Proc. Nati. Acad. Sci. USA Vol. 74, No. 11, pp. 4905-4908, November 1977

Isopeptide linkage between N-a-monomethylalanine and in ribosomal protein S 1 from Escherichia coli (ribosome structure/protein modification/dansyl amino acids/solid-state peptide synthesis) ROBERT CHEN* AND URSULA CHEN-SCHMEISSER Max-Planck-Institut fur Molekulare Genetik, Abt. Wittmann, D-1000 Berlin-Dahlem, Germany Communicated by Stanford Moore, August 29, 1977

ABSTRACT Protein SI1 from the Escherichia coli ribosome peptides were digested with the (100:1, by weight) in has a unique NHrterminal structure not previously observed 0.2 M N-methylmorpholine, pH 8.1, for 1 hr at 37'. among ribosomal proteins. Owing to the formation of an iso- analyses were performed under standard conditions (12) on a between a secondary amino acid (N-a-mono- methylalanine) and the eamino group of the NH2-terminal ly- Durrum D500 amino acid analyzer. sine residue, a "branching point" is formed. Therefore, two Sequence Determinations. The NH2-terminal sequence of amino acids are seen when the NH2 terminus of the protein is the intact protein was determined by the automatic Edman determined. degradation process (13) (model PS 110, Socosi, Paris, France). The thiazolinone derivatives of the amino acids were converted is a result of a number of highly coordi- to the phenylthiohydantoins in 1 M HCI at 800 for 10 min and nated events in which the proper codon-anticodon interaction extracted from the aqueous solution with ethyl acetate. They between mRNA and aminoacylated tRNAs guarantees the were identified by chromatography on silica gel thin-layer correct translation of the genetic message (reviewed in refs. 1 plates (HPTLC silica gel6o, 10 X 10, Merck, Darmstadt, GFR) and 2). This interaction is governed by several ribosomal in system I [chloroform/1-propanol/2-propanol, 98:1:1 (vol/ components, one of which is protein S1i from the Escherichia vol)] and in system II [dichloroethane/acetic acid, 6:1 (vol/vol)]. coli ribosome. This protein has been shown to have a strong In addition, they were hydrolyzed in HI to generate the free influence on the fidelity of translation (3). amino acids (14) and were then subjected to amino acid analysis. Recently the occurrence of an unusual modification, N-a- The refined micro dansyl-Edman technique (10) was used to monomethylation, of the NH2-terminal alanine in Sil was establish the sequences of the tryptic peptides. Micro polyamide reported (4). This residue was determined by mass spectrom- plates were photographed with Agfa-ortho film. etry, amino acid analysis, and chromatography in various sys- Synthesis of the Tripeptide L-Lys(eN-methyl-L-Ala)LAla. tems. In a search for similarly modified ribosomal proteins, The tripeptide was synthesized in solid phase attached to a N-a-monomethylation was also established for the NH2 termini chloromethyl resin (15). After the synthesis was completed the of proteins L16 (4, 5) and L33 (4, 6, 7). The NH2-terminal peptide was cleaved from the resin with HBr in trifluoroacetic residues of these proteins are completely methylated (4). N- acid. It was purified with the micro Dowex 50W-X7 column a-Monomethylation was not measured for protein Si 1 because system (11). Its homogeneity was judged by analytical peptide the Edman degradation process always indicated the presence maps (10), NH2 terminus determination (16) and amino acid of lysine in addition to the presence of N-a-monomethylalanine analysis (12). t-Butoxycarbonyl (Boc)-alanine and a-L-benzy- (NaMeAla). This suggested some "heterogeneity" in the pro- loxycarbonyl (Cbz)-eBoc-L-lysine were purchased from Serva tein. The experiments reported in this communication were (Heidelberg, GFR). Boc-NaMeAla was synthesized with t- designed to investigate this observation. butylazidoformate and was recrystallized from ethyl acetate/ hexane (17). MATERIALS AND METHODS Protein SlI used throughout these experiments was provided RESULTS by H. G. Wittmann. Its identity and purity were judged NHrTerminal Sequence of SlI. Determination of the NH2 by one-dimensional sodium dodecyl sulfate/gel electrophoresis terminus of protein SIl by the dansylation technique gave one (8) and two-dimensional polyacrylamide gel electrophoresis spot in the position of dansyl (Dns)-NaMeAla and a second spot (9). in the position of a- or eDns-lysine on the polyamide plate. This Preparation of Peptides. Protein Si1 was digested with second spot is always seen when proteins (or peptides) that trypsin (TPCK-trypsin, Merck, Darmstadt, GFR). The peptides contain lysine residues react with dansyl chloride. This is due were purified by peptide mapping on cellulose thin-layer plates to the reactivity of the e-amino group of lysine. Bis-Dns-lysine or by ion exchange chromatography on a Dowex 50W-X7 is seen when lysine occurs at the NH2 terminus of the protein micro column (10, 11). A more detailed report of the prepara- (or peptide). Therefore, it was concluded that the sample tion of the peptides will appear elsewhere. contained pure protein SIl and that the NH2-terminal residue Amino Acid Analyses. Hydrolyses of the peptides were was NaMeAla. performed in 6 M HCI in the presence of 0.02% 2-mqrcapto- This conclusion, however, conflicted with the data from the ethanol or by digestion with leucine aminopeptidase (Serva, Heidelberg, GFR). Amounts corresponding to 200 pmol of the Abbreviations: NaMeAla, N-a-monomethylalanine; Boc, t-butoxy- carbonyl; Cbz, benzyloxycarbonyl; Dns, dansyl (1-dimethylamino- The costs of publication of this article were defrayed in part by the naphthalene-5-sulfonyl); PhNCS, 3-phenyl-2-thiohydantoin; PhNHCS, payment of page charges. This article must therefore be hereby marked phenylthiocarbamyl. "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate * Present address: Max-Planck-Institut ffir Biologie, Corrensstrasse 38, this fact. D-7400 Tubingen 1, Germany. 4905 Downloaded by guest on September 24, 2021 4906 Biochemistry: Chen and Chen-Schmeisser Proc. Natl. Acad. Sci. USA 74 (1977)

Table 1. Amino acid composition of the NH2-terminal tryptic peptide of S11 1 NMA p Amino Standard amino Calibration with acid acid analysis Lys(E-N-MeAla)-Ala # S > 3* I NaMeAla + 1.10 A * A Ala 1.10 0.95 o K 6***t * t Ile 1.00 &.. Pro 0.95 Lys 1.01 1.00 I * * 9~~~~~~~R R R Arg 0.92 O A.- V.**ae..+A. . STEP: 1 2 3 4 5 6 7 8 addition, it revealed that the second amino group of lysine FIG.....1. Automatic * Edman degradation of S11. The phenylthio- became accessible to dansyl chloride when the sample was hydantoin (PhNCS)*A*..-....derivatives of the amino acids released in every hydrolyzed before it was treated with the reagent. step of the analysis were identified on silica gel thin-layer plates. Standard PhNCS-amino acid mixtures were chromatographed par- Analysis with Leucine Aminopeptidase. Digestion with allel to them (18). Note that two amino acids are seen in every step. leucine aminopeptidase was used to determine whether one A = Ala; I = Ile; K = Lys; P = Pro; R = Arg; NMA = NaMeAla. amino group of the NH2-terminal lysine in Sli was blocked by alkylation, , or , because leucine automatic degradation of Sil in the sequenator (Fig. 1). In the aminopeptidase would not hydrolyze these residues from lysine. first step of the analysis, both NaMeAla and lysine were re- The NH2-terminal tryptic peptide, which contained a single leased from the protein. Two amino acids also were seen in each lysine residue, was treated with the enzyme. From the digest of the following steps, indicating that apparently two poly- an aliquot was dansylated and chromatographed on polyamide peptide chains were present in the sample. In addition, the data plates, which revealed that both bis-Dns-lysine and Dns- suggested, that the "two" chains were homologous, because an NaMeAla were present on the chromatogram. This result amino acid seen in one step of the analysis also occurred in the showed that alkylation, acylation, or glycosylation of the lysine next step. Hence, the difference between the "two" chains residue had to be excluded. seemed to be an additional amino acid at the NH2 terminus of Measurement of NaMeAla in the NH2-Terminal Tryptic one of them that induced a shift of the entire sequence. Peptide. No standard amino acid mixtures are available that Sequence of the NH2-Terminal Tryptic Peptide. To resolve contain NaMeAla. Therefore, the synthetic peptide L-Lys(E- the discrepancy between the dansylation results and the se- N-methyl-L-Ala)-L-Ala was used to calibrate the absorptivity quenator data, protein SlI was digested with trypsin. The of NaMeAla relative to equimolar amounts of lysine and ala- NH2-terminal tryptic peptide was isolated from the digest on nine. Amino acid analyses of both the synthetic peptide and the peptide maps. This peptide was sequenced by the micro dan- NH2-terminal tryptic peptide of Si 1 were conducted on a syl-Edman technique which gave Dns-NaMeAla and a- or Durrum D500 amino acid analyzer at the sensitivity of 2 OD, e-Dns-lysine in the first cycle (Fig. 2). Bis-Dns-lysine was not at which the standard error of the analysis is minimal. The data seen in the first step nor was lysine identified in any further step are given in Table 1. The molar ratio of NaMeAla to lysine and of the analysis. alanine was the same in both peptides, which showed that In order to investigate why a- or E-Dns-lysine was seen rather NaMeAla and lysine occurred in every molecule of the than bis-Dns-lysine in the first cycle, the NH2-terminal tryptic NH2-terminal tryptic peptide. The data confirmed that the peptide was submitted to a subtractive micro dansyl-Edman peptide was pure and that we were not dealing with a mixture technique. Before each step of the degradation of the peptide, of two almost identical peptides differing only in their NH2- a fraction of the sample was hydrolyzed in 6 M HCL. The lib- terminal residues. erated amino acids then were dansylated and identified on Structure of the NHrTerminal Tryptic Peptide. Amino acid micro polyamide plates. In these experiments, bis-Dns-lysine analyses of the NH2-terminal tryptic peptide had shown that was found before the first residue was cleaved from the peptide we had isolated a pure peptide that contained equimolar but not in any succeeding step (Fig. 3). This result confirmed amounts of NaMeAlal, Ala,, Ile,, Pro1, Lysj, and Arg1 (Table that lysine occurred at the NH2 terminus of the peptide. In 1). Sequencing the peptide by the micro dansyl-Edman tech- nique (both direct and subtractive) gave NaMeAla and lysine NMA in the first step followed by one single residue in each of the succeeding steps (Fig. 3). Therefore, lysine and NaMeAla must occur together at the NH2 terminus of the peptide. These data were strong evidence for the existence of an isopeptide bond between NaMeAla and the e-amino group of the NH2-terminal lysine residue. The proposed structure of the NH2-terminal tryptic peptide is shown in Fig. 4. The isopeptide bond between lysine and NaMeAla explains why only one amino group of lysine reacted when the peptide was treated with dansyl chlo- eamino of this residue. DNS-OH .C/C-Lys ride-NaMeAla blocked the group .f Hydrolysis of the peptide with hydrochloric acid followed by dansylation of the liberated amino acids (subtractive technique) FIG. 2. NH2-terminus determination of the NH2-terminal tryptic gave bis-Dns-lysine. peptide of Sli. Two spots corresponding to Dns-amino acids- Analogous Results from the Synthetic Peptide L-Lys(e- Dns-NaMeAla (NMA) and a- or E-Dns-lysine-are seen on the N-methyl-L-Ala)L-Ala. This synthetic peptide was used to in- polyamide plate. DNS-OH is the free sulfonic acid. vestigate three general problems in order to substantiate the Downloaded by guest on September 24, 2021 Biochemistry: Chen and Chen-Schmeisser Proc. Nati. Acad. Sci. USA 74 (1977) 4907

A B C

N MA. lie _ Pro lie Pro Pro lie _ _7 AMa Ala bis-Lys

DNS-OH Arg * DNS-OH DNS-OH ~./ Arg Arg lk <, _

D E

Aw *m lie NO 4dPv

DNS-OH , Arg DNS-OH Arg n s .F~~~~~~A

FIG. 3. Subtractive micro dansyl-Edman degradation of the NH2-terminal tryptic peptide of Sl1. After each step, an aliquot of the sample was hydrolyzed in 6 M HCl and dansylated. The Dns-amino acids were chromatographed in 1.5% formic acid (first dimension) and in toluene/glacial acetic acid, 10:1, (vol/vol) (second dimension). Dns-NaMeAla (NMA) and bis-Dns-lysine (bis-Lys) were only seen before the peptide was submitted to the first cycle of the Edman degradation. proposed NH2-terminal structure of S11. (i) The specificity of migrated in the position of a-PhNCS-NE-phenylthiocarbamyl leucine aminopeptidase was tested, because it was previously (PhNHCS)-lysine on silica gel thin-layer plates (Fig. 1). Because unknown that the enzyme could hydrolyze isopeptide bonds. lysine and NaMeAla formed an isopeptide bond in the intact (ii) The accessibility of the isopeptide bond to the Edman protein, the PhNCS-amino acid in the position of a-PhNCS- degradation process was investigated. (iii) The chromatographic NE-PhNHCS-lysine must have been a-PhNCS-lysine. To in- properties of a-PhNCS-lysine on silica gel thin-layer plates were vestigate this, the synthetic peptide was degraded by the determined. manual Edman degradation process. The released PhNCS- Digestion of the synthetic peptide with leucine aminopep- amino acids were chromatographed on silica gel thin-layer tidase and analysis of the liberated amino acids revealed that plates and two spots were seen in the first cycle, one in the po- the enzyme can hydrolyze isopeptide bonds even when an sition of PhNCS-NaMeAla and the other in the position of a- N-a-monomethylated amino acid is involved. Similarly, we PhNCS-Nf-PhNHCS-lysine, indicating that a-PhNCS-lysine were able to show that NaMeAla is cleaved from the e-amino and a-PhNCS-NE-PhNHCS-lysine migrate to the same posi- group of lysine by the Edman degradation procedure. tion. Two PhNCS-amino acids were released from protein SI1 in In Fig. 5 the mobility of a synthetic a-PhNCS-lysine is the first cycle of the automatic sequence analysis. One of them compared to that of a-PhNCS-Nf-PhNHCS-lysine. In the first solvent system, a-PhNCS-lysine migrated slightly below a- (1) 0 (2) (3) (4) (5) PhNCS-NE-PhNHCS-lysine. When rechromatographed in the (Lys) H2N-CH-C-Ala-Pro-Ile-Arg-COOH second system, both amino acid derivatives had the same RF value. Therefore, they were not distinguished by this tech- (H2)4 nique. Incomplete Cleavage of the NH2-Terminal Lysine from C-NH SIl. NaMeAla was quantitatively cleaved from Sil when the H I protein was degraded in the sequenator, whereas lysine strongly (NMA) H3C-N-CH overlapped in the second cycle (Fig. 1). The same observation was made when SI1 was degraded by the manual Edman OH3 degradation procedure. However, when the protein was sub- FIG. 4. Primary structure of the NH2-terminal tryptic peptide mitted to a double cleavage, the overlap almost disappeared. of S11. The numbers in parentheses represent cycles of the Edman This result suggested that, under the conditions used in the se- degradation. NMA = NaMeAla. quenator, the cleavage of lysine from the polypeptide chain was Downloaded by guest on September 24, 2021 4908 Biochemistry: Chen and Chen-Schmeisser Proc. Natt. Acad. Sci. USA 74 (1977)

A B 51 a biological significance of its unique NH2-terminal structure has not been established. However, the new structural L L A.s 4w data are especially interesting in view of the strong implication M i translation of the K that protein SIl participates in the correct Y genetic message in the ribosome (3). T The authors thank Dr. J. Littlefield for carefully reading the man- K _ Q 0 uscript of this publication. T 1. Lengyel, P. (1974) in Ribosomes, eds. Nomura, M., Tissieres, A. & Lengyel, P. (Cold Spring Harbor Laboratory, Cold Spring a b c a b c Harbor, NY), pp. 13-52. FIG. 5. Mobilities of a-PhNCS-lysine and a-PhNCS-N'- 2. Pongs, O., Nierhaus, K. H., Erdmann, V. A. & Wittmann, H. G. PhNHCS-lysine on silica gel thin-layer plates. The PhNCS-amino (1974) FEBS Lett. 40, 28-37. acids were chromatographed in chloroform/1-propanol/2-propanol, 3. Nomura, M., Mizushima, S., Ozaki, M., Traub, P. & Lowry, C. 98:1:1 (vol/vol) (A) and then rechromatographed in dichloroethane/ V. (1969) Cold Spring Harbor Symp. Quant. Biol. 34, 49-61. acetic acid, 6:1 (vol/vol) (B). Lanes: a, standard PhNCS-amino acid 4. Chen, R., Brosius, J., Wittmann-Liebold, B. & Schifer, W. (1977) mixture; b, a-PhNCS-lysine; c, a-PhNCS-Ne-PhNHCS-lysine. K, J. Mol. Biol. 111, 173-181. Lys; L, Leu; M, Met; Q, Gln; T, Thr; Y, Tyr. 5. Brosius, J. & Chen, R. (1976) FEBS Lett. 68,105-109. 6. Wittmann-Liebold, B. & Pannenbecker, R. (1976) FEBS Lett. incomplete. A similar observation was made by Goldknopf and 68, 115-118. Busch (19). They observed a strong overlap of lysine when they 7. Chang, D. N., Schwartz, M. & Chang, F. N. (1976) Biochem. degraded a peptide in which lysine formed an isopeptide bond Blophys. Res. Commun. 73,283-293. with . The reason for the incomplete cleavage reaction 8. Weber, K. & Osborn, M. (1969) J. Biol. Chem. 244, 4406- has not yet been established and requires further investiga- 4412. 9. Kaltschmidt, E. & Wittmann, H. G. (1970) Anal. Biochem. 36, tion. 401-412. 10. Chen, R. (1976) Hoppe-Seyler's Z. Physiol. Chem. 357, 873- DISCUSSION 886. In prokaryotic as well as eukaryotic organisms, several proteins 11. Chen, R. (1977) Hoppe-Seyler's Z. Physiol. Chem. 358, 1415- have previously been found to contain lysine residues that form 1431. W. H. & Moore, S. (1958) Anal. Chem. isopeptide bonds with or glycine. The isodipep- 12. Spackman, D. H., Stein, isolated from collagen (20), 30, 1190-1206. tide e-(y-glutamyl)lysine has been 13. Edman, P. & Begg, G. (1967) Eur. J. Biochem. 1, 80-91. hair medulla protein (21), and fibrin (22, 23). A nonhistone 14. Smithies, O., Gibson, D., Fanning, E. M., Goodfliesh, R. M., protein was found to be bound through an e(glycyl)lysine bond Gilman, J. G. & Ballantyne, D. L. (1971) Biochemistry 10, to histone 2A from calf thymus (19). Isopeptide bonds between 4912-4921. adjacent peptidoglycans are known to stabilize the structure 15. Marshall, G. R. & Merrifield, R. B. (1965) Biochemistry 4, of the murein sacculus in bacteria (24). 2394-2401. Protein S11 is the first protein from the E. coil ribosome 16. Gray, W. R. & Hartley, B. S. (1963) Biochem. J. 89,379-380. shown to have a "branching point" and hence to have two 17. Schnabel, E. (1967) Liebigs Ann. Chem. 702, 188-196. NHrterminal amino acid residues. This finding indicates that 18. Edman, P. (1970) in Protein Sequence Determination, ed. B. Verlag, Berlin-Heidelberg-New isopeptide bonds between and other amino acids occur Needleman, S. (Springer than has been York), pp. 211-255. more often among different types of proteins 19. Goldknopf, I. L. & Busch, H. (1977) Proc. Natl. Acad. Sci. USA shown thus far. The isopeptide bond in S11 differs from the 74,864-868. isopeptide linkages observed in other proteins in that it does not 20. Mechanic, G. L. & Levy, M. (1958) J. Am. Chem. Soc. 81, form an interchain or intrachain crosslink. Instead, an unusual 1889-1892. secondary amino acid, NaMeAla, is bound to the NH2-terminal 21. Harding, H. W. J. & Rogers, G. E. (1976) Biochim. Biophys. Acta lysine residue, suggesting that this modification may serve some 427,315-324. other, as yet unknown, function. 22. Pisano, J. J., Finlayson, J. S. & Peyton, M. (1968) Science 160, Some proteins of the E. coil ribosome have NH2 termini 892-893. Matacic, S. & Loewy, A. G. (1968) Biochem. Btophys. Res. modified in a different manner. Proteins L16 and L33 are 23. and alanine residues, Commun. 20,356-362. N-a-monomethylated at their 24. Lehninger, A. (1970) in Biochemistry (Worth Publishers, Inc., respectively (4-7), and N-a- was established for New York), pp. 232-235. proteins S18 (25) and L7 (26). As yet it is unknown why proteins 25. Yaguchi, M. (1975) FEBS Lett. 59,217-220. L16 and L33 are methylated, whereas large variations in the 26. Terhorst, C., Wittmann-Liebold, B. & M6ller, W. (1970) Eur. amount of L7 present relative to its nonacetylated form, L12, J. Biochem. 25, 13-19. in the ribosome during the growth cycle suggest some specific 27. Ramagopal, S. & Subramanian, A. R. (1974) Proc. Natl. Acad. control mechanism for the acetylation process (27). For protein Sci. USA 71,2136-2140. Downloaded by guest on September 24, 2021