the amino acid sequence and interaction with the nucleosome core dna of transition protein 4 from...

Eur. J. Biochem. 233, 179-185 (1995) 0 FEBS 1995

The amino acid sequence and interaction with the nucleosome core DNA of transition protein 4 from boar late spermatid nuclei Kuniko AKAMA’, Hirokazu ICHIMURA’, Hiroki SATO’, Shuichi KOJIMA’, Kin-ichiro MlURA ’, Hiroaki HAYASHI‘, Yasuhiko KOMATSU’ and Minoru NAKANO’ ’ Department of Chemistry, Faculty of Science, Chiba University, Japan

Graduate School of Science and Technology, Chiba University, Japan ’ Institute for Biomolecular Science, Gakushuin University, Tokyo, Japan ‘ Gunma Prefectural College of Health Sciences, Gunma, Japan

National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology, 1-1, Ibaraki, Japan

(Received 10 July 1995) - EJB 95 1123/3

The primary structure of transition protein 4 (TP4) from boar late spermatid nuclei was determined by automated Edman degradation of S-pyridylethylated protein and of peptides generated by cleavage with Staphylococcus aureus V8 protease, lysyl endopeptidase and CNBr. Boar TP4 is a basic protein consisting of a highly basic amino-terminal half (residues 1-73) and a less basic carboxy-terminal half (residues 74-1 38). The latter half includes a highly hydrophobic segment, a four-times tandemly repeated sequence, N(G)QNKR(K)X, and a carboxy-terminal segment containing Trpl26. Ultraviolet absorption and CD spectra of TP4 - rat-liver-nucleosome-core-DNA (double-stranded DNA) complexes suggest a TP4-induced local melting of DNA. Although at 1 mM NaCl TP4 brought about a slight stabilization of the DNA against thermal melting, a destabilization of the DNA was observed at 50 mM NaCl. From the results of quenching of tryptophan (Trp126) fluorescence of TP4 upon its binding to double-stranded and single-stranded boar liver nucleosome-core DNA at 50 mM NaCl, the apparent association constants for the binding of TP4 to double-stranded and single-stranded DNA were calculated to be 7.3X10’ M-’ and 4.1 X 10’ M- ’, respectively. These results suggest that TP4, having different domain structures from TP1-3 and a higher affinity for double-stranded DNA, induces a local destabilization of DNA probably through the stacking of Trp126 with nucleic acid bases.

Keywords: transition protein ; amino acid sequence; DNA-protein interaction; late spermatid nuclei.

The mechanism of transformation of nucleosome-type chromatin into a nucleoprotamine fiber is rather poorly understood. A direct transition from a nucleosome-type chromatin organiza- tion into a nucleoprotamine fiber operates in most species with the exception of mammals [ I , 21. In mammals, nucleosomal histones are transiently replaced by small basic proteins called transition proteins (TPI -4) and, finally, by protamines [3-61. At the same time, transformation of the nucleosomal-type chromatin into a smooth chromatin fiber, initiation of chromatin condensation and cessation of transcription occur [7-121. The amino acid sequences of TPI and TP2, and the nucleotide sequences of their genes are known [ 13 - 161, and attempts have been made to understand how they are transcriptionally and translationally regulated [17, 181. It has been postulated that rat TP1 is a DNA melting protein, mediated through the intercala- tion of its tyrosine residue between the nucleic acid bases [19], that it destabilizes the compact nucleosome core particles 1201, and that rat TP2, with two possible zinc fingers in the amino-

Correspondence to K. Akama, Department of Chemistry, Faculty of Science, Chiba University, Chiba, Japan 263

F a : +81 043 290 2874. Abbreviations. TP, transition protein ; AKP, CAMP-dependent and

cGMP-dependent protein kinase phosphorylation-site motif; KCP, protein kinase C phosphorylation-site motif; NTS, bipartite nuclear- targeting sequence ; t,,,, melting temperature.

Enzynzes. Staphylococcus uureus V8 protease (EC 3.4.21 -19); lysyl endopeptidase (EC 3.4.21 S O ) ; carboxypeptidase B (EC 3.4.17.2); pronase E (EC 3.4.24.4).

terminal region, is a DNA stabilizing protein [21, 221. Recently, we have developed methods for isolating intact boar TP1, TP3 and TP4 123, 241, and have reported that boar TP3 has 27% sequence similarity to boar TPI [25]. In this paper, we describe the amino acid sequence of boar TP4 and the nature of interaction of TP4 with nucleosome-core DNA in vitro.

MATERIALS AND METHODS Materials. staphylococcus aureus V8 protease and lysyl en-

dopeptidase were purchased from Boehringer Mannheim and Wako Pure Chemical Industries, respectively. Carboxypeptidase B was prepared as described earlier [26]. All other chemicals were of reagent grade for biochemical reaearch.

Isolation of TP4. Boar TP4 was isolated by our method 1241 with a slight modification. Basic proteins (850 mg), extracted from boar late spermatid nuclei, were chromatographed on a Fractogel EMD SO; 650 (M) column (2.8 cmX24 cm) with a linear gradient of 450 ml 0.2-0.3 M NaCl in 0.1 M sodium ace- tate/HCl, pH 2.0, at a flow rate of 78 m l h ; Then, the TP4-containing fraction (1.7 mg) was chromatographed on a Nucleosil 300 7C18 column (0.76 cmx 15 cm) in 0.1 % trifluoroacetic acid with a linear gradient of 0-80% acetonitrile at a flow rate of 3.6 ml/min at 35°C. The purity of TP4 was confirmed by SDS/ PAGE on a 15% gel by the method of O’Farrell [27]. The reduced and carboxymethylated boar TP4 was prepared by our method [28].

S-Pyridylethylation of TP4. Cystine residues of purified TP4 were reduced with a 500-fold molar excess of dithiothreitol for 2.5 h, then modified with a 600-fold molar excess of 4- vinylpyridine for 1.5 h in 6 M guanidine/HCl, 0.1 M TrisIHCl, 10 mM EDTA, pH 8.3, at 37°C. S-Pyridylethylated TP4 was purified by reverse-phase HPLC on a Asahipak C4-P50 column (0.46 cmX15 cm) in 0.1 %I trifluoroacetic acid with a gradient of acetonitrile at a flow rate of 0.8 ml/min.

Enzymic and chemical digestion. S. u~ireus V8 protease and lysyl endopeptidase digestions of 2 nmol S-pyridylethylated TP4 were carried out at 37°C for 1 6 h with an enzyme/substrate ratio of 1 :50 (by mol) in 100 pl 1 M urea, 0.1 M NH,HCO,, pH 7.8, and 1 M urea, 0.1 M Tris/HCI, pH 8.6, respectively. Chemi- cal digestion of 2 nmol S-pyridylethylated TP4 by CNBr was performed with a 1000-fold molar excess of CNBr in 100 pl 70% HCOOH at 37°C for 16 h. The solvent for chemical digestion was removed by centrifugal evaporation, and the sample was dissolved in 0.1 % tritluoroacetic acid. Enzymically or chemically digested peptides were separated by reverse-phase HPLC on a L-column ODS column. Peptides were designated by a serial number prefixed by a letter, representing the type of digestion. V, S. aureus V8 protease; L, lysyl endopeptidase; C , CNBr.

Amino acid composition and sequence analyses. The amino acid composition was analyzed using a Hitachi amino acid analyzer model L-8500 or model 655, after hydrolysis in vucuo with 5.7 M HCI containing 0.1 % phenol at 110°C for 24 h. The cysteine residue was detected as S-pyridylethylcys- teine. TP4 was also hydrolyzed in vacuo with 3 M p-toluenesulfonic acid containing 0.2 % 3-(2-aminoethyl)indole at 110°C for 24 h (291. Automated sequence analysis by Edman degradation was performed using Applied Biosystems model 476A protein sequencer.

Carboxypeptidase B treatment. Intact TP4 (1.5 nmol) was treated with 15 pmol carboxypeptidase B in 120 pl 50 mM Tris/ HC1, pH 8.0, at 37°C for 4 h, then applied to a Hitachi 655 amino acid analyzer.

Preparation of rat and boar liver nucleosome-core DNA. Rat and boar liver nucleosome-core particles were prepared from the micrococcal nuclease digest of their liver nuclei by a slight modification of the method of Rill et al. 1301. The nucleosome- core-particle DNA was obtained from the nucleosome-core particles by pronase E treatment, phenolkhloroform extraction, and subsequent ethanol precipitation. This DNA was chrnmato- graphed on Sepharose 4B (1.5 cmX75 cm) in 0.15 M NaCU 15 mM sodium citrate, pH 7.0. An aliquot of each eluted fraction was analyzed by 3.5 % polyacrylamide/0.5 % agarose gel electro- phoresis in 2 mM EDTA, 40 mM Tris/HCI, 20 mM sodium ace- tate, pH 7.8. The fraction containing about 145 bp DNA was dialyzed against 1 mM NaCI, 0.1 mM EDTA, 1 mM Tricine/ NaOH, p H 7 . 4. The dialyzed DNA was used as a source of double-stranded DNA. Single-stranded DNA was prepared by heating the nucleosome-core-particle DNA at 90°C for 15 min and rapidly cooling on ice.

Preparation of complexes of TP4 with DNA. TP4 was renatured as described earlier [ 24). Rat-liver-nucleosome-core- DNA-TP4 complex was prepared by direct mixing. To a solution of DNA in 1 mM NaCI, 0.l mM EDTA, 1 mM Tricine/ NaOH, pH 7. 4, the same buffer or 5 0 0 m M NaCI, 0.1 mM EDTA, 1 mM Tricine/NaOH, pH 7.4, and TP4 in 1 mM NaCI, 0.1 mM EDTA, 1 mM k ricine/NaOH, pH 7.4, was added with gentle stirring. The mixture was incubated at 37°C for 12 h. The final concentrations of the nucleic acids added were calculated using the following absorption coefficients/mol phosphate at 260 nm and 25°C: double-stranded DNA, 6.5X10'; single- stranded DNA, 9.4X 10'. The concentration of DNA was in the

range 40 yM with respect to phosphorus. The concentration of TP4 was determined by amino acid analysis. TP4/DNA molar ratios were calculated using 16044 as the M , of TP4 and 97 150 as the M , of 145-bp DNA.

Ultraviolet difference absorption and CD spectroscopy. The ultraviolet difference absorption spectra for binding of TP4 to DNA were recorded on the double-beam Hitachi U-2000 spectrophotometer. CD spectra for binding of TP4 to DNA were measured with a Jasco 5-500 spectropolarimeter at 25°C with nitrogen flushing. The results were expressed as the mean residue ellipticity [(>I.

Thermal melting of DNA and the DNA-TP4 complex. Thermal denaturation of DNA and its complexes with TP4 at low (1 mM NaCI) and at high (50 mM NaCI) ionic strengths were recorded on a Beckman DU-8 spectrophotometer. The heating rate was set to 1 .5 min for every degree rise in the temperature. Absorbance was recorded at 1 "C intervals in the temperature range 30-90°C. The TP4 concentrations were below the range which resulted in the precipitation of the nucleoprotein complexes. There was no change in the A12,1/Alh,) ratio during the course of the experiment. The first-derivative values were calculated from the absorbance data by the three-point average method described by Li 1311 and Ansevin 1321.

Fluorescence measurements. The fluorescence spectra of TP4 and its complex with double-stranded and single-stranded boar liver nucleosome-core DNA were recorded in a Hitachi 204 fluorescence spectrophotometer. The tryptophan fluorescence spectrum for free TP4 in solution (50 mM NaCI, 0.1 rnM EDTA, 1 mM Tricine/NaOH, pH 7.4) was recorded in a cuvette of I-cm path length after exciting at 295 nm. The fluorescence emission maximum was at 345 nm. The spectra were recorded for a fixed amount of TP4 (3.6 pM) with increasing concentrations of nucleic acids in 50 mM NaCl, 0.1 mM EDTA, 1 mM Tricine/ NaOH, pH 7.4. The screening effect of DNA on fluorescence was corrected using N-acetyltryptophan ethylester at a concentration that had the same reelative fluorescence intensity as the TP4 solution. The final concentrations of the nucleic acids added were expressed as molar concentrations of bases. In order to calculate the binding constant, K , the fluorescence quenching data were plotted according to the equation described by Kelley et al. 1331:

l / A F = 1/(K[N A F , ) + l / A F , ,

where A F = decrease of fluorescence intensity at emission maximum in the presence of concentration N of DNA, and A F, = decrease in fluorescence intensity at infinite ligand concentration. When 1idF is plotted against I/"], a straight line is obtained for which the slope = 1 / K A F , and the intercept = I / A F,.

RESULTS

Isolation of TP4. TP4 was prepared on large scale and purified to homogeneity (Fig. 1) from boar late sperinatid nuclei. The yield of TP4 was 110 nmoM.5 kg testes. The amino acid composition of TP4 thus obtained (data not shown) indicated that the content of proline, glycine and methinnine was somewhat lower, and that the composition of tyrosine was somewhat higher than that which we have previously reported [28]. This may be due to the higher purity of the TP4 prepared on a large scale than that of the previous TP4 prepared on a small scale.

Determination of complete amino acid sequence of TP4. Puri- fied S-pyridylethylated TP4 was applied to an automated protein sequencer and the amino acid sequence up to the 49th residue

181 Akama et al. (Eur: J . Biochem. 233)

31.0 .

21.5 I 4.4

(-1

-TP4

(+I 1 2 3 4 5

Fig. 1. SDSPAGE of boar TP4. The proteins were run in the presence of 2-mercaptoethanol. Lanes 1 and 5 , molecular-mass markers [phospho- rylase b (91 400 Da), bovine serum albumin (66200 Da), ovalbumin (45000 Da). carbonic anhydrase (31 DO0 Da), soybean trypsin inhibitoi- (21 500 Da) and lysozyme (14400 Da)!: lanes 2 and 4, intact boar TP4, 1 pg and 8 pg, respectively; lane 3, reduced and carboxymethylated boar

was determined. Then, the S-pyridylethylated TP4 was subjected to digestions with S. uureu.s V8 protease or lysyl endopeptidase, and the digested peptides were separated by reverse-phase HPLC. Amino acid sequences of V-1 -V-5 peptides up to their carboxy termini and that of V-6 peptide up to the 25th residue were determined (Fig. 2). The sequences indicate that V-2 and V-3 peptides and V-1 and V-4 peptides were derived from V-6 and V-5 peptides, respectively, by cleavages with S. ciureus V8 protease at serine residues. Each of the determined carboxy-terminal sequences of V-4 and V-5 peptidec was consistent with the result of carboxypeptidase B digestion; 1.1 nmoI Tyr, 2.9 nmol Arg and 0.2 nmol Gly released from 1.5 nmol TP4 and, thus, V-4 and V-5 peptides were concluded to be carboxy-terminal fragments.

Sequence analysis of L-l peptide showed that V-3 and V-5 peptides were arranged in the order V-3-Gin-Glu-V-5, with a connecting peptide of Gln-Glu (Fig. 2). Thus, the sequences of the amino-terminal portion ofTP4, composed of 49 amino acids, and its carboxy-terminal portion, composed of 78 amino acids, were determined.

In order to determine the sequence between the amino-terminal and carboxy-terminal portions, S-pyridylethylated TP4 was cleaved by CNBr at the methionine residues. The amino acid

sequence of C-l peptide up to the 27th residue was determined. The revealed sequence of C-l peptide up to the sixth residue was identical to that of intact TP4 at positions 44-49, and the sequence from the 18th residue of C-1 peptide was identical to that from the amino termini of V-2 and V-6 peptides.

Thus, the amino-terminal and carboxy-terminal portions of TP4 are connected by a 1 1-residue sequence, and the entire sequence of TP4, composed of 138 amino acids, was determined (Fig. 2). The amino acid composition of TP4 obtained by sequence analysis was in good agreement with that obtained by acid hydrolysis (data not shown). The presence of a serine residue at position 60 indicates that V-2 and V-6 peptides were pro- duced by S. aureus V8 protease cleavage at the serine residue. The phosphorylated serine residues in ram TP1 [I31 and boar TP1 [34] were not found in TP4 during sequence determination.

Binding properties of TP4 to nucleosome-core DNA. To study binding properties of TP4 to DNA by ultraviolet difference absorption spectroscopy, CD, and thermal denaturation, we used rat liver nucleosome-core DNA as a model DNA, since TP4- boar-liver-nucleosome-core-DNA complexes were precipitated at a TP4/DNA molar ratio of over 0.5.

The ultraviolet difference absorption spectra for complexes of TP4 with DNA in the presence of 1 mM NaCl showed a positive band at about 265 nm. There was an increase in the absorbance at 260 nm with increasing concentrations of TP4, suggesting that TP4 has a DNA melting activity. In contrast, a minimum peak at about 260 nm and a positive shoulder at 295 nm were observed in the presence of 50 mM NaCI, suggesting that a tryptophan residue is involved in the interaction of the protein with double-stranded DNA 1351.

TP4 binding to DNA shifted the CD crossover point to a longer wavelength in the presence of 1 mM NaCl and 5 0 m M NaCl (Fig. 3). The band at 260 nm decreased markedly in intensity with an increase in the amount of TP4, and the band near 270 nm and 280 nm was decreased rather weakly by the interaction. The spectral contributions by TP4 itself were negligible at wavelengths longer than 245 nm at these concentrations [24]. The CD positive band of the TP4 complex at a TP4/DNA molar ratio of0.54 increased slightly at 280-300 nm (Fig. 3, curve 2). The CD spectra of the TP4 -double-stranded-DNA complexes

1 10 20 3 0 Ala-Lys-Val-Ser-Arg-Lys-Pro-Arg-Glu-Pro-Arg-Thr-Ala-Val-Thr-Gln-Ser-Thr-Arg-Arg-Ile-Lys-Arg-Lys-Lys-Thr-Leu-Ser-Lys-Pro-

intact

40 50 6 0

intact Arg-Ser-Arg-Gly-Gl~-Val-Lys-Ala-Pro-L~s-Thr-Thr-Met-Lys-Ile-Lys-Arg-Ala-Leu-Arg-Arg-Asn-Le~-Ar~-Arg-Lys-IIe-GI~-Th~-Ser-

c- 1

L- 1 >

130 138 Thr-Thr-Ser-Cys-Lys-Trp-Cys-Ser-Gln-Gly-Val-Thr-Arg-Arg-Gly-Arg-Arg-Tyr - v-1 + v-4 >

v-5 > c C P a s eB-

Fig. 2. Complete amino acid sequence and sequence strategy of TP4. Long arrows show the amino acid sequences identified by the sequencer and dashed lines indicate the rcmaining regions. Amino acids released by carboxypeptidase B are indicated by CPase B.

182 Akama et al. ( E m J. Biochem. 233)

l o l . , , . , , , , ~

: 20 I B

-20 240 260 260 300

X (nm)

Fig. 3. Effect of TP4 on the CD spectrum of double-stranded DNA. (A) In 1 mM NaCI, 0.1 mM EDTA, 1 mM Tricine/NaOH, pH7.4; 1 (......), DNA alone; 2-4 (-), TP4/DNA molar ratios of 0.54, 0.72 and 1 . 1 , respectively. (B) In 50mM NaCI, 0.1 mM EDTA, 1 mM Tricine/NaOH, pH 7.4; 1, (. . . . . .), DNA alone; 2-4, (-), TP41DNA molar ratios of 0.54, 0.72 and 0.90, respectively.

were not like those of the TP4- single-stranded-DNA complexes, but rather resemble the spectra obtained with DNA- melting-gene-32-protein complexes with polynucleotides [36, 371. Accordingly, the CD changes observed in the TP4-double- stranded-DNA complexes may be due to the formation of TP4 complexes with the DNA containing locally induced single strand.

The melting temperature (tm) of the DNA at low ionic strength was slightly increased from 59°C to 63°C at a final TP4/DNA molar ratio of 1.1, with the appearance of another small peak at 75°C (Fig. 4 B , curves 1 and 3). In addition, there was a 4-5% decrease in the overall hyperchromicity in the TP4-DNA complexes (Fig. 4A, curves 2 and 3), suggesting that DNA is slightly stabilized against thermal denaturation upon TP4 binding at low ionic strength. In the presence of 50 mM NaCI, the t , value for DNA was decreased by TP4 binding from 73°C to 69°C at a TP4/DNA molar ratio of 0.54 (Fig. 5 A and B, curves 1 and 2). At a TP4/DNA molar ratio of 0.90, the overall hyperchromicity was decreased by 5 % (Fig. 5 A, curve 3) and the slope of the hyperchromicity curves for DNA and the TP4-DNA complex at higher temperatures were similar (Fig. 5 A and B, curves 1 and 3). These results indicate that TP4 destabilizes the DNA at a low TP4/DNA molar ratio, and at a higher TPWDNA molar ratio both of its DNA-destabilizing and stabilizing effects appear.

Quenching of tryptophan fluorescence of TP4 upon binding to nucleosome-core DNA. TP4 contains only one tryptophan residue (Trpl26). When TP4 was irradiated at 295 nm, under which condition only the tryptophan residue is excited, TP4 yielded a fluorescence emission spectra with maximum wavelength at 345 nm, while the emission maximum of the free tryptophan was at 350 nm. The fluorescence intensity at 345 nm of TP4 was quenched upon binding to two types of DNA. No peak shift was observed when increasing amounts of double-stranded and single-stranded boar liver nucleosome-core DNA were

30 50 70 90 Temperature (OC)

3 P " 30 5 0 70 90

Temperature (OC)

Fig.4. Effect of binding of TP4 on the thermal melting of douhle- stranded DNA under low salt condition (1 mM NaCI, 0.1 mM EDTA, 1 mM TricinemaOH, pH 7.4). (A) Hyperchromicity profile; (B) first- derivative profile. 1 (......), DNA alone; 2 (-) and 3 (-), TP4/ DNA molar ratios of 0.54 and 1.1, respectively.

30 50 70 90 Temperature (OC)

I 1

4 L B 2 1

30 50 70 90 Temperature ("c)

Fig. 5. Effects of binding of TP4 on the thermal melting of douhle- stranded DNA at high salt concentrations (50 mM NaCI, 0.1 mM EDTA, 1 mM TricineNaOH, pH 7.4). (A) Hyperchromicity profile; (B) first-derivative profile. 1 (. . . . .), DNA alone; 2 (-) and 3 (-), TP4DNA molar ratios of 0.54 and 0.9, respectively.

added. The fluorescence quenching data were plotted according to the method of Kelley et al. [33] (Fig. 6). The association constants, K, for binding of TP4 to double-stranded and single- stranded DNA were estimated to be 7.3X103M-' and 4.1 X 3 0' M- ', respectively, indicating that the affinity of TP4 for double-stranded DNA is higher than that for single-stranded DNA. Maximums of 84% and 62% of the fluorescence quenching were obtained with 18 pM (molar concentration of bases) double-stranded and single-stranded DNA, respectively. The large quenching of the tryptophan fluorescence with low concentrations of nucleic acids may be due to a conformational change

Akama et al. ( E m

o.o+ 0 0.5 1.0 1.5

VIDNA3 x lo* (M'I 1 Fig. 6. Dependence of fluorescence quenching of TP4 on the concentration of nucleic acids. For calculating the binding constant, 11 quenching was plotted against the reciprocal molar concentration of nucleic acid bases. (-O-) double-stranded DNA. (-0-) single-stranded DNA.

of TP4 upon binding to nucleic acids. These suggest that Trpl26 of TP4 interacts with these nucleic acids.

DISCUSSION The amino acid sequence of TP4 was determined (Fig. 2).

TP4 is composed of 138 amino acid residues. Its calculated M, of 16044 is in reasonable agreement with the value (16000) estimated by SDS/PAGE using boar TPI , TP3, and calf thymus histone HI as M,- markers [28]. The fact that TP4 migrated between carbonic anhydrase (MT 31 000) and soybean trypsin in- hibitor (Mr 21 500), corresponding to a protein with M , of 28000 in SDS/PAGE (Fig. I), may be due to its higher basic amino acid content than that of M, standards.

A computer-(DNA Data Bank of Japan)-assisted similarity search of the National Biomedical Research Foundations and SWISS-PROT data bases did not show any protein with significant similarity to boar TP4. TP1 and TP2 and the amino-terminal portion of rat TP4 (residues 1-25) [6] have no sequence similarity to boar TP4. The mobilities of boar, rat and ram TP4 in acid urea/PAGE have been reported to vary slightly with the species [28]. TP4 has 21 % sequence similarity to boar TP3 [25]. A part of TP4, the sequence at residues 14-37, showed 58% similarity to that at residues 11 -34 of boar TP3. In this sequence, TP4 has one CAMP-dependent and cCMP-dependent protein kinase phosphorylation-site motif (AKP; residues 23 - 26) [38-401 and at least four protein kinase C phosphorylation- site motif(KCP; residues 4-6, 17-19 or 18-20, 123-125 and 132-134) [41, 421, while TP3 also has one AKP (residues 20- 23) and at least one KCP (residues 14 - 16 or 15 - 17). The phosphorylation of serine (threonine) residues i s known to modulate interaction of histones and protamines with DNA 141 -451. The highly conserved sequence at residues 29-42 of boar and ram TPI containing two and three phosphorylatable serine residues, respectively, has been suggested to be involved in the interaction with DNA [13, 341. TP2 [16] also has one AKP (residues 127- 130) and one KCP (residues 131 - 133) in its conserved carboxy- terminal basic region. Accordingly, there i s a possibility that the region with phosphorylation-site motifs in each TP is involved in the interaction with DNA, and interaction of the TP with DNA is regulated by phosphorylation and dephosphorylation, although phosphorylation sites of TP2-4 have not been reported yet.

J . Biocliem. 233) 183

TP4 has two bipartite nuclear-targeting sequences (NTS) (residues 5-23 and 54-72); (a) two adjacent basic amino acids (Arg or Lys), (b) a spacer region of any ten residues and (c) at least three basic residues (Arg or Lys) in five positions after the spacer region [46, 471. In this connection, each of TP1 and TP2 [13, 161 has one NTS in its conserved region (residues 24-42 for TPI , and residues 1 13 - 129 for TP2), and TP3 [25] has two NTS (residues 16-32 or 19-35 and 49-65). The transitory incorporation of lysine into condensing spermatid nuclei has been suggested to be a consequence of the synthesis, then loss of the TPs, which contain significant amounts of lysine, uncom- mon or absent in the protamines (48-511. However, the mecha- nisms involved in the nuclear translocation of the TP have not yet been fully elucidated.

TP4 is composed of a highly basic amino-terminal half (residues 1-73) and a less basic carboxy-terminal half (residues 74-138) (Fig. 2). Two-thirds of the basic amino acids of TP4 are located in the amino-terminal half as a single, di-, tri- or tetrapeptide, each alternating with non-basic amino acids. TP4 has a highly hydrophobic segment (residues 74-80) with the putative p-structure 1521. In addition, there is a region consisting of a four-times tandemly repeated sequence with the putative P-turn structure [52], ([N(G)QNKR(K)X,]; residues 84-107). However, the significance of this structure is not known at present. TP4 has a carboxy-terminal segment with the putative /?- turn structure [52] including Trpl26. Cys124 and Cys127 are thought to be essentially not crosslinked [24]. The carboxy-terminal Tyr138 is preceded by two arginine residues. Thus, TP4 has no tyrosine (tryptophan) residue flanked with two basic clus- ters such as in the TPI family and boar TP3 (13, 251.

Ultraviolet difference absorption and CD spectroscopy suggest that TP4 has a DNA melting activity. The very weak CD signal that the TP4-DNA complex exhibits at a TP4/DNA molar ratio of 0.54 (Fig. 3 B, curve 2) resembles the CD signal that the Lys-Trp-Lys -DNA complex exhibits at a peptide/DNA molar ratio of 1 [53] . Addition of rat TP1 (a DNA melting protein) induces a marginal increase in the positive band at 275 nm at both molar ratios of 0.5 and 1 (TPI/DNA) [19]. Thus, TP4 and TPI induce the different conformational changes in DNA. The binding mode of TP4 to DNA is probably different from that of TPI .

Thermal denaturation studies substantiated the DNA destabilization by TP4 at 50 mM NaC1. TP4 brought about a decrease in the tr,t of DNA from 73 "C to 69°C at a TP4/DNA molar ratio of 0.54. In addition, TP4 brought about a decrease ( 5 % ) in the overall hyperchromicity in the final phase of melting at a TP4/ DNA molar ratio of 0.9, profiles being similar to those reported for binding of rat TPI to native DNA, which have been ex- plained as equilibrium destabilization [ 191. However, the decrease (4°C) in the t,,, of DNA upon binding to TP4 is smaller than that (6°C) upon binding to rat TPI 1191, suggesting that the melting activity of TP4 is somewhat weaker than that of TP1.

The tryptophan fluorescence of TP4 was quenched upon binding to double-stranded and single-stranded DNA. The interaction of tripeptide Lys-Trp-Lys with double-stranded and single-stranded DNA resulted in the quenching of the tryptophan fluorescence due to a stacking of the indole ring with bases [54]. A comparison of the association constants, K , showed that the affinity of TP4 for double-stranded DNA was about 1.8-fold higher than that for single-stranded DNA. In contrast, rat TP1 has a higher affinity for single-stranded DNA than for double- stranded DNA, and has been suggested to bring about a local melting of the DNA through interaction of its tyrosine residues with the DNA bases [19]. TP4 brought about a local melting of the DNA through interaction of its tryptophan residue with the DNA bases, while the basic residues, binding electrostatically to

184 Akania et al. (Eur. J. Bioc,hern. 233)

the DNA phosphates, have a DNA-stabilizing effect. Although TP4 is more basic than histone H1 [28], the increase ( 5 ° C ) in the t,,, of DNA upon binding to TP4 at low ionic strength is less than that brought about by binding of histone HI (32°C) [19] . The net result of the local melting and stabilizing effects of interaction may manifest in this slight increase in the t,,, value upon binding to TP4 at low ionic strength. The size of the indole ring is similar to that of purine bases so that the stacking of tryptophan with DNA bases possibly involves only one strand of the double helix. If base pairing is locally disrupted in regions where a tryptophyl ring o f TP4 is inserted, bending of D N A may be induced 1531, and this could introduce new hydrogen bonding for the recognition of bases by other amino acid side chains of the protein 1.541. The large quenching of the tryptophan fluorescence of TP4 with low concentrations of nucleic acids (Fig. 6) may be due to such a conformational change of TP4 upon binding to nucleic acids.

Baskaran et al. [21, 221 have proposed the possibility that TP2, with its zinc-finger structures, interacts with DNA in a sequence-specific manner which, in turn, can influence the other two major events, namely, cessation of the transcription process and initiation of chromatin condensation, and that T P I , with its more even distribution of the basic amino acid residues along the length of the molecules, interacts with the chromatin randomly, facilitating local destabilization of nucleosome core particles. Rat and ram TP4 are known to be present in their late spermatids along with TPI and TP2 [6, 9, 55, 561. Moreover, low levels of TP4 were also detected in rat epididymal sperm by a TP4-specific antibody 161. The binding of TP4 to DNA induced conformational changes in the DNA molecules, including a local melting of D N A which possibly introduces a bending of DNA, as described above. Accordingly, TP4 is probably involved in the modulation of the structure of the nucleosome-core particles to facilitate chromatin transformation to a nucleoprotamine structure in a different manner from T P I and TP2.

The authors are indebted to Professor Takaxhi Obinata. and DI- Na- ruki Sato of Chiba University, Japan, for a computer-assisted similarity search. The authors are also indebted to Dr Akihito Yamaguchi of Chiba University, Japan, for a protein sequence motif search and ultracentrifu- gal treatment in the preparation of rat and boar liver nucleosome-core DNA.

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Supplemerztciry material. The amino acid sequence and interaction with the nucleosome core DNA of transition protein 4 from boar late spermatid nuclei. Fig. S 1 . Ultraviolet difference absorption spectroscopy of double-stranded DNA upon binding to transition protein 4 (TP4). Fig. S2. Comparison of the effect of TP4 on the CD spectrum of single-stranded DNA with that of double-stranded DNA. Fig. S3. Fluorescence quenching of TP4 by nucleic acids. Four pages are available.

the amino acid sequence and interaction with the nucleosome core dna of transition protein 4 from...

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