BiP, HSP70, NDK and PDI in wheat endosperm. II. Effects of high temperature on protein and mRNA accumulation

The effects of high temperature on accumulation of the 70‐kDa heat shock protein (HSP70) and nucleoside diphosphate kinase (NDK) as well as two other proteins that have roles in the biosynthesis of storage proteins were examined during grain development. An HSP70 homolog and a 17‐kDa NDK were co‐purified from wheat endosperm, their identity verified, and a cDNA for an HSP70 expressed in endosperm was isolated. Wheat plants ( Triticum aestivum , cvs Butte and Vulcan) were heat shocked at 40°C or exposed to maximum daily temperatures of 37 or 40°C during early or mid‐grain fill. Antibodies and cDNA probes for BiP, HSP70, NDK and PDI were used to examine the effect of high temperatures on the accumulation of protein and mRNA in the endosperm. HSP70 mRNA levels increased substantially when plants were exposed to heat shock or to a 1‐day gradual increase to 40°C. The effects of a 5‐day heat treatment on mRNA levels were more complicated and depended on the developmental stage of the grain. A treatment that began at 7 days post‐anthesis (DPA) decreased the level of mRNA for HSP70, BiP, PDI and NDK, whereas a treatment that began at 14 DPA slightly increased mRNA levels. The same treatments increased the accumulation of HSP70 but did not affect BiP, PDI, or NDK protein levels. This is the first detailed report on the effects of heat on mRNA and protein levels for HSP70 in a developing seed storage tissue.     

Dimeric structure of nucleoside diphosphate kinase from moderately halophilic bacterium: contrast to the tetrameric Pseudomonas counterpart

Light scattering and chemical cross-linking analyses of nucleoside diphosphate kinase (NDK) from moderate halophile, Halomonas sp. 593 (HaNDK), unambiguously demonstrated that this enzyme formed a dimeric structure, in contrast to the Pseudomonas NDK (PaNDK), a nonhalophilic counterpart, and other NDKs from Gram-negative bacteria, which all formed a tetrameric structure. Comparison of HaNDK and PaNDK showed that the HaNDK was less thermally stable than the PaNDK: the optimum temperature of PaNDK enzyme activity was 20 degrees C higher than that of HaNDK. However, the HaNDK readily refolded and reassembled back to the active dimeric structure, upon heat denaturation at 0.2 M NaCl, as soon as the temperature was lowered. On the contrary, the thermally more stable PaNDK was irreversibly denatured at its melting temperature.     

A structural mechanism for dimeric to tetrameric oligomer conversion in Halomonas sp. nucleoside diphosphate kinase

Nucleoside diphosphate kinase (NDK) is known to form homotetramers or homohexamers. To clarify the oligomer state of NDK from moderately halophilic Halomonas sp. 593 (HaNDK), the oligomeric state of HaNDK was characterized by light scattering followed by X‐ray crystallography. The molecular weight of HaNDK is 33,660, and the X‐ray crystal structure determination to 2.3 and 2.7 Å resolution showed a dimer form which was confirmed in the different space groups of R3 and C2 with an independent packing arrangement. This is the first structural evidence that HaNDK forms a dimeric assembly. Moreover, the inferred molecular mass of a mutant HaNDK (E134A) indicated 62.1–65.3 kDa, and the oligomerization state was investigated by X‐ray crystallography to 2.3 and 2.5 Å resolution with space groups of P2₁ and C2. The assembly form of the E134A mutant HaNDK was identified as a Type I tetramer as found in Myxococcus NDK. The structural comparison between the wild‐type and E134A mutant HaNDKs suggests that the change from dimer to tetramer is due to the removal of negative charge repulsion caused by the E134 in the wild‐type HaNDK. The higher ordered association of proteins usually contributes to an increase in thermal stability and substrate affinity. The change in the assembly form by a minimum mutation may be an effective way for NDK to acquire molecular characteristics suited to various circumstances.     

Characterization of the 3' --> 5' exonuclease activity found in human nucleoside diphosphate kinase 1 (NDK1) and several of its homologues

Nucleoside diphosphate kinases (NDKs), an evolutionarily conserved family of proteins, synthesize nucleoside triphosphates from nucleoside diphosphates and ATP. Here, we have characterized the kinase activity and DNA processing functions of eight human proteins that contain at least one domain homologous to Escherichia coli NDK. Not all human proteins with NDK-like domains exhibited NDK activity when expressed as recombinant proteins in E. coli. Human NDK1 (NM23-H1) has been reported to have 3' --> 5' exonuclease activity. In addition to human NDK1, we also find that human NDK5, NDK7, and NDK8 contain 3' --> 5' exonuclease activity. Site-directed mutagenesis, competition assays between wild-type and mutant NDK proteins, and NMR studies confirmed that the DNA-binding and 3' --> 5' exonuclease activity of human NDK1 is an intrinsic activity of the protein. Using double-stranded DNA substrates containing modified bases, human NDK1 efficiently excised nucleotides from the single-strand break produced by APE1 or Nth1. When human cells were treated with various DNA-damaging agents, human NDK1 translocated from the cytoplasm to the nucleus. These results suggest that, in addition to maintenance of nucleotide pool balance, the human NDK-like proteins may have previously unrecognized roles in DNA nucleolytic processing.     

Engineering of halophilic enzymes: two acidic amino acid residues at the carboxy-terminal region confer halophilic characteristics to Halomonas and Pseudomonas nucleoside diphosphate kinases

Nucleoside diphosphate kinase from Halomonas sp. 593 (HaNDK) exhibits halophilic characteristics. Residues 134 and 135 in the carboxy-terminal region of HaNDK are Glu-Glu, while those of its homologous counterpart of non-halophilic Pseudomonas NDK (PaNDK) are Ala-Ala. The double mutation, E134A-E135A, in HaNDK results in the loss of the halophilic characteristics, and, conversely, the double mutation of A134E-A135E in PaNDK confers halophilic characters to this enzyme, indicating that the charged state of these two residues that are located in the C-terminal region plays a critical role in determining halophilic characteristics. The importance of these two residues versus the net negative charges will be discussed in relation to the halophilicity of NDK.     

Regulatory GTP-binding proteins (ADP-ribosylation factor, Gt, and RAS) are not activated directly by nucleoside diphosphate kinase.

The expression of nucleoside diphosphate kinase (NDK) genes has been implicated as a negative regulator of murine and human tumor metastases and is critical to proper development in Drosophila melanogaster. Molecular mechanisms for the role(s) of NDK in these complex processes have not yet been elucidated, but several reports have suggested that these and many other signal transduction pathways may be activated by NDK acting directly on a regulatory GTP-binding protein(s). To test this hypothesis, we examined the ability of NDK to catalyze the phosphorylation of the GDP bound to the following three members of the superfamily of regulatory GTP-binding proteins: Gt, Ha-ras p21, and ARF. We have found no evidence to support the hypothesis that NDK can directly activate any GTP-binding protein. Rather, evidence is presented which clearly shows that all of the GTP formed upon incubation of GTP-binding proteins with NDK is the result of NDK utilizing free GDP as substrate. The GDP bound to the regulatory proteins is not a substrate for NDK under conditions in which free nucleotides are rapidly and efficiently phosphorylated. The importance of appropriate controls for dissociation of GDP from the regulatory proteins both during the NDK reaction and during the analysis of product is demonstrated. We believe there is currently no experimental evidence to support the hypothesis that NDK can directly activate a regulatory GTP-binding protein.     

Computer-aided design of the stability of pyruvate formate-lyase from Escherichia coli by site-directed mutagenesis

Using computer-aided design of single-site mutations, three amino acid residues determined by changes in folding free energy between wild-type (wt) and mutant proteins were exchanged to enhance the stability of pyruvate formate-lyase (PFL). The mutant enzymes were tested for properties such as optimum temperature, optimum pH, kinetic parameters, and stability to temperature. There were two mutant variants, Glu336Cys and Glu400Ile, that exhibited increased thermostability as compared to the wt enzyme. The melting temperatures (T(m), the temperature at which 50% inactivation occurs after heat treatment for 20 min) of Glu336Cys and Glu400Ile increased by 3.7 and 2.2 respectively. They also showed an increase in half life of about 1.80 and 2.21-fold, whereas Ala273Cys showed a slight decrease as compared with the wt enzyme.     

Selection of putative colorectal cancer markers by applying PCA on the soluble proteome of tumors: NDK A as a promising candidate

Aiming to find new tumor markers for colorectal cancer (CRC), we applied proteomic methodologies to compare the soluble sub-proteome of healthy and tumoral colorectal mucosa. Out of 91 differentially expressed proteins, 23 were selected by principal component analysis (PCA) as the major contributors to the overall difference detected. After MS/MS analysis, 16 proteins were identified. From those, we chose 14-3-3-zeta/delta, retinoblastoma-binding protein 4 (RBBP-4), DJ-1, and nucleoside diphosphate kinase A (NDK A) for further studies, on the basis of their levels and known implication in cancer. Specific immunodetection demonstrated only the NDK A levels allowed to differentiate healthy mucosa from tumor tissue in all the patients. Hence, we used the colon cancer cell line Caco-2 to study the relationship between NDK A and colon cell tumorigenesis, finding it over-expressed in undifferentiated (tumor-like) cells regarding the differentiated ones. Noticeably, we also found increased levels of the NDK A in the secretome of tumor-like cells and, as expected, indications of higher levels of NDK A in the serum of CRC patients. In conclusion, the four validated proteins could constitute a panel of tissue markers for CRC, being the NDK A the most interesting candidate for further serum biomarker studies.     

Sensitivity to a nonpeptidic compound (RPR103611) blocking human immunodeficiency virus type 1 Env-mediated fusion depends on sequence and accessibility of the gp41 loop region

The triterpene RPR103611 is an efficient inhibitor of membrane fusion mediated by the envelope proteins (Env, gp120-gp41) of CXCR4-dependent (X4) human immunodeficiency virus type 1 (HIV-1) strains, such as HIV-1(LAI) (LAI). Other X4 strains, such as HIV-1(NDK) (NDK), and CCR5-dependent (R5) HIV-1 strains, such as HIV-1(ADA) (ADA), were totally resistant to RPR103611. Analysis of chimeric LAI-NDK Env proteins identified a fragment of the NDK gp41 ectodomain determining drug resistance. A single difference at position 91, leucine in LAI and histidine in NDK, apparently accounted for their sensitivity or resistance to RPR103611. We had previously identified a mutation of isoleucine 84 to serine in a drug escape LAI variant. Both I84 and L91 are located in the "loop region" of gp41 separating the proximal and distal helix domains. Nonpolar residues in this region therefore appear to be important for the antiviral activity of RPR103611 and are possibly part of its target. However, another mechanism had to be envisaged to explain the drug resistance of ADA, since its gp41 loop region was almost identical to that of LAI. Fusion mediated by chimeric Env consisting of LAI gp120 and ADA gp41, or the reciprocal construct, was fully blocked by RPR103611. The gp120-gp41 complex of R5 strains is stable, relative to that of X4 strains, and this stability could play a role in their drug resistance. Indeed, when the postbinding steps of ADA infection were performed under mildly acidic conditions (pH 6.5 or 6.0), a treatment expected to favor dissociation of gp120, we achieved almost complete neutralization by RPR103611. The drug resistance of NDK was partially overcome by preincubating virus with soluble CD4, a gp120 ligand inducing conformational changes in the Env complex. The antiviral efficacy of RPR103611 therefore depends on the sequence of the gp41 loop and the stability of the gp120-gp41 complex, which could limit the accessibility of this target.     

Computer-Aided Design of the Stability of Pyruvate Formate-Lyase from Escherichia coli by Site-Directed Mutagenesis

Using computer-aided design of single-site mutations, three amino acid residues determined by changes in folding free energy between wild-type (wt) and mutant proteins were exchanged to enhance the stability of pyruvate formate-lyase (PFL). The mutant enzymes were tested for properties such as optimum temperature, optimum pH, kinetic parameters, and stability to temperature. There were two mutant variants, Glu336Cys and Glu400Ile, that exhibited increased thermostability as compared to the wt enzyme. The melting temperatures (T _m, the temperature at which 50% inactivation occurs after heat treatment for 20 min) of Glu336Cys and Glu400Ile increased by 3.7 and 2.2 respectively. They also showed an increase in half life of about 1.80 and 2.21-fold, whereas Ala273Cys showed a slight decrease as compared with the wt enzyme.     

Arabidopsis NDK1 is a component of ROS signaling by interacting with three catalases

Plants sense various environmental stimuli and have specific signaling pathways to respond to these cues. We focused on light responsive components and found that NDKs were phosphorylated specifically after red light irradiation in Pisum sativum [Tanaka et al. (1998) J. Photochem. Photobiol. B 45: 113] and after blue light irradiation in Neurospora crassa [Oda and Hasunuma (1997) Mol. Gen. Genet. 256: 593, Ogura et al. (2001) J. Biol. Chem. 276: 21228]. We performed yeast two-hybrid screening using AtNDK1, the counterpart of NDK-P1 (Pisum sativum NDK1) in Arabidopsis, as bait, and isolated catalase3 (AtCat3). Interactions between AtNDK1-AtCAT1 and AtNDK1-AtCAT2 were also detected with the two-hybrid system. Non-denaturing two-dimensional gel electrophoresis of crude extracts from plants revealed that catalase and NDK activities co-migrated in the same area of the gel. Transgenic plants expressing AtNDK1 under control of the CaMV 35S promoter exhibited tolerance to paraquat and high ability to eliminate exogenous H2O2. These results indicate that AtNDK1 has a role in ROS response.     

Activation of halophilic nucleoside diphosphate kinase by a non-ionic osmolyte,trimethylamine N-oxide

The folding and activity of halophilic enzymes are believed to require the presence of salts at high concentrations. When the inactivated nucleoside diphosphate kinase (NDK) from extremely halophilic archaea was incubated with low salt media, no activity was regained over the course of 8 days. When it was incubated with 2 M NaCl or 3 M KCl, however, it gradually regained activity. To our surprise, trimethylamine N-oxide (TMAO) also was able to induce activation at 4.0 M. The enzyme activity and secondary structure of refolded NDK in 4 M TMAO were comparable with those of the native NDK or the refolded NDK in 3.8 M NaCl. TMAO is not an electrolyte, meaning that the presence of concentrated salts is not an absolute requirement, and that charge shielding or ion binding is not a sole factor for the folding and activation of NDK. Although both NaCl and TMAO are effective in refolding NDK, the mechanism of their actions appears to be different: the effect of protein concentration and pH on refolding is qualitatively different between these two, and at pH 8.0 NDK could be refolded in the presence of 4 M TMAO only when low concentrations of NaCl are included.     

NDK Interacts with FtsZ and Converts GDP to GTP to Trigger FtsZ Polymerisation - A Novel Role for NDK

Introduction

Nucleoside diphosphate kinase (NDK), conserved across bacteria to humans, synthesises NTP from NDP and ATP. The eukaryotic homologue, the NDPK, uses ATP to phosphorylate the tubulin-bound GDP to GTP for tubulin polymerisation. The bacterial cytokinetic protein FtsZ, which is the tubulin homologue, also uses GTP for polymerisation. Therefore, we examined whether NDK can interact with FtsZ to convert FtsZ-bound GDP and/or free GDP to GTP to trigger FtsZ polymerisation.

Methods

Recombinant and native NDK and FtsZ proteins of Mycobacterium smegmatis and Mycobacterium tuberculosis were used as the experimental samples. FtsZ polymersation was monitored using 90° light scattering and FtsZ polymer pelleting assays. The γ³²P-GTP synthesised by NDK from GDP and γ³²P-ATP was detected using thin layer chromatography and quantitated using phosphorimager. The FtsZ bound ³²P-GTP was quantitated using phosphorimager, after UV-crosslinking, followed by SDS-PAGE. The NDK-FtsZ interaction was determined using Ni²⁺-NTA-pulldown assay and co-immunoprecipitation of the recombinant and native proteins in vitro and ex vivo, respectively.

Results

NDK triggered instantaneous polymerisation of GDP-precharged recombinant FtsZ in the presence of ATP, similar to the polymerisation of recombinant FtsZ (not GDP-precharged) upon the direct addition of GTP. Similarly, NDK triggered polymerisation of recombinant FtsZ (not GDP-precharged) in the presence of free GDP and ATP as well. Mutant NDK, partially deficient in GTP synthesis from ATP and GDP, triggered low level of polymerisation of MsFtsZ, but not of MtFtsZ. As characteristic of NDK’s NTP substrate non-specificity, it used CTP, TTP, and UTP also to convert GDP to GTP, to trigger FtsZ polymerisation. The NDK of one mycobacterial species could trigger the polymerisation of the FtsZ of another mycobacterial species. Both the recombinant and the native NDK and FtsZ showed interaction with each other in vitro and ex vivo, alluding to the possibility of direct phosphorylation of FtsZ-bound GDP by NDK.

Conclusion

Irrespective of the bacterial species, NDK interacts with FtsZ in vitro and ex vivo and, through the synthesis of GTP from FtsZ-bound GDP and/or free GDP, and ATP (CTP/TTP/UTP), triggers FtsZ polymerisation. The possible biological context of this novel activity of NDK is presented.     

Down regulation effect of Rosmarinus officinalis polyphenols on cellular stress proteins in rat pheochromocytoma PC12 cells

Polyphenols are known to exhibit wide spectrum of benefit for brain health and to protect from several neurodegenerative diseases. The present study was sought to determine the neuroprotective effects of Rosmarinus officinalis' polyphenols (luteolin, carnosic acid, and rosmarinic acid) through the investigation of stress-related proteins. We carried out measurement of the expression of heat-shock protein (Hsp) 47 promoter in heat stressed Chinese hamster ovary transfected cells. We performed proteomic analysis and confirmed gene expression by real time PCR in PC12 cells. Results showed that these compounds modulated significant and different effects on the expression of 4 stress-related proteins: heat shock protein 90 α (Hsp90), Transitional endoplasmic reticulum ATPase (VCP/p97), Nucleoside diphosphate kinase (NDK), and Hypoxia up-regulated protein 1 (HYOU1)) at translational and post translational levels in PC12 cells and they downregulated the expression of Hsp47 activity in Chinese hamster transformed cells. These findings suggest that luteolin, carnosic acid, and rosmarinic acid may modulate the neuroprotective defense system against cellular stress insults and increase neuro-thermotolerance.     

Resistance to a drug blocking human immunodeficiency virus type 1 entry (RPR103611) is conferred by mutations in gp41.

A triterpene derived from betulinic acid (RPR103611) blocks human immunodeficiency virus type 1 (HIV-1) infection and fusion of CD4+ cells with cells expressing HIV-1 envelope proteins (gp120 and gp41), suggesting an effect on virus entry. This compound did not block infection by a subtype D HIV-1 strain (NDK) or cell-cell fusion mediated by the NDK envelope proteins. The genetic basis of drug resistance was therefore addressed by testing envelope chimeras derived from NDK and a drug-sensitive HIV-1 strain (LAI, subtype B). A drug-resistant phenotype was observed for all chimeras bearing the ectodomain of NDK gp41, while the origins of gp120 and of the membrane anchor and cytoplasmic domains of gp41 had no apparent role. The envelope gene of a LAI variant, fully resistant to the antiviral effect of RPR103611, was cloned and sequenced. Its product differed from the parental sequence at two positions in gp41, with changes of arginine 22 to alanine (R22A) and isoleucine 84 to serine (I84S), the gp120 being identical. In the context of LAI gp41, the I84S substitution was sufficient for drug resistance. Therefore, in two different systems, differences in gp41 were associated with sensitivity or resistance to RPR103611. Modifications of gp41 can affect the quaternary structure of gp120 and gp41 and the accessibility of gp120 to antiviral agents such as neutralizing antibodies. However, a direct effect of RPR103611 on a gp41 target must also be envisioned, in agreement with the blocking of apparently late steps of HIV-1 entry. This compound could be a valuable tool for structure-function studies of gp41.     

Human nucleoside diphosphate kinase B (Nm23-H2) from melanoma cells shows altered phosphoryl transfer activity due to the S122P mutation.

The Ser122 --> Pro mutation in human nucleoside diphosphate kinase (NDK)-B/Nm23-H2 was recently found in melanoma cells. In comparison to the wild-type enzyme, steady state activity of NDKS122P with ATP and TDP as substrates was slowed down 5-fold. We have utilized transient kinetic techniques to analyze phosphoryl transfer between the mutant enzyme and various pairs of nucleoside triphosphates and nucleoside diphosphates. The two half-reactions of phosphorylation and dephosphorylation of the active site histidine residue (His118) were studied separately by making use of the intrinsic fluorescence changes which occur during these reactions. All apparent second order rate constants are drastically reduced, falling 5-fold for phosphorylation and 40-200-fold for dephosphorylation. Also, the reactivity of the mutant with pyrimidine nucleotides and deoxy nucleotides is more than 100-fold reduced compared with the wild-type. Thus, the rate-limiting step of the NDK-BS122P-catalyzed reaction is phosphoryl transfer from the phospho-enzyme intermediate to the nucleoside diphosphate and not phosphoryl transfer from the nucleoside triphosphate to the enzyme as was found for the wild-type protein. This results in a pronounced shift of the equilibrium between unphosphorylated and phosphorylated enzyme. Moreover, like the Killer-of-prune mutation in Drosophila NDK and the neuroblastoma Ser120 --> Gly mutation in human NDK-A/Nm23-H1, the Ser122 --> Pro substitution in NDK-B affects the stability of the protein toward heat and urea. These significantly altered properties may be relevant to the role of the mutant enzyme in various intracellular processes.     

Conformational stability of legume lectins reflect their different modes of quaternary association: solvent denaturation studies on concanavalin A and winged bean acidic agglutinin

Thermodynamic parameters associated with the unfolding of the legume lectin, WBA II, were determined by isothermal denaturation. The analysis of isothermal denaturation data provided values for conformational stability and heat capacity for WBA II unfolding. To explore the role of intersubunit contact in stability, we carried out similar studies under identical conditions on Concanavalin A, a legume lectin of nearly similar size, buried hydrophobic surface area and tertiary structure to that of WBA II but with a different oligomerization pattern. Both proteins showed a reversible two-state unfolding with guanidine hydrochloride. As expected, the change in heat capacity upon unfolding was similar for both proteins at 3.5 and 3.7 kcal mol(-1) K(-1) for Concanavalin A and WBA II, respectively. Although the deltaG(H20) at the maximum stability of both proteins is around 16 kcal/mol, Concanavalin A exhibits greater stability at higher temperatures. The T(g) obtained for Concanavalin A and WBA II were 21 degrees C apart at 87.2 and 66.6 degrees C, respectively. The higher conformational stability at higher temperatures and the T(g) of Concanavalin A as compared to that of WBA II are largely due to substantial differences in the degree of subunit contact in these dimeric proteins. Ionic interactions and hydrogen bonding between the monomers of the two proteins also seem to play a significant role in the observed stability differences between these two proteins.     

家蝇幼虫抗菌物质组成及其理化性质

用抑菌圈、酸碱性聚丙烯酰胺凝胶电泳(PAGE)等方法,对家蝇幼虫免疫血淋巴抗菌物质组成及其理化特性进行了研究。结果表明,家蝇抗菌物质是蛋白质,呈碱性和近中性,由5种成分组成。家蝇抗菌物质有较稳定的理化特性。如对热,对较高浓度盐溶液,较极端的pH溶液,室温放置6h及冻融12次等处理均有较高的稳定性。     

Thermodynamic basis for the stabilities of three CutA1s from Pyrococcus horikoshii,Thermus thermophilus, and Oryza sativa, with unusually high denaturation temperatures

In order to elucidate the stabilization mechanism of CutA1 from Pyrococcus horikoshii (PhCutA1) with a denaturation temperature of nearly 150 degrees C, GuHCl denaturation and heat denaturation were examined at neutral and acidic pHs. As a comparison, CutA1 proteins from Thermus thermophilus (TtCutA1) and Oryza sativa (OsCutA1) were also examined, which have lower optimum growth temperatures of 75 and 28 degrees C, respectively, than that (98 degrees C) of P. horikoshii. GuHCl-induced unfolding and refolding curves of the three proteins showed hysteresis effects due to an unusually slow unfolding rate. The midpoints of refolding for PhCutA1, TtCutA1 and OsCutA1 were 5.7 M, 3.3 M, and 2.3 M GuHCl, respectively, at pH 8.0 and 37 degrees C. DSC experiments with TtCutA1 and OsCutA1 showed that the denaturation temperatures were remarkably high, 112.8 and 97.3 degrees C, respectively, at pH 7.0 and that the good heat reversibility was amenable to thermodynamic analyses. At acidic pH, TtCutA1 showed higher stability to both heat and denaturant than PhCutA1. Combined with the data for DSC and denaturant denaturation, the unfolding Gibbs energy of PhCutA1 could be depicted as a function of temperature. It was experimentally revealed that (1) the unusually high stability of PhCutA1 basically originates from a common trimer structure of the three proteins, (2) the stability of PhCutA1 is superior to those of the other two CutA1s over all temperatures above 0 degrees C at neutral pH, due to the decrease in both enthalpy and entropy, and (3) ion pairs of PhCutA1 contribute to the unusually high stability at neutral pH.