Separation of hexahistidine fusion proteins with immobilized metal ion affinity chromatographic (IMAC) sorbents derived from MN+‐tacn and its derivatives

The capabilities of a new class of immobilized (im) metal ion chelate complexes (IMCCs), derived from 1,4,7‐triazacyclononane (tacn), bis(1,4,7‐triazacyclononyl) ethane (dtne) and bis(1,4,7‐triazacyclononyl)propane (dtnp) complexed with the borderline metal ions Cu²⁺, Ni²⁺, Zn²⁺, Mn²⁺, Co²⁺, and Cr³⁺, for the purification of proteins have been investigated. In particular, the binding behavior of a model protein, the C‐terminal hexahistidine tagged recombinant fusion protein Schistosoma japonicum glutathione S‐transferase‐Saccharomyces cerevisiae mitochondrial ATP synthase δ‐subunit (GST‐δATPase‐His₆), with these new immobilized metal ion affinity chromatographic (IMAC) sorbents was compared to the properties of a conventional sorbent, derived from immobilized Ni(II)‐nitrilotriacetic acid (im‐Ni²⁺‐NTA). Investigations using the recombinant GST‐δATPase‐His₆ and recombinant S. japonicum glutathione S‐transferase (GST) lacking a hexahistidine tag have confirmed that the C‐terminal tag hexahistidine residues were required for the binding process to occur with these IMAC systems. The results also confirm that recombinant fusion proteins such as GST‐δATPase‐His₆ can be isolated in high purity with these IMAC systems. Moreover, these new macrocyclic systems manifest different selectivity features as a function of pH or ionic strength when compared to the conventional, unconstrained iminodiacetic acid (IDA) or NTA chelating ligands, complexed with borderline metal ions such as Cu²⁺ or Ni²⁺, as IMAC systems. Biotechnol. Bioeng. 2009;103: 747–756. © 2009 Wiley Periodicals, Inc.     

The solution structure of the Mg2+ form of soybean calmodulin isoform 4 reveals unique features of plant calmodulins in resting cells

Soybean calmodulin isoform 4 (sCaM4) is a plant calcium‐binding protein, regulating cellular responses to the second messenger Ca²⁺. We have found that the metal ion free (apo‐) form of sCaM4 possesses a half unfolded structure, with the N‐terminal domain unfolded and the C‐terminal domain folded. This result was unexpected as the apo‐forms of both soybean calmodulin isoform 1 (sCaM1) and mammalian CaM (mCaM) are fully folded. Because of the fact that free Mg²⁺ ions are always present at high concentrations in cells (0.5–2 mM), we suggest that Mg²⁺ should be bound to sCaM4 in nonactivated cells. CD studies revealed that in the presence of Mg²⁺ the initially unfolded N‐terminal domain of sCaM4 folds into an α‐helix‐rich structure, similar to the Ca²⁺ form. We have used the NMR backbone residual dipolar coupling restraints ¹D_NH, ¹D_CαHα, and ¹D_C′Cα to determine the solution structure of the N‐terminal domain of Mg²⁺‐sCaM4 (Mg²⁺‐sCaM4‐NT). Compared with the known structure of Ca²⁺‐sCaM4, the structure of the Mg²⁺‐sCaM4‐NT does not fully open the hydrophobic pocket, which was further confirmed by the use of the fluorescent probe ANS. Tryptophan fluorescence experiments were used to study the interactions between Mg²⁺‐sCaM4 and CaM‐binding peptides derived from smooth muscle myosin light chain kinase and plant glutamate decarboxylase. These results suggest that Mg²⁺‐sCaM4 does not bind to Ca²⁺‐CaM target peptides and therefore is functionally similar to apo‐mCaM. The Mg²⁺‐ and apo‐structures of the sCaM4‐NT provide unique insights into the structure and function of some plant calmodulins in resting cells.     

CIDNP study of the aromatic side chain interactions in myotoxina

CIDNP and COSY measurements were applied to study aromatic side chain interactions and conformations in myotoxina, aCrotalus venom toxin which acts as blocker of the Ca²⁺ influx in the sarcoplasmic reticulum calcium pump. New evidence for the existence of a hydrophobic aromatic cluster at the amino terminus was obtained. This cluster consists of Tyr₁, His₅, His₁₀, and (possibly) F₁₂. The CIDNP data clearly establish that the usual order of the tyrosine 2, 6 and 3, 5 proton signals of Tyr, is inverted, because of the large diamagnetic shielding effects of one ring on the other. The lines of the 2, 6 ring protons of Tyr₁, and proton 4 in each of His₅ and His₁₀ are significantly broadened, an outcome of the side-chain hydrophobic interaction. The aromatic cluster could possibly present a hydrophobic sticky patch for binding of toxin by Ca²⁺ ATPase.     

CIDNP study of the aromatic side chain interactions in myotoxina

CIDNP and COSY measurements were applied to study aromatic side chain interactions and conformations in myotoxina, aCrotalus venom toxin which acts as blocker of the Ca²⁺ influx in the sarcoplasmic reticulum calcium pump. New evidence for the existence of a hydrophobic aromatic cluster at the amino terminus was obtained. This cluster consists of Tyr₁, His₅, His₁₀, and (possibly) F₁₂. The CIDNP data clearly establish that the usual order of the tyrosine 2, 6 and 3, 5 proton signals of Tyr, is inverted, because of the large diamagnetic shielding effects of one ring on the other. The lines of the 2, 6 ring protons of Tyr₁, and proton 4 in each of His₅ and His₁₀ are significantly broadened, an outcome of the side-chain hydrophobic interaction. The aromatic cluster could possibly present a hydrophobic sticky patch for binding of toxin by Ca²⁺ ATPase.     

The Identification of Histidine 712 as a Critical Residue for Constitutive TRPV5 Internalization

The epithelial Ca²⁺ channel TRPV5 constitutes the apical entry gate for Ca²⁺ transport in renal epithelial cells. Ablation of the trpv5 gene in mice leads to a reduced Ca²⁺ reabsorption. TRPV5 is tightly regulated by various calciotropic hormones, associated proteins, and other factors, which mainly affect channel activity via the C terminus. To further identify the role of the C terminus in TRPV5 regulation, we expressed channels harboring C-terminal deletions and studied channel activity by measuring intracellular Ca²⁺ concentration ([Ca²⁺]_i) using fura-2 analysis. Removal of amino acid His⁷¹² elevated the [Ca²⁺]_i, indicating enlarged TRPV5 activity. In addition, substitution of the positively charged His⁷¹² for a negative (H712D) or neutral (H712N) amino acid also stimulated TRPV5 activity. This critical role of His⁷¹² was confirmed by patch clamp analysis, which demonstrates increased Na⁺ and Ca²⁺ currents for TRPV5-H712D. Cell surface biotinylation studies revealed enhanced plasma membrane expression of TRPV5-H712D as compared with wild-type (WT) TRPV5. This elevated plasma membrane presence also was observed with the Ca²⁺-impermeable TRPV5-H712D and TRPV5-WT pore mutants, demonstrating that the elevation is not due to the increased [Ca²⁺]_i. Finally, using an internalization assay, we demonstrated a delayed cell surface retrieval for TRPV5-H712D, likely causing the increase in plasma membrane expression. Together, these results demonstrate that His⁷¹² plays an essential role in plasma membrane regulation of TRPV5 via a constitutive endocytotic mechanism.     

The myristoylation of guanylate cyclase-activating protein-2 causes an increase in thermodynamic stability in the presence but not in the absence of Ca²⁺

Guanylate cyclase activating protein‐2 (GCAP‐2) is a Ca²⁺‐binding protein of the neuronal calcium sensor (NCS) family. Ca²⁺‐free GCAP‐2 activates the retinal rod outer segment guanylate cyclases ROS‐GC1 and 2. Native GCAP‐2 is N‐terminally myristoylated. Detailed structural information on the Ca²⁺‐dependent conformational switch of GCAP‐2 is missing so far, as no atomic resolution structures of the Ca²⁺‐free state have been determined. The role of the myristoyl moiety remains poorly understood. Available functional data is incompatible with a Ca²⁺‐myristoyl switch as observed in the prototype NCS protein, recoverin. For the homologous GCAP‐1, a Ca²⁺‐independent sequestration of the myristoyl moiety inside the proteins structure has been proposed. In this article, we compare the thermodynamic stabilities of myristoylated and non‐myristoylated GCAP‐2 in their Ca²⁺‐bound and Ca²⁺‐free forms, respectively, to gain information on the nature of the Ca²⁺‐dependent conformational switch of the protein and shed some light on the role of its myristoyl group. In the absence of Ca²⁺, the stability of the myristoylated and non‐myristoylated forms was indistinguishable. Ca²⁺ exerted a stabilizing effect on both forms of the protein, which was significantly stronger for myr GCAP‐2. The stability data were corroborated by dye binding experiments performed to probe the solvent‐accessible hydrophobic surface of the protein. Our results strongly suggest that the myristoyl moiety is permanently solvent‐exposed in Ca²⁺‐free GCAP‐2, whereas it interacts with a hydrophobic part of the protein's structure in the Ca²⁺‐bound state.     

Crystal structure of the trimeric N‐terminal domain of ciliate Euplotes octocarinatus centrin binding with calcium ions

Centrin is a member of the EF‐hand superfamily of calcium‐binding proteins, a highly conserved eukaryotic protein that binds to Ca²⁺. Its self‐assembly plays a causative role in the fiber contraction that is associated with the cell division cycle and ciliogenesis. In this study, the crystal structure of N‐terminal domain of ciliate Euplotes octocarinatus centrin (N‐EoCen) was determined by using the selenomethionine single‐wavelength anomalous dispersion method. The protein molecules formed homotrimers. Every protomer had two putative Ca²⁺ ion‐binding sites I and II, protomer A, and C bound one Ca²⁺ ion, while protomer B bound two Ca²⁺ ions. A novel binding site III was observed and the Ca²⁺ ion was located at the center of the homotrimer. Several hydrogen bonds, electrostatic, and hydrophobic interactions between the protomers contributed to the formation of the oligomer. Structural studies provided insight into the foundation for centrin aggregation and the roles of calcium ions.     

Ca2+ influx into identified leech neurons induced by 5‐hydroxytryptamine

The role of 5‐hydroxytryptamine (5‐HT, serotonin) in the control of leech behavior is well established and has been analyzed extensively on the cellular level; however, hitherto little is known about the effect of 5‐HT on the cytosolic free calcium concentration ([Ca²⁺]_i) in leech neurons. As [Ca²⁺]_i plays a pivotal role in numerous cellular processes, we investigated the effect of 5‐HT on [Ca²⁺]_i (measured by Fura‐2) in identified leech neurons under different experimental conditions, such as changed extracellular ion composition and blockade of excitatory synaptic transmission. In pressure (P), lateral nociceptive (N1), and Leydig neurons, 5‐HT induced a [Ca²⁺]_i increase which was predominantly due to Ca²⁺ influx since it was abolished in Ca²⁺‐free solution. The 5‐HT‐induced Ca²⁺ influx occurred only if the cells depolarized sufficiently, indicating that it was mediated by voltage‐dependent Ca²⁺ channels. In P and N1 neurons, the membrane depolarization was due to Na⁺ influx through cation channels coupled to 5‐HT receptors, whereby the dose‐dependency suggests an involvement in excitatory synaptic transmission. In Leydig neurons, 5‐HT receptor‐coupled cation channels seem to be absent. In these cells, the membrane depolarization activating the voltage‐dependent Ca²⁺ channels was evoked by 5‐HT‐triggered excitatory glutamatergic input. In Retzius, anterior pagoda (AP), annulus erector (AE), and median nociceptive (N2) neurons, 5‐HT had no effect on [Ca²⁺]_i. © 2004 Wiley Periodicals, Inc. J Neurobiol, 2005     

Allosteric effects of the antipsychotic drug trifluoperazine on the energetics of calcium binding by calmodulin

Trifluoperazine (TFP; Stelazine?) is an antagonist of calmodulin (CaM), an essential regulator of calcium‐dependent signal transduction. Reports differ regarding whether, or where, TFP binds to apo CaM. Three crystallographic structures (1CTR, 1A29, and 1LIN) show TFP bound to (Ca²⁺)₄‐CaM in ratios of 1, 2, or 4 TFP per CaM. In all of these, CaM domains adopt the “open” conformation seen in CaM‐kinase complexes having increased calcium affinity. Most reports suggest TFP also increases calcium affinity of CaM. To compare TFP binding to apo CaM and (Ca²⁺)₄‐CaM and explore differential effects on the N‐ and C‐domains of CaM, stoichiometric TFP titrations of CaM were monitored by ¹⁵N‐HSQC NMR. Two TFP bound to apo CaM, whereas four bound to (Ca²⁺)₄‐CaM. In both cases, the preferred site was in the C‐domain. During the titrations, biphasic responses for some resonances suggested intersite interactions. TFP‐binding sites in apo CaM appeared distinct from those in (Ca²⁺)₄‐CaM. In equilibrium calcium titrations at defined ratios of TFP:CaM, TFP reduced calcium affinity at most levels tested; this is similar to the effect of many IQ‐motifs on CaM. However, at the highest level tested, TFP raised the calcium affinity of the N‐domain of CaM. A model of conformational switching is proposed to explain how TFP can exert opposing allosteric effects on calcium affinity by binding to different sites in the “closed,” “semi‐open,” and “open” domains of CaM. In physiological processes, apo CaM, as well as (Ca²⁺)₄‐CaM, needs to be considered a potential target of drug action. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Oligohis‐tags: mechanisms of binding to Ni2+‐NTA surfaces

Since immobilized metal ion affinity chromatography (IMAC) was first reported, several modifications have been developed. Among them, Ni²⁺ immobilized by chelation with nitrilotriacetic acid (NTA) bound to a solid support has become the most common method for the purification of proteins carrying either a C‐ or N‐terminal histidine (His) tag. Despite its broad application in protein purification, only little is known about the binding properties of the His‐tag, and therefore almost no thermodynamic and kinetic data are available. In this study, we investigated the binding mechanism of His‐tags to Ni²⁺‐NTA. Different series of oligohistidines and mixed oligohistidines/oligoalanines were synthesized using automated solid‐phase peptide synthesis (SPPS). Binding to Ni²⁺‐NTA was analyzed both qualitatively and quantitatively with surface plasmon resonance (SPR) using commercially available NTA sensor chips from Biacore. The hexahistidine tag shows an apparent equilibrium dissociation constant (K_D) of 14 ± 1 nM and thus the highest affinity of the peptides synthesized in this study. Furthermore, we could demonstrate that two His separated by either one or four residues are the preferred binding motifs within hexahis tag. Finally, elongation of these referred motifs decreased affinity, probably due to increased entropy costs upon binding. Copyright © 2009 John Wiley & Sons, Ltd.     

Effects of IMAC specific peptide tags on the stability of recombinant green fluorescent protein

Immobilized metal ion affinity chromatography (IMAC) using peptide affinity tags has become a popular tool for protein purification. An important feature dictating the use of a specific affinity tag is whether its structure influences the properties of the target protein to which it is attached. In this work we have studied the influence on protein stability of two novel peptide affinity tags, namely NT1A and HIT2, and compared their effect to the commonly used hexa‐histidine tag, all attached to the C‐terminus of a enhanced green fluorescent protein (eGFP). A comparison of the influence of C‐ or N‐terminal orientation of the tags was also carried out by studying the NT1A tag attached at either terminus of the eGFP. Protein stability was studied utilising guanidine hydrochloride equilibrium unfolding procedures and CD and fluorescence spectroscopy. The novel peptide affinity tags, NT1A and HIT2, and the His₆ tag were found to not affect the stability of eGFP. Although these results are protein specific, they highlight, nevertheless, the need to employ suitable characterisation tools if the impact of a specific peptide tag on the folded status or stability of a recombinant tagged protein, purified by immobilized metal ion affinity chromatographic methods, are to be rigorously evaluated and the appropriate choice of peptide tag made. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Progesterone triggers rapid transmembrane calcium influx and/or calcium mobilization from endoplasmic reticulum,via a pertussis-insensitive G-protein in granulosa cells in relation to luteinization process

We investigated the early effects (5–60 s) of progesterone (1 pM–0.1 μM) on cytosolic free calcium concentration ([Ca²⁺]_i) and inositol 1,4,5-trisphosphate (InsP₃) formation in nonluteinized and in vitro luteinized porcine granulosa cells (pGCs). Progesterone increased [Ca²⁺]_i and InsP₃ formation within 5 s in both cell types. Progesterone induced calcium mobilization from the endoplasmic reticulum via the activation of a phospholipase C linked to a pertussis-insensitive G-protein. This process was controlled by protein kinases C and A. In contrast, only nonluteinized pGCs showed a Ca²⁺ influx via dihydropyridine-insensitive calcium channel. In both cell types, the nuclear progesterone receptor antagonist RU-38486 did not inhibit the progesterone-induced increase in [Ca²⁺]_i; progesterone immobilized on bovine serum albumin, which did not enter the cell, increased [Ca²⁺]_i within 5 s and was a full agonist, but less potent than the free progesterone; pertussis toxin did not inhibit progesterone effect on InsP₃. In conclusion, progesterone may interact with membrane unconventional receptors that belong to the class of membrane receptors coupled to a phospholipase C via a pertussis toxin-insensitive G-protein. The source of the Ca²⁺ for the progesterone-induced increase in [Ca²⁺]_i also depends on the stage of cell luteinization. © 1996 Wiley-Liss, Inc.     

Calcium transients in response to salinity and osmotic stress in the nitrogen-fixing cyanobacterium Anabaena sp. PCC7120, expressing cytosolic apoaequorin

We show here that both salinity and osmotic stress trigger transient increases in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) in cells of the nitrogen‐fixing filamentous cyanobacterium Anabaena sp. PCC7120, which constitutively expresses apoaequorin. Isoosmolar concentrations of salt (NaCl) and osmoticum (sucrose) induced calcium transients of similar magnitude and shape, suggesting that cells sense, via Ca²⁺ signalling, mostly osmotic stress. The Ca²⁺ transients induced by NaCl and sucrose were completely blocked by the calcium chelator ethylene glycol‐bis(b‐aminoethylether)N,N,N¢,N¢‐tetraacetic acid (EGTA) and were partially inhibited by the calcium channel blocker verapamil. Increased external Ca²⁺ and the Ca²⁺ ionophore calcimycin (compound A23187) enhanced Ca²⁺ influx further, suggesting the involvement of extracellular Ca²⁺ in the observed response to salinity and osmotic stress. However, the plant hormone abscisic acid (ABA) did not provoke any effect on the Ca²⁺ transients induced by both stresses, indicating that it may not be acting upstream of Ca²⁺ in the signalling of salinity and/or osmotic stress in Anabaena sp. PCC7120.     

Mapping the in vitro interactome of cardiac sodium (Na+)‐calcium (Ca2+) exchanger 1 (NCX1)

The sodium (Na⁺)‐calcium (Ca²⁺) exchanger 1 (NCX1) is an antiporter membrane protein encoded by the SLC8A1 gene. In the heart, it maintains cytosolic Ca²⁺ homeostasis, serving as the primary mechanism for Ca²⁺ extrusion during relaxation. Dysregulation of NCX1 is observed in end‐stage human heart failure. In this study, we used affinity purification coupled with MS in rat left ventricle lysates to identify novel NCX1 interacting proteins in the heart. Two screens were conducted using: (1) anti‐NCX1 against endogenous NCX1 and (2) anti‐His (where His is histidine) with His‐trigger factor‐NCX1_cyt recombinant protein as bait. The respective methods identified 112 and 350 protein partners, of which several were known NCX1 partners from the literature, and 29 occurred in both screens. Ten novel protein partners (DYRK1A, PPP2R2A, SNTB1, DMD, RABGGTA, DNAJB4, BAG3, PDE3A, POPDC2, STK39) were validated for binding to NCX1, and two partners (DYRK1A, SNTB1) increased NCX1 activity when expressed in HEK293 cells. A cardiac NCX1 protein–protein interaction map was constructed. The map was highly connected, containing distinct clusters of proteins with different biological functions, where “cell communication” and “signal transduction” formed the largest clusters. The NCX1 interactome was also significantly enriched with proteins/genes involved in “cardiovascular disease” which can be explored as novel drug targets in future research.     

Immobilized biocatalysts for detoxification of neurotoxic organophosphorous compounds

New biocatalysts were developed using organophosphorus hydrolase (OPH, EC 3.1.8.1) with a polyhistidine tag at the N-terminus of the protein (His₆-OPH). The use of His₆-OPH together with previously developed approaches for the entrapment of cells into poly(vinyl alcohol) cryogels and covalent immobilization of enzymes into porous fabric materials, impregnated with chemically cross-linked chitosan sulphate gel, enabled dramatic improvement of catalytic characteristics against various organophosphorous compounds (OPCs; Paraoxon, Coumaphos, Methyl parathion, etc.). The polyhistidine tag of OPH was used to create a new immobilized biocatalyst using metal-chelating carriers, such as Ni²⁺-nitrilotriacetic acid-agarose and Co²⁺-iminodiacetic acid-polyacrylamide cryogel. The latter biocatalyst had high activity and stability for the continuous hydrolysis of OPCs.     

Binding of calcium is sensed structurally and dynamically throughout the second calcium‐binding domain of the sodium/calcium exchanger

We report the effects of Ca²⁺ binding on the backbone relaxation rates and chemical shifts of the AD and BD splice variants of the second Ca²⁺‐binding domain (CBD2) of the sodium–calcium exchanger. Analysis of the Ca²⁺‐induced chemical shifts perturbations yields similar K_D values of 16–24 μM for the two CBD2‐AD Ca²⁺‐binding sites, and significant effects are observed up to 20 Å away. To quantify the Ca²⁺‐induced chemical shift changes, we performed a comparative analysis of eight Ca²⁺‐binding proteins that revealed large differences between different protein folds. The CBD2 ¹⁵N relaxation data show the CBD2‐AD Ca²⁺ coordinating loops to be more rigid in the Ca²⁺‐bound state as well as to affect the FG‐loop located at the opposite site of the domain. The equivalent loops of the CBD2‐BD splice variant do not bind Ca²⁺ and are much more dynamic relative to both the Ca²⁺‐bound and apo forms of CBD2‐AD. A more structured FG‐loop in CBD2‐BD is suggested by increased S² order parameter values relative to both forms of CBD2‐AD. The chemical shift and relaxation data together indicate that, in spite of the small structural changes, the Ca²⁺‐binding event is felt throughout the molecule. The data suggest that the FG‐loop plays an important role in connecting the Ca²⁺‐binding event with the other cytosolic domains of the NCX, in line with in vivo and in vitro biochemical data as well as modeling results that connect the CBD2 FG‐loop with the first Ca²⁺‐binding domain of NCX. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

The role of calcium in the interaction between calmodulin and a minimal functional construct of eukaryotic elongation factor 2 kinase

Eukaryotic elongation factor 2 kinase (eEF‐2K) regulates protein synthesis by phosphorylating eukaryotic elongation factor 2 (eEF‐2), thereby reducing its affinity for the ribosome and suppressing global translational elongation rates. eEF‐2K is regulated by calmodulin (CaM) through a mechanism that is distinct from that of other CaM‐regulated kinases. We had previously identified a minimal construct of eEF‐2K (TR) that is activated similarly to the wild‐type enzyme by CaM in vitro and retains its ability to phosphorylate eEF‐2 efficiently in cells. Here, we employ solution nuclear magnetic resonance techniques relying on Ile δ1‐methyls of TR and Ile δ1‐ and Met ε‐methyls of CaM, as probes of their mutual interaction and the influence of Ca²⁺ thereon. We find that in the absence of Ca²⁺, CaM exclusively utilizes its C‐terminal lobe (CaM_C) to engage the N‐terminal CaM‐binding domain (CBD) of TR in a high‐affinity interaction. Avidity resulting from additional weak interactions of TR with the Ca²⁺‐loaded N‐terminal lobe of CaM (CaM_N) at increased Ca²⁺ levels serves to enhance the affinity further. These latter interactions under Ca²⁺ saturation result in minimal perturbations in the spectra of TR in the context of its complex with CaM, suggesting that the latter is capable of driving TR to its final, presumably active conformation, in the Ca²⁺‐free state. Our data are consistent with a scenario in which Ca²⁺ enhances the affinity of the TR/CaM interactions, resulting in the increased effective concentration of the CaM‐bound species without significantly modifying the conformation of TR within the final, active complex.     

The Ca2+‐binding protein PCaP2 located on the plasma membrane is involved in root hair development as a possible signal transducer

Plasma membrane‐associated Ca²⁺‐binding protein–2 (PCaP2) of Arabidopsis thaliana is a novel‐type protein that binds to the Ca²⁺/calmodulin complex and phosphatidylinositol phosphates (PtdInsPs) as well as free Ca²⁺. Although the PCaP2 gene is predominantly expressed in root hair cells, it remains unknown how PCaP2 functions in root hair cells via binding to ligands. From biochemical analyses using purified PCaP2 and its variants, we found that the N–terminal basic domain with 23 amino acids (N23) is necessary and sufficient for binding to PtdInsPs and the Ca²⁺/calmodulin complex, and that the residual domain of PCaP2 binds to free Ca²⁺. In mutant analysis, a pcap2 knockdown line displayed longer root hairs than the wild‐type. To examine the function of each domain in root hair cells, we over‐expressed PCaP2 and its variants using the root hair cell‐specific EXPANSIN A7 promoter. Transgenic lines over‐expressing PCaP2, PCaP2^G2A (second glycine substituted by alanine) and ?23PCaP2 (lacking the N23 domain) exhibited abnormal branched and bulbous root hair cells, while over‐expression of the N23 domain suppressed root hair emergence and elongation. The N23 domain was necessary and sufficient for the plasma membrane localization of GFP‐tagged PCaP2. These results suggest that the N23 domain of PCaP2 negatively regulates root hair tip growth via processing Ca²⁺ and PtdInsP signals on the plasma membrane, while the residual domain is involved in the polarization of cell expansion.