Oligomerization, Conformational Stability and Thermal Unfolding of Harpin, HrpZPss and Its Hypersensitive Response-Inducing C-Terminal Fragment, C-214-HrpZPss

Tarafdar, Pradip K.; Vedantam, Lakshmi Vasudev; Sankhala, Rajeshwer S.; Purushotham, Pallinti; Podile, Appa Rao; Swamy, Musti J.

doi:10.1371/journal.pone.0109871

Cited by 7 publications

(4 citation statements)

References 35 publications

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“…This denaturation step putatively results in a residual (βα) 4 β 5 structure but leads to a loss of substrate binding capabilities. 35 Other well-examined examples of proteins with a non-two-state nature of thermal denaturation are harpin protein HrpZ 36 and GrpE from Thermus thermophilus. 37 In both cases, the first transitions (according to DSC experiments) are invisible in CD spectroscopy.…”

Section: ■ Discussionmentioning

confidence: 99%

Hexamerization of Geranylgeranylglyceryl Phosphate Synthase Ensures Structural Integrity and Catalytic Activity at High Temperatures

et al. 2018

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The cell membranes of all archaea contain ether lipids, and a number of archaea are hyperthermophilic. Consequently, the enzymes that catalyze the synthesis of membrane ether lipids had to adopt to these rough conditions. Interestingly, the enzyme that establishes the first ether bond in these lipids, the geranylgeranylglyceryl phosphate synthase (GGGPS), forms hexamers in many hyperthermophilic archaea, while also dimeric variants of this enzyme exist in other species. We used Methanothermobacter thermautotrophicus GGGPS (mtGGGPS) as a model to elucidate the benefit of hexamerization. We studied the oligomerization interfaces in detail by introducing disturbing mutations and subsequently compared the stability and activity of generated dimeric and monomeric variants with the wild-type enzyme. Differential scanning calorimetry revealed a biphasic denaturation of mtGGGPS. The temperature of the first transition varies and rises with increasing oligomerization state. This first phase of denaturation leads to catalytic inactivation, but CD spectroscopy indicated only minor changes on the secondary structure level. The residual part of the fold is extremely thermostable and denatures in a second phase at temperatures >120 °C. The analysis of another distant native GGGPS enzyme affirms these observations. Molecular dynamics simulations revealed three structural elements close to the substrate binding sites with elevated flexibility. We assume that hexamerization might stabilize these structures, and kinetic studies support this hypothesis for the binding pocket of the substrate glycerol 1-phosphate. Oligomerization might thus positively affect the thermostability-flexibility trade-off in GGGPS by allowing a higher intrinsic flexibility of the individual protomers.

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Section: ■ Discussionmentioning

confidence: 99%

Hexamerization of Geranylgeranylglyceryl Phosphate Synthase Ensures Structural Integrity and Catalytic Activity at High Temperatures

et al. 2018

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“…The DSC data shown here demonstrate that there are two species of invertase with unique denaturation peaks. Previous studies have demonstrated that two peaks in a DSC scan could be due to a conformational change or oligomerization …”

Section: Discussionmentioning

confidence: 99%

“…Previous studies have demonstrated that two peaks in a DSC scan could be due to a conformational 21 change or oligomerization. 22 The decrease in the delay time as the invertase concentration and temperature increase suggests the more active invertase state is more stable at higher temperatures and higher invertase concentrations. These two observations are at odds with each other as higher invertase concentrations favor the formation of the octamer, 23 but higher temperatures favor the dimer.…”

Section: ■ Discussionmentioning

confidence: 99%

Employing Calorimetric Methods to Determine the Mechanism of the Invertase Maximal Activity Delay

Chan

Hicks

Kaliappan

et al. 2020

ACS Food Sci. Technol.

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Invertase catalyzes the hydrolysis of sucrose to glucose and fructose. Invertase from Saccharomyces cerevisiae has been used in studies of the enzyme catalytic mechanism for more than a century. Isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC) were used to determine the kinetics and physical characteristics of invertase, respectively. Two subspecies of invertase with unique melting points of ∼57 and ∼65 °C are identified using DSC. The rate of invertase-catalyzed sucrose hydrolysis was measured with ITC. ITC has the distinct advantage of directly measuring the rate of reaction, which makes it easy to determine when the enzyme exhibits maximal activity. ITC revealed a previously unknown significant delay after the injections of sucrose before invertase reaches maximal activity. The delay in maximum activity was decreased by a statistically significant (p > 0.05) 85% by an increase in invertase concentration from 1 to 47 μg/mL. Additionally, increasing the temperature of the reaction mixture from 25 to 55 °C resulted in a 66% decrease in the delay to reach maximum activity. However, increasing the sucrose concentration had no effect of the delay in maximum activity. These data are consistent with a change in the conformational or oligomeric state preceding the enzyme reaching full catalytic activity.

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“…The parameters of HpaG-Xcm were computed using the ProtParam tool (https://web.expasy.org/protparam/?_ga=1.7545010.1849019704.1487649425) 30 . The secondary structure, solvent accessibility and tertiary structure were predicted using I-TASSER (https://zhanglab.ccmb.med.umich.edu/I-TASSER/) 31,32 . The coiled-coil (CC) structure was predicted using Coiled-coil Prediction (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_lupas.html) 33 .…”

Section: Methodsmentioning

confidence: 99%

High temperatures affect the hypersensitive reaction, disease resistance and gene expression induced by a novel harpin HpaG-Xcm

Zhou

Liu

Huang

et al. 2019

Sci Rep

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Harpin proteins are produced by plant-pathogenic Gram-negative bacteria and regulate bacterial pathogenicity by inducing plant growth and defence responses in non-hosts. HpaG-Xcm, a novel harpin protein, was identified from Xanthomonas citri pv. mangiferaeindicae, which causes bacterial black spot of mango. Here, we describe the predicted structure and functions of HpaG-Xcm and investigate the mechanism of heat resistance. The HpaG-Xcm amino acid sequence contains seven motifs and two α-helices, in the N- and C-terminals, respectively. The N-terminal α-helical region contains two heptads, which form the coiled-coil (CC) structure. The CC region, which is on the surface of HpaG-Xcm, forms oligomeric aggregates by forming hydrophobic interactions between hydrophobic amino acids. Like other harpins, HpaG-Xcm was heat stable, promoted root growth and induced a hypersensitive response (HR) and systemic acquired resistance in non-host plants. Subjecting HpaG-Xcm to high temperatures altered the gene expression induced by HpaG-Xcm in tobacco leaves, probably due to changes in the spatial structure of HpaG-Xcm. Phenotypic tests revealed that the high-temperature treatments reduced the HR and disease resistance induced by HpaG-Xcm but had little effect on growth promotion. These findings indicate that the stability of interactions between CC and plants may be associated with thermal stability of HpaG-Xcm.

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Oligomerization, Conformational Stability and Thermal Unfolding of Harpin, HrpZPss and Its Hypersensitive Response-Inducing C-Terminal Fragment, C-214-HrpZPss

Cited by 7 publications

References 35 publications

Hexamerization of Geranylgeranylglyceryl Phosphate Synthase Ensures Structural Integrity and Catalytic Activity at High Temperatures

Hexamerization of Geranylgeranylglyceryl Phosphate Synthase Ensures Structural Integrity and Catalytic Activity at High Temperatures

Employing Calorimetric Methods to Determine the Mechanism of the Invertase Maximal Activity Delay

High temperatures affect the hypersensitive reaction, disease resistance and gene expression induced by a novel harpin HpaG-Xcm

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