Studies on the function mechanism of a Formosan grey mullet protamine—mugiline β M6: interaction of the M6 and M6 fragments with DNA

Studies on the function mechanism of a Formosan grey mullet protamine-mugiline/ M6: interaction of the M6 and M6 fragments with DNA Yoshiko Okamoto, Eiko Muta and Shoshi Ota Department of Biochemistry, Daiichi College of Pharmaceutical Sciences, Minami-ku, Fukuoka 815, Japan

and Norio Nishi Department of Polymer Science, Faculty of Science, Hokkaido University, Kita-ku, Sapporo 060, Japan

(Received 6 January 1992; revised 11 March 1992) The interaction of three peptide segments of one component of Formosan grey mullet protamine (mugiline fl M6), obtained by chemical and enzymatic cleavage, with DNA was studied by spectroscopic measurement, thermal denaturation and circular dichroism. The data obtained were then compared with those of whole M6 and other fish protamines such as salmine of salmon and clupeine of herring. M6-B-L which lacks C-terminal 11 amino acids in M6, showed significantly different properties. It showed remarkably high DNA aggregating ability which was due to a conformational change of DNA from B to/I form. The conformational change of DNA induced by the binding of M6-B-I was reproduced by the carboxypeptidase B digestion of DNA-M6 complex. From these results, the arginine-rich, C-terminal domain of the M6 molecule was estimated to be essential for natural DNA binding. Keywords: Protamine;mugilinefl M6; Formosangreymullet;M6 fragments;DNA aggregation;circulardichroism

Introduction Protamines are extremely basic small proteins found in the spermatic cells of eukaryotic organisms. During spermatogenesis protamines displace the nucleosomal core histones and compact the DNA to form nucleoprotamine in many species 1'2. In this compact and very condensed structure the transcription process is completely inhibited 3 and DNA i s protected from enzymatic hydrolysis. Sequence studies for the protamines included in cephalopods4, fish5'6, birds 7,s and mammals9'~° have demonstrated that they differ from each other in size, constituent amino acids and molecular structure, reflecting specialization in the function of the protamine molecules. Thus, it seems likely that the structure of nucleoprotamine varies with the different species. Different models have been proposed for the mode of protamine binding to DNA 11-16. In contrast to the well defined structure of somatic chromatin, the mode of packaging and organization of DNA in the sperm as well as the mechanisms of DNA unpackaging after fertilization are still not fully understood. Mugiline r, the protamine of Formosan grey mullet is composed of several components similar to the protamines involved in other fish sperm. We have determined the amino acid sequences of M6 and M7 of mugiline fls. We also obtained three kinds of M6 fragments (M6-B-I, M6-B-II, M6-S-II) by cleaving M6 with cyanogen bromide or staphylococcal V8 protease (Figure 1). As a further contribution to the mode of the interaction of protamine with DNA, we herein report the 0141-8130/92/040215-06 © 1992 Butterworth-HeinemannLimited

binding properties of M6 and M6 fragments to DNA and discuss the meaning and function of the protamine segments in the complex with D N A .

Experimental Materials

The M6, M7 and M6 fragments (M6-B-I, M6-B-II, M6-S-II) of mugiline /~ were prepared from Formosan grey mullet (Mugiljaponicus) according to our methods reported previouslys. Clupeine sulphate (Clupea palasii), salmine sulphate (Oncorhynus keta) and poly Arg hydrochloride (MW 5000-15 000) were purchased from Sigma. Protamine sulphate was converted to protamine hydrochloride as follows; protamine sulphate (10-20 mg) dissolved in small volume of distilled water was applied to an Amberlite IRA-400 (C1 form) column (2 x 30 cm) and eluted with distilled water as hydrochloride. Protamine hydrochloride was recovered by lyophilization. The purity of the protamine hydrochloride was confirmed by elementary analysis. Herring testes DNA (Type XIV, Sigma) was dissolved in 13.3 mM Tris-HC1 (pH 7.4) containing 200 mM NaC1 and 1.3 mM EDTA, and then sonicated twice at 0°C for 2min using a Branson B-15 Sonifier at 32W. The molecular weight of DNA was determined as 105-5 x 105 on the basis of agarose gel electrophoresis using Marker II (Nippon Gene) as a standard. The DNA nucleotide concentration was determined by using c~raoNn ,._,,x.,258 20 cm 2 mg- 1 and the mean nucleotide residue molecular weight of 330.9317 .

Int. J. Biol. Macromol., 1992, Vol. 14, August 215

Interaction of M6 and M6 fragments with DNA." Y. Okamoto et al. 1

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Preparation of DNA-protamine complexes D N A - p r o t a m i n e complex was prepared by direct mixing ~8. To a solution of DNA in 13.3 mM Tris-HC1 (pH 7.4) containing 200 mM NaC1 and 1.3 mM EDTA, a solution of protamine in water was added while stirring gently using a vortex mixer. The final concentrations of DNA, Tris-HC1 (pH 7.4), NaC1 and EDTA were 50 #M, 10mM, 150mM and 1 mM, respectively. The molar ratio of protamine to DNA is expressed as that of arginine residues of protamine to nucleotide phosphate (R = Arg/Pi).

Digestion of DNA- M6 complexes with CPB and trypsin D N A - M 6 complexes were prepared as described above. After incubation at 25°C for 30 min, the complexes were digested with diisopropylfluorophosphate-treated carboxypeptidase B (CPB) (substrate:enzyme = 2:1, mol/mol) at 37°C or trypsin (substrate:enzyme = 15:1, w/w) at 25°C. The time course of the digestion was followed by measuring the absorption at 320 nm (A32o) and the c.d. spectrum.

Analytical procedures The DNA aggregating ability was estimated by monitoring A32 o after the D N A - p r o t a m i n e complexes had been incubated at 25°C for 30 min. The melting profiles of the complexes were measured on a Hitachi U-2000 spectrometer equipped with a SPR-10 temperature controller. The complexes were heated at a rate of 0.5°C/min and the absorbance was recorded at 260 nm. The melting temperature (Tin) was defined as the temperature at which the maximum value of hyperchromicity was attained according to Li and Bonner 19. The c.d. spectra of protamines and D N A - p r o t a m i n e complexes were obtained with a Jasco 720A spectropolarimeter. The results were expressed as the mean residue ellipticity [0]4 for protamines, and the mean nucleotide residue ellipticity [0]4 for D N A - p r o t a m i n e complexes in deg. cm 2- d m o l - 1.

Results

DNA aggregating ability In the absorption spectra of the D N A - p r o t a m i n e complex, absorption at around 260 nm, which is due to the DNA base, undergoes a slight red shift, and absorption at around 300-400 nm increases with the increase of R (Arg/Pi) value owing to the aggregate formation2°'2L The degree of DNA aggregation thus induced reflects the formation of a soluble D N A protamine complex, and furthermore A32o values reflect the aggregation state and conformation of DNA protamine complex. Therefore, the DNA aggregating ability is indirectly represented by the A32 o values. Since DNA aggregate formation was induced inmediately after

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mixing and reached a plateau in 20-30 min, and also showed a dependence on temperature, the A320 value was determined after incubation at 25°C for 30 min 21. The DNA aggregating ability for M6 and M6 fragments as a function of R are shown in Figure 2a. The aggregation by M6 reaches a maximum at R values about 1.2, just as in other fish protamines, as is also shown in Figure 3a. The highly efficient DNA aggregating abilities of protamines have been previously explained by their ability to link several DNA molecules. This may not hold true for the M6 fragments because of their limited size, hence the DNA aggregate formation by M6 fragments is supposed to be less than that by M6. Actually the degrees of aggregation induced by M6-B-II and M6-S-II were lower than with M6, but that of M6-B-I was

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Figure 3 DNA aggregating ability of protamines as a function of R (a) or NaC1 concentration (b). DNA-protamine complexes were prepared in 50/~M DNA, 10 mM Tris-HC1 (pH 7.4 ) containing 1 mM EDTA and 150 mM NaC1 (a) or at R = 1.0 (b). Protamine: (O) mugiline fl M6; ((3) mugiline fl M7; (&) clupeine; (A) salmine; (m) poly Arg

Interaction of M6 and M6 fragments with DNA." Y. Okamoto et al. unexpectedly higher than M6. M6-B-I seems to have high DNA aggregating ability despite its short peptide length. The DNA aggregating property of M6 and M6 fragments was investigated using a different method. The contribution of electrostatic interaction to the aggregate formation was estimated by measuring it at various NaC1 concentrations (Fipure 2b). A strong inhibition by NaC1 was found in the DNA aggregation by M6 fragments; the DNA aggregations produced by M6-B-I and M6-B-II were inhibited at a much lower concentraton of NaC1 (300mM), than those by M6-S-II (500mM) and M6 (600 mM). Generally, the aggregate formations by the other fish protamines were inhibited at 700 mM NaC1, although poly Arg formed a DNA aggregate even at 800mM (Figure3b). Another characteristic found in Fioure 2b was strikingly high A32 o values induced by M6-B-I at 100-200 mM NaCI. As shown in Fioure 4,

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the absorption spectrum of D N A - M 6 - B - I undergoes a remarkable red shift at 100-200 mM NaCI, compared with D N A - M 6 , D N A - M 6 - B - I I and D N A - M 6 - S - I I complexes. These results (Figure2a, b and Figure4) indicated that some conformational change of DNA must be induced by M6-B-I under suitable ionic conditions, and this conformational change must be responsible for the results obtained for the DNA M6-B-I complex in

Figure 2b. Thermal melting profile D N A - M 6 and D N A - M 6 fragment complexes were used in thermal denaturation studies to investigate their ability to stabilize the DNA molecule. As shown in Figure 5 and Table 1, all the complexes Show a biphasic shape with a lower melting point (Tin 1 ) attributed to the denaturation of naked DNA and a second transition (Tin 2) at higher temperatures assigned to proteincovered DNA. The Tm 1 values (48.6-49.1) are similar to the value of DNA alone, while the Tm 2 values (85.7-87.2) are about 37-38°C higher than the Tm 1 values. The slightly higher Tm 2 values obtained for the D N A - M 6 - B - I I complex are probably due to the high arginine content of M6-B-II, suggesting the high D N A stabilizing ability. No significant difference was found among M6 and M6 fragments in contrast to the case for salmine and its fragments 22.

C.d. studies Fipure 6a shows the c.d. spectra in the 280-190 nm region of M6 and M6 fragments in water. Two negative

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Int. J. Biol. Macromol., 1992, Vol. 14, August

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Interaction of M6 and M6 fragments with DNA." Y. Okamoto et al. Table 1 Melting transitions of DNA-M6 and DNA-M6 fragments complexes

maxima at 233 nm (weak) and at 198 nm (very strong) and a negative minimum at 218 nm are apparent, with a single exception of M6-B-I where the negative minimum at 218 nm is replaced by a positive maximum. The c.d. spectra indicate that the M6 and M6 fragments exist as unordered structures in water. The general features of the c.d. curves of M6 and M6 fragments are in close agreement with those already reported for clupeine 23 and salmine 24, Bonora et al. reported that the positive Cotton effect is more intense in shorter fragments, while the negative Cotton effect is more intense in the longer fragments of clupeine in water 25. The analogous results however were not obtained for M6 and M6 fragments. The c.d. spectra of M6 and M6 fragments in methanol were also examined. As shown in Figure 6b, M6, M6-B-I and M6-S-II show the c.d. spectra with negative maxima at 207 nm and 222-224 nm, a characteristic feature of the right-handed a-helix. Since we can ignore the effect of dissociation of arginine residues in such non-aqueous solutions, these results may suggest the conformation of M6 and M6 fragments in the complexes with DNA, in which arginine residues are bound tightly to the phosphate group in DNA. C.d. spectra of D N A - M 6 and D N A - M 6 fragments complexes were measured as a function of R and compared with that of DNA alone (Figure 7a, b). Free DNA in neutral solution showed the typical c.d. spectrum characteristic of the B form 26. The spectra of D N A - M 6 complexes (Figure 7a), DNA M6-B-II and D N A - M 6 S-II (data not shown) show a minor reduction in the positive 275 nm band and little change at the second negative band around 245 nm. Nishi et al. reported similar results for the DNA-clupeine complex while a noticeable change was observed for the D N A - p o l y Arg complex 27. They concluded that the protamine molecule induces D N A aggregation in a very efficient manner without drastically affecting the conformation of DNA. On the other hand. in the present experiments, the c.d. spectra of the D N A - M 6 - B - I complex showed a drastic change; namely a blue shift of the positive 275 nm peak accompanied by an increase in intensity of the positive peak (Figure 7b). Figure 8a and b show the )'max and the

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Int. J. Biol. Macromol., 1992, Vol. 14, August

Interaction of M6 and M6 fragments with DNA." Y. Okamoto et al. 290

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magnitude of 1-01268 of the DNA-protamine complexes as a function of R. These results also elucidate the difference between the DNA-M6-B-I complex and other complexes. Since the c.d. spectrum of the A form of DNA displays a negative band at 290 nm and a positive band around 260-265 nm 2s, the conformational change of DNA complexed with M6-B-I would be the transition from the B to the A form of DNA.

Digestion of DNA- M6 complexes with CPB and trypsin D N A - M 6 complexes were digested with two kinds of proteases to investigate the effects of fragmentation of M6 on the DNA aggregate and DNA conformation. The D N A - M 6 complexes prepared by direct mixing were incubated at 25°C for 30 min and then digested with CPB or trypsin. The time course of digestion was first followed by DNA aggregation, i.e. by A320 as shown in Figure 9. Owing to their different substrate specificities, the A32o value for trypsin digestion decreased very rapidly compared with that for CPB and reached a plateau after 40 rain. However, slightly more DNA aggregate remained after CPB digestion than after trypsin digestion.

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prepared in 50 gM DNA, 10 mM Tris-HC1 (pH 7.4) containing 10mM NaC1 at R = 1.0. Enzyme: ( 0 ) CPB; (©) trypsin

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The effect of protease digestion on the c.d. spectrum of the DNA-M6 complex was also studied. As shown in Figure lOa, after a 30 min digestion with CPB, a blue shift of the positive 275 nm peak and an increase in intensity were observed. The result was the same as with the c.d. spectra of the DNA-M6-B-I complex (Figure 7b, curve 6). Neither the blue shift of the positive 275 nm peak (Figure lOb) nor the increase in intensity were observed in trypsin digestion. These results suggest that digestion from the C-terminus of M6 leads a conformational change of DNA in the D N A - M 6 complex from the B to the A form.

Discussion Protamine generally had a far higher DNA aggregating ability than poly Arg (Figure 3a), although protamine and poly Arg had a similar DNA aggregating ability to that of denatured DNA 21. However, the conformational change of DNA induced by protamine (Figure 7a) was found to be less than that by poly Arg in the c.d. studies of the complexes. Thus, the protamine molecule seems to induce DNA aggregation in a very efficient manner without substantially affecting the conformation of DNA. Some special DNA aggregating mechanisms with protamine seem to exist in addition to the general electrostatic interaction between the Arg residues in protamine and the phosphodiester group in DNA 29. M6 and M6 fragments in water gave clear c.d. spectra indicating an unordered structure. These results can be explained by the electrostatic repulsion of Arg residues in the molecules. No gross difference could be detected in the secondary structures of M6 and M6 fragments, whereas, when M6 and M6 fragments were complexed with DNA, the difference between the D N A - M 6 - B - I complex and the other complexes could be elucidated. M6-B-I showed an extraordinarily high DNA aggregating ability (Figure 2a) in spite of its short peptide length. This is probably because of the conformational transition of DNA from B to A form induced by M6-B-I (Figures 7b, 8a, b). On the other hand, M6-S-II did not show any unusual DNA aggregating ability, although it is comprised of about two-thirds of the M6 molecules

Int. J. Biol. Macromol., 1992, Vol. 14, August

219

Interaction of M6 and M6 fragments with DNA." Y. Okamoto et al.

like M6-B-I; M6-B-I lacks the highly basic C-terminal 11 amino acids and M6-S-II lacks the N-terminal six amino acids. Our data thus indicate that the C-terminal highly basic region of M6 is essential for natural binding to DNA. A similar observation was reported for clupeine YII. Fragment ABC of clupeine YII, which lacks the C-terminal region, showed a high DNA aggregating ability; on the other hand fragment CD, which lacks the N-terminal region did not show such an ability 22. The mode of protamine binding to DNA is not still fully understood. Some different models have been proposed for the structure of nucleoprotamine 11-16. Warrant and Kim investigated the structure of the tRNA-protamine complex in an X-ray diffraction study and suggested that a-helical segments of protamines are arranged along a major groove of DNA and protamine molecules cross link plural DNA double helices together. In this DNA-protamine complex all protamines have a structure composed of three or four a-helical domains connected by two or three flexible joints which are well-known helix-breaking residues such as Pro, Ser and Gly 14. In simulated environmental conditions as in the complex with DNA, where charge repulsion from neighbouring positive side chains are minimized, a-helical conformation was induced in M6 and M6 fragments (Fi#ure 6b). Therefore, our results closely parallel those already described for clupeine and salmine 23'2a. Many reports have recently described the leucine zipper class of DNA-binding proteins 3°-32. These proteins contain a bipartite structural motif consisting of a 'leucine zipper' dimerization domain and a segment rich in basic residues responsible for DNA interaction. The basic region "is approximately 25 residues in length and composed of numerous positively charged residues as well as neutral residues that appear essential for sequence specific interactions with the base pairs. O'Neil et al. found that a-helical conformation of the basic region was only induced by DNA containing the target site, and binding to non-specific DNA induced considerably less ~-helix content 33. In the case of protamine binding to DNA, even though its binding is non-specific, a-helical conformation of protamine could be induced by a DNA-like basic region of leucine zipper proteins. The a-helical, arginine-rich C-terminal domain in the protamine molecule would thus function to maintain the B form of DNA.

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