least four different antigenic determinants. Immunofluorescence staining of unfixed detergent-extracted cells showed that antibodies to determinants on C-terminal domains labelled microtubules, but these were not decorated with antibodies to N-terminal domains. The same results were obtained after microinjection of antibodies into living cells. The unchanged distribution of microtubules in injected cells was confirmed by double-label immunofluorescence with polyclonal antibodies. The data indicate that while parts of C-terminal domains of both subunits are exposed on the exterior of the microtubules, considerable regions of the N-terminal domains are either not exposed on the surface of cytoplasmic microtubules, or are masked by interacting proteins.

Molecular analysis of the deletion mutants in the E homeotic complex of the silkworm Bombyx mori

Development ◽  
1992 ◽  
Vol 114 (3) ◽  
pp. 555-563 ◽  
Author(s):  
K. Ueno ◽  
C.C. Hui ◽  
M. Fukuta ◽  
Y. Suzuki
Keyword(s):  
Molecular Structure ◽  
Amino Acid ◽  
Bombyx Mori ◽  
Structural Changes ◽  
Amino Acid Sequences ◽  
Homeotic Gene ◽  
Deletion Mutants ◽  
Gene Complex ◽  
Bithorax Complex ◽  
Silkworm Bombyx Mori

The E loci in Bombyx mori are expected to contain a homeotic gene complex specifying the identities of the larval abdominal segments. However, the molecular structure of this complex remains to be determined. We have started to analyze the structural changes in the E complex mutations. We used three newly isolated Bombyx homeobox genes as probes. These genes are probably homologues of the Ultrabithorax (Ubx), abdominal-A (abd-A) and Abdominal-B (Abd-B) in the Drosophila bithorax complex, because the amino-acid sequences of the homeobox regions in these Bombyx genes are almost identical to those of Drosophila genes. We found that the Bombyx Ubx and abd-A genes are deleted in the EN chromosome, and the Bombyx abd-A gene is deleted in the ECa chromosome. From these results, we conclude that the Bombyx E complex consists of the Ubx, abd-A and possibly Abd-B genes, which may play similar roles to their homologues in the Drosophila bithorax complex.

The FHA domain mediates phosphoprotein interactions

2000 ◽  
Vol 113 (23) ◽  
pp. 4143-4149 ◽  
Author(s):  
J. Li ◽  
G.I. Lee ◽  
S.R. Van Doren ◽  
J.C. Walker
Keyword(s):  
Sequence Similarity ◽  
Sequence Motif ◽  
Amino Acid Residues ◽  
Fha Domain ◽  
Tertiary Structures ◽  
Cellular Processes ◽  
Forkhead Transcription Factors ◽  
Binding Domains ◽  
Cycle Arrest ◽  
Protein Kinase Signaling

The forkhead-associated (FHA) domain is a phosphopeptide-binding domain first identified in a group of forkhead transcription factors but is present in a wide variety of proteins from both prokaryotes and eukaryotes. In yeast and human, many proteins containing an FHA domain are found in the nucleus and involved in DNA repair, cell cycle arrest, or pre-mRNA processing. In plants, the FHA domain is part of a protein that is localized to the plasma membrane and participates in the regulation of receptor-like protein kinase signaling pathways. Recent studies show that a functional FHA domain consists of 120–140 amino acid residues, which is significantly larger than the sequence motif first described. Although FHA domains do not exhibit extensive sequence similarity, they share similar secondary and tertiary structures, featuring a sandwich of two anti-parallel (beta)-sheets. One intriguing finding is that FHA domains may bind phosphothreonine, phosphoserine and sometimes phosphotyrosine, distinguishing them from other well-studied phosphoprotein-binding domains. The diversity of proteins containing FHA domains and potential differences in binding specificities suggest the FHA domain is involved in coordinating diverse cellular processes.

scholarly journals Insights into histidine kinase activation mechanisms from the monomeric blue light sensor EL346

2019 ◽  
Vol 116 (11) ◽  
pp. 4963-4972 ◽  
Author(s):  
Igor Dikiy ◽  
Uthama R. Edupuganti ◽  
Rinat R. Abzalimov ◽  
Peter P. Borbat ◽  
Madhur Srivastava ◽  
...  
Keyword(s):  
Blue Light ◽  
Conformational Changes ◽  
Histidine Kinase ◽  
Structural Changes ◽  
Molecular Mechanisms ◽  
Catalytic Domain ◽  
Response Regulator ◽  
Phosphoryl Group ◽  
Dark State ◽  
Light Sensing

Translation of environmental cues into cellular behavior is a necessary process in all forms of life. In bacteria, this process frequently involves two-component systems in which a sensor histidine kinase (HK) autophosphorylates in response to a stimulus before subsequently transferring the phosphoryl group to a response regulator that controls downstream effectors. Many details of the molecular mechanisms of HK activation are still unclear due to complications associated with the multiple signaling states of these large, multidomain proteins. To address these challenges, we combined complementary solution biophysical approaches to examine the conformational changes upon activation of a minimal, blue-light–sensing histidine kinase from Erythrobacter litoralis HTCC2594, EL346. Our data show that multiple conformations coexist in the dark state of EL346 in solution, which may explain the enzyme’s residual dark-state activity. We also observe that activation involves destabilization of the helices in the dimerization and histidine phosphotransfer-like domain, where the phosphoacceptor histidine resides, and their interactions with the catalytic domain. Similar light-induced changes occur to some extent even in constitutively active or inactive mutants, showing that light sensing can be decoupled from activation of kinase activity. These structural changes mirror those inferred by comparing X-ray crystal structures of inactive and active HK fragments, suggesting that they are at the core of conformational changes leading to HK activation. More broadly, our findings uncover surprising complexity in this simple system and allow us to outline a mechanism of the multiple steps of HK activation.

Conformational Alterations of α2-Antiplasmin Induced by Complex Formation with Plasmin

1979 ◽  
Author(s):  
E.F. Plow ◽  
B. Wiman ◽  
D. Collen
Keyword(s):  
Complex Formation ◽  
Structural Changes ◽  
Limited Proteolysis ◽  
Terminal Region ◽  
The Other ◽  
Antigenic Determinants ◽  
Chemical Analyses ◽  
Large Fragment ◽  
Reactive Site ◽  
Immunodiffusion Analysis

The conformational and structural changes induced in the α2-antiplasmin (AP) molecule by complex formation with plasmin have been analyzed utilizing quantitative radioimmuno-chemical analyses. Complexes prepared in plasmin excess -(PAP-P) and therefore subjected to limited proteolysis and complexes prepared in AP excess (PAP-A) have been compared with free AP. With AP antiserum, PAP-A, PAP-P and AP yielded reactions of complete identity by immunodiffusion analysis. In radioimmunoassay, however, these were clearly distinguished, and four distinct sets of antigenic determinants were delineated. Set I determinants were expressed equivalently by PAP-P, PAP-A and AP and were, therefore, not altered by complex formation. This set was recognized by 90% of the antibodies, and the determinants were all included within a large fragment of Mr 60,000 derived from the NH2-terminal region of AP. The other three sets of determinants were modulated by complex formation. Set II was expressed by PAP-A and AP but not by PAP-P, and these were sensitive to proteolysis by plasmin. Set III determinants were expressed only by AP and were localized to a peptide of Mr 8,000 derived from the COOH-terminal region of AP. Set IV determinants were also present only on AP but were not present in the peptide and required an intact reactive site in AP for expression. Thus, there is evidence for multiple conformational modulations in AP induced by complex formation, and these modulations can be pinpointed to specific loci within the AP molecule.

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