Free Access Zinc binding site in PICK1 is dominantly located at the CPC motif of its PDZ domain Yawei Shi, Institute of Biotechnology, Key Laboratory of Chemical Biology and Molecular Engineering of Education Ministry, Shanxi University, Taiyuan, ChinaSearch for more papers by this authorLei Zhang, Institute of Biotechnology, Key Laboratory of Chemical Biology and Molecular Engineering of Education Ministry, Shanxi University, Taiyuan, ChinaSearch for more papers by this authorJingming Yuan, Institute of Biotechnology, Key Laboratory of Chemical Biology and Molecular Engineering of Education Ministry, Shanxi University, Taiyuan, ChinaSearch for more papers by this authorHong Xiao, Institute of Biotechnology, Key Laboratory of Chemical Biology and Molecular Engineering of Education Ministry, Shanxi University, Taiyuan, ChinaSearch for more papers by this authorXiuqing Yang, Institute of Biotechnology, Key Laboratory of Chemical Biology and Molecular Engineering of Education Ministry, Shanxi University, Taiyuan, ChinaSearch for more papers by this authorLixi Niu, Institute of Biotechnology, Key Laboratory of Chemical Biology and Molecular Engineering of Education Ministry, Shanxi University, Taiyuan, ChinaSearch for more papers by this author Yawei Shi, Institute of Biotechnology, Key Laboratory of Chemical Biology and Molecular Engineering of Education Ministry, Shanxi University, Taiyuan, ChinaSearch for more papers by this authorLei Zhang, Institute of Biotechnology, Key Laboratory of Chemical Biology and Molecular Engineering of Education Ministry, Shanxi University, Taiyuan, ChinaSearch for more papers by this authorJingming Yuan, Institute of Biotechnology, Key Laboratory of Chemical Biology and Molecular Engineering of Education Ministry, Shanxi University, Taiyuan, ChinaSearch for more papers by this authorHong Xiao, Institute of Biotechnology, Key Laboratory of Chemical Biology and Molecular Engineering of Education Ministry, Shanxi University, Taiyuan, ChinaSearch for more papers by this authorXiuqing Yang, Institute of Biotechnology, Key Laboratory of Chemical Biology and Molecular Engineering of Education Ministry, Shanxi University, Taiyuan, ChinaSearch for more papers by this authorLixi Niu, Institute of Biotechnology, Key Laboratory of Chemical Biology and Molecular Engineering of Education Ministry, Shanxi University, Taiyuan, ChinaSearch for more papers by this author Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URLShare a linkShare onEmailFacebookTwitterLinked InRedditWechat Abstract PICK1 (protein interacting with Ckinase 1) containing a PDZ domain, a BAR domain, and two short acidic regions is as an adaptor protein that plays an important role in α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor trafficking, cell morphology and migration, as well as in some diseases such as cancer, schizophrenia and pain. To better understand the physiological function of PICK1, we expressed the recombinant PICK1 and its truncated mutants in E.coli, and measured their zinc binding properties by fluorescence and competition assay. It is shown that PICK1 has one Zn2+-binding site. The Zn2+-binding properties of PICK1 are not appreciably affected after the removal of BARC domain (involving BAR domain and C-terminal acidic region). Deleting the N-terminal acidic region of NPDZ domain (involving PDZ domain and N-terminal acidic region) in PICK1 impairs its Zn2+-binding capacity.The mutation of the CPC (Cys-Pro-Cys) motif in the PDZ domain of PICK1 abolishes the ability of Zn2+-binding. In addition, Zn2+ can enhance the lipid-binding ability of PDZ domain as observed in both protein-lipid overlay assay and fluorescence analysis. The results presented in this report suggested that Zn2+ plays a regulatory role in the trafficking of PICK1 from the cytoplasm to cell membrane. Abbreviations used CD circular dichroism GST Glutathione S-transferase MBP Maltose-binding protein PAR 4-(2-pyridylazo)resorcinol Zincon 2-carboxy-2-hydroxy-5-(sulfoformazyl)benzene Zinc is an abundant divalent metal ion in the brain and involved in a variety of metabolic processes (Coleman 1992; Cowan 1998; Dudev and Lim 2003). In human, zinc deficiency has been known to cause many neurological and neuropsychiatric disorders (Frederickson 1989). The role of zinc in the central neuronal function and signaling is increasingly being appreciated (Aedo etal. 2007). In the brain, zinc, like calcium which is another important divalent cation in the central nervous system (CNS), is unevenly distributed with the highest level in hippocampus (Donaldson etal. 1973). PICK1 (Protein Interacting with Ckinase 1) was first identified to interact with the catalytic domain of protein kinase Cα (Staudinger etal. 1995). PICK1 as an adaptor protein is expressed in many tissues, with high level in the brain and testis. PICK1 as a peripheral membrane protein, is also located at the neuronal synapses and associated with a wide range of proteins via its functional domains, including a PDZ domain and a BAR domain (Xu and Xia 2007). PICK1 is implicated in targeting the activated Protein Kinase Cα to regulate the phosphorylation of many PICK1-interacting partners, thereby regulating the synaptic clustering and trafficking (Dev 2007). Although its exact contribution to diseases remains unclear, PICK1 does interact with a number of proteins which play an important role in certain diseases such as cancer, schizophrenia and pain (Dev 2004). However, the metal-binding properties of PICK1 have not been understood fully and studies reported so far have been limited to the role of PICK1 as a calcium-sensor for NMDA-induced α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor trafficking (Hanley and Henley 2005). In addition, it is still unclear whether acidic amino acid residues located at both N-terminal and C-terminal regions of PICK1 are related to binding with other divalent metal ions except Ca2+, Mg2+ (Hanley and Henley 2005; Shi etal.2007). Therefore, we prepared a recombinant PICK1 as well as its truncated mutants to study its Zn2+-binding property. Based on the spectroscopic analysis, we found that the CPC motif in the PDZ domain of PICK1 is a dominant binding site for Zn2+. Strains E.coli DH5α or E.coli BL21 stored in this laboratory were used as host strains for cloning and protein expression. Ni2+–NTA agarose, Glutathione-Sepharose 4FF, Sephacryl S-200 and pre-packed Superose 12 (10/30) and P10 (5 mL) columns were products of GE Healthcare. All restriction enzymes, T4 ligase, Taq polymerase, were purchased from Takala Co. Dalian Branch (Dalian,China). FITC protein Labeling kit (F6434) was purchased by Invitrogen. (d,l)-1,4-Dithiothreitol, Hepes, and all other chemicals used were all analytical grade reagents. DNA fragments encoding PICK1 (from rat) and its truncated forms i.e. NPDZ (1-110 aa), PDZ (20-110 aa), Bar domain and C-terminal acidic region (BARC) (128-416 aa), as illustrated in Fig.1(a), were cloned by PCR and ligated into pGEX-6P-1 or pMal-s (Factor Xa cleavage site in pMAL-c2 was replaced by protease 3C cleavage site) as described in refs 12,13. In addition, the mutant I (C44C46/G44G46 in PDZ domain) was generated by primers (FW: 5′GGAGGGGCCCAGTACGGTCCTGGTCTC3′) and (RV: 5′CTGGACAATGTAGAGACCAGGACCGTA3′). Glutathione S-transferase (GST)-PICK1, GST-NPDZ, GST-PDZ and Maltose-binding protein (MBP)-BARC were expressed and purified as described previously (Pan etal. 2007; Shi etal. 2007). PICK1 and its PDZ domain alignment. (a) Schematic diagram of PICK1 and its fragments generated in this study. (b) Protein sequence alignment of PDZ domains from a known similar PDZ domain. PICK1 (PDB: 2GZV), MAGI-1: MAGUK with the inverted domain structure-1 (PDB: 2I04), GOPC: Golgi-associated PDZ and coiled-coil motif-containing protein (PDB: 2DC2), Af6: AF6 protein (2AIN). FITC was conjugated to PDZ, NPDZ and BARC by using the method described in the instruction from vendor (FluoReport®FITC protein Labelling, Molecular Probes). Briefly, FITC was dissolved in dimethylsulfoxide and diluted with the conjugation buffer (0.1 M NaHCO3-NaOH, pH 9.0), and then immediately added to protein solution with slowly stirring at 4°C overnight in the dark (FITC/protein = 20 : 1). Then the conjugated protein was exchanged into Hepes buffer (20 mM Hepes, 100 mM NaCl, 1 mM (d,l)-1,4-Dithiothreitol, 0.1%Triton X-100) using a pre-packed desalting P10 column. Fluorescence spectra were recorded on a HITACHI F-2500 fluorescence spectrophotometer using a 10 nm slit width. All measurements were performed in a total sample volume of 1 mL in 20 mM Hepes, 100 mM NaCl, pH 7.4 in a 10 mm path length cuvette at a 280 nm excitation wavelength for PICK1 and 495 nm excitation wavelengths for FITC-PDZ, FITC-NPDZ, and FITC-BARC. The stock solution of ZnCl2 is 10 mM, which was preparated in Milli-Q H2O adjusted pH value to pH 5.5 to dissolve the zinc salt by HCl. To study the relationship between zinc and liposome for PDZ, the concentration of zinc and liposome used were 20 μM and 15 μg/mL, respectively. During the titration experiment, the sample was maintained at 25°C by using a jacketed cell holder connected to an external circulating water bath. In the competition test between Zn2+-chelator, PAR or Zincon, and the protein (Ciuculescu etal. 2005; Armas etal. 2006), the decrease in absorbance of Zn2+-(PAR)2 complex was measured upon the addition of protein at 500 nm(at 620 nm for Zn-Zincon) on a Hitachi UV-2010 spectrophotometer. All measurements were performed using 5 μM Zn2+, 120 μM PAR or 10 μM Zn2+, 20 μM Zincon in 20 mM Hepes, pH 7.4 containing 100 mM NaCl. As a routine test to evaluate protein aggregation, the turbidity measurement was conducted at 350 nm on a Hitach UV-2010 spectrophotometer. Before each sample measurement, 20 μM GST, GST-PDZ, GST-mutant I, in 20 mM Hepes buffer containing 100 mM NaCl (pH 7.4), was incubated at 25°C for 2 min in the presence and absence of Zn2+. Brain-lipid extracts (Folch fraction I, Sigma B1502) were re-suspended at 2 mg/mL in 20 mM Hepes, pH 7.4, containing 100 mM NaCl. The protein sample (10 μM) was incubated with 0.6 mg/mL. Brain-lipid extracts in 40 μL buffer for 15 min at 25°C and then ultra-centrifuged at 65 000 g for 15 min at 4°C in a Beckman TLA100.1 rotor. The supernatant removed was used to determine the unbound proteins. The pellet was washed twice with the same buffer and then brought up to the same volume before electrophoresis. Both the supernatant and pellet were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and visualized by Coomassie blue staining. Bovine brain lipid extracts (Type I, Folch Fraction I, Sigma) were dissolved in a small volume of chloroform/methanol (2 : 1 vol/vol), and then diluted by ethanol to achieve the desired concentration. Each sample well in the 96-well microplate was coated with lipid extracts (50 μL/well) and then incubated at 25°C until all solvents were evaporated. To block non-specific sites, each well was coated with 2% bovine serum albumin in 10 mM Tris-HCl, pH 7.5, 150 mM NaCl (Tris-buffered saline), incubated for 1 h at 25°C and then washed with Tris-buffered saline buffer. The GST, GST-PDZ, and GST-Mutant I (0.2 mg/mL) with different concentrations of Zn2+ were added to the lipid-coated well (100 μL/well) and incubated for 1 h at 25°C, then washed as described above. One hundred microliter of GST-antibody at 1 : 2000 dilutions was then added into the microplate and incubated at 25°C for 1 h. After being thoroughly washed, an anti-rat IgG conjugated with alkaline phosphatase and 3-ethylbenzthiazoline-6-sulfonic acid substrate were added into the wells and the developed color was detected using a microplate reader at 405 nm. It was reported that Ca2+ could bind to two acidic regions of PICK1 located at the N-terminus and C-terminus to regulate the interaction between the GluR2 and PICK1 (Hanley and Henley 2005; Shi etal. 2007). However, there has been no report about other divalent ions binding to PICK1. On the basis of the fluorescence measurement, it is first time to demonstrate that Zn2+ can bind tightly to PICK1, accompanied by a decreasing of the fluorescence intensity. A family of fluorescence spectra produced by the addition of sequential aliquots of Zn2+ to PICK1 solution is shown in Fig.2(a), the fluorescence emission intensity of PICK1 is decreased in parallel with an increasing of Zn2+ concentration until reaching a saturation level. To correct the sample dilution, the fluorescence intensity at 338 nm was converted to molar fluorescence intensity by dividing the fluorescence intensity by the protein concentration of PICK1. Titration curve has been prepared by plotting (F-F0)/ [PICK1] versus equivalent of zinc, [Zn2+]/[PICK1], as shown in Fig.2(a)-inset. The initial part of this titration curve is linear. This indicates that the early part of the titration, essentially 100% of each aliquot of zinc is binding to the protein. The zinc titration curve eventually levels off at a molar fluorescence intensity at 338 nm (F-F0)/ [PICK1] of about 3.5, which corresponds to the completely occupation of PICK1 binding site(s). The linear segment of initial part of titration and the line at (F-F0)/[PICK1] = 3.5 crosses at [Zn2+]/[PICK1] = 1.12, suggesting a 1 : 1 stoichiometry of the PICK1-Zn2+ complex. Figure 2(b) is the zinc titration curves at different concentrations of PICK1, which are decreased gradually from a to d. From Fig.2(b), the association constant (Ka) of the PICK1-Zn2+ complex is (8.17 ± 0.23) × 105 /M which calculated by fitting (F-F0) to [Zn2+] with a one-site-binding model using a GraphPad Prism 4 program. There is a maximum absorbance at 500 nm for Zn2+-PAR. The addition of sequential aliquots of PICK1 to Zn2+-PAR leads to the decrease of absorbance at 500 nm. The competition test between proteins and zinc-chelator, PAR are shown in Fig.2(c). It indicates that Zn and PICK1 can form 1 : 1 complex. This is consistent with the result by fluorescence titration. In 5,5′-dithiobis-(2-nitrobenzoic acid) assay, our result further demonstrated that the higher concentration of zinc did not retard 5,5′-dithiobis-(2-nitrobenzoic acid) oxidation any more, suggesting that PICK1 can bind no more than two Zn2+ ions per molecule (data no shown). Association constants of Zn2+ with PICK1 and its domains. (a) Fluorescence emission spectra of PICK1 with Zn2+. Condition: 5 μM of PICK1 in 20 mM Hepes, pH 7.4 containing 100 mM NaCl. The fluorescence emission of PICK1 is decreased by increasing concentrations of Zn2+. The inset shows a plot of the fluorescence intensity (F-F0)/[PICK1] vs. [Zn2+]/[PICK1]. (b) Stoichiometry of Zn2+ to PICK1 was assessed by zinc fluorescence titration at different PICK1 concentrations. Condition: Concentration of PICK1 is 0.5 μM, 1 μM, 2 μM, 5 μM, respectively. (c) Estimation of Zn2+-binding capacity by a competition test with a Zn2+ chelator, PAR. Zn2+ binding capacity of PICK1 and its PDZ, NPDZ, BARC domains was measured by adding a Zn2+ chelator, PAR at 500 nm. The experimental conditions are in 120 μM Zincon, 5 μM Zn2+ with different concentrations of samples in 20 mM Hepes, pH 7.4 containing 100 mM NaCl. With the same method described above, the association constant of Ca2+ or Mg2+ for PICK1 was also measured. Table1 shows that the association constant of Zn2+ is close to that of Ca2+ or Mg2+, indicating that PICK1 may be a Zn2+-binding protein. As described earlier, PICK1 can be expressed in high level in the brain (Xia etal. 1999; Xu and Xia 2007). The average concentration of Zn2+ ion in brain is estimated to be approximately 150 μM (Takeda 2000) and Zn2+ concentration in synaptic vesicles of some neurons in forebrain regions, including the hippocampus, was found to be approximately more than 1 mM (Frederickson etal. 2000). However, the physiological role of synaptic Zn2+ is relatively little understood. Zinc may bind to receptors on the post-synaptic neuron and modulate the flow of ions through channels, or it may act as an intracellular signal following entry of post-synaptic neuron through Zn2+-permeable channels (Burdette and Lippard 2003). Therefore, in synapse, Zn2+ may regulate the function of PICK1. Table 1. Association constants of PICK1 and its three domains in the presence of Ca2+ Mg2+ and Zn2+ aThe association constant of each protein for Ca2+, Mg2+, Zn2+ was calculated by the fluorescence titration. bDeleting N terminal acidic region of NPDZ distorts its Ca2+, Mg2+ binding site. cDeleting the NPDZ of PICK1 distorts its Zn2+, Mg2+ binding site. Based on the same experimental method as described in above section, we measured the association constant of Zn2+ to three truncated forms of PICK1, i.e. NPDZ (1-110 aa), PDZ (20-110 aa) and BARC (128-416 aa) as shown in Fig.1(a). Table1 shows that the association constant of Zn2+ to NPDZ is similar to that of PICK1, while there is not any Zn2+-binding occurred in BARC domain, implying that the Zn2+-binding site in PICK1 may be only located at the N-terminus other than C-terminus of PICK1. This is quite different from the Ca2+ binding sites in PICK1, located both at the N-terminus and C-terminus (Hanley and Henley 2005; Shi etal. 2007). Furthermore, we used a Zn2+-chelator, PAR or Zincon, to examine the Zn2+-binding capacity between PICK1 and its truncated mutants. When the amount of PICK1 (1-416 aa), PDZ domain (20-110 aa), or NPDZ domain (1-110 aa) was increased, the absorbance at 500 nm for Zn2+(PAR) 2 (at 620 nm for Zn-Zincon, data no shown) was correspondingly decreased. In contrast, there was no obvious change for BARC domain (128-416 aa) during the titration (Fig.2c). The decrease of the absorption at 500 nm may reflect the transfer of Zn2+ from PAR to NPDZ or PDZ domain, but not to BARC domain. These results confirmed that zinc binding site is located at the N-half portion of PICK1. When N-terminal acidic amino acids (1-19 aa) were deleted, the PDZ domain produced a slightly lower affinity for Zn2+, compared to NPDZ. Therefore, the PDZ domain of PICK1 is most likely a high affinity binding site for Zn2+. From an analysis of the amino acid sequence of the PDZ domain, CPC motif at positions 44-46 may be a binding site for Zn2+ (Liu etal. 2006). To further examine the hypothesis, C44C46/GG mutant in PDZ (Mutant I) was generated and purified as described in ‘Materials and Methods’. The change in the absorption at around 220 nm is typical for the ligation of thiolates to Zn2+ (Armas etal. 2006).As the concentration of Zn2+ in solution was increased, the absorption at around 220 nm was only increased for PDZ domain and not for its mutant I (Fig.3a). This result indicated that the Zn2+ binding site is predominantly located at the CPC motif in the PDZ domain of PICK1. In addition, the circular dichroism (CD) band of the mutant I at 220 nm was no any changeable compared to wild-type PDZ in the presence of 50 μM Zn2+ as shown in Fig.3(b). From the CD spectrum of wild type PDZ, the band was also changed at 220 nm that may be attributed to the Zn2+ binding, due to the charge transfer band of Cys-Zn2+ (Armas etal. 2006). The circular dichroism (CD) band of mutant I at 220 nm has no any change either in or none of 50 μM Zn2+, indicating that C44 and C46 in PDZ domain may be as the crux residues for Zn2+-binding site. CPC motif involved in Zn2+ binding. (a) UV-absorption changes of PDZ and its Mutant I upon titration of Zn2+ at 220 nm. (b) Comparison of the CD spectrum for PDZ and Mutant I proteins in the presence or absence of Zn2+. (c) Analysis of Zn2+-dependent aggregation of PDZ domain by the turbidimetric assay at 350 nm. We also used the GST-fusion protein to measure the aggregation state of the PDZ domain and the mutant I in the presence of Zn2+. The aggregation degree of the PDZ domain is obviously higher than that of mutant I or GST alone, meaning that Zn2+ may induce the aggregation of the PDZ domain when the Zn2+ binds to the CPC motif (Fig.3c). As soon as the PDZ domain loses its Zn2+ binding capacity, such as the mutant I, it reverts to its normal form in solution even in the high concentration of Zn2+. In our previous results, we have demonstrated that the PDZ domain in PICK1 can bind to lipids by the liposome-protein binding test (Pan etal. 2007). The result in Fig.4(a) shows that PDZ domain and its mutant I are always in the soluble form in the absence of liposome. On contrary, the PDZ domain produces a precipitate while the mutant I is still in the soluble form in the presence of liposome after ultracentrifugation at 65 000 g for 15 min, implying that the PDZ domain can tightly bind to liposome, while the mutant I is not. The data further support the notion that the lipid-binding site on the PDZ domain is located at the CPC motif as the same as Zn2+-binding site. Therefore, it is necessary to further probe the relationship between the lipid-binding and Zn-binding site. Effect of Zn2+ on the binding capacity of the PDZ domain with liposome. (a) Brain liposome-binding test of the PDZ domain and its mutant I by a sedimentation ion assay. ‘S’ and ‘P’ denote proteins recovered in the supernatant and pellet of the reaction mixture after ultra-centrifugation respectively. (b) Solid phase interaction between PDZ domain and liposome. Each well of a 96-well microplate was coated with 50 μL. Brain lipid extracts dissolved in ethanol (0.17 mg/mL), and then mixed with 20 μL/well of GST-PDZ, GST-mutant I and GST (0.2 mg/mL), respectively, finally 10 μL/well of Zn2+ at different concentrations were added. The quantitative analysis of the PDZ-Zn2+ complex was measured by an anti-rat IgG conjugated with alkaline phosphatase and 3-ethylbenzthiazoline-6-sulfonic acid (ABTS) as the substrate. (c) Fluorescence analysis of the interaction between PDZ domain and liposome in the presence or absence of Zn2+. Condition: 5 μM of PDZ domain in 20 mM Hepes, pH 7.4 containing 100 mM NaCl, 1 mM (d,l)-1,4-Dithiothreitol (DTT). The concentration of Zn2+ is 20 μM. In Protein-Lipid-Overlay assay, the PDZ domain can bind to lipids, regardless of the absence or presence of Zn2+. With increasing the concentration of Zn2+, the interaction between the PDZ domain and lipids was significantly enhanced until 100 μM Zn2+. In the case of mutant I, there is no any change occurred just as the control GST alone (Fig.4b). We also examined the effect of zinc on the PDZ-liposome interaction by fluorescence spectroscopy. The PDZ domain (15 μg) mixed with or without zinc (20 μM) was titrated with 0–50 μg/mL liposome as shown in Fig.4(c). The addition of zinc promotes the PDZ-liposome interaction. However, when the CPC motif is replaced with the GPG, the apparent effect with zinc is disappeared. With the above aggregation test, it is suggested that the effect of zinc on the PDZ domain of PICK1 is probably occurred through stimulating or stabilizing the PDZ-lipid membrane interaction and/or PDZ-PDZ interactions. The effect of liposome on the PDZ-zinc binding was also examined by fluorescence spectroscopy. The PDZ domain (15 μg) mixed with or without liposome (15 μg/mL) was titrated with 0–50 μM zinc (Fig.5a). The addition of liposome decreases the binding capacity of zinc to the PDZ domain. In order to further examine the role of liposome on the PDZ domain aggregation, a turbidimetric assay was performed for the PDZ domain in the presence and absence of 15 μg/mL liposome. The result indicated that liposome also decreases the PDZ aggregation by the induction of zinc (Fig.5b). Effect of liposome on the PDZ domain bound to Zn2+. The interaction between PDZ domain and Zn2+ in the presence or absence of 15 μg/mL liposome was conducted by fluorescence analysis. Condition: 5 μM of PDZ domain in 20 mM Hepes, pH 7.4 containing 100 mM NaCl, 1 mM (d,l)-1,4-Dithiothreitol (DTT). Aggregation of PDZ domain induced by Zn2+ in the presence or absence of 15 μg/mL liposome. In this study, the binding capacity of PICK1 with metal ions such as Zn2+, Mg2+ and Ca2+ was investigated. As a Ca2+-binding protein, PICK1 has two binding sites located at acidic regions of N- and C-terminus (Hanley and Henley 2005; Shi etal. 2007). Although Ca2+ and Mg2+, as ‘hard’ ion, prefer to bind to the ‘hard’ ligand such as Asp and Glu, Mg2+ binds preferentially to the N-terminal acidic region of PICK1. The different affinity of Ca2+ and Mg2+ for the acidic region of PICK1 indicates that the domain specificity in the interaction of PICK1 with its relevant ligand may be modulated by Ca2+ or Mg2+ like in the Calmodulin (CaM) (Martin etal. 2000). Unlike Ca2+ or Mg2+, Zn2+ prefers ‘softer’ ligands such as Cys and His, although it may coordinate with Asp and Glu. Zn2+-binding site in PICK1 seem to be different from Ca2+, Mg2+-binding sites, which dominantly locate at the CPC motif in the PDZ domain of PICK1. The stoichiometry of PICK1-Zn2+ is 1 : 1 at mole ratio with a binding constant of Ka = (8.17 ± 0.23) × 105/M. The PDZ domain is well-characterized for the protein-protein interaction (Sheng and Sala 2001). The PDZ domain of PICK1 was found to interact with over 40 different proteins, which contained both type I and type II PDZ-binding motifs (Dev 2007). In comparison of the known three-dimensional structure of human PICK1 PDZ domain (Protein Data Bank code. 2 GZV) with other known PDZ domains, we found that there was a low sequence identity among these PDZ domains (Fig.1b), and that CPC motif in the PDZ domain was a unique sequence in these PDZ domains. To our knowledge, this is the first report that zinc can bind to the CPC motif of PDZ domain. It was reported that the CPC motif located at the transmembrane region in CPx-type ATPase could bind to Cu2+ (Liu etal. 2006). Several types of Zn2+-binding motifs such as Cys2His2, Cys3His, Cys4 were occurred as well. (Krizek etal. 1993; Laity etal. 2001; Payne etal. 2003). Compared to those motifs, the CPC motif mentioned in this study is the shortest one as a Zn2+-binding site. The interaction between the PDZ domain and lipid membrane is mediated by both a polybasic amino acid cluster and the CPC motif located away from the peptide ligand binding groove (Pan etal. 2007). When two Cys residues at position 44 and 46 in the PDZ domain were changed to Gly by site-directed mutagenesis, not only the Zn2+ binding capacity for the PDZ domain was lost, but also its lipid-binding capacity was abolished, indicating that both Zn2+ and lipid-binding may regulate PICK1’s function. Furthermore, questions remain whether the CPC motif contributes not only to lipid binding site but also to the Zn2+ binding site or whether Zn2+ can regulate the affinity between the PDZ domain and lipid. The Protein Lipid Overlay and fluorescence analysis show that Zn2+ can enhance the interaction between the PDZ domain and membrane lipids. Compared to the CPC motif at the transmembrane region in CPx-type ATPase, the CPC motif in the PDZ domain of PICK1 is located in the loop between the βB and βC (Fig.6).The Zn2+ may induce a dimerization of PDZ domain so as to improve the lipid-binding capacity of the PDZ domain in PICK1 (Maret 2004; Ciuculescu etal. 2005). The three-dimensional structure of PDZ domain of human PICK1 is shown in ribbon diagram. Helix, sheet and loop were shown red color, blue and yellow respectively. For this diagram, the atomic coordinates for PDZ of PICK1 which obtained from the Brookhaven Protein Data Bank (2GZV) were modified to add Y43C44 residues and ligated two half fragments together using DeepView program. It was reported that the Ca2+ bound to the N-terminal acidic region of PICK1 affects its interaction with receptors such as GluR2, N-ethylmaleimide sensitive fusion protein (NSF) and Soluble NSF attachment protein (SNAP) (Hanley 2007). The subsequently physiological event has not to be elucidated yet when Zn2+ binds to PICK1. The fact that zinc also induces the aggregation of the PDZ domain and increases the affinity with lipid membrane suggests that zinc may be involved in stimulating the PDZ-PDZ and/or PDZ-lipid membrane interaction. On the other hand, the lipid membrane impairs the interaction between the PDZ domain and zinc. To certain extent, the lipid membrane also results in the destabilization of the PDZ-PDZ interaction. Our data lead us to have a hypothesis model of Zn-triggered PICK1 clustering on the membrane. The key feature of this model is as follows: (i) the zinc in cytoplasm promotes the PDZ-PDZ and PDZ-lipid membrane interaction to cluster PICK1 on the membrane, (ii) once clustered on the membrane, PICK1 will release the zinc and then associate with lipid membrane in the same site with a higher affinity and (iii) zinc is released to weaken PDZ-PDZ interaction and to strengthen the PICK1-lipid membrane association. The finding that PICK1 is a Zn2+ binding protein can be served as a framework in which the mechanism of the interaction between the PICK1 and its receptors in the presence of Zn2+ might be elucidated in future. 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