Identification of Protein-Protein Contacts between alpha /beta -Type Small, Acid-soluble Spore Proteins of Bacillus Species Bound to DNA*

Christopher S. Hayes and Peter SetlowDagger

From the Department of Biochemistry, University of Connecticut Health Center, Farmington, Connecticut 06030

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Small, acid-soluble spore proteins (SASP) of the alpha /beta -type from several Bacillus species were cross-linked into homodimers, heterodimers and homooligomers with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) in the presence of linear plasmid DNA. Significant protein cross-linking was not detected in the absence of DNA. In all four alpha /beta -type SASP examined, the amino donor in the EDC induced amide cross-links was the alpha -amino group of the protein. However, the carboxylate containing amino acid residues involved in cross-linking varied. In SASP-A and SASP-C of Bacillus megaterium two conserved glutamate residues, which form part of the germination protease recognition sequence, were involved in cross-link formation. In SspC from Bacillus subtilis and Bce1 from Bacillus cereus the acidic residues involved in cross-link formation were not in the protease recognition sequence, but at a site closer to the N terminus of the proteins. These data indicate that, although there are likely to be subtle structural differences between different alpha /beta -type SASP, the N-terminal regions of these proteins are involved in protein-protein interactions while in the DNA bound state.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Between 5 and 10% of the total protein in spores of the Bacillus and Clostridium species of bacteria is alpha /beta -type small, acid-soluble spore protein (alpha /beta -type SASP)1 (1, 2). These proteins are encoded by four to seven monocistronic genes in each species, and their amino acid sequences are highly conserved both within and between Bacillus species (1, 2). The alpha /beta -type SASP are nonspecific DNA-binding proteins which are synthesized only within the forespore compartment during sporulation (3, 4). Typically, two major alpha /beta -type SASP accumulate to high levels within the spore, while the minor alpha /beta -type SASP are found at much lower levels. The level of total alpha /beta -type SASP in spores is sufficient to saturate the spore chromosome, and the binding of these proteins to spore DNA is the major determinant of spore resistance to UV radiation and a significant determinant of spore heat resistance (1, 2). Bacillus subtilis spores which lack the two major alpha /beta -type SASP (alpha  and beta ) are much more sensitive to UV radiation and heat than are wild type spores (5). During the first few minutes of spore germination, alpha /beta -type SASP are quickly degraded by a sequence-specific protease termed germination protease (GPR) (1, 2).

Structural studies of purified alpha /beta -type SASP and alpha /beta -type SASP·DNA complexes have shown that significant changes in these proteins' structure occur upon binding to DNA, as alpha /beta -type SASP are predominantly unfolded in solution but acquire significant alpha -helical content upon binding to DNA2 (6). The alpha /beta -type SASP cover 4-6 base pairs of DNA, and binding of these proteins to DNA is highly cooperative, particularly to DNAs bound with low affinity. (7). Electron micrographs of alpha /beta -type SASP·DNA complexes indicate that the protein forms a helical coat along the DNA (8), suggesting that there are extensive interactions between alpha /beta -type SASP when bound to DNA, although these proteins are monomers in solution3 (9). Consequently, it is possible that interactions between adjacent alpha /beta -type SASP along the DNA backbone may be important for the alpha /beta -type SASP/DNA binding interaction.

To determine which regions of the proteins are involved in interactions between alpha /beta -type SASP bound to DNA, we have performed protein cross-linking studies with the zero-length cross-linking reagent 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). We have identified EDC-catalyzed protein cross-links in four different alpha /beta -type SASP from Bacillus species, and the identification of these cross-links has yielded new insights into the interaction of alpha /beta -type SASP on DNA.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Bacterial Strains and Growth Conditions-- The Escherichia coli strains used include: JM107 (F' traD36 proA+ proB+ lacIq lacZ Delta M15/endA1 gyrA96 (Nalr) thi hsdR17 supE44 relA1 Delta (lac-proAB) mcrA) (Life Technologies, Inc.), JM83 (ara Delta (lac-proAB) rpsL phi 80 lacZDelta M15) (10), BL21(DE3) (T7 RNA polymerase under control of the lac promoter) (11), and BMH 71-18 (F' proAB lacIq lacZDelta M15 thi supE Delta (lac-proAB) mutS::Tn10) (CLONTECH Laboratories, Inc.). The Bacillus strains used were Bacillus cereus T (originally obtained from H. O. Halvorson) and Bacillus megaterium QMB1551, ATCC no. 12872 (originally obtained from H. Levinson).

E. coli strains were routinely grown in 2× YT medium (16 g of tryptone, 10 g of yeast extract, 5 g of NaCl per liter) at 37 °C with shaking. For the overexpression of cloned genes encoding alpha /beta -type SASP, the medium was supplemented with 100 µg/ml ampicillin (JM107) or 200 µg/ml ampicillin and 0.5% glucose (BL21). B. megaterium was sporulated at 30 °C in supplemented nutrient broth, and spores were harvested and cleaned as described previously (12).

Polymerase Chain Reaction Amplification and Site-directed Mutagenesis-- Oligonucleotides were designed to polymerase chain reaction amplify a 512-base pair fragment containing the gene encoding Bce1 (13) from B. cereus genomic DNA; the amplified fragment contained the gene's ribosome binding site and transcription terminator (13). The upstream primer, BCE1-1 (5'-AAAGGATCCTTATTATTTCATAATTTGTAGC; complementary to nucleotides 119-140) (13) and downstream primer, BCE1-2 (5'-AAAGGATCCTTTTAAGTATGCTTTTTCCTGC; complementary to nucleotides 592-613) (13), each contained BamHI restriction sites and 5'-flanking sequences (underlined residues) for cloning purposes. The BamHI-digested polymerase chain reaction product was agarose gel-purified and ligated into BamHIdigested plasmid pET3 (11), generating plasmid pPS2532 in which BclI digestion confirmed that the gene encoding Bce1 was under the control of the T7 promoter. Plasmid pPS2532 was used to transform E. coli strain DE3(BL21) to ampicillin resistance.

The E10K mutant form of Bce1 was generated with the TransformerTM site-directed mutagenesis kit from CLONTECH according to manufacturer's instructions. Phosphorylated primers complementary, except for designed mismatches (underlined bases), to the unique AlwNI restriction site of pET3 (5'-CCTGTTACTAGTGGATGCTGC) and the Gly6-Gly15 coding region of the gene encoding Bce1 (5'-GGAAGTCGTAATAAAGTATTAGTTCGAGGC) (13) were used with plasmid pPS2532 as a template to synthesize a mutagenized plasmid lacking the AlwNI site, and with a lysine codon replacing the codon for glutamate 10 of bce1. The mutagenized plasmid was digested with AlwNI prior to transformation into the mismatch repair deficient E. coli strain (mutS::Tn10, Tetr) supplied with the mutagenesis kit. Mutagenized plasmid was enriched by plasmid isolation, digestion with AlwNI and retransformation into E. coli strain JM83. One clone was isolated and the identity of the mutagenized plasmid, termed pPS2734, was confirmed by DNA sequencing.

SASP Expression and Purification-- SspC and SASP-C were overexpressed in E. coli strain JM107 from pDG148 derived plasmids containing the alpha /beta -type SASP genes under control of an isopropyl beta -D-thiogalactopyranoside inducible promoter as described previously (14, 15). Bce1 and Bce1E10K were overexpressed in E. coli strain DE3(BL21) from plasmids pPS2532 and pPS2734, respectively. SspC, Bce1, and Bce1E10K were extracted from dry ruptured E. coli cells with 3% acetic acid/30 mM HCl as described previously (16). SASP-C and SASP-A were extracted with 3% acetic acid (9) from dry ruptured E. coli cells and dry ruptured spores of B. megaterium strain QMB1551, respectively. All alpha /beta -type SASP were purified as described previously (9).

Cross-linking of alpha /beta -Type SASP with EDC-- alpha /beta -Type SASP (0.5 mg/ml) with or without cesium chloride gradient-purified, EcoRI-linearized pUC19 plasmid DNA (100 µg/ml) were incubated in 1 ml of 5 mM sodium phosphate (pH 7.5) at 24 °C for 20 min prior to addition of EDC to 25 mM. The 5:1 (w/w) ratio of protein to DNA is sufficient to saturate the DNA with alpha /beta -type SASP, although SASP-A binds more weakly than do the other alpha /beta -type SASP tested (17). Under these conditions, approximately 50% of the alpha /beta -type SASP are bound to the DNA. The cross-linking reactions were incubated for 30 min at 24 °C, followed by dialysis in Spectrapor 3 tubing against 1 liter of 10 mM sodium phosphate (pH 7.5) at 4 °C for 18 h. Dialyzed cross-linking reactions were frozen, lyophilized, dissolved in sample buffer and run on Tris-Tricine SDS-PAGE (18). Gels were stained with Coomassie Blue, destained, and monomeric and cross-linked proteins were excised with a clean razor blade. Proteins were electroeluted from polyacrylamide gel slices into 50 mM NH4HCO3, 0.1% SDS using ElutrapTM separation chambers (Schleicher & Schuell). Gel purified proteins were frozen, lyophilized, dissolved in 100 µl of MilliQ-H2O, and precipitated with 800 µl of cold acetone. Precipitated proteins were washed with 500 µl of cold acetone and dissolved in freshly prepared 8 M urea prior to trypsin digestion.

Cross-linked Peptide Purification and Analysis-- EDC-treated proteins (~20-40 µg) were digested with trypsin (Worthington, 5 µg) in 100 µl of 0.2 M NH4HCO3, 10 mM CaCl2, 1.2 M urea at 37 °C for 15-18 h. Tryptic digests were run on reverse-phase high performance liquid chromatography (HPLC) using a Waters 680 gradient controller, two Waters 501 pumps, a Waters U6K injector, and a Vydac protein C4 column (3.9 × 150 mm). Tryptic digests were loaded onto the reverse phase column in 100% buffer A (0.06% trifluoroacetic acid) followed by 5 min of washing with 100% buffer A. Peptides were eluted at a flow rate of 1 ml/min with a discontinuous linear gradient as follows: 5-30 min, 0-30% buffer B (0.052% trifluoroacetic acid in 80% acetonitrile); 30-50 min, 30-40% buffer B; 50-70 min, 40-100% buffer B. Peptides were detected by their UV absorption at 214 nm with a Waters 481 spectrophotometer, and fractions containing peptides were collected with an Isco 2150 peak separator and an Isco Foxy fraction collector.

Molecular masses of HPLC-purified peptides were determined by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry using a Perseptive Biosystems Linear MALDI-TOF instrument. External calibration consisting of two standards (angiotensin, 1297.5 Da; and ACTH(18-39), 2466.7 Da) was used for all determinations, resulting in ±0.15% mass accuracy. Peptides from HPLC fractions (1 µl, ~1-10 pmol/µl) were mixed and dried on the instrument stage with an equal volume of alpha -cyano-4-hydroxycinnamic acid (10 mg/ml) in 50% acetonitrile. Amino acid analysis was conducted as described previously (9). Peptide sequences were determined with an ABI model 492A Procise automated protein sequencer.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

EDC Cross-linking of alpha /beta -Type SASP Is DNA-dependent-- In an effort to identify the interacting regions of alpha /beta -type SASP bound to DNA, we decided to use protein cross-linking to trap interacting amino acid residues for subsequent biochemical analysis. Most alpha /beta -type SASP do not contain cysteine residues (1) and therefore several protein cross-linking reagents which rely upon thiol chemistry could not be used for this study. The alpha /beta -type SASP are small proteins (6-7.6 kDa) which are monomeric in solution3 (9) and appear to interact with one another only when bound to DNA. We were interested in regions of close contact between alpha /beta -type SASP and therefore decided to use cross-linking reagents with short- or zero-length linker arms. EDC, a water-soluble carbodiimide, gave efficient cross-linking of alpha /beta -type SASP only in the presence of DNA (see below). Consequently, we chose this reagent for further work. Protein cross-linking with EDC usually involves the formation of an amide bond between either an N-terminal alpha -amino or lysine epsilon -amino group and the carboxyl side chain of aspartate/glutamate residues. Therefore, in contrast to cross-linkers that contain flexible linker arms several angstroms in length, EDC induced cross-links should occur only between residues that are in very close proximity to one another.

The proteins chosen for this study were SASP-A and SASP-C from B. megaterium, SspC from B. subtilis, and Bce1 from B. cereus (Fig. 1). Most of the variability between these proteins occurs near the N termini which vary both in length and amino acid sequence (Fig. 1) (1). All alpha /beta -type SASP lack the N-terminal methionine residue which is the only residue that is removed post-translationally (Fig. 1) (1). SASP-A and SASP-C are major alpha /beta -type SASP in spores, whereas SspC and Bce1 are minor proteins. For all four proteins little or no protein-protein cross-linking was detected in the absence of added DNA, while significant protein-protein cross-linking was detected in reactions containing alpha /beta -type SASP and DNA (Fig. 2, and data not shown). The extent of protein-protein cross-link formation and the number of higher order oligomers formed varied for each protein tested (Fig. 2). By overloading polyacrylamide gels, decamers could be easily detected in cross-linking reactions with Bce1 and DNA, whereas only small amounts of trimer were detected in reactions with SASP-A and DNA (Fig. 2 and data not shown). The efficiency of protein cross-linking corresponded roughly to the affinity of each protein for linear plasmid DNA (Bce1 > SspC approx  SASP-C > SASP-A) as determined by DNase protection assays (17) (data not shown), although the observed cross-linking efficiency of SASP-A was lower than expected.


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Fig. 1.   Amino acid sequence alignment of some alpha /beta -type SASP. Amino acid sequences are from Setlow (1) and are given in the one-letter code with the amino-terminal residue called residue 1; note that the N-terminal methionine residues are removed post-translationally. The arrow indicates the peptide bond cleaved by the GPR. Trypsin cleavage occurs at peptide bonds following the underlined residues. Amino acid residues above asterisks (*) are completely conserved in all alpha /beta -type SASP identified thus far from Bacillus, Sporosarcina, and "Thermoactinomyces" species.


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Fig. 2.   EDC cross-linking of alpha /beta -type SASP is DNA-dependent. Protein (0.5 mg/ml) with or without linear plasmid DNA (100 µg/ml) was reacted with 25 mM EDC for 30 min at 22 °C, followed by dialysis against 10 mM sodium phosphate (pH 7.5) at 4 °C as described under "Experimental Procedures." Samples were lyophilized, dissolved in sample buffer, and run on Tris-Tricine SDS-PAGE (16.7% acrylamide) and stained with Coomassie Brilliant Blue R-250. Numbers on the left margin indicate the positions of molecular mass markers in kDa.

Different alpha /beta -Type SASP Interact on DNA-- The DNA dependence of cross-link formation between alpha /beta -type SASP suggested that the EDC-generated protein-protein cross-links are formed only between alpha /beta -type SASP that are adjacent to one another on the DNA backbone. These in vitro experiments used only a single alpha /beta -type SASP. However, there are multiple alpha /beta -type SASP in spores, with two proteins present at high levels. Consequently, an obvious question is whether the different alpha /beta -type SASP interact when bound to DNA. To obtain data pertinent to this question we analyzed protein-protein cross-link formation in reactions with two different alpha /beta -type SASP bound to DNA. SASP-A and SASP-C from B. megaterium were chosen for the initial hetero-cross-linking experiments because they are the two major alpha /beta -type SASP found in spores of B. megaterium (9). These proteins also differ sufficiently in molecular mass (SASP-A = 6,260.1 Da and SASP-C = 7,423.3 Da) to allow resolution of the three possible dimeric forms by Tris-Tricine SDS-PAGE. Electrophoretic analysis of cross-linking reactions containing SASP-A, SASP-C, and DNA revealed the presence of a new predominant band that migrated at the position expected for a SASP-A/SASP-C heterodimer (Fig. 3, lane A + C), and this band is indeed a SASP-A/SASP-C heterodimer (see below). Titration experiments demonstrated that the ratio of SASP-A to SASP-C that produces the most heterodimer is ~3:1 (w/w) (data not shown). This latter ratio approximates the relative levels of these two proteins in B. megaterium spores (9). Heterodimers were also formed between SASP-A and SspC from B. subtilis (data not shown). However, in contrast to the SASP-A/SASP-C cross-linking reaction in which the SASP-A/SASP-C heterodimer was the predominant cross-linked product (Fig. 3), SASP-A/SspC heterodimer formation was much less efficient than formation of the SspC homodimer in these reactions (data not shown). Since SspC and SASP-C have similar affinities for linear plasmid DNA in solution, the difference in their formation of heterodimers with SASP-A is presumably due to differences in the amino acid sequences of SspC and SASP-C.


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Fig. 3.   Heterodimer formation between SASP-A and SASP-C. Protein (0.5 mg/ml) with linear plasmid DNA (100 µg/ml) was reacted with 25 mM EDC for 30 min at 22 °C, followed by dialysis against 10 mM sodium phosphate (pH 7.5) at 4 °C. Samples were lyophilized, dissolved in sample buffer, and run on Tris-Tricine SDS-PAGE (16.7% acrylamide) and stained with Coomassie Brilliant Blue R-250. Lane A, SASP-A; lane C, SASP-C; and lane A + C, SASP-A and SASP-C at a 3:1 (w/w) ratio. Proteins labeled A-A, A-C, and C-C are SASP-A homodimers, SASP-A/SASP-C heterodimers, and SASP-C homodimers, respectively. Numbers on the left margin indicate the positions of molecular mass markers in kDa.

Identification of Cross-links between alpha /beta -Type SASP-- There is presently very little detailed structural information available on alpha /beta -type SASP or the complex they form with DNA. Therefore, identification of the amino acid residues involved in EDC-dependent cross-link formation was undertaken to determine which regions of alpha /beta -type SASP are involved in protein-protein interactions that occur in the DNA bound state. Purified monomeric and oligomeric alpha /beta -type SASP from EDC cross-linking reactions were digested with trypsin and the products resolved by reverse phase-HPLC. Two types of differences should be detected between the HPLC tryptic maps of dimeric (or oligomeric) and monomeric (but EDC treated) alpha /beta -type SASP. First, the digests of alpha /beta -type SASP dimers should show decreases (~50%) in the relative yield of some peptide(s) as compared with the monomer, because amino acid residues within this peptide(s) will be in a cross-linked peptide in the dimer. Second, there should be a new peptide peak(s) in HPLC tryptic maps of alpha /beta -type SASP dimers, which should be the peptide containing the cross-link. Detailed analyses, including mass spectrometry, amino acid analysis and amino acid sequencing of the latter peptides should then allow both the unambiguous identification of the peptides in the cross-link, as well as the specific amino acid residues involved. Intramolecular cross-links could also be formed by EDC, as alpha /beta -type SASP go from an unfolded to a more ordered structure on binding to DNA. Intramolecular cross-links could be found within both monomeric and oligomeric proteins, and this modification could be detected by comparing HPLC tryptic maps of EDC treated monomers and untreated protein. However, we never saw evidence for intramolecular cross-link formation in these analyses (data not shown).

HPLC analysis identified two unique, closely eluting peptides in the tryptic digest of dimeric SASP-A (Fig. 4B, peptides labeled 1 and 2) which were not present in the digest of the SASP-A monomer (Fig. 4A). A substantial reduction in the amount of one peptide was also noted in the digest of dimeric SASP-A when compared with that of monomeric SASP-A (Fig. 4B, peptide labeled 3). No other significant differences were observed between digests of the monomeric and dimeric species. Because the relative amounts of all other peptides appeared to be approximately the same between digests of monomeric and dimeric SASP-A, these data suggested that the cross-link occurred between an amino acid residue in peptide 3 and a residue within a small peptide which has very little UV absorbance. The tryptic digests of the SASP-C monomer and dimer exhibited differences that were very similar to those seen with SASP-A (data not shown). HPLC analysis of the tryptic digest of the SASP-A/SASP-C heterodimer also identified two unique peptides which were not present in digests of SASP-A or SASP-C monomers (data not shown). Both of these unique peptides from the SASP-A/SASP-C heterodimer had HPLC retention times that differed from those of the putative cross-linked peptides identified from the SASP-A and SASP-C homodimers (data not shown).


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Fig. 4.   A and B, HPLC analysis of cross-linked SASP-A. Polyacrylamide gel-purified monomeric and dimeric SASP-A were digested with trypsin and peptides resolved by reverse-phase HPLC as described under "Experimental Procedures." A, tryptic digest of EDC-treated monomeric SASP-A. B, tryptic digest of EDC-treated dimeric SASP-A. The cross-linked peptides are labeled 1 and 2. The peak labeled 3 is peptide Y20-R37.

Only two additional significant tryptic peptides were detected in the Bce1 dimer that were not present in the Bce1 monomer (Fig. 5, A and B). One of these peptides (Fig. 5B, peptide labeled with an asterisk) was an oxidized form of Bce1 tryptic peptide Lys55-Arg66 which contained a methionine sulfoxide residue (data not shown). The other unique peptide, presumably the cross-linked peptide, eluted early in the HPLC gradient (Fig. 5B, peptide labeled 1). No obvious reduction in the level of any major peptide peak was observed when the HPLC profile of the tryptic digest of monomeric Bce1 was compared with that of dimeric Bce1, suggesting that the cross-link occurred between amino acid residues from two small tryptic peptides. In contrast to SASP-A, SASP-C, and Bce1, analysis of the tryptic digest of the SspC dimer identified only one unique peptide in comparison to the digest of the SspC monomer (data not shown). However, as was found with SASP-A and SASP-C, the amount of one major peptide was decreased significantly in the digest of the SspC dimer as compared with the digest of the monomer (data not shown). Presumably this large peptide is involved in cross-link formation with a rather small peptide.


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Fig. 5.   A and B, HPLC analysis of cross-linked Bce1. Polyacrylamide gel-purified monomeric and dimeric Bce1 were digested with trypsin and peptides resolved by reverse-phase HPLC as described under "Experimental Procedures." A, tryptic digest of EDC-treated monomeric Bce1. B, tryptic digest of EDC-treated dimeric Bce1. The cross-linked peptide is labeled 1. The peak labeled with an asterisk (*) is peptide L55-R66 containing an oxidized methionine.

The relatively high efficiency of SspC and Bce1 cross-linking (Fig. 2) also allowed the purification and analysis of cross-linked trimeric and tetrameric species of these proteins. The HPLC profiles of the tryptic digests of the trimeric and tetrameric species of both SspC and Bce1 were essentially identical to the tryptic map of the dimeric forms, with the exception of greater reductions in the larger peptide partner in the cross-link in the higher oligomers of SspC (data not shown). These data suggest that identical EDC catalyzed cross-links occur between each protein in higher oligomers of cross-linked alpha /beta -type SASP.

Elucidation of Amino Acid Residues Involved in Cross-link Formation-- Various types of information were used to determine the amino acid residues involved in cross-link formation in the different alpha /beta -type SASP. For SASP-A, SASP-C, and SspC, mass spectrometry and amino acid analysis identified the large peptides whose level was decreased in tryptic digests of the dimeric species as Tyr20-Arg37, Phe29-Arg46, and Ser8-Lys27, respectively. This identified one probable partner in the major cross-link formed in these three proteins. Determination of the mass of each peptide tentatively identified as a cross-linked species from tryptic digests of both homo- and heterodimers (Table I), as well as amino acid analyses (data not shown) allowed assignment of the two tryptic peptides in the various cross-links. In all cases, the site of cross-linking was tentatively identified as between the alpha -amino group of the protein and an acidic group on a separate tryptic peptide. The peptides in the two new peaks from tryptic digests of cross-linked dimers of either SASP-A or SASP-C had virtually identical observed molecular masses (Table I), suggesting that each peak contained the same two tryptic peptides linked together, but cross-linked at a different site. Analyses of the two unique peptides isolated from the tryptic digest of the SASP-A/SASP-C heterodimer predicted that the cross-links were between the alpha -amino group of one protein and a tryptic peptide in the other protein (Table I).

                              
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Table I
Mass determination of cross-linked peptides by MALDI-MS
Mass spectrometry was performed as described under "Experimental Procedures."

Amino acid sequence analysis definitively identified the cross-linked peptides from the four alpha /beta -type SASP as well as the SASP-A/SASP-C heterodimer (Table II). Only one amino acid sequence was obtained from the cross-linked peptides, consistent with the involvement of the alpha -amino group of each protein in these cross-links. Sequencing of the two cross-linked peptides from SASP-A confirmed that one of the peptides involved in the cross-link was indeed Tyr20-Arg37. The differences in molecular mass and amino acid composition between the cross-linked peptides and Tyr20-Arg37 were consistent with the cross-linking of Tyr20-Arg37 to Ala1-Lys5 of SASP-A (Table I and data not shown). Peptide 1 from the SASP-A dimer had a reduced yield of glutamate in cycle 6, while peptide 2 had a dramatically reduced yield of glutamate in cycle 2 (Table II). Thus, both of these glutamate residues are involved in cross-links to the amino terminus of the protein, and reduced yields of these glutamate residues were observed instead of blank cycles, because the two cross-linked peptides were not completely separated from one another by HPLC (Fig. 2B). A similar result was obtained in sequencing unique peptide 2 from the SASP-C dimer, as a reduced yield of glutamate was found in cycle 2 (Table II). We presume that unique peptide 1 from SASP-C dimer, which had the same mass and amino acid composition as peptide 2, contains a cross-link to the glutamate residue at position 6 in Phe29-Arg46. Sequencing of the two unique peptide peaks from the SASP-A/SASP-C heterodimer revealed cross-links at the same residues as in the SASP-A and SASP-C homodimers (Table II). However, the decreases in glutamate yields were not as pronounced as with the cross-linked peptides from the SASP-A and SASP-C homodimers, indicating that each of the unique HPLC peaks from the heterodimers were mixtures of two different cross-linked peptides (Table II).

                              
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Table II
Amino acid sequencing of cross-linked peptides
Peptides are as described in Table I.

As was found with the cross-linked peptide from SASP-A and SASP-C, only a single amino acid sequence was obtained with the cross-linked peptide from SspC and Bce1 (Table II). This is again consistent with the involvement of the alpha -amino group of the latter two proteins in cross-link formation. However, the sequencing results indicated that the glutamate residues involved in cross-link formation in SASP-A and SASP-C are not involved in formation of the major cross-link found in SspC and Bce1. Instead, blank sequencing cycles were obtained at positions corresponding to aspartate 13 of SspC and glutamate 10 of Bce1 (Table II and Fig. 1). These residues are at identical positions in alpha /beta -type SASP amino acid sequence alignments (Fig. 1). However, the residues at this position are not as highly conserved as are other residues in alpha /beta -type SASP (1).

Generation and Analysis of Bce1E10K-- The acidic residues identified as sites of cross-link formation in Bce1 and SspC are not highly conserved among alpha /beta -type SASP (1), and therefore their involvement in EDC cross-linking was somewhat unexpected. Consequently, we decided to investigate the role these specific acidic residues play in DNA binding and to determine whether in Bce1 the observed cross-link between the alpha -amino group and glutamate 10 was the only one generated by EDC. A site-directed mutant form of Bce1 was generated in which glutamate 10 was changed to a lysine residue. This change should not destroy Bce1/DNA binding, because most other alpha /beta -type SASP contain either a lysine (e.g. SASP-A and SASP-C) or a glutamine residue at this position (1). This type of mutagenesis was not conducted with SASP-A or SASP-C, because previous work has shown that changes in the glutamate residues involved in cross-link formation in these proteins significantly diminish DNA binding (19).

Purified Bce1E10K bound to DNA and gave approximately twice the protection against DNase I to linear plasmid as did equivalent amounts of Bce1 (data not shown). Bce1E10K also displayed DNA-dependent EDC cross-linking, but with much lower efficiency (<20% based on Coomassie staining) than wild type Bce1 (Fig. 6). The large difference in cross-linking efficiency between Bce1 and Bce1E10K indicates that glutamate 10 is the major site of EDC cross-link formation in Bce1, but that there is at least one additional cross-linking site. Therefore, cross-linked dimeric Bce1E10K was purified and analyzed to identify the residues involved in cross-linking. HPLC analysis of a tryptic digest of the Bce1E10K dimer revealed two unique peptides when compared with the digest of monomeric Bce1E10K (data not shown). One of these peptides contained an EDC-induced modification but not a cross-link, while the other peptide was identified as a cross-linked peptide by mass spectrometry and amino acid analysis. The cross-link appeared to be between the alpha -amino group of the protein and one of the two conserved glutamate residues which participate in the cross-links in SASP-A and SASP-C (data not shown).


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Fig. 6.   EDC cross-linking of Bce1 and Bce1E10K. Protein (0.5 mg/ml) with or without linear plasmid DNA (100 µg/ml) was reacted with 25 mM EDC for 30 min at 22 °C as described under "Experimental Procedures." Samples were lyophilized, dissolved in sample buffer, and run on Tris-Tricine SDS-PAGE (16.7% acrylamide) and stained with Coomassie Brilliant Blue R-250. Numbers on the left margin indicate the positions of molecular mass markers in kDa. The bands marked with asterisks (*) are contaminants in the Bce1 and Bce1E10K preparations.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Previous studies have demonstrated that alpha /beta -type SASP bind to DNA in a cooperative fashion and that protein-protein interactions probably occur during binding (7). The data reported in this communication confirm that direct protein-protein interactions occur between alpha /beta -type SASP bound to DNA. The protein-protein interaction detected by EDC cross-linking is not seen in the absence of DNA, which is consistent with previous data that suggest that alpha /beta -type SASP exist as monomers in solution3 (9). It appears that alpha /beta -type SASP/DNA binding involves the polymerization of the monomeric proteins along the backbone of the DNA double helix. This is suggested both by direct visualization of alpha /beta -type SASP·DNA complexes by electron microscopy (8) and by the ability of alpha /beta -type SASP to protect the DNA backbone from attack by hydroxyl radicals and orthophenanthroline-Cu2+ (17). It further appears that alpha /beta -type SASP assemble in a polarized fashion along the DNA backbone, because EDC cross-linked trimers and tetramers of SspC and Bce1 appear to contain only one type of cross-link, which is consistent with a head-to-tail arrangement. However, it is not clear whether DNA polarity itself has any role in organizing alpha /beta -type SASP binding.

The DNA binding region of alpha /beta -type SASP has been postulated to be within the C-terminal half of these proteins. This region of alpha /beta -type SASP is more highly conserved than the N-terminal region, and a 29-amino acid residue synthetic peptide corresponding to residues Thr43-Phe70 of SspC has been shown to bind to DNA and change the DNA's UV photochemistry (6). However, the binding of this synthetic peptide to DNA was much weaker than that of full-length SspC (6). The EDC cross-link sites found in alpha /beta -type SASP suggest that the N-terminal region of these proteins is involved in protein-protein interactions. These protein-protein interactions presumably increase the binding affinity of alpha /beta -type SASP for DNA, and therefore account, at least in part, for the difference in DNA binding affinities between the synthetic peptide and full-length SspC. The N-terminal region of alpha /beta -type SASP is the most variable region of these proteins, with variations in both sequence and length (Fig. 1) (1). In fact, many alpha /beta -type SASP are virtually identical proteins with the exception of their N-terminal regions. This is best exemplified by SASP-A and SASP-C of B. megaterium, which are essentially identical with the exception of the longer N terminus of SASP-C (Fig. 1). However, despite their similarity in primary sequence, SASP-C has a higher affinity for DNA in solution (9). In light of the results reported here, it appears that the N-terminal regions of alpha /beta -type SASP are involved in protein-protein interactions, and therefore could be a major factor in determining the binding affinity of these proteins for DNA in solution. It is important to note that other regions of alpha /beta -type SASP may also be involved in protein-protein interactions, which may not be detected because the reagent used in this study cross-links only amino and carboxyl groups.

The identification of cross-linked acidic residues within the GPR recognition sequences of SASP-A and SASP-C is particularly interesting because previous in vitro studies have demonstrated that alpha /beta -type SASP are resistant to GPR cleavage when bound to DNA (20). The data presented in this communication suggest that the N termini of SASP-A and SASP-C are close to the GPR cleavage site, while these proteins are in the DNA bound state. Thus, the GPR cleavage site may be inaccessible to the GPR protease due to steric interference by the N terminus of an adjacent protein. However, other structural changes are probably also important because purified cross-linked dimeric and trimeric SspC (which both contain unmodified GPR cleavage sequences) are partially resistant to GPR cleavage (data not shown). Thus, EDC cross-linking may stabilize a protein conformation of alpha /beta -type SASP in which GPR cleavage is inhibited.

The identification of two different sites of DNA dependent cross-link formation in the alpha /beta -type SASP examined was unexpected based upon the large degree of primary sequence conservation between members of this protein family. Although the position corresponding to Asp-13 and Glu-10 in SspC and Bce1, respectively, is far removed from the GPR recognition site in primary sequence, it is possible that these two regions are near one another in the three dimensional structure of alpha /beta -type SASP bound to DNA. Because SASP-A and SASP-C contain a lysine residue at the position corresponding to Asp13-Glu10, these proteins are unable to form cross-links with the alpha -amino group at this position and instead form cross-links with the glutamate residues of the GPR recognition sequence. The observed cross-links between the alpha -amino group and the two glutamate residues of the GPR recognition sequence in SASP-A and SASP-C, indicate that the N terminus of each protein is fairly mobile and may interact electrostatically with an acidic patch that is formed by the glutamate residues of the GPR recognition sequence. This acidic patch may also contain Asp13 and Glu10 in SspC and Bce1, respectively, and could explain the apparent shift in the cross-link formation site from Glu10 in Bce1 to the GPR recognition site in Bce1E10K. Although, we believe that we have identified the major sites of EDC cross-linking in each alpha /beta -type SASP studied, we cannot of course exclude the possibility that other minor cross-linking sites exist which were not detected by our HPLC analysis.

Another property of alpha /beta -type SASP that has been established in this study is that different alpha /beta -type SASP make functional protein-protein contacts with one another in the DNA bound state. Indeed, proteins from different species were found to interact as demonstrated by cross-linking of SspC from B. subtilis to SASP-A from B. megaterium. However, it appears that the interaction between SspC and SASP-A is not as favorable as the interaction between SASP-C and SASP-A. In fact, heterodimers of SASP-A and SASP-C were the predominant cross-linking products when the ratio of the two proteins approximated the in vivo ratio. Preferential heterodimer formation may be due to preferential association of the two proteins while bound to DNA, or merely to a greater probability of successful cross-linking occurring between these two particular proteins. Because SASP-A and SASP-C had identical EDC cross-linking sites, which were different from those of SspC and Bce1, it is reasonable to assume that the precise nature of the protein-protein interactions in these two groups of alpha /beta -type SASP is similar, yet distinct from one another. Thus, subtle variations in protein structure may allow some alpha /beta -type SASP to interact with one another more easily than others. These apparently minor differences in primary sequence between alpha /beta -type SASP may be important for efficient binding to different regions of the spore chromosome, and account for the need to maintain multiple alpha /beta -type SASP in each species.

We are currently using another cross-linking strategy to identify amino acid residues in alpha /beta -type SASP which make close contacts with DNA. However, the structural information obtained during these types of studies is limited and efforts to obtain a high resolution structure of an alpha /beta -type SASP·DNA complex are ongoing. A high resolution structure of an alpha /beta -type SASP·DNA complex should confirm the results obtained in cross-linking experiments and also illustrate the nature of the change in DNA conformation which underlies the change in UV photochemistry of spore DNA and ultimately spore UV resistance.

    ACKNOWLEDGEMENTS

We thank Barbara Setlow and Dave Hutcheson for purification of SASP-A and SspC, respectively. All mass spectrometry and amino acid sequencing were performed by Dr. John Leszyk at the Worcester Foundation for Biomedical Research.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant GM19698.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel. 860-679-2607; Fax: 860-679-3408; E-mail: setlow{at}sun.uchc.edu

1 The abbreviations used are: SASP, small, acid-soluble spore proteins; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; HPLC, high performance liquid chromatography; MALDI-TOF, matrix-assisted laser desorption/ionization-time of flight; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; PAGE, polyacrylamide gel electrophoresis; GPR, germination protease.

2 S. C. Mohr and P. Setlow, unpublished results.

3 B. Setlow and P. Setlow, unpublished results.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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