THEJOURNAL
OF BIOLOGICAL CHEMISTRY 0 1991 by The American Society for Biochemistry and Molecular Biology, Inc
Vol. 266, No. 13, Issue of May 5, pp. 8184-8191,1991 Printed in U.S.A.
Primary, Secondary, and Tertiary Structure of the Core of a Histone MytiZus* H1-like Protein from the Sperm of (Received for publication, November 26,
1990)
Laia JutglarS, Jose I. Borrellg, and Juan AusioTII From the $Unidad de Quimica Macromolecular, Escola TecnicaSuperior d’Enginyers Industrials de Barcelona Diagonal647, Barcelona E-08028, Spain, the §Centra de Enseizanza Ticnica Superior Instituto Quimico de Sarria, (Centra Asociado CSIC) Departamento de Quimica Organica,Barcelona E-08017, Spain, and the VDepartment of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia V8 W 3P6, Canada
We have analyzed the structure of the trypsin-resistant core of the protein PL-11* of the sperm from M y tilus californianus. The peptide has a molecular mass of 8436 Da and its primary sequence is ATGGAKKP STLSMIVAAIQAMKNRKGSSVQAIRKYILANNKG INTSRLGSAMKLAFAKGLKSGVLVRPKTSAGA SGATGSFRVG. This sequence bears an enormous homology and fulfills the constraints of the consensus sequence of the trypsin-resistant peptides of the proteins of the histone H1 family. Secondary structure analysis using Fourier-transform infared spectroscopy as well as predictive methods indicate the presence of 20-30%&structure and -25% a-helix for this peptide. As in the case of histone H1 proteins, the protein PL11* core exhibits a compact globular structure as deduced from hydrodynamic measurements. The presence of a histone H1 protein with protamine-like features, seems to be thus, a common general feature of the chromatin composition in the sperm of the bivalve molluscs.
many years, a true sperm-specific histone H2B (Ausio and Subirana, 1982). Furthermore, its electrophoretic mobility in urea-acetic acid gels is almost indistinguishable from that of a canonical histone H2B. However, in a recent study Uschewa et al. (1985) using an immunological approach have questioned the identityof PL11* and have identified it with the members of the histoneH1 family, althoughthisisnotsurprising considering that a highly specific histone HI-like, protamine-like (PL-I) component has been recently isolated and characterized indifferent species of bivalve molluscs (Giancotti et al., 1983; Ausio et al., 1987; Ausio, 1988). One of the most significant features of the proteinmembers of the histone H1 family is the presence of a folded trypsinresistant internal core (Allan et al., 1980) which is also the more evolutionarily conserved region of the molecule (Cole, 1987). This region usually contains 82 k 4 amino acids and is organized in aglobular structure which results from the spatial organization of a discrete number of domains having a well-defined secondary structure (Clore et al., 1987; CraneRobinson and Ptitsyn, 1989). Also, it has been shown that both the core and its proper folding areessential for the The nucleus of the sperm of Mytilus contains three major functional role of these molecules in DNA and chromatin sperm-specificproteins: 92B (PL-II*), 91 (PL-III), and 43 condensation (Losaet al., 1984; Chan et al., 1984).Therefore, (PL-IV). (Ausio and Subirana, 1982; Ausio, 1986). The first this may explain why the structural featuresof this region are nomenclatureadopted for theseproteins(Subirana et al., highly conserved. Should the PL-11* protein from the sperm of Mytilus cali1973) was based on their differential solubility in the solvent fornianus indeed be a true member of the histone H1 family, mixtures used by Johns (1964) for the selective fractionation of histones. Accordingly, protein 92B was the major compo- it shouldhave an internalcore with the structural constraints nent of the protein fraction solubilized under the conditions imposed by this group of proteins. In the present study, we describe the isolation and complete used to extract histone F2B (now H2B). Substitution of the structural characterizationof a trypsin-resistant peptidefrom letter F by 4 was proposed in order to distinguish these sperm proteins from their somatic histone counterparts. More re- PL-II*. In addition, the possible relationship of this protacently (Ausio, 1986), a different nomenclature was proposed mine-like protein to histoneH1 is also discussed. inorderto emphasize thefunctional role of these highly MATERIALS ANDMETHODS sperm-specific proteins. It was based on their simple protamine-like (PL)’ aminoacid composition and on theirrelative Living Organisms mobility in urea-acetic acid gels. It was, however, because of The specimens of M. californianus were collected in Newport, OR, its aminoacid composition and the aforementioned solubility, and the sperm was collected and prepared as described elsewhere that the PL-11* protein of Mytilus was considered to be for (Ausio, 1986).
* This work was supported in part by Natural Sciences and Engineering Research Council of Canada Grant OGP0046399 to (J. A.) and by Grant BT 009/87 to Professor J. A. Subirana. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. I)Towhom all correspondence should be addressed. The abbreviations used are: PL, protamine-like; NBS, N-bromo succinimide;HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid.
Gel Electrophoresis High resolution polyacrylamide gel electrophoresis (acrylamide/ bisacrylamide, 30:l)in the presence of 2.5 M urea and 5% acetic acid was performed as described elsewhere (Ausioet al., 1986).Urea-acetic acid gels (6 M urea, 5% acetic acid) at acrylamide/bisacrylamide (1501)without stacking were prepared according to Panyim and Chalkley (1969)as modified by Hurley (1977).Briefly, 40 mlof a stock solution containing 30% acrylamide and 0.2% bisacrylamide was mixed with 10 ml of 43.2% acetic acid, 30 g of urea, and 70 mg of thiourea. The solution was adjusted to 79.5 ml with distilled water.
8184
Structure the of
Core of a Sperm Protein from Mytilus
Once the urea was completely solubilized, 0.5 ml of 30% H202were added to the mixture, and the resulting solution was immediately poured. Column Chromatography Ionic exchange chromatography was routinely carried out on 1 x 20-cm columns using CM-Sephadex C-25. Samples were disolved in 1 M NaCI, 50 mM sodium acetate buffer, pH 6.7, and eluted with linear 1-2 M NaCl gradients (60 ml each) in the presence of the same acetate buffer as described elsewhere (Ausio, 1986). Protein Digestion Trypsin-Protein PL-11* was digested with trypsin in thepresence of 2 M NaCI, 10 mM (Tris-HCI), pH 7.5, as described before (Ausio et al., 1987). Endoproteinme Glu-C-Protein PL-11* initially dissolved in water (OD 230 E 6) was mixed with one volume of 100 mM ammonium acetate,pH 4.2. After 10min at 37 "C, one-tenth volume of an endoproteinase glu-C (Protease V8) (Boehringer) solution a t -2 mg/ ml, in 50 mM ammonium acetate, pH 4.2, was added to thatsolution, and the resulting mixture was incubated a t 37 "C for 1 h 50 min. A t that time, the sample was immediately loaded onto a CM-Sephadex C-25 column without any further treatment. Cyanogen Bromide Cleauage-Protein PL-II* a t -7 mg/ml in 0.1 N HCI was incubated in the dark and at room temperature in the presence of cyanogen bromide a t a w/w ratio of 1:3 (cyanogen bromide/protein). After 24 h, the sample was lyophilized, dissolved in 1 M NaCl sodium acetate buffer, and loaded onto a CM-Sephadex C25 column. NBS Cleauage-Protein PL-II* was dissolved in 8 M urea, 5% acetic acid a t -20 mg/ml. One-fourth volume of NBS (N-bromo succinimide) at 20 mg/ml in the samebuffer was then added to that solution, and the reaction was allowed to proceed for 1 h a t room temperature. The reaction was stopped by diluting the reaction mixture with a 10-fold excess of 50 mM sodium acetate buffer, pH 6.7. The solution thus obtainedwas then immediately loaded onto a CMSephadex C-25 column.
8185 Sedimentation Analysis
Sedimentation velocity runs were carried out on a Beckman model E analytical ultracentrifuge equipped with electronic speed and temperature control and photoelectric scanner. Sedimentation velocity analysis was performed in an An-F four-hole aluminum rotor using double sector charcoal-filled Epon cells and sapphire windows. The boundaries were recorded routinely at 282 nm. The analysis of the scans was carried out according to themethod described by van Holde and Weischet (1978). The temperatureof the rotor was held constant during each run with the help of the RTIC temperature regulation unit. The buffer used in all the sedimentation experiments was 0.2 M NaCI, 20 mM Tris-HCI, pH 7.5, 0.5 mM EDTA. RESULTS
Presence of a Trypsin-resistant Core-The three major sperm-specific nuclear proteinsof M.californianus are shown in Fig. 1A. These proteins can be easily fractionated in one step, usingionic exchangechromatography (Ausio, 1986).The protein PL-11* wasisolatedin this way (Fig. IC) and was further digested with trypsin to yield a trypsin-resistant peptide (Fig. 1B).The time course of digestion is shown in Fig. 2. As it can be seen there, the peptide obtained exhibits a clear microheterogeneity (see arrows in Fig. 1B).In fact after 30 min of digestion, three peptides can be clearly distinguished
A
B
C
HI
Primary StructureAnalysis Peptide sequencing was performed on an AB1 model 470A gasphase protein sequencer. The standard AB1 02C s e r program was used for coupling and cleavage with the cartridge set a t 40 "C. Anilinothiazolinone amino acid derivatives were converted to phenylthiohydantoin derivatives with trifluoroacetic acid a t a conversion temperature of 55 "C. These derivatives were analyzed on a Beckman microflow HPLC (Downing and Mann, 1976) system equipped with an IBM cyano column, 2-3 nmol protein/peptide were used in the analysis.
PL- IV ( 0 J ,
Fourier-transform Infrared Spectroscopy Infrared spectra were recorded on a BOMEMMichelson 100, FTIR spectrometer using ahigh speed deuterated triglycine sulfate detector. All experiments were performed a t room temperature (-23 "C). 256 interferograms were collected for each spectrum as in Holloway and Mantsch (1989). The protein PL-11* core was first dissolved in DzO and then an equal volume of 2 M NaCI, 100 mM HEPES, pH 7.5 buffer in DzO was added. The final protein concentration was -30 mg/ml (E 3.5 mM). The sample was assembled between AgCl windows, using a 50pm Teflon spacer. Nominal instrument resolution was set at 4 cm". Deconvolution of the spectra was carried out according to Mantsch et al. (1986). Fourier self-deconvolution was performed with a BOMEM Easy Version 1.41 program using a Lorentzian of 25 cm" bandwidth with a narrowing factor (nf) = 2 and a Lorentz fraction (If) = 0.9. Alternatively, the deconvolution was also carried out with a BOMEM-CALCFTIR Software (Spectra Calc and Collect Arithmetic C2.12) from Galactic Industries Corp. (1988), which was generously provided to us by MICROBEAM S.A. (Barcelona, Spain). The deconvolution in this latter case was carried out with a Bessel apodization function using gamma 1.23 and filter 0.77. Secondary Structure Prediction fromAmino Acid Sequence The secondary structure was predicted using eachof the following methods: Chou and Fasman (1974a, 1974b, 1976) and Garnier et al., (1978) and with the help of MacVector program (IBI).
FIG. 1. Acetic acid, urea (6 M) gel electrophoresis of total of M. californianus ( A ) ;trypsinnuclear proteins of the sperm resistant peptide of PL-11* ( B ) ;PL-11* (C). 1 2 3 4 5 6
-. -I
FIG. 2. Time course of trypsin digestion of thePL-11* protein from M. californianus carried out at room temperature i n 2 M NaCl, 10 mM Tris-HC1, pH 7.5, at a trypsin/protein ratio of 1:lOOO. The time for each lane is as follows: I , 0 min; 2 , 5 min; 3, 15 min; 4,30 min; 5,60 min; 6, 120 min.
8186
Structure the of
Core of a Sperm Protein from Mytilus
in the trypsin-resistant mixture (see I-111 in Fig. 2). As the sequencing yields after amino acid number 12 (from the N time course of digestion increases, the size distribution and terminus) of theC-terminalfragmentsobtained by NBS the stoichiometry of the mixture changes slightly. After 120 cleavage of the whole PL-11* protein. Such a sudden drop at min, two major pepetides (I1and I I I ) are only present. Fur- this arginine residue could not be ascribed to the end of the ther digestion (results not shown) changes the relative stoi- peptide, and most likely reflects the presence of an unusual chiometry of these two bandsat theexpense of a diminishing secondary and/or tertiary structure featurebeyond that resiyield of the overall mixture. This behavior suggests a precur- due. Anotherinterestingfeature was the cleavage site of sor relationship among these peptides in the order I+II+III. endoproteinase Glu-C within the trypsin-resistant peptide of Considering that the mobility of peptides in the urea-acetic PL-II*. Under the experimental conditions chosen here, the acid gels shown in Fig. 2 depends both on the charge and the enzyme cleaves a peptide bond between alanine and lysine. size of the molecules, the precursor relationship observed In fact, when the whole PL-11* protein was digested with this among these peptides (in particular 11411) must arise from enzyme at pH 4.0, only two major fragments were obtained, a small reduction in size accompanied by a charge reduction. corresponding to thecleavage of the protein a t this unusual Indeed, attempts to fractionate and isolate peptide I1 from site of cleavage (resultsnotshown). However, this is not 111, using conventional molecular sieve chromatography with surprising considering the high degree of atypical cleavage different gel filtration media, proved to be completely unsuc- exhibited by this enzyme (Crimminset al., 1989).Indeed, cessful suggesting a very small difference in size between I1 anomalous major sites of cleavage, in addition to the more and 111. The isolation and purification of these peptides was specific gentamicsites, have been observed bothinnononly possible by ionic exchange chromatography asshown in histone and histone proteins. For example, a major chymoFig. 3. tryptic-like site of cleavage was found in the case of troutAmino Acid Sequence-The presence of a trypsin-resistant testis non-histone protein H6 (Watson et al., 1979). Also, a peptide together with the high lysine content of the PL-11* cleavage C-terminal to serine was observed a t several posicomponent (Ausio and Subirana,1982) and itsimmunological tions (Ser-Ala) for non-histone proteins HMG 17 and HMG cross-reactivity with histone H 1 (Uscherva et al., 1985) sug- 14 (Walker etal., 1977; Walker etal., 1979). A similarcleavage gest there is a relationship between PL-11* and the proteins C-terminal to serine was observed in thecase of Tetrahymena of the histone H1 family. However, conclusive proof of this histone H 1 (Kayashiet al., 1987). Also, goose erythrocyte relationship can only be obtained from the direct comparison histone H5 was found to be cleaved at the carboxyl site of of the primary structure of the PL-11* trypsin-resistant pep- glycine (Gly-Ala) (Yaguchi et al., 1979). tide with thatof other H1histones. Secondary Structure-The analysis of the secondary strucFig. 4 shows the complete amino acidsequence of the ture of the trypsin-resistant peptidewas carried out by Fourtrypsin-resistant core ofPL-11* (peptide 111) as well as the ier-transform infrared spectroscopy. The results from such strategy followed in the sequencing procedure. Contrary to analysis (the spectrum and its deconvolution in the amide I what happened with the trypsin-resistant core of PL-I from frequency region) are shown in Figs. 5 and 6, and the quanthe sperm of Spisula solidissima (Ausio et al., 1987), it was titative data for the predicted secondary structure are prenot possible here to obtain thewhole sequence of the peptide sented in TablesI1 and 111. Special attention in this analysis in one single sequencing run. Despite the comparablehomo- was given to the criteria for the successful substraction of geneity of peptide I11 with that of the PL-I corefrom absorption bands due to liquid and gaseous water (Dong et solidissima, direct automaticsequence analysis of this peptide al., 1990). Theassignment of thebands was carriedout was not possible beyond the first30 residues. It was therefore basically according to Byler and Susi (1986), and the quantinecessary to cleave this trypsin fragment into smaller pep- fication was performed as described by Mantsch etal. (1986). tides. Table I shows some of the sequencing yields obtained There is certainambiquity, however, in the assignmentof the in thecourse of the sequencingprocedure. 1660 cm" frequency which although usually assignedto turns, Two additional important points deserve special mention is also characteristic of a-helices for proteins in thepresence within this section. First, a sudden drop was observed in the of nonaqueous solvents (Wasacz et al., 1987).
s.
OB FIG. 3. Ionic-exchange purification and fractionation of the trypsin-resistant protein fragmentson a (1 X 20 cm) CM-Sephadex C-25 column. Elution was with a linear 1-2 M NaCl gradient in 50 mM sodium acetate, pH 6.7, buffer. The flow rate was 5 ml/h and fractions were collected every 12 min. The inset shows a urea-acetic acid gel electrophoresis analysis of different fractions (A-K) from the elution profile. S = starting sample.
0.6 -
x z 0.4 -
Fraction Ne
S t r u c t u r e of the Core of a Sperm Protein from Mytilus Also shown in Table I11 is the the secondary structure of the trypsin-resistant peptide predicted from its amino acid sequence. Except for the @-turns there appears to bea good agreement between the theoretical and experimentally predicted values. Tertiary Structure-Fig. 7 shows the detailed sedimentation velocity analysis of the whole PL-11* protein in comparison to that of its trypsin-resistant fragment. Notice that despite the considerable size reduction of the protein upon trypsinization, the sedimentation coefficient remains virtually unchanged. This clearly suggests a more compact structure (K) m10 (Who 50 40 I T 6 6 I K K P S T L S M I U I A I O I M K N ~ K 6 S S U O ~ l ~ K ~ l l ~ N N
"_
TRC
I
c "CNB
60
6 NBS
10
00
-
-
NBS
v-8
forthetrypsin-resistant peptide.Indeed, it is possible to evaluatethefrictional coefficient of theprotein from its sedimentation velocity coefficient. A relative frictional ratio f / f o can also be calculated, f a being the frictional coefficient of a hypothetical equivalent sphere of radius R, and having the samemolecular mass. This relative frictional ratio canbe to the three-dimensional shape of the molecule in turn related (Tanford, 1961). In the case of ellipsoidal shapes (prolate or oblate) with volume Vh,the semiaxial ratio a/b can be determined and the axial parameters a,b can thus be obtained as well as the corresponding Stokes radius. The data obtained from this analysis are summarized in Table IV. They provide the experimental support atocompact globular conformation for the trypsin-resistant core of the PL-11* protein. DISCUSSION
00
K 6 I N T S l l 6 S l M K L I F I K S L K S 6 U l U ~ P K l S ~ 6 ~ S 6 ~ T 6 S
- CNB-
8187
"_ ~
Flu6
- v-8 FIG. 4. Amino acid sequence of the trypsin-resistant core of PL-11'. Also shown are the peptide fragments used to establish the complete sequence. TRC, whole trypsin-resistant core; CNB, cyanogen bromide peptide; NBS, N-bromo succinimide peptide; V-8, peptide obtained upon digestion with endoproteinase Glu-C.
The Primary Structure of the Trypsin-resistant Fragment of PL-II* from Mytilus Bears a n Enormous Similitude to the Trypsin-resistant Cores of the Histones from the Histone H1 Family-The comparison between the amino acid sequences of several trypsin-resistant peptidesfrom histone H1 proteins and thatof protein PL-11* is shown in Fig. 8. The quantitative data for the conserved amino acids among all these peptides is presented in Table V. As it can be seen in this table and also in Fig. 8, there is an important sequencecoincidence between the PL-11* core and the trypsin-resistant cores of the
TABLEI Sequencing yields for some of the peptides used to determine the primarystructure of the twpsin resistant peptide from PL-ZZ* peptide Amino Residue
Whole
peptide acid
Yield
Glu-C Endoproteinase NBS peptide Residue
Amino acid
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
Ala Thr/Lys Gly Gly Ala LY9 LY8 Pro Ser/Thr Thr Leu Ser Met Ile Val Ala Ala Ile Gln/Asn Ala Met LYS Asn Arg LYs
865 330/280 285 300 370 245 490 280 120165 180 160 70 50 50 40 50 60 45 47/20 50 40 45 35 25 28
Yield
Residue
Amino acid
pmol
pmol
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35
Ile Leu Ala Asn Asn LY8 GlY Ile Asn Thr Ser Arg Leu Gly Ser Ala Met LYS Leu Ala Phe Ala LYs GlY Leu (LYS) Ser Glr Val Leu Val Arg Pro (LYS) Thr
576 696 1213 465 647 538 634 336 472 553 332 10 70 72 40 49 22 28 29 43 18 37 14 18 10 9 19 6 5 7 3 4 3 7 2
Yield pmol
10
U 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
LYs GlY Leu LYs Ser GlY Val Leu Val Arg Pro LYS Thr Ser Ala G~Y Ala Ser Gly Ala Thr GlY Ser Phe Arg Val GlY LYs Ala Pro
2980 5180 2200 1800 2700 1800 900 600 650 900 1000 600 1000 590 775 400 350 440 310 200 80 60 40 20 70 20 40 20
Structure of the Core of a Sperm Proteinfrom Mytilus
8188
TABLEI1 Amide I frequencies and secondary structure assignments of the deconvoluted spectra of the protein PL-II* core Frequency Assignment cm"
Bends Bends Turns 8-Sheet Turns a-Helix" (turns)* a-Helix Random coil 8-Sheet 8-Sheet a Wasacz et al. (1987). Byler and Susi (1986).
1697 1689 1682 1674 1666 1660 1651 1643 1633 1626
TABLE 111 Quantitatiue (%) secondary structure analysisof PL-II' trypsinresistant peptide in comparison to the trypsin-resistant core of PL-I from the sperm of S. solidissima and to the core of histone H5 from chicken erythrocytes PL-11'
I
I
1
0
1650 1600 1550 40 10 25 WAVELENGTH, cm-l FIG.5. Infrared spectrum of the trypsin-resistant core of PL-II*. The solid line shows the original infrared spectrum in D,O, and thedotted line shows the same spectrum after band enhancement by Fourier self-deconvolution according to Mantsch et al. (1986).
a-Helix14 26 29 29 22 33 8-Sheet Turns 17 29 4 18 14 Random coil 16 16 Other 12 37 12 49 46 "Assigning the spectral band at 1660 to a-helix (see Table 11). Assigning the spectral band at 1660 to turns (see Table 11). Prediction analysis according to Chou and Fasman (1974) and Garnier et al. (1978). The percentages shown in this column correspond to the overlapping regions predicted by both methods. From Ausio et al. (1987). e Calculated from Zarbock et al. (1986).
.t
A
5c 4-
I
1100
I
l680
I
I
l660 1640 WAVELENGTH, cm-'
1
1620
1
1600
FIG. 6. Deconvolution and curve fitting of the region of the amide I band of the infrared spectrum of the PL-II* core, using a Bessel apodization function (see "Materialsand Methods"). The dashed line shows the starting infrared spectrum.
sperm H1 histones from other marine invertebrates. Additionally, although the extentof coincidence varies among the H1 histones from different species, a strikingly high extent of similarity (- 40%) is observed between histone H5 and each of the sperm H1 histonespresentedinthis table.Consequently,this provides supporttotheexistence of a true homology in all these cases. In otherwords, it emphasizes the specific functional role of the "H1 cores" in these terminally differentiated systems. The sequence of the PL-11* core shown in Fig. 8 clearly fulfills the amino acid distribution pattern of what, based on the most conserved residueswithin thisregion (Crane-Robinson and Ptitsyn,1989),could be considered as the consensus
3-
I
1
05
1.0
"L
15
2.0
as
lo~.t-'%-~'~l
FIG. 7. Sedimentation velocity of protein PL-11* ( A ) and protein PL-11* trypsin-resistant core ( B ) .The data were analyzed according to the method of van Holde and Weischet (1978). In this kind of analysis, the number of lines convergingtoward a common s value is proportional to thefraction of sample represented. The run was performed at 48,000 rpm and 24 "C.
S t r u c t u r e of the Core of a Sperm Protein from Mytilus sequence of the histone H1 core (see Fig. 8). This sequence arrangement eventually sets the constraints which allow (as will be discussed later) for the proper folding of the peptide into its higher levels of structure (Crane-Robinson and PtitTABLEIV Physical and conformationalparameters of protein PL-ZZ* and its tqpsin resistant core Core
..
PL-III
14,000 Da Molecular mass" 8,436 Da 0.728 cm3/g 0.736 cm3/g fit 1.18 s 1.27 S S201W 0.316 g H,O/g protein 0.404 g HnO/g protein S1c 1.40 1.05 flf 18.5 A 15.2 R O 16.7 A 26.6 A R,d 2.1 7.6 alb" 71.3 A 24.9 A ae 11.9 A 9.4 A be Based on the amino acid composition. *Based on the amino acid composition, calculated according to Cohn and Edsall (1943). e Calculated from the amino acid composition according to Kuntz (1974). Stokes radius. e Assuming a prolate ellipsoid shape.
8189
syn, 1989). These in turn may be responsible forthe suggested functional constraints of this protein domain (Cole, 1987) within the general role of histone H1 in chromatin condensation. Chromatin condensation is indeed one of the most important features associated with the processes of cell differentiation during spermatogenesis. Thus, the presence of highly specialized histone H1 variants in these kind of cells should not, therefore, be surprising. The Secondary and Tertiary Structureof PL-IF Fulfills the General Constraints of the Histone H1 Core-The results from the analysisof the secondary structure of the trypsin-resistant peptide of PL-11* are summarized in Table 111. As it can be seen in this table, the experimental results obtainedfor PL11* using circulardichroism are very similar to those obtained with the PL-I core of the sperm from the clam S. solidissima (Ausio et al., 1987). Thus, there is already an indication of a relationship between these two proteins. As pointed out before, the experimental data obatined from the FTIR analysis agree reasonablywell with the theoretically predicted values from the primary structure (see Table 111). Notwithstanding, the presence of a high content of &sheet (- 30%) in PL proteins contrasts with its complete absence in histone H1 (Bradbury et al., 1975). Nevertheless, taking advantage of the reasonable agreement between the experi-
M.C.PfII
10 20 30 40 50 A 1 G G A K K P S T L S - M I V A A I Q A M K N R K G S S V Q A I R K Y l L A N N - - - K - G - - I
S.S. PL-I
-
l?d.lal H1
- -
A - A A 3 P - - V A T M V V T A I L G L K E R K G S S M V A I K K Y I A A N - Y - - R V D - - V
"
A - S T H P - P V L E M V Q A A I T A M K E R K G S S A A K I K S Y M A A N - Y - - R V D - - M
"
S - A S H P - T Y S E M I A A A I R A E K S R G G S S R Q S I Q K Y I K S - H Y - - K V G - - H
Pa. H1 H5
-
- K G S S G - M M S - M V A A A I A A N R T K K G A S A Q A I X K Y V A A - H S S L K - G A V L
ct H1
_"
M.c.PL?II
60 70 80 90 N T S R - - L G S A M K L A F A K G ; L K S G V L V R P K T S A G A S G A T G S F R V G
Pd'lalH1
A R L A P F I R K F I R K A V - - - - - K Q T - - - - K G T A S G - " S F R V N K T A V
pa'H1
K V L A P H V R R A L R N G V A S G A L K Q V - - - - - T G T - G A S G - - - R F R V G A V A K
H5
N - A D L Q I K L S I R R L L A A G V L K Q T - - - - K G - G A S G - - - S F R L A K S D K
ct H1
K - N N S R I K ~ G L K S L V S K G T L V E T - - - - - K G T - G A S G - - - S F K L N K K A A T G E A K P K
K A S G P - P V S E L I T K A V A A S K E R S G V S L A A L K K A L A A A G Y - - D V - - - E
100
a 111 FIG. 8. Sequence conparison between the PL-11* core and several trypsin-resistant peptides of proteins from the histone H1 family. The shaded boxes shown below indicate of the experimentally determined H5 a-helices (a-Z-a-IZZ) (Zarbock et al., 1986). Also shown are the major conserved features of histone H1 core. Hydrophobic (shaded circles) and charged (circles with a sign) are represented as in Crane-Robinson and Ptitsyn (1989). Other conserved residues are also shown in bold letters. Mc. PL-ZP, M.californianus, PL-II*; Ss PL-I, S. solidissima (surf clam sperm) PL-I (Ausio et al., 1987); P.d.(a) H I , Platynereis dumerilii (annelid sperm) histone H1 (fraction a) (Kmiecik et al., 1985); P.a. H I , Parechinus angulosus (sea urchin sperm) histone H1 (Strickland et al., 1980); H5, chicken erythrocyte histone H5 (Briand et al., 1980);ct. H I , calf thymus histone H1 (fraction CTL2) (Liao and Cole, 1981).
Structure of the Core of a Si3erm Protein from Mytilus
8190
TABLEV Comparison of the percentages of conserved amino acids between different sperm HI histones of some marine invertebrates and somatic histones HI and H5 Substitution of isoleucine by leucine or vice versa was considered to be conserved. Sp, spisula; An, annelid; Su, sea urchin; H1 from calf thymus; H5 from chicken erythroc-ytes. PL-11*
PL-11*
100 27
SP 32
An
su
An
Sp
H5
H1
4232 40 43 38 100 75 40 100 57 40 35 100 42
20 41
Su
84
H5 H1
A.
-.--=
‘G
ze
No. of residues
75 80 79 86
100 5.003
I
I
I
I
I
I
4.00
.”
I
. ..... .....
3.004 2.00
1.00
0.00 -1.00 0, -2.00 I -3.00
-4.00 -5.00
+--“I
B.u
I
I
I
I
I
10
20
30
40
50
60
I 70
80
10
20
30
40
50
60
70
80
I
I
I
CF Helix CF Sheet CF Turns Helix 5 = RG RG Sheet L RG Turns 0 CfRg Hlx 8 CfRg S h t 8 CfRg T r n
z
v)
FIG. 9. Hydrophilicity (A) and secondary structure prediction for the PL-11* core ( B ) as a function of the residue number. A , hydrophilicity profile predicted according to Kyte and Doolittle (1982). Notice that in the plot used here, values above the axis denote hydrophilic regions whereas those below correspond to hydrophobic regions. B, two predictive methods were used CF, Chou and Fasman (1974a, 1974b, 1976) and RG, Robson and Garnier (see Garnier et al.,1978). The black boxes represent the regions of overlapping prediction combining both methods(Cf Rg).
H5
A
PL-II*
FIG. 10. Hypothetical tertiary structure model of the PL11* trypsin-resistant core in comparison to the tertiary structure of the trypsin-resistant core stablished by NMR (Clore et al., 1987).TheexperimentallydeterminedStokesradius of the globular domain is shown in both instances.
and about residue 43). With regards to the a-helices, these regionscoincide and overlapsurprisingly well with helix I (residues 7-17) and helix I11 (residues 44-55) of the histone H5 core as determined by NMR analysis (Clore et al., 1987) (see Fig. 8). Helix I in PL-11* has an amphipathic character as can beclearly seen in Fig. 9A. Although no particular role has been ascribed yet to helix I, helix I11 has been suggested to be involved in DNA binding (Crane-Robinson and Ptitsyn, 1989) by interaction with the minor groove (Turnell et al., 1988). It is therefore not surprising to find a highly conserved secondary structure within this region. Also, the 0-turn predicted in position 25-27 agrees very well with the half-turn structure observed in residues 24-27of the H5 when using NMR (Zarbock et al., 1986). The presence of a conserved turn in thisregion is noteworthy. In fact, the amino acid consensus sequence of that region: KXRXG S*S, has been shown to contain the unique site of sperm H1 phosphorylation induced by egg jelly in sea urchin (Porter et al., 1988). The phosphorylation a t such a hinge region has been postulated to play an important role inthealteration of thespermchromatin structure during fertilization (Porter et al., 1988). The only possibleevidencefor /3-sheet in the NMR analysis of the histone H5 core was found between residues 71 to 73 and 74 (Zarbock et al., 1986). Afterallowing for the proper alignment of the sequences (H5 uersus PL-II*, see Fig. 8), this region again coincides with the predicted P-sheet between residues 63-68 of PL-II*. The major discrepancybetween the predicted secondary structure domains of PL-11* and those experimentallyobserved in histone H5 exists from residues 28-40 of the PL-11* core. In fact this region coincidentally overlaps with helix I1 of the “H5-core” which has been suggested to be involved in protein-protein interactions through a leucine-zipper (Mannermaa and Oikarinen, 1990). A p-structure substitution in that region in the case of the sperm proteins such as PL-11* could easily fulfill or even enhance the same functional role without substantially altering the DNA binding constraints of the whole “histone-core.” As a matter of fact, histonehistone interactions may play a very important role in the processes of chromatin condensation and aggregation that take place during spermatogenesis. With the exception of the first one-third of the sequence, which comprises the first a-helix, the trypsin-resistant core ofPL-11* does notexhibitany well-defined regions with marked extentsof either hydrophilicity or hydrophobicity (see Fig. 9A). The hydrodynamic measurements carried out in comparison with the whole PL-11* protein are clearly indicative of a globular structure for that core region. Fig. 10 shows a hypothetical model for thetertiarystructure of thispeptide (adapted from Clore et al., 1987) based on the folding of the globular domain of chicken H5 determined by nuclear magnetic resonance (Clore et al., 1987). CONCLUDING REMARKS
In this paper we have characterized and analyzed the different levels of structure of the trypsin-resistant core of the sperm-specific protein PL-11* from Mytilus. The analysis of mental values andthosetheoreticallypredicted from the each of these levels (primary, secondary, and tertiary struccore of primary structure analysis, we decided to carry out this analy-ture) together with the fact that the trypsin-resistant sis further and analyze the sequence distribution of the pre- histone H1 is the most conserved region of this protein (Cole, dicted secondary structure regions of the PL-11* core. The 1987) leadsus to conclude that PL-11* is indeeda highly results of such analysis are shown in Fig. 9 (see also Fig. 8). specialized member of the histone H1family. Similar spermAccording to this, there aretwo major a-helix domains (resi- specific H1 histone have been already described in the sperm dues 15-26 and 48-60) and two major P-sheet domains (resi- of other bivalve molluscs (Giancotti et al., 1984; Ausio et al., 1987; Ausio et al., 1988). According to the recent nomenclature dues 28-40 and 63-68) as well as two p-turns (residues25-27
Structure of the Core of a Sperm Protein from Mytilus adopted for these kindof proteins (Ausio, 1986),all the spermspecific H1-like members characterized previously belonged to the PL-I (protaimine-like) class. The protein PL-11* from Mytilus, described here, exhibits a higher electrophoretic mobility and presumablya lower molecular mass than anyof the PL-I analyzed so far. Nevertheless, because of its structural composition it shouldclearly be considered as a PL-I protein. Therefore, the presence of a highly specialized histone H1like protein, (PL-I) in the sperm of the bivalve molluscs seems t o be a general feature shared by all members of this taxonomicgroup, despite the different extent of complexity of their nuclear protein composition (Ausio, 1986). REFERENCES Allan,J., Hartman, P. G., Crane-Robinson, C., and Avilis,F. X. (1980) Nature 288, 675-679 Ausio, J . (1986) Comp. Biochem. Physiol.B Comp. Biochem. 85,439449 Ausio, J. (1988) J. Biol. C k m . 263, 10141-10150 Ausio, J. and Subirana, J . A. (1982) Exp. Cell Res. 141, 39-45 Ausio, J., Sasi, R., and Fasman, G. D. (1986) Biochemistry 25, 19811988 Ausio, J., Toumadje, A,, McParland, R., Becker, R. R., Johnson, W. C., Jr., and van Holde, K. E. (1987) Biochemistry 26, 975-982 Bradbury, E. M., Cary, P. D., Chapman, G. E., Crane-Robinson, C., Danby, S. E., and H. W.E. Rattle (1975) Eur. J. Biochem 52,605613 Briand, G., Kmiecik, D., Sautiere, P., Wouters,D.,Borie-Loy, O., Biserte, G., Mazen, A,, and Champagne, M. (1980) FEBS Lett. 112,147-151 Byler, M. D., and Susi, H.(1986) Biopolymers 25,469-487 Chan, D. C., Biard-Roche, J, Gorka, C., Girardet, J. L. Lawrence, J. J., and Piette L. M. (1984) J. Biomol. Struct. & Dyn. 2, 319-332 Chou, P. Y., and Fasman, G. D. (1974a) Biochemistry 13, 211-222 Chou, P. Y., and Fasman G. D. (1974b) Biochemistry 13, 222-245 Chou P. Y., and Fasman, G. D. (1976) Adu. Enzymol. Relat. Areas Mol. Bid. 47, 45-148 Clore, G. M., Gronenhorn, A. M., Nilges, M., Sukumaran, D. K., and Zarbock, J . (1987) EMBO J. 6, 1833-1842 Cohn, E. J.,andEdsall, J. T. (1943) ProteinsAminoAcidsand Peptides, pp. 370-381, Van Nostrand-Reinhold, Princeton, NJ Cole, R. D. (1987) Int. J. Pept. Protein Res. 30, 433-449 Crane-Robinson, C., and Ptitsyn, C. B. (1989) Protein Eng. 2, 577582 Crimmins, D. L., Thoma, R. S., McCourt, D. W., and Schwarz, B. D. (1989) Anal. Biochem. 176, 255-260 Dong, A., Huang, P., and Caughey, W. S. (1990) Biochemistry 29, 3303-3308 Downing, M. R, and Mann,K. G. (1976) Anal. Biochem. 74,298-319
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