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Identification of zinc-binding sites of proteins: Zinc binds to the amino-terminal region of tubulin
ANALYTICALBIOCHEMISTRY
Identification
172,210-218
(1988)
of Zinc-Binding Sites of Proteins: Zinc Binds to the Amino-Terminal Region of Tubulin L. SERRANO,J.E. DOMINGUEZ,AND J. AVILA
Centro de Biologia Molecular (CSIC-UAM),
Universidad Authma.
Canto Blanco, 28049 Madrid, Spain
Received November 25, 1987 The discovery that certain proteins may require zinc for their activity, and the fact that several of them cannot be purified in large amounts, has led us to develop a rapid. sensitive method to detect these proteins in samples. This method is based on the fractionation of the proteins by gel electrophoresis, blotting onto nitrocellulose paper, and overlaying with 65Zn. We have tested the procedure with well-characterized zinc-binding proteins. In the case of tubulin, we have used this method to localize its zinc-binding site. It was found that zinc binds to the first I50 amino acids of both OI-and fl-tubulin subunits. D 1988 Academic press, IX. KEY WORDS: zinc: binding; proteins; tubulin.
Zinc is an essential component for the functional activity of some proteins involved in animal metabolism (1). It has recently been indicated that zinc could be needed for the interaction of some proteins with nucleic acids (2), in which case the presence of certain cysteine and histidine residues seem to be required for the binding of zinc (3-5). Two classes of zinc-binding proteins have been suggested: the CzH2 class in which pairs of closed cysteines and histidines are separated by a loop of 12 amino acids, and the Cx class containing a variable number of conserved cysteines available for metal chelation (6). An example for the first class is the Xenopus luevis transcription factor TFIIIA (3). while a good example of the second class is metallothionein (6). Zinc also seems to maintain the polymeric structure of liver alcohol dehydrogenase (7) and is required for its catalytic activity. In the presence of zinc, tubulin polymerizes into sheet-like polymers rather than forming microtubules (8). As indicated above, zinc appears to play a role in the function of different proteins involved in various kinds of cell activities. The detection of possible zinc-binding proteins 0003.2697/S
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210
usually requires their purification in milligram amounts, a task which is difficult for certain proteins. Moreover, once the protein is purified and its zinc-binding properties analyzed, the region of the protein involved in the binding of this cation remains to be localized. A simple method for identifying calciumbinding proteins in a protein mixture has been described (9). This method is based on the fractionation of proteins by electrophoresis, their transfer to nitrocellulose sheets, and incubation with radiolabeled calcium, followed by a washing step. We describe here a rapid and specific method, based on the above protocol, to identify zinc-binding proteins in a protein mixture and also. in combination with limited proteolysis, to identify zinc-binding regions. MATERIALS
AND
METHODS
Materials. 65ZnC12 (2 mCi/pmol) and 45CaC12 (2 mCi/pmol) were purchased from the Radiochemical Center, Amersham UK. Protein pur(jicution. Tubulin from porcine brain was prepared by polymerization-depolymerization cycles ( 10) and phosphocellu-
ZINC-BINDING
SITES
OF
211
PROTEINS
lose chromatography (11). Nuclear extracts from human lymphoma cells were obtained as indicated (12). Briefly, human lymphoblastoma DHL- 100 cells were minced and homogenized in 0.05% Triton X- 100, 10 mM sodium, 1,4piperazinediethanesulfonic acid (Pipes),’ pH 6.8, 5 mM MgCl*, 300 mM sucrose, and 1 rnM PMSF on ice. The mixture was centrifuged for 5 min at 25OOg and the pellet resuspended in 10 mM Pipes, pH 6.8,2 M NaCl, 0.3 M sucrose, and 1 KtM PMSF. The preparation was homogenized and centrifuged at 100,OOOg for 2 h and the supernatant was taken for zinc-binding test. Carboxypeptidase A from bovine pancreas, malate dehydrog,enase from pig heart, lactate dehydrogenase from rabbit muscle, alkaline phosphatase firom calf intestine, chymotrypsin A from bolvine pancreas, leucine aminopeptidase from hog kidney, trypsin from bovine pancreas,, and alcohol dehydrogenase from yeast were purchased from Boehringer. Carbonic anhydrase (CA) from bovine erythrocytes and bovine serum albumin (BSA) were purchased from Sigma. Electrophoresis and blotting onto nitrocelIulose paper. Sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis was carried out according to the method of Laemmli (13). A 5- 15RI polyacrylamide gradient was normally used. The samples were boiled for 1 min at 100°C in 0.1 M Tris-HCl, pH 6.8, 30% glycerol, 1% SDS, and 5 KIM P-mercaptoethanol (electrophoresis buffer) prior to their application to the gel. After electrophoresis. duplicates of the samples were stained with Coomassie blue in some cases. Electrophoretic transfer to nitrocellulose sheets was performed as indicated by Towbin et al. ( 14) at a constant current of I25 mA for 12 h after the gel was washed with transfer buffer (14) with or without methanol and plus or minus 4 M urea.
Incubation with 6’ZnC12. After the transfer procedure, nitrocellulose sheets were soaked in a buffer containing phosphate-buffered saline, pH 7.0 (PBS), with 0.05% Tween 20 for 2-4 h at room temperature. The sheets were subsequently soaked for an additional 2-4 h in 10 mM Pipes, pH 6.9, 50 mM NaCl, 0.5 mM MgC& or 0.5 mM MnC12 with or without 5 mM DTT (dithiothreitol) (renaturation buffer). The paper was then soaked for 2 h in the same buffer with 5 X 10m6 M 65ZnC12 (1 pCi/ml) at room temperature. Finally, the paper was washed once for 1 min (unless otherwise indicated) in the above buffer without zinc and twice more for 30 s with distilled water. The sheets were dried on filter paper and then exposed overnight to Kodak XAR-5 X-ray film. The exposed nitrocellulose sheets were then stained with Ponceau red (15) or amido black (9). Exposed films or stained nitrocellulose sheets were occasionally subjected to densitometry to quantify protein or the associated radioactivity.
’ Abbreviations used: DTT. DL-dithiothreitol: BSA. bovine serum albumin; CA, carbonic anhydrase: Pipes, 1.4-piperazinediel hanesulfonic acid; PMSF, phenylmethylsulfonyl fluoride; SDS. sodium dodecyl sulfate; PBS, phosphate-buffered saline.
Identification by the Method of Known Zinc-Binding Proteins
RESULTS
Detection qf Zinc-Biflding in a Protein Mi.~ture
Proteins
A nuclear protein preparation (see Materials and Methods) from human lymphoma cells (Fig. 1) was fractionated by gel electrophoresis, transferred to nitrocellulose paper, and incubated with 65ZnC12. Figure 1 shows that only some proteins from the whole extract bound zinc (i.e., histones), while others present in amounts higher than those of the previous proteins were unable to bind it. This result indicates that zinc does not bind to proteins in a nonspecific way and that some proteins in a whole extract could be readily identified as potential zinc-binding proteins.
Several proteins previously identified in vivo or in vitro as zinc-binding proteins. such
212
SERRANO,
DOMINGUEZ,
AND AVILA
hemoglobin (data not shown), cytochrome C, trypsin, or chymotrypsin bind 65Zn only in very low amounts or not at all (Fig. 2). 97 K
66K 46K
29K
14 K
S
A
FIG. 1. Zinc overlay on a nuclear extract. A nuclear extract from human lymphoma cells (100 wg) obtained as indicated under Materials and Methods was applied to a lo-20% polyacrylamide gradient. The gel was run, the proteins were transferred to nitrocellulose, and the nitrocellulose paper was processed as indicated under Materials and Methods. The figure shows the Coomassie blue staining of the transferred proteins (S) and the corresponding autoradiography (A) following zinc overlay. Molecular mass markers are indicated: phosphorylase B, 97 kDa; bovine serum albumin, 67 kDa; ovalbumin, 45 kDa, carbonic anhydrase, 29 kDa; and lysozyme, 14 kDa. The histones band may correspond to that close to the position of lysozyme.
as BSA (16), carbonic anhydrase, alkaline phosphatase ( 17) alcohol dehydrogenase (17), lactate dehydrogenase (16), carboxypeptidase A (2), leucine aminopeptidase (I 6) and tubulin (8) as well as four nonzinc-binding proteins (hemoglobin, cytochrome C, chymotrypsin, and trypsin) were subjected to electrophoresis, transferred to nitroceliulose, and incubated with ‘j5Zn. Those proteins described as interacting with zinc were found to bind the isotope, while
Washing Conditions in the Zinc-Binding Procedure Metals (Mn’+, Fe*+, Co2+, Ni2+, Cu*+, Zn2+, Ca’+, and Hg*+) form highly stable complexes with proteins (16), with a Kd ranging from 2 X 10e5 in zinc enolase ( 18) to 1 X lo-*’ in some copper or iron proteins (19). To form these stable complexes, zinc must form a tetrahedral complex with the protein (16). Using the overlay method described here, zinc might be nonspecifically trapped on denatured protein, and consequently the stability of the interaction would be low. To test this possibility, we have used different times for the first washing step (OS-5 min). There is a rapid decrease in the amount of bound 65Zn when the first washing step is extended from 0.5 to 1 min; thereafter a plateau is reached for all proteins tested (Fig. 3). We have therefore used a 1-min washing time for this first washing step. Sensitivity of the Method The advantage of this method is the possibility of detecting zinc-binding proteins
‘,“- FIG. 2. Zinc overlay on purified proteins. A group of several zinc-binding proteins, alcohol dehydrogenase (30 pg, ADH), leucine aminopeptidase (LA), bovine serum albumin (25 pg, BSA), carboxypeptidase A (10 rg, CA), alkaline phosphatase (25 pg, Ap), carbonic anhydrase (20 pg, Ca), tubulin (50 pg, Tub), and three negative controls: cytochrome C (30 fig, Cytc), trypsin (30 pg), and chymotrypsin (40 pgg),were treated as indicated in Fig. 1. The figure shows the Ponceau red staining of transferred proteins (5) and the corresponding autoradiograPnY.
ZINC-BINDING
SITES
FIG. 3. Effect of the interval time of the first washing step, on zinc binding. Twenty micrograms of leucine aminopeptidase (LA) (top). 20 pg of bovine serum albumin (BSA), 20 pg of tubulin (a, p). and 10 +g of lactate dehydrogenase (LDH) (center) were applied to a 5- 15% polyacrylamide gel and treated as indicated in Fig. 1. Here. four identical lanes of each sample were used and each was subjected to a different washing time in the first washing step (0.58-5 min). The figure shows the Ponceau red staining (S) of each of the samples and the corresponding autoraldiograph after the transferred proteins are washed for different times (0.5, 1, 3, and 5 min). At the right, the amount of bound radioactive zinc, from the above proteins, in relation to the time of the first washing step is shown. The amount of bound zinc is indicated in arbiirary units (100% for a 0.5-min washing step) and was measured by densitometry.
present in low amounts in a mixture of proteins. Consequently, we have determined the minimum amount of protein detectable by this method. ‘Varying amounts of three zincbinding protek (BSA, tubulin, and carbonic anhydrase), ranging from 1O-5 to 2 X 1O-4 pmol were used (Fig. 4). At least 10e5 pmol of a zinc-binding protein could be easily detected by this method: it is possible that, by decreasing the amount of cold zinc, and/or by increasing that of labeled zinc, even lower amounts of protein might be detected. Competition of Zinc with Calcium and Cobalt.for Binding to Zinc-Binding Proteins It is possible to replace the “native” metal atom of metalloenzymes with others not as-
OF
213
PROTEINS
sociated with them in nature, yielding an enzymatically active product (16). In the case of zinc, it has been found that cobalt can, in many cases, replace or compete for the binding of zinc to zinc-binding proteins (20). For example, it has been found that cobalt and zinc have almost the same affinity for tubulin, while for calcium a concentration two orders of magnitude higher than that of zinc is required to displace it (2 I ). We have analyzed competition between labeled 65Zn (5 X lop6 M) and cold zinc, cobalt, or calcium ( 1OP5- 1Om3M), in binding to leucine aminopeptidase (Fig. 5A) and tubulin (Fig. 5B). For both proteins, using concentrations up to 1 InM calcium displaces a maximum of 10% of the original zinc binding. However, when cobalt is used as the competitor for labeled zinc, differences are observed for the two proteins. In the case of leucine aminopeptidase, cold zinc displaces labeled zinc more efficiently than cobalt (Fig. 5A), while in the
1 Protem
2 lj~Ml x lo-&
FIG. 4. Determination of the minimum amount of protein detectable by this method. Different amounts of BSA (1, 2. 5, 10 fig), tubulin (a, 8) (2, 5, 10, 20 pg), and carbonic anhydrase (CA) (0.5, 1. 2, 5 pg) were subjected to electrophoresis on a 5- 15% polyacrylamide gradient and treated as indicated in Fig. I. On the right, the Ponceau red staining of the transferred proteins (S) and the corresponding autoradiograph (A) are shown. On the left, the relationship between the binding of radioactive zinc in arbitrary units (ordinates) and the amount of protein (abcissas) is shown. Bovine serum albumin (0); cu-tubulin (A); fi-tubulin (Cl); carbonic anhydrase (0). The amount of bound zinc was determined by densitometry scanning of the autoradiograph. The zinc that was bound by the higher amount of cu-tubulin subunit was considered as 100%.
214
SERRANO,
DOMINGUEZ,
AND AVILA
Comparison between the Binding of Radioactive Calcium and Zinc to Proteins It is possible that zinc binding to these proteins could be due to the presence of nonspecific metal-binding sites on these proteins, We have therefore compared a calcium (45Ca) with a zinc (65Zn) overlay (Fig. 6). While BSA, tubulin, carbonic anhydrase, lactate dehydrogenase, and alcohol dehydrogenase were found to bind zinc, only the (Yand &subunits of tubulin, which contain a high-affinity calcium-binding site (22), are able to bind radioactive calcium efficiently. Efect of the Use of Reducing Agents in the Binding of Zinc
FIG. 5. Competition between zinc and other metals or their binding to transferred proteins. Samples containing 50 pg of tubulin or 35 pg of leucine aminopeptidase were subjected to electrophoresis on a 5- 1S% polyacrylamide gradient gel. The proteins were transferred to nitrocellulose. After the renaturation step, the nitrocellulose papers containing either tubulin or leucine aminopeptidase were incubated with the zinc buffer (see Materials and Methods) containing increasing amounts of calcium (10e5 to 2 X lo-’ mM) (A, A), cobalt (10m5 to 2 X 10m3 mM) (0, 0), or cold zinc (IO-’ to 2 X 10m3mM) (m, 0) and washed as indicated under Materials and Methods. (A) The relationship between increasing amounts of the above metals and the binding of zinc to leucine aminopeptidase. The amount of zinc bound to leucine aminopeptidase in the presence of 10 mM Pipes, pH 6.9, 50 mM NaCI, 0.5 mM MgClr, and 5 X 1Om6M 65ZnC1, (zinc buffer) is considered 100% binding. (B) The relationship is shown between increasing amounts of the above metals and the binding of zinc to both (Y-and &tubulin subunits considered together. As in (5A), 100% binding is that for zinc buffer with no added metal.
case of tubulin, the displacement is almost the same for cobalt and zinc. This is in agreement with previous observations with native tubulin (2 1).
For certain proteins, the correct formation and breakage of disulfide bridges (17) is important for the folding of denatured proteins. We have tested the effect of a reducing agent such as DTT to analyze changes in the amount of zinc bound to different proteins. The data in Table 1 indicate that an increase
AB
C
A
B
C
FIG. 6. Binding of radioactive zinc or calcium to transferred proteins. Twenty micrograms of bovine serum albumin (BSA), 20 fig of tubulin (a and @),and 10 pg of carbonic anhydrase (CA) or 40 fig of alcohol dehydrogenase (ADH) and 30 fig of lactate dehydrogenase (LDH) were run in duplicate on a 5-15% polyacrylamide gel as indicated under Materials and Methods. After renaturation the nitrocellulose papers were incubated with zinc buffer (B) or calcium buffer (C) (see Materials and Methods). The papers incubated with zinc buffer were processed as indicated under Materials and Methods, while those incubated with calcium buffer were processed as indicated in Ref. (9). The figure shows Ponceau red staining (A) of the above samples and the autoradiographs corresponding to the papers incubated with radioactive zinc (B) or radioactive calcium (C).
ZINC-BINDING
SITES TABLE
RELATIONSHIP
Bovine serum albumin a-Tubulin /STubulin Alkaline phosphatase Alcohol dehydrogenase Carbonic anhydrase Carboxypeptidase A
BETWEEN BOUND
OF
215
PROTEINS
1 ZINC AND AMOUNT
OF PROTEIN
- DTT
+DTT
-DTT/+DTT
0.4 0.5 0.45 0.4 0.3 0.5 0.3
0.9 0.2 0.15 0.3 0.4 1 0.15
2.25 0.4 0.35 0.75 1.33 2 0.5
Note. Fifty micrograms of bovine serum albumin, 100 pg of tubulin, 25 pg of carboxypeptidase A. 20 bg of carbonic anhydrase. 40 pg of alkaline phosphatase, and 50 pg of alcohol dehydrogenase were applied in duplicate to a S- 15% poiyacrylamide gradient gel. The fractionated proteins were transferred to nitrocellulose and the nitrocellulose papers processed as indicated under Materials and Methods with 0.5 mM MnCI? in the renaturing buffer. One of the duplicates was incubated with 5 mM DTT (+DTT) in the renaturation process. The papers were then incubated with “ZnClz as Indicated under Materials and Methods, dried, and exposed to kodak X-Omat X-ray film for different times. A densitometric scanning of the autoradiographs was done. The relationship between bound zinc in arbitrary units and the amount of protein is shown for the different proteins.
in zinc binding is found in some proteins (BSA, alcohol dehydrogenase, CA), while the opposite effect is observed for others (tubulin, alkaline phosphatase, and carboxypeptidase A).
ure 7). However, when radioactive zinc is used, only the amino-terminal fragments (olN, ON) and the intact subunits bind zinc (Fig. 7). Further analysis by limited proteolysis using pronase (25) (Fig. 8) indicates that zinc Zn
S
One possible application of this method is its use in combination with limited proteolysis to locate zinc-binding regions. Under certain conditions, tubulin digested with trypsin is cleaved only at its a-subunit, yielding a fragment of 35 kDa which corresponds to the amino terminal (aN) and a I6-kDa fragment which is quickly degraded and corresponds to the carboxyl terminal of the protein (&) (22,23). When chymotrypsin is used, only the B-subunit .is cleaved, rendering a 30-kDa fragment corresponding to the amino terminal (PN) and a 20-kDa fragment corresponding to the carboxyl terminal (PC) (23.24). When the cal,:ium overlay method (22) is used with tubulin digested with trypsin or chymotrypsin, only the intact subunits and the carboxyl-terminal fragments (cvC, PC) are found to bind radioactive calcium (22) (Fig-
Tub-@)
ld)
-Tub
Tzrhr
;
Co _Tub
Tub-.
-Tub
PN WC-
s - pc
a
b
WC -
a b
PC
a b
FIG. 7. Zinc binds to the amino-terminal domain of tubulin. Fifty micrograms of tubulin digested with trypsin (a) or chymotrypsin (b) as indicated in Refs. (27) and (30) were subjected to electrophoresis on a 10% polyacrylamide gel. The digested protein was transferred to nitrocellulose and subjected to the zinc-overlay method (see Materials and Methods) (Zn) or to the calciumoverlay method (Ref. (22)) (Ca). The figure shows Ponceau red staining (S) of the trypsin (a) or chymotrypsindigested (b) tubulin and the autoradiographs of the nitrocellulose papers incubated with radioactive 6’Zn (Zn) or “5Ca (Ca). The positions of the undigested tubulin subunits (Tub), of the a-subunit amino (aN) and carboxy1 (&)-terminal tryptic fragments. and of the P-subunit amino (@N) and carboxyl-terminal (DC) chymotryptic fragments are indicated.
216
SERRANO.
DOMINGUEZ.
AND
AVILA
acrylamide gels (26) after removal of the detergent by washing with an aqueous baffer. Also protein renaturation has been achieved on proteins blotted onto nitrocellulose. By removing the detergent with an aqueous buffer, containing urea, before the transfer of protein from gel to nitrocellulose, the acetylcholine receptor was renatured (27). Similar FIG. 8. Binding of zinc to pronase-digested tubulin. methods have been used for the renaturation Tubulin (2 mg/ml) digested with 0.1% (w/w) pronase for of other proteins (15.28) and in the present 30 min at 30°C as indicated in Ref. (25) were subjected work. to electrophoresis on a 10% polyacrylamide gel. The digested protein was transferred to nitrocellulose and subThere are three different ways in which a jected to the zinc-overlay method (Zn) or to the calmetal could interact with a protein: the first cium-overlay method (Ca). The figure shows the Poninvolves the interaction of a cation with resiceau red staining (S) of the pronase-digested tubulin (a) dues located in a short segment of a protein and the corresponding autoradiographs of the nitrocelcomprising less than 30 amino acids (i.e., the lulose papers incubated with radioactive 65Zn (b) or 45Ca (C). The positions of the undigested cy- and /J-subunits. so-called “zinc fingers” (2)): the second inthe amino-terminal fragments of o1- (nN) and p-subunits volves the interaction of the metal with resi(@N. B’N). and the corresponding carboxyl-terminal dues distantly located from each other in the fragments ((UC. DC) are shown. The scheme shows the primary sequence and not belonging to a relative positions of the cleavages of pronase in both unique secondary structural element (29.30); subunits and the molecular weight markers of the corresponding fragments. and the third involves the interaction in which the metal is bound between two subunits of a protein (17). In the first case, the binds to a fragment of 35 kDa containing the metal could bind to the transferred proteins amino terminal of the a-subunit (aN), to a with no requirement for extensive renaturafragment of 30 kDa containing the amino tion. In the second case, extensive renaturaterminal of the P-subunit (PN), and to an tion is needed. It is evident in the third case 1%kDa fragment that comprises amino acids that zinc binding to transferred proteins 1 to 130- 150 (B’N). Zinc does not bind to a could not be detected by this method. Pro3 I-kDa fragment containing the carboxyl teins belonging to the first two groups have terminal of the P-subunit (S’C) and to two been tested here, and zinc binding has been fragments of 20 and 16 kDa, containing the detected in all cases. carboxyl termini of &PC) and the a-subunits There are some data obtained from the ex(&), respectively. Zinc therefore binds to periments we have performed that indicate the first 130- 150 amino acids of the P-subthat the zinc binding to the transferred prounit and probably also to those of the oc-subteins is highly specific and occurs at the same unit. site as in the native structure. Only a few proteins of a whole nuclear extract were DISCUSSION found to bind zinc in significant amounts, proteins tested (cyIdentification of zinc-binding proteins by and all non-zinc-binding chymotrypsin, the overlay procedure described is a specific tochrome C. hemoglobin, and trypsin) were negative or bind zinc in and rapid method in our hands. Although negligible amounts, although they contain this procedure requires the use of denaturing agents such as ionic detergents, the recovery cysteine and histidine residues. The metal of the native structure (as determined by the competition experiments gave results the recovery of protein function) of proteins same as those found for native soluble proboiled with SDS has been achieved in poly- teins. in which calcium hardly displaces zinc,
ZINC-BINDING
SITES
while cobalt efficiently competes with zinc. Moreover, the competition kinetics of cobalt with zinc were not the same for leucine aminopeptidase as for tubulin; in this latter case, they are equal to the kinetics described for native soluble tubulin (2 1). Reduction of cysteine residues, which may be implicated1 in the interaction of zinc with some proteins but not with others, results in an increase or a decrease of the zinc binding to proteins. ‘This clearly indicates that the binding of zinc is not due to a nonspecific trapping of zinc by cysteine residues, since in some cases a decrease in zinc binding is found. Rather it seems that the zinc binding to transferred proteins requires a renaturation process, which may or may not be favored by the use of reducing agents. Finally. the comparison between the calcium overlay technique and that of zinc indicates that zinc-binding proteins can be clearly distinguished from those which bind calcium, thus confirming the specificity of our method. The use of limited proteolysis in combination with the zinc-overlay method could assist in the localization of putative zinc-binding regions in proteins. The results obtained here with tubulin indicate that zinc binds to the first 130- 150 amino acids of both subunits. Only histidines 28, 107, 139. 192, 227. 264, 307, and 396 and cysteines 129, 213. and 303 are conserved in both N- and B-tubulin subunits (31). None of these residues are located on a sequence that fulfills the fingerprint sequence of zinc fingers (2). Consequently we cannot determine to which residues zinc is bound. This result again suggests that zinc does not bind nonspecifically to transferred proteins, since there are histidine and cysteine residues scattered though both subunits together with a very acidic C terminal in both subunits (3 1), but zinc binds only to a fragment comprising the first 130-l 50 residues. In summary, the zinc-overlay method has been tested. not only for well-known zincbinding proteins, but also for some proteins
OF
217
PROTEINS
in which the evidence for zinc binding was preliminary (i.e.. tubulin). Thus, this method should facilitate the search for new zincbinding proteins and the localization of zincbinding regions in those proteins. ACKNOWLEDGMENTS This work FIS (Spain).
was supported
by grants from
CAICYT
and
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Identification
172,210-218
(1988)
of Zinc-Binding Sites of Proteins: Zinc Binds to the Amino-Terminal Region of Tubulin L. SERRANO,J.E. DOMINGUEZ,AND J. AVILA
Centro de Biologia Molecular (CSIC-UAM),
Universidad Authma.
Canto Blanco, 28049 Madrid, Spain
Received November 25, 1987 The discovery that certain proteins may require zinc for their activity, and the fact that several of them cannot be purified in large amounts, has led us to develop a rapid. sensitive method to detect these proteins in samples. This method is based on the fractionation of the proteins by gel electrophoresis, blotting onto nitrocellulose paper, and overlaying with 65Zn. We have tested the procedure with well-characterized zinc-binding proteins. In the case of tubulin, we have used this method to localize its zinc-binding site. It was found that zinc binds to the first I50 amino acids of both OI-and fl-tubulin subunits. D 1988 Academic press, IX. KEY WORDS: zinc: binding; proteins; tubulin.
Zinc is an essential component for the functional activity of some proteins involved in animal metabolism (1). It has recently been indicated that zinc could be needed for the interaction of some proteins with nucleic acids (2), in which case the presence of certain cysteine and histidine residues seem to be required for the binding of zinc (3-5). Two classes of zinc-binding proteins have been suggested: the CzH2 class in which pairs of closed cysteines and histidines are separated by a loop of 12 amino acids, and the Cx class containing a variable number of conserved cysteines available for metal chelation (6). An example for the first class is the Xenopus luevis transcription factor TFIIIA (3). while a good example of the second class is metallothionein (6). Zinc also seems to maintain the polymeric structure of liver alcohol dehydrogenase (7) and is required for its catalytic activity. In the presence of zinc, tubulin polymerizes into sheet-like polymers rather than forming microtubules (8). As indicated above, zinc appears to play a role in the function of different proteins involved in various kinds of cell activities. The detection of possible zinc-binding proteins 0003.2697/S
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210
usually requires their purification in milligram amounts, a task which is difficult for certain proteins. Moreover, once the protein is purified and its zinc-binding properties analyzed, the region of the protein involved in the binding of this cation remains to be localized. A simple method for identifying calciumbinding proteins in a protein mixture has been described (9). This method is based on the fractionation of proteins by electrophoresis, their transfer to nitrocellulose sheets, and incubation with radiolabeled calcium, followed by a washing step. We describe here a rapid and specific method, based on the above protocol, to identify zinc-binding proteins in a protein mixture and also. in combination with limited proteolysis, to identify zinc-binding regions. MATERIALS
AND
METHODS
Materials. 65ZnC12 (2 mCi/pmol) and 45CaC12 (2 mCi/pmol) were purchased from the Radiochemical Center, Amersham UK. Protein pur(jicution. Tubulin from porcine brain was prepared by polymerization-depolymerization cycles ( 10) and phosphocellu-
ZINC-BINDING
SITES
OF
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PROTEINS
lose chromatography (11). Nuclear extracts from human lymphoma cells were obtained as indicated (12). Briefly, human lymphoblastoma DHL- 100 cells were minced and homogenized in 0.05% Triton X- 100, 10 mM sodium, 1,4piperazinediethanesulfonic acid (Pipes),’ pH 6.8, 5 mM MgCl*, 300 mM sucrose, and 1 rnM PMSF on ice. The mixture was centrifuged for 5 min at 25OOg and the pellet resuspended in 10 mM Pipes, pH 6.8,2 M NaCl, 0.3 M sucrose, and 1 KtM PMSF. The preparation was homogenized and centrifuged at 100,OOOg for 2 h and the supernatant was taken for zinc-binding test. Carboxypeptidase A from bovine pancreas, malate dehydrog,enase from pig heart, lactate dehydrogenase from rabbit muscle, alkaline phosphatase firom calf intestine, chymotrypsin A from bolvine pancreas, leucine aminopeptidase from hog kidney, trypsin from bovine pancreas,, and alcohol dehydrogenase from yeast were purchased from Boehringer. Carbonic anhydrase (CA) from bovine erythrocytes and bovine serum albumin (BSA) were purchased from Sigma. Electrophoresis and blotting onto nitrocelIulose paper. Sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis was carried out according to the method of Laemmli (13). A 5- 15RI polyacrylamide gradient was normally used. The samples were boiled for 1 min at 100°C in 0.1 M Tris-HCl, pH 6.8, 30% glycerol, 1% SDS, and 5 KIM P-mercaptoethanol (electrophoresis buffer) prior to their application to the gel. After electrophoresis. duplicates of the samples were stained with Coomassie blue in some cases. Electrophoretic transfer to nitrocellulose sheets was performed as indicated by Towbin et al. ( 14) at a constant current of I25 mA for 12 h after the gel was washed with transfer buffer (14) with or without methanol and plus or minus 4 M urea.
Incubation with 6’ZnC12. After the transfer procedure, nitrocellulose sheets were soaked in a buffer containing phosphate-buffered saline, pH 7.0 (PBS), with 0.05% Tween 20 for 2-4 h at room temperature. The sheets were subsequently soaked for an additional 2-4 h in 10 mM Pipes, pH 6.9, 50 mM NaCl, 0.5 mM MgC& or 0.5 mM MnC12 with or without 5 mM DTT (dithiothreitol) (renaturation buffer). The paper was then soaked for 2 h in the same buffer with 5 X 10m6 M 65ZnC12 (1 pCi/ml) at room temperature. Finally, the paper was washed once for 1 min (unless otherwise indicated) in the above buffer without zinc and twice more for 30 s with distilled water. The sheets were dried on filter paper and then exposed overnight to Kodak XAR-5 X-ray film. The exposed nitrocellulose sheets were then stained with Ponceau red (15) or amido black (9). Exposed films or stained nitrocellulose sheets were occasionally subjected to densitometry to quantify protein or the associated radioactivity.
’ Abbreviations used: DTT. DL-dithiothreitol: BSA. bovine serum albumin; CA, carbonic anhydrase: Pipes, 1.4-piperazinediel hanesulfonic acid; PMSF, phenylmethylsulfonyl fluoride; SDS. sodium dodecyl sulfate; PBS, phosphate-buffered saline.
Identification by the Method of Known Zinc-Binding Proteins
RESULTS
Detection qf Zinc-Biflding in a Protein Mi.~ture
Proteins
A nuclear protein preparation (see Materials and Methods) from human lymphoma cells (Fig. 1) was fractionated by gel electrophoresis, transferred to nitrocellulose paper, and incubated with 65ZnC12. Figure 1 shows that only some proteins from the whole extract bound zinc (i.e., histones), while others present in amounts higher than those of the previous proteins were unable to bind it. This result indicates that zinc does not bind to proteins in a nonspecific way and that some proteins in a whole extract could be readily identified as potential zinc-binding proteins.
Several proteins previously identified in vivo or in vitro as zinc-binding proteins. such
212
SERRANO,
DOMINGUEZ,
AND AVILA
hemoglobin (data not shown), cytochrome C, trypsin, or chymotrypsin bind 65Zn only in very low amounts or not at all (Fig. 2). 97 K
66K 46K
29K
14 K
S
A
FIG. 1. Zinc overlay on a nuclear extract. A nuclear extract from human lymphoma cells (100 wg) obtained as indicated under Materials and Methods was applied to a lo-20% polyacrylamide gradient. The gel was run, the proteins were transferred to nitrocellulose, and the nitrocellulose paper was processed as indicated under Materials and Methods. The figure shows the Coomassie blue staining of the transferred proteins (S) and the corresponding autoradiography (A) following zinc overlay. Molecular mass markers are indicated: phosphorylase B, 97 kDa; bovine serum albumin, 67 kDa; ovalbumin, 45 kDa, carbonic anhydrase, 29 kDa; and lysozyme, 14 kDa. The histones band may correspond to that close to the position of lysozyme.
as BSA (16), carbonic anhydrase, alkaline phosphatase ( 17) alcohol dehydrogenase (17), lactate dehydrogenase (16), carboxypeptidase A (2), leucine aminopeptidase (I 6) and tubulin (8) as well as four nonzinc-binding proteins (hemoglobin, cytochrome C, chymotrypsin, and trypsin) were subjected to electrophoresis, transferred to nitroceliulose, and incubated with ‘j5Zn. Those proteins described as interacting with zinc were found to bind the isotope, while
Washing Conditions in the Zinc-Binding Procedure Metals (Mn’+, Fe*+, Co2+, Ni2+, Cu*+, Zn2+, Ca’+, and Hg*+) form highly stable complexes with proteins (16), with a Kd ranging from 2 X 10e5 in zinc enolase ( 18) to 1 X lo-*’ in some copper or iron proteins (19). To form these stable complexes, zinc must form a tetrahedral complex with the protein (16). Using the overlay method described here, zinc might be nonspecifically trapped on denatured protein, and consequently the stability of the interaction would be low. To test this possibility, we have used different times for the first washing step (OS-5 min). There is a rapid decrease in the amount of bound 65Zn when the first washing step is extended from 0.5 to 1 min; thereafter a plateau is reached for all proteins tested (Fig. 3). We have therefore used a 1-min washing time for this first washing step. Sensitivity of the Method The advantage of this method is the possibility of detecting zinc-binding proteins
‘,“- FIG. 2. Zinc overlay on purified proteins. A group of several zinc-binding proteins, alcohol dehydrogenase (30 pg, ADH), leucine aminopeptidase (LA), bovine serum albumin (25 pg, BSA), carboxypeptidase A (10 rg, CA), alkaline phosphatase (25 pg, Ap), carbonic anhydrase (20 pg, Ca), tubulin (50 pg, Tub), and three negative controls: cytochrome C (30 fig, Cytc), trypsin (30 pg), and chymotrypsin (40 pgg),were treated as indicated in Fig. 1. The figure shows the Ponceau red staining of transferred proteins (5) and the corresponding autoradiograPnY.
ZINC-BINDING
SITES
FIG. 3. Effect of the interval time of the first washing step, on zinc binding. Twenty micrograms of leucine aminopeptidase (LA) (top). 20 pg of bovine serum albumin (BSA), 20 pg of tubulin (a, p). and 10 +g of lactate dehydrogenase (LDH) (center) were applied to a 5- 15% polyacrylamide gel and treated as indicated in Fig. 1. Here. four identical lanes of each sample were used and each was subjected to a different washing time in the first washing step (0.58-5 min). The figure shows the Ponceau red staining (S) of each of the samples and the corresponding autoraldiograph after the transferred proteins are washed for different times (0.5, 1, 3, and 5 min). At the right, the amount of bound radioactive zinc, from the above proteins, in relation to the time of the first washing step is shown. The amount of bound zinc is indicated in arbiirary units (100% for a 0.5-min washing step) and was measured by densitometry.
present in low amounts in a mixture of proteins. Consequently, we have determined the minimum amount of protein detectable by this method. ‘Varying amounts of three zincbinding protek (BSA, tubulin, and carbonic anhydrase), ranging from 1O-5 to 2 X 1O-4 pmol were used (Fig. 4). At least 10e5 pmol of a zinc-binding protein could be easily detected by this method: it is possible that, by decreasing the amount of cold zinc, and/or by increasing that of labeled zinc, even lower amounts of protein might be detected. Competition of Zinc with Calcium and Cobalt.for Binding to Zinc-Binding Proteins It is possible to replace the “native” metal atom of metalloenzymes with others not as-
OF
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PROTEINS
sociated with them in nature, yielding an enzymatically active product (16). In the case of zinc, it has been found that cobalt can, in many cases, replace or compete for the binding of zinc to zinc-binding proteins (20). For example, it has been found that cobalt and zinc have almost the same affinity for tubulin, while for calcium a concentration two orders of magnitude higher than that of zinc is required to displace it (2 I ). We have analyzed competition between labeled 65Zn (5 X lop6 M) and cold zinc, cobalt, or calcium ( 1OP5- 1Om3M), in binding to leucine aminopeptidase (Fig. 5A) and tubulin (Fig. 5B). For both proteins, using concentrations up to 1 InM calcium displaces a maximum of 10% of the original zinc binding. However, when cobalt is used as the competitor for labeled zinc, differences are observed for the two proteins. In the case of leucine aminopeptidase, cold zinc displaces labeled zinc more efficiently than cobalt (Fig. 5A), while in the
1 Protem
2 lj~Ml x lo-&
FIG. 4. Determination of the minimum amount of protein detectable by this method. Different amounts of BSA (1, 2. 5, 10 fig), tubulin (a, 8) (2, 5, 10, 20 pg), and carbonic anhydrase (CA) (0.5, 1. 2, 5 pg) were subjected to electrophoresis on a 5- 15% polyacrylamide gradient and treated as indicated in Fig. I. On the right, the Ponceau red staining of the transferred proteins (S) and the corresponding autoradiograph (A) are shown. On the left, the relationship between the binding of radioactive zinc in arbitrary units (ordinates) and the amount of protein (abcissas) is shown. Bovine serum albumin (0); cu-tubulin (A); fi-tubulin (Cl); carbonic anhydrase (0). The amount of bound zinc was determined by densitometry scanning of the autoradiograph. The zinc that was bound by the higher amount of cu-tubulin subunit was considered as 100%.
214
SERRANO,
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Comparison between the Binding of Radioactive Calcium and Zinc to Proteins It is possible that zinc binding to these proteins could be due to the presence of nonspecific metal-binding sites on these proteins, We have therefore compared a calcium (45Ca) with a zinc (65Zn) overlay (Fig. 6). While BSA, tubulin, carbonic anhydrase, lactate dehydrogenase, and alcohol dehydrogenase were found to bind zinc, only the (Yand &subunits of tubulin, which contain a high-affinity calcium-binding site (22), are able to bind radioactive calcium efficiently. Efect of the Use of Reducing Agents in the Binding of Zinc
FIG. 5. Competition between zinc and other metals or their binding to transferred proteins. Samples containing 50 pg of tubulin or 35 pg of leucine aminopeptidase were subjected to electrophoresis on a 5- 1S% polyacrylamide gradient gel. The proteins were transferred to nitrocellulose. After the renaturation step, the nitrocellulose papers containing either tubulin or leucine aminopeptidase were incubated with the zinc buffer (see Materials and Methods) containing increasing amounts of calcium (10e5 to 2 X lo-’ mM) (A, A), cobalt (10m5 to 2 X 10m3 mM) (0, 0), or cold zinc (IO-’ to 2 X 10m3mM) (m, 0) and washed as indicated under Materials and Methods. (A) The relationship between increasing amounts of the above metals and the binding of zinc to leucine aminopeptidase. The amount of zinc bound to leucine aminopeptidase in the presence of 10 mM Pipes, pH 6.9, 50 mM NaCI, 0.5 mM MgClr, and 5 X 1Om6M 65ZnC1, (zinc buffer) is considered 100% binding. (B) The relationship is shown between increasing amounts of the above metals and the binding of zinc to both (Y-and &tubulin subunits considered together. As in (5A), 100% binding is that for zinc buffer with no added metal.
case of tubulin, the displacement is almost the same for cobalt and zinc. This is in agreement with previous observations with native tubulin (2 1).
For certain proteins, the correct formation and breakage of disulfide bridges (17) is important for the folding of denatured proteins. We have tested the effect of a reducing agent such as DTT to analyze changes in the amount of zinc bound to different proteins. The data in Table 1 indicate that an increase
AB
C
A
B
C
FIG. 6. Binding of radioactive zinc or calcium to transferred proteins. Twenty micrograms of bovine serum albumin (BSA), 20 fig of tubulin (a and @),and 10 pg of carbonic anhydrase (CA) or 40 fig of alcohol dehydrogenase (ADH) and 30 fig of lactate dehydrogenase (LDH) were run in duplicate on a 5-15% polyacrylamide gel as indicated under Materials and Methods. After renaturation the nitrocellulose papers were incubated with zinc buffer (B) or calcium buffer (C) (see Materials and Methods). The papers incubated with zinc buffer were processed as indicated under Materials and Methods, while those incubated with calcium buffer were processed as indicated in Ref. (9). The figure shows Ponceau red staining (A) of the above samples and the autoradiographs corresponding to the papers incubated with radioactive zinc (B) or radioactive calcium (C).
ZINC-BINDING
SITES TABLE
RELATIONSHIP
Bovine serum albumin a-Tubulin /STubulin Alkaline phosphatase Alcohol dehydrogenase Carbonic anhydrase Carboxypeptidase A
BETWEEN BOUND
OF
215
PROTEINS
1 ZINC AND AMOUNT
OF PROTEIN
- DTT
+DTT
-DTT/+DTT
0.4 0.5 0.45 0.4 0.3 0.5 0.3
0.9 0.2 0.15 0.3 0.4 1 0.15
2.25 0.4 0.35 0.75 1.33 2 0.5
Note. Fifty micrograms of bovine serum albumin, 100 pg of tubulin, 25 pg of carboxypeptidase A. 20 bg of carbonic anhydrase. 40 pg of alkaline phosphatase, and 50 pg of alcohol dehydrogenase were applied in duplicate to a S- 15% poiyacrylamide gradient gel. The fractionated proteins were transferred to nitrocellulose and the nitrocellulose papers processed as indicated under Materials and Methods with 0.5 mM MnCI? in the renaturing buffer. One of the duplicates was incubated with 5 mM DTT (+DTT) in the renaturation process. The papers were then incubated with “ZnClz as Indicated under Materials and Methods, dried, and exposed to kodak X-Omat X-ray film for different times. A densitometric scanning of the autoradiographs was done. The relationship between bound zinc in arbitrary units and the amount of protein is shown for the different proteins.
in zinc binding is found in some proteins (BSA, alcohol dehydrogenase, CA), while the opposite effect is observed for others (tubulin, alkaline phosphatase, and carboxypeptidase A).
ure 7). However, when radioactive zinc is used, only the amino-terminal fragments (olN, ON) and the intact subunits bind zinc (Fig. 7). Further analysis by limited proteolysis using pronase (25) (Fig. 8) indicates that zinc Zn
S
One possible application of this method is its use in combination with limited proteolysis to locate zinc-binding regions. Under certain conditions, tubulin digested with trypsin is cleaved only at its a-subunit, yielding a fragment of 35 kDa which corresponds to the amino terminal (aN) and a I6-kDa fragment which is quickly degraded and corresponds to the carboxyl terminal of the protein (&) (22,23). When chymotrypsin is used, only the B-subunit .is cleaved, rendering a 30-kDa fragment corresponding to the amino terminal (PN) and a 20-kDa fragment corresponding to the carboxyl terminal (PC) (23.24). When the cal,:ium overlay method (22) is used with tubulin digested with trypsin or chymotrypsin, only the intact subunits and the carboxyl-terminal fragments (cvC, PC) are found to bind radioactive calcium (22) (Fig-
Tub-@)
ld)
-Tub
Tzrhr
;
Co _Tub
Tub-.
-Tub
PN WC-
s - pc
a
b
WC -
a b
PC
a b
FIG. 7. Zinc binds to the amino-terminal domain of tubulin. Fifty micrograms of tubulin digested with trypsin (a) or chymotrypsin (b) as indicated in Refs. (27) and (30) were subjected to electrophoresis on a 10% polyacrylamide gel. The digested protein was transferred to nitrocellulose and subjected to the zinc-overlay method (see Materials and Methods) (Zn) or to the calciumoverlay method (Ref. (22)) (Ca). The figure shows Ponceau red staining (S) of the trypsin (a) or chymotrypsindigested (b) tubulin and the autoradiographs of the nitrocellulose papers incubated with radioactive 6’Zn (Zn) or “5Ca (Ca). The positions of the undigested tubulin subunits (Tub), of the a-subunit amino (aN) and carboxy1 (&)-terminal tryptic fragments. and of the P-subunit amino (@N) and carboxyl-terminal (DC) chymotryptic fragments are indicated.
216
SERRANO.
DOMINGUEZ.
AND
AVILA
acrylamide gels (26) after removal of the detergent by washing with an aqueous baffer. Also protein renaturation has been achieved on proteins blotted onto nitrocellulose. By removing the detergent with an aqueous buffer, containing urea, before the transfer of protein from gel to nitrocellulose, the acetylcholine receptor was renatured (27). Similar FIG. 8. Binding of zinc to pronase-digested tubulin. methods have been used for the renaturation Tubulin (2 mg/ml) digested with 0.1% (w/w) pronase for of other proteins (15.28) and in the present 30 min at 30°C as indicated in Ref. (25) were subjected work. to electrophoresis on a 10% polyacrylamide gel. The digested protein was transferred to nitrocellulose and subThere are three different ways in which a jected to the zinc-overlay method (Zn) or to the calmetal could interact with a protein: the first cium-overlay method (Ca). The figure shows the Poninvolves the interaction of a cation with resiceau red staining (S) of the pronase-digested tubulin (a) dues located in a short segment of a protein and the corresponding autoradiographs of the nitrocelcomprising less than 30 amino acids (i.e., the lulose papers incubated with radioactive 65Zn (b) or 45Ca (C). The positions of the undigested cy- and /J-subunits. so-called “zinc fingers” (2)): the second inthe amino-terminal fragments of o1- (nN) and p-subunits volves the interaction of the metal with resi(@N. B’N). and the corresponding carboxyl-terminal dues distantly located from each other in the fragments ((UC. DC) are shown. The scheme shows the primary sequence and not belonging to a relative positions of the cleavages of pronase in both unique secondary structural element (29.30); subunits and the molecular weight markers of the corresponding fragments. and the third involves the interaction in which the metal is bound between two subunits of a protein (17). In the first case, the binds to a fragment of 35 kDa containing the metal could bind to the transferred proteins amino terminal of the a-subunit (aN), to a with no requirement for extensive renaturafragment of 30 kDa containing the amino tion. In the second case, extensive renaturaterminal of the P-subunit (PN), and to an tion is needed. It is evident in the third case 1%kDa fragment that comprises amino acids that zinc binding to transferred proteins 1 to 130- 150 (B’N). Zinc does not bind to a could not be detected by this method. Pro3 I-kDa fragment containing the carboxyl teins belonging to the first two groups have terminal of the P-subunit (S’C) and to two been tested here, and zinc binding has been fragments of 20 and 16 kDa, containing the detected in all cases. carboxyl termini of &PC) and the a-subunits There are some data obtained from the ex(&), respectively. Zinc therefore binds to periments we have performed that indicate the first 130- 150 amino acids of the P-subthat the zinc binding to the transferred prounit and probably also to those of the oc-subteins is highly specific and occurs at the same unit. site as in the native structure. Only a few proteins of a whole nuclear extract were DISCUSSION found to bind zinc in significant amounts, proteins tested (cyIdentification of zinc-binding proteins by and all non-zinc-binding chymotrypsin, the overlay procedure described is a specific tochrome C. hemoglobin, and trypsin) were negative or bind zinc in and rapid method in our hands. Although negligible amounts, although they contain this procedure requires the use of denaturing agents such as ionic detergents, the recovery cysteine and histidine residues. The metal of the native structure (as determined by the competition experiments gave results the recovery of protein function) of proteins same as those found for native soluble proboiled with SDS has been achieved in poly- teins. in which calcium hardly displaces zinc,
ZINC-BINDING
SITES
while cobalt efficiently competes with zinc. Moreover, the competition kinetics of cobalt with zinc were not the same for leucine aminopeptidase as for tubulin; in this latter case, they are equal to the kinetics described for native soluble tubulin (2 1). Reduction of cysteine residues, which may be implicated1 in the interaction of zinc with some proteins but not with others, results in an increase or a decrease of the zinc binding to proteins. ‘This clearly indicates that the binding of zinc is not due to a nonspecific trapping of zinc by cysteine residues, since in some cases a decrease in zinc binding is found. Rather it seems that the zinc binding to transferred proteins requires a renaturation process, which may or may not be favored by the use of reducing agents. Finally. the comparison between the calcium overlay technique and that of zinc indicates that zinc-binding proteins can be clearly distinguished from those which bind calcium, thus confirming the specificity of our method. The use of limited proteolysis in combination with the zinc-overlay method could assist in the localization of putative zinc-binding regions in proteins. The results obtained here with tubulin indicate that zinc binds to the first 130- 150 amino acids of both subunits. Only histidines 28, 107, 139. 192, 227. 264, 307, and 396 and cysteines 129, 213. and 303 are conserved in both N- and B-tubulin subunits (31). None of these residues are located on a sequence that fulfills the fingerprint sequence of zinc fingers (2). Consequently we cannot determine to which residues zinc is bound. This result again suggests that zinc does not bind nonspecifically to transferred proteins, since there are histidine and cysteine residues scattered though both subunits together with a very acidic C terminal in both subunits (3 1), but zinc binds only to a fragment comprising the first 130-l 50 residues. In summary, the zinc-overlay method has been tested. not only for well-known zincbinding proteins, but also for some proteins
OF
217
PROTEINS
in which the evidence for zinc binding was preliminary (i.e.. tubulin). Thus, this method should facilitate the search for new zincbinding proteins and the localization of zincbinding regions in those proteins. ACKNOWLEDGMENTS This work FIS (Spain).
was supported
by grants from
CAICYT
and
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