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

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.

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

Biochemical Journal ◽

10.1042/bj20041119 ◽

2005 ◽

Vol 385 (2) ◽

pp. 355-361 ◽

Cited By ~ 17

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.

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The Only Chemoreceptor Encoded by che Operon Affects the Chemotactic Response of Agrobacterium to Various Chemoeffectors

Microorganisms ◽

10.3390/microorganisms9091923 ◽

2021 ◽

Vol 9 (9) ◽

pp. 1923

Author(s):

Jingyang Ye ◽

Miaomiao Gao ◽

Qingxuan Zhou ◽

Hao Wang ◽

Nan Xu ◽

...

Keyword(s):

Signal Transduction ◽

Cellular Localization ◽

Deletion Mutant ◽

Helical Structure ◽

Chemotactic Response ◽

Signaling System ◽

Chemotactic Migration ◽

Exact Function ◽

Encoding Gene ◽

Key Residues

Chemoreceptor (also called methyl-accepting chemotaxis protein, MCP) is the leading signal protein in the chemotaxis signaling pathway. MCP senses and binds chemoeffectors, specifically, and transmits the sensed signal to downstream proteins of the chemotaxis signaling system. The genome of Agrobacterium fabrum (previously, tumefaciens) C58 predicts that a total of 20 genes can encode MCP, but only the MCP-encoding gene atu0514 is located inside the che operon. Hence, the identification of the exact function of atu0514-encoding chemoreceptor (here, named as MCP514) will be very important for us to understand more deeply the chemotaxis signal transduction mechanism of A. fabrum. The deletion of atu0514 significantly decreased the chemotactic migration of A. fabrum in a swim plate. The test of atu0514-deletion mutant (Δ514) chemotaxis toward single chemicals showed that the deficiency of MCP514 significantly weakened the chemotactic response of A. fabrum to four various chemicals, sucrose, valine, citric acid and acetosyringone (AS), but did not completely abolish the chemotactic response. MCP514 was localized at cell poles although it lacks a transmembrane (TM) region and is predicted to be a cytoplasmic chemoreceptor. The replacement of residue Phe328 showed that the helical structure in the hairpin subdomain of MCP514 is a direct determinant for the cellular localization of MCP514. Single respective replacements of key residues indicated that residues Asn336 and Val353 play a key role in maintaining the chemotactic function of MCP514.

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Structural insights into flagellar stator–rotor interactions

eLife ◽

10.7554/elife.48979 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 14

Author(s):

Yunjie Chang ◽

Ki Hwan Moon ◽

Xiaowei Zhao ◽

Steven J Norris ◽

MD A Motaleb ◽

...

Keyword(s):

Conformational Change ◽

High Speed ◽

Electron Tomography ◽

Deletion Mutants ◽

Flagellar Motor ◽

Wild Type ◽

Cryo Electron Tomography ◽

Flagellar Filament ◽

Structural Insights

The bacterial flagellar motor is a molecular machine that can rotate the flagellar filament at high speed. The rotation is generated by the stator–rotor interaction, coupled with an ion flux through the torque-generating stator. Here we employed cryo-electron tomography to visualize the intact flagellar motor in the Lyme disease spirochete, Borrelia burgdorferi. By analyzing the motor structures of wild-type and stator-deletion mutants, we not only localized the stator complex in situ, but also revealed the stator–rotor interaction at an unprecedented detail. Importantly, the stator–rotor interaction induces a conformational change in the flagella C-ring. Given our observation that a non-motile mutant, in which proton flux is blocked, cannot generate the similar conformational change, we propose that the proton-driven torque is responsible for the conformational change required for flagellar rotation.

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Only One of the Five CheY Homologs in Vibrio cholerae Directly Switches Flagellar Rotation

Journal of Bacteriology ◽

10.1128/jb.187.24.8403-8410.2005 ◽

2005 ◽

Vol 187 (24) ◽

pp. 8403-8410 ◽

Cited By ~ 43

Author(s):

Akihiro Hyakutake ◽

Michio Homma ◽

Melissa J. Austin ◽

Markus A. Boin ◽

Claudia C. Häse ◽

...

Keyword(s):

Vibrio Cholerae ◽

Gene Clusters ◽

Swimming Behavior ◽

Phosphoryl Group ◽

Flagellar Motor ◽

Response Regulators ◽

E Coli ◽

Control Functions ◽

Flagellar Rotation ◽

Principal Roles

ABSTRACT Vibrio cholerae has three sets of chemotaxis (Che) proteins, including three histidine kinases (CheA) and four response regulators (CheY) that are encoded by three che gene clusters. We deleted the cheY genes individually or in combination and found that only the cheY3 deletion impaired chemotaxis, reinforcing the previous conclusion that che cluster II is involved in chemotaxis. However, this does not exclude the involvement of the other clusters in chemotaxis. In other bacteria, phospho-CheY binds directly to the flagellar motor to modulate its rotation, and CheY overexpression, even without CheA, causes extremely biased swimming behavior. We reasoned that a V. cholerae CheY homolog, if it directly controls flagellar rotation, should also induce extreme swimming behavior when overproduced. This was the case for CheY3 (che cluster II). However, no other CheY homolog, including the putative CheY (CheY0) protein encoded outside the che clusters, affected swimming, demonstrating that these CheY homologs cannot act directly on the flagellar motor. CheY4 very slightly enhanced the spreading of an Escherichia coli cheZ mutant in semisolid agar, raising the possibility that it can affect chemotaxis by removing a phosphoryl group from CheY3. We also found that V. cholerae CheY3 and E. coli CheY are only partially exchangeable. Mutagenic analyses suggested that this may come from coevolution of the interacting pair of proteins, CheY and the motor protein FliM. Taken together, it is likely that the principal roles of che clusters I and III as well as cheY0 are to control functions other than chemotaxis.

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The functional importance of the extreme C-terminal tail in the gene 2 organellar Ca2+-transport ATPase (SERCA2a/b)

Biochemical Journal ◽

10.1042/bj3030979 ◽

1994 ◽

Vol 303 (3) ◽

pp. 979-984 ◽

Cited By ~ 62

Author(s):

H Verboomen ◽

F Wuytack ◽

L Van den Bosch ◽

L Mertens ◽

R Casteels

Keyword(s):

Amino Acids ◽

Splice Variant ◽

Turnover Rate ◽

Peptide Chain ◽

Deletion Mutants ◽

Functional Difference ◽

Critical Importance ◽

Transmembrane Segment ◽

Functional Importance ◽

Transport Atpase

Ca(2+)-uptake experiments in microsomal fractions from transfected COS-1 cells have revealed a functional difference between the non-muscle SERCA2b Ca2+ pump and its muscle-specific SERCA2a splice variant. Structurally, the two pumps differ only in their C-terminal tail. The last four amino acids of SERCA2a are replaced in SERCA2b by a 49-residue-long peptide chain containing a very hydrophobic stretch which could be an additional transmembrane segment. The functionally important subdomains in the SERCA2b tail were analysed by constructing three SERCA2b deletion mutants lacking 12, 31 or 49 amino acids. The mutants and the parental SERCA2 pumps were expressed in COS-1 cells and analysed for functional difference. SERCA2b had a twofold higher Ca2+ affinity, a twofold lower turnover rate and a 10-fold lower vanadate-sensitivity than SERCA2a and the mutants. Since each of the three truncated versions of SERCA2b acquire the characteristic properties of SERCA2a, it is concluded that the stretch of the last 12 residues of SERCA2b is of critical importance.

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Azorhizobium caulinodans chemotaxis is controlled by an unusual phosphorelay network

Journal of Bacteriology ◽

10.1128/jb.00527-21 ◽

2021 ◽

Author(s):

Emily N. Kennedy ◽

Sarah A. Barr ◽

Xiaolin Liu ◽

Luke R. Vass ◽

Yanan Liu ◽

...

Keyword(s):

Competitive Advantage ◽

Bacterial Colonization ◽

Root Nodules ◽

Bacterial Species ◽

Root Surface ◽

Phosphoryl Group ◽

Flagellar Motor ◽

Azorhizobium Caulinodans ◽

Concerted Mechanism ◽

Phosphoryl Groups

Azorhizobium caulinodans is a nitrogen-fixing bacterium that forms root nodules on its host legume, Sesbania rostrata . This agriculturally significant symbiotic relationship is important in lowland rice cultivation, and allows for nitrogen fixation under flood conditions. Chemotaxis plays an important role in bacterial colonization of the rhizosphere. Plant roots release chemical compounds that are sensed by bacteria, triggering chemotaxis along a concentration gradient toward the roots. This gives motile bacteria a significant competitive advantage during root surface colonization. Although plant-associated bacterial genomes often encode multiple chemotaxis systems, A. caulinodans appears to encode only one. The che cluster on the A. caulinodans genome contains cheA , cheW , cheY2 , cheB , and cheR . Two other chemotaxis genes, cheY1 and cheZ , are located independently from the che operon. Both CheY1 and CheY2 are involved in chemotaxis, with CheY1 being the predominant signaling protein. A. caulinodans CheA contains an unusual set of C-terminal domains: a CheW-like/Receiver pair (termed W2-Rec), follows the more common single CheW-like domain. W2-Rec impacts both chemotaxis and CheA function. We found a preference for transfer of phosphoryl groups from CheA to CheY2, rather than to W2-Rec or CheY1, which appears to be involved in flagellar motor binding. Furthermore, we observed increased phosphoryl group stabilities on CheY1 compared to CheY2 or W2-Rec. Finally, CheZ enhanced dephosphorylation of CheY2 substantially more than CheY1, but had no effect on the dephosphorylation rate of W2-Rec. This network of phosphotransfer reactions highlights a previously uncharacterized scheme for regulation of chemotactic responses. IMPORTANCE Chemotaxis allows bacteria to move towards nutrients and away from toxins in their environment. Chemotactic movement provides a competitive advantage over non-specific motion. CheY is an essential mediator of the chemotactic response with phosphorylated and unphosphorylated forms of CheY differentially interacting with the flagellar motor to change swimming behavior. Previously established schemes of CheY dephosphorylation include action of a phosphatase and/or transfer of the phosphoryl group to another receiver domain that acts as a sink. Here, we propose A. caulinodans uses a concerted mechanism in which the Hpt domain of CheA, CheY2, and CheZ function together as a dual sink system to rapidly reset chemotactic signaling. To the best of our knowledge, this mechanism is unlike any that have previously been evaluated. Chemotaxis systems that utilize both receiver and Hpt domains as phosphate sinks likely occur in other bacterial species.

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Divergent Effects of G2019S and R1441C LRRK2 Mutations on LRRK2 and Rab10 Phosphorylations in Mouse Tissues

Cells ◽

10.3390/cells9112344 ◽

2020 ◽

Vol 9 (11) ◽

pp. 2344

Author(s):

Lucia Iannotta ◽

Alice Biosa ◽

Jillian H. Kluss ◽

Giulia Tombesi ◽

Alice Kaganovich ◽

...

Keyword(s):

Cellular Localization ◽

Disease Risk ◽

Brain Area ◽

Artificial Chromosome ◽

Mouse Tissues ◽

Lrrk2 G2019s ◽

Substrate Phosphorylation ◽

Lrrk2 Kinase ◽

Key Residues

Mutations in LRRK2 cause familial Parkinson’s disease and common variants increase disease risk. LRRK2 kinase activity and cellular localization are tightly regulated by phosphorylation of key residues, primarily Ser1292 and Ser935, which impacts downstream phosphorylation of its substrates, among which Rab10. A comprehensive characterization of LRRK2 activity and phosphorylation in brain as a function of age and mutations is missing. Here, we monitored Ser935 and Ser1292 phosphorylation in midbrain, striatum, and cortex of 1, 6, and 12 months-old mice carrying G2019S and R1441C mutations or murine bacterial artificial chromosome (BAC)-Lrrk2-G2019S. We observed that G2019S and, at a greater extent, R1441C brains display decreased phospho-Ser935, while Ser1292 autophosphorylation increased in G2019S but not in R1441C brain, lung, and kidney compared to wild-type. Further, Rab10 phosphorylation, is elevated in R1441C carrying mice, indicating that the effect of LRRK2 mutations on substrate phosphorylation is not generalizable. In BAC-Lrrk2-G2019S striatum and midbrain, Rab10 phosphorylation, but not Ser1292 autophosphorylation, decreases at 12-months, pointing to autophosphorylation and substrate phosphorylation as uncoupled events. Taken together, our study provides novel evidence that LRRK2 phosphorylation in mouse brain is differentially impacted by mutations, brain area, and age, with important implications as diagnostic markers of disease progression and stratification.

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Identification of an immunodominant functional domain on human CD36 antigen using human-mouse chimaeric proteins and homologue-replacement mutagenesis

Biochemical Journal ◽

10.1042/bj3050221 ◽

1995 ◽

Vol 305 (1) ◽

pp. 221-224 ◽

Cited By ~ 28

Author(s):

L Daviet ◽

R Buckland ◽

M D Puente Navazo ◽

J L McGregor

Keyword(s):

Amino Acids ◽

Monoclonal Antibody ◽

Monoclonal Antibodies ◽

Low Density Lipoprotein ◽

Density Lipoprotein ◽

Functional Domain ◽

Oxidized Low Density Lipoprotein ◽

Key Residues ◽

Antibody Epitopes ◽

Human And Mouse

The human CD36 antigen is a multifunctional membrane glycoprotein that acts as a receptor for thrombospondin, malaria-infected erythrocytes and oxidized low-density lipoprotein, as well as being implicated in the recognition of apoptotic neutrophils by macrophages. OKM5 and other anti-CD36 monoclonal antibodies have been shown to inhibit these CD36 adhesive functions, suggesting that the monoclonal-antibody epitopes and the domains that mediate these events are closely related. Analysis of a series of chimaeric exchanges between human and mouse CD36 shows that six anti-CD36 monoclonal antibodies (OKM5, FA6-152, L103, 5F1, SM phi and 10/5) recognize epitopes within the domain comprising amino acids 155-183. A seventh monoclonal antibody (13/10) binds to another domain that spans amino acids 30-76. Homologue-replacement mutagenesis performed within the human 155-183 immunodominant sequence identifies key residues for the binding of three functional monoclonal antibodies (OKM5, FA6-152 and L103). The fact that antibodies directed against the 155-183 domain can inhibit adhesion suggests that this domain is directly involved in CD36-ligand binding.

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Membrane curvature and the Tol-Pal complex determine polar localization of the chemoreceptor Tar inE. coli

10.1101/212670 ◽

2017 ◽

Author(s):

Terrens N. V. Saaki ◽

Henrik Strahl ◽

Leendert W. Hamoen

Keyword(s):

Cell Division ◽

Cell Membrane ◽

Cellular Localization ◽

Cluster Formation ◽

Signal Transduction Pathway ◽

Membrane Curvature ◽

Deletion Mutants ◽

E Coli ◽

Polar Localization ◽

Curvature Driven

AbstractChemoreceptors are localized at the cell poles ofEscherichia coliand other rod-shaped bacteria. Over the years different mechanisms have been put forward to explain this polar localization; from stochastic clustering, membrane curvature driven localization, interactions with the Tol-Pal complex, to nucleoid exclusion. To evaluate these mechanisms, we monitored the cellular localization of the aspartate chemoreceptor Tar in different deletion mutants. We did not find any indication for either stochastic cluster formation or nucleoid exclusion. However, the presence of a functional Tol-Pal complex appeared to be essential to retain Tar at cell poles. This finding also implies that the curvature of cell poles does not attract chemoreceptor complexes. Interestingly, Tar still accumulated at midcell intoland inpaldeletion mutants. In these mutants, the protein appears to gather at the base of division septa, a region characterised by strong membrane curvature. Chemoreceptors, like Tar, form trimer-of-dimers that bend the cell membrane due to a rigid tripod structure with an estimated curvature of approximately 37 nm. This curvature approaches the curvature of the cell membrane generated during cell division, and localization of chemoreceptor tripods at curved membrane areas is therefore energetically favourable as it lowers membrane tension. Indeed, when we introduced mutations in Tar that abolish the rigid tripod structure, the protein was no longer able to accumulate at midcell or cell poles. These findings favour a model where chemoreceptor localization inE. coliis driven by strong membrane curvature and association with the Tol-Pal complex.ImportanceBacteria have exquisite mechanisms to sense and to adapt to the environment they live in. One such mechanism involves the chemotaxis signal transduction pathway, in which chemoreceptors specifically bind certain attracting or repelling molecules and transduce the signals to the cell. In different rod-shaped bacteria, these chemoreceptors localize specifically to cell poles. Here, we examined the polar localization of the aspartate chemoreceptor Tar inE. coli, and found that membrane curvature at cell division sites and interaction with the Tal-pol protein complex, localize Tar at cell division sites, the future cell poles. This study shows how membrane curvature can guide localization of proteins in a cell.

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Characterization of Adenovirus 5 E1A Exon 1 Deletion Mutants in the Viral Replicative Cycle

Viruses ◽

10.3390/v12020213 ◽

2020 ◽

Vol 12 (2) ◽

pp. 213

Author(s):

Rita Costa ◽

Nikolas Akkerman ◽

Drayson Graves ◽

Leandro Crisostomo ◽

Scott Bachus ◽

...

Keyword(s):

Viral Replication ◽

Virus Replication ◽

Cellular Localization ◽

Negative Impact ◽

S Phase ◽

Viral Fitness ◽

Deletion Mutants ◽

Gene And Protein Expression ◽

Exon 1 ◽

Replicative Cycle

Human adenovirus infection is driven by Early region 1A (E1A) proteins, which are the first proteins expressed following the delivery of the viral genome to the cellular nucleus. E1A is responsible for reprogramming the infected cell to support virus replication alongside the activation of expression of all viral transcriptional units during the course of the infection. Although E1A has been extensively studied, most of these studies have focused on understanding the conserved region functions outside of a full infection. Here, we investigated the effects of small deletions in E1A exon 1 on the viral replicative cycle. Almost all deletions were found to have a negative impact on viral replication with the exception of one deletion found in the mutant dl1106, which replicated better than the wild-type E1A expressing dl309. In addition to growth, we assessed the virus mutants for genome replication, induction of the cytopathic effect, gene and protein expression, sub-cellular localization of E1A mutant proteins, induction of cellular S-phase, and activation of S-phase specific cellular genes. Importantly, our study found that virus replication is likely limited by host-specific factors, rather than specific viral aspects such as the ability to replicate genomes or express late proteins, after a certain level of these has been expressed. Furthermore, we show that mutants outside of the conserved regions have significant influence on viral fitness. Overall, our study is the first comprehensive evaluation of the dl1100 series of exon 1 E1A deletion mutants in viral fitness and provides important insights into the contribution that E1A makes to viral replication in normal human cells.

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