Protein Splicing and Auto-cleavage of Bacterial Intein-like Domains Lacking a C′-flanking Nucleophilic Residue
2004; Elsevier BV; Volume: 279; Issue: 31 Linguagem: Inglês
10.1074/jbc.m404562200
ISSN1083-351X
AutoresBareket Dassa, Haim Haviv, Gil Amitai, Shmuel Pietrokovski,
Tópico(s)RNA Research and Splicing
ResumoBacterial intein-like (BIL) domains are newly identified homologs of intein protein-splicing domains. The two known types of BIL domains together with inteins and hedgehog (Hog) auto-processing domains form the Hog/intein (HINT) superfamily. BIL domains are distinct from inteins and Hogs in sequence, phylogenetic distribution, and host protein type, but little is known about their biochemical activity. Here we experimentally study the auto-processing activity of four BIL domains. An A-type BIL domain from Clostridium thermocellum showed both protein-splicing and auto-cleavage activities. The splicing is notable, because this domain has a native Ala C′-flanking residue rather than a nucleophilic residue, which is absolutely necessary for intein protein splicing. B-type BIL domains from Rhodobacter sphaeroides and Rhodobacter capsulatus cleaved their N′ or C′ ends. We propose an alternative protein-splicing mechanism for the A-type BIL domains. After an initial N-S acyl shift, creating a thioester bond at the N′ end of the domain, the C′ end of the domain is cleaved by Asn cyclization. The resulting amino end of the C′-flank attacks the thioester bond next at the N′ end of the domain. This aminolysis step splices the two flanks of the domain. The B-type BIL domain cleavage activity is explained in the context of the canonical intein protein-splicing mechanism. Our results suggest that the different HINT domains have related biochemical activities of proteolytic cleavages, ligation and splicing. Yet the predominant reactions diverged in each HINT type according to their specific biological roles. We suggest that the BIL domain cleavage and splicing reactions are mechanisms for post-translationally generating protein variability, particularly in extracellular bacterial proteins. Bacterial intein-like (BIL) domains are newly identified homologs of intein protein-splicing domains. The two known types of BIL domains together with inteins and hedgehog (Hog) auto-processing domains form the Hog/intein (HINT) superfamily. BIL domains are distinct from inteins and Hogs in sequence, phylogenetic distribution, and host protein type, but little is known about their biochemical activity. Here we experimentally study the auto-processing activity of four BIL domains. An A-type BIL domain from Clostridium thermocellum showed both protein-splicing and auto-cleavage activities. The splicing is notable, because this domain has a native Ala C′-flanking residue rather than a nucleophilic residue, which is absolutely necessary for intein protein splicing. B-type BIL domains from Rhodobacter sphaeroides and Rhodobacter capsulatus cleaved their N′ or C′ ends. We propose an alternative protein-splicing mechanism for the A-type BIL domains. After an initial N-S acyl shift, creating a thioester bond at the N′ end of the domain, the C′ end of the domain is cleaved by Asn cyclization. The resulting amino end of the C′-flank attacks the thioester bond next at the N′ end of the domain. This aminolysis step splices the two flanks of the domain. The B-type BIL domain cleavage activity is explained in the context of the canonical intein protein-splicing mechanism. Our results suggest that the different HINT domains have related biochemical activities of proteolytic cleavages, ligation and splicing. Yet the predominant reactions diverged in each HINT type according to their specific biological roles. We suggest that the BIL domain cleavage and splicing reactions are mechanisms for post-translationally generating protein variability, particularly in extracellular bacterial proteins. Bacterial intein-like (BIL) 1The abbreviations used are: BIL, bacterial intein-like; B, BIL; Rsp, Rhodobacter sphaeroides; Rca, Rhodobacter capsulatus; Cth, C. thermocellum; HINT, Hog/intein; MS, mass spectrometry; MALDI, matrix-assisted laser desorption/ionization; M, maltose-binding protein; C, Chitin-binding domain. 1The abbreviations used are: BIL, bacterial intein-like; B, BIL; Rsp, Rhodobacter sphaeroides; Rca, Rhodobacter capsulatus; Cth, C. thermocellum; HINT, Hog/intein; MS, mass spectrometry; MALDI, matrix-assisted laser desorption/ionization; M, maltose-binding protein; C, Chitin-binding domain. domains are newly identified protein homologs of intein protein-splicing domains (1Amitai G. Belenkiy O. Dassa B. Shainskaya A. Pietrokovski S. Mol. Microbiol. 2003; 47: 61-73Google Scholar). The two known types of BIL domains together with inteins and hedgehog-like (Hog) auto-processing domains form the HINT (Hog/Intein) domain superfamily (2Paulus H. Annu. Rev. Biochem. 2000; 69: 447-496Google Scholar). Inteins and Hogs have related auto-catalytic protein-processing activities. Hog domains rearrange their N′-peptide bond into a thioester bond. This thioester is cleaved by a nucleophilic attack of a cholesterol molecule bound by a downstream domain (3Porter J.A. Ekker S.C. Park W.J. von Kessler D.P. Young K.E. Chen C.H. Ma Y. Woods A.S. Cotter R.J. Koonin E.V. Beachy P.A. Cell. 1996; 86: 21-34Google Scholar, 4Perler F.B. Cell. 1998; 92: 1-4Google Scholar). A similar nucleophilic attack occurs during the protein splicing of inteins out of their protein hosts. The rearranged ester/thioester bond at the intein N′ end is attacked by the nucleophilic side chain of the intein C′-flanking residue followed by additional splicing reactions (5Xu M.Q. Comb D.G. Paulus H. Noren C.J. Shao Y. Perler F.B. EMBO J. 1994; 13: 5517-5522Google Scholar). Intein protein splicing thus depends on an invariable Cys, Ser, or Thr nucleophilic C′-flanking residue (+1) for the trans-esterification and acyl rearrangement steps (2Paulus H. Annu. Rev. Biochem. 2000; 69: 447-496Google Scholar, 6Perler F. Noren C. Wang J. Angew. Chem. Int. Ed. Engl. 2000; 39: 450-466Google Scholar).BIL domains are distinct from inteins and Hogs in sequence, phylogenetic distribution, and host protein type (1Amitai G. Belenkiy O. Dassa B. Shainskaya A. Pietrokovski S. Mol. Microbiol. 2003; 47: 61-73Google Scholar). Each of the two BIL types has characteristic and unique sequence features that cluster them separately from other HINT types. Although inteins are integrated in highly conserved sites of essential proteins and Hogs are present in hedgehog and related nematode proteins, BIL domains are integrated in variable regions of non-conserved diverse bacterial proteins, some of which have extracellular motifs. This leads to the hypothesis that BIL domains may have biological roles different from those of other HINT domains (1Amitai G. Belenkiy O. Dassa B. Shainskaya A. Pietrokovski S. Mol. Microbiol. 2003; 47: 61-73Google Scholar). Yet little is known regarding the biochemical activity of each BIL type.We previously described (1Amitai G. Belenkiy O. Dassa B. Shainskaya A. Pietrokovski S. Mol. Microbiol. 2003; 47: 61-73Google Scholar) the catalytic activity of an A-type and a B-type BIL domain. The A-type BIL domain was shown to have protein-splicing and C′-cleavage activities. However, this domain was naturally flanked by a Thr +1 residue, which is typical of inteins but not of A-type BIL domains. Only 15% of known A-type BIL domains is followed by Ser or Thr, and none is followed by Cys residues. An A-type BIL domain with +1 Tyr residue was shown recently by Southworth et al. (7Southworth M.W. Yin J. Perler F.B. Biochem. Soc Trans. 2004; 32: 250-254Google Scholar) to have N′-terminal cleavage but no protein-splicing activity. The B-type BIL domain was examined previously by us only in a cell-free system. It was shown to be active with preliminary evidence for cleavage and protein splicing. Peptide splicing outside the context of intein-like domains also was shown recently to occur in the proteasome, generating variant peptides to be displayed on major histocompatibility complex class I proteins (8Vigneron N. Stroobant V. Chapiro J. Ooms A. Degiovanni G. Morel S. Van Der Bruggen P. Boon T. Van Den Eynde B.J. Science. 2004; 304: 587-590Google Scholar).Here we examine in detail the auto-cleavage and splicing activity of four BIL domains: one A-type BIL domain with a native non-nucleophilic C′-flanking residue (Ala +1) and three different B-type BIL domains. We also show that BIL domains are present in more major groups of bacteria and in proteins likely to be secreted. The probable functions and chemical reaction mechanisms of BIL domains and their relation to inteins are discussed.EXPERIMENTAL PROCEDURESBacterial Strains and DNA Primers—Rhodobacter sphaeroides 2.4.1 (Rsp) genome was a kind gift from Dr. Steven L. Porter (University of Oxford). Rhodobacter capsulatus (Rca) MD1 genome was a kind gift from Dr. Fevzi Daldal (University of Pennsylvania), and Clostridium thermocellum (Cth) genome was a kind gift from Dr. Ying Tsai (University of Rochester). The following BIL domains were cloned: BIL4-Cth (NCBI gi code 23020817); BIL1-Rsp (NCBI gi code 22959584); BIL2-Rsp (NCBI gi code 22959191); and 1522-Rca (1Amitai G. Belenkiy O. Dassa B. Shainskaya A. Pietrokovski S. Mol. Microbiol. 2003; 47: 61-73Google Scholar). The BIL domains were amplified by PCR using the primers in Table I and cloned between two protein tags in a plasmid termed pC2C (as described by Amitai et al. (1Amitai G. Belenkiy O. Dassa B. Shainskaya A. Pietrokovski S. Mol. Microbiol. 2003; 47: 61-73Google Scholar)). This plasmid is a modification of the pMALC2 vector (New England BioLabs, Beverly, MA) containing the malE gene for maltose-binding protein (M) from Escherichia coli and a downstream cbd gene coding for chitin-binding domain (C) from Bacillus circulans).Table IPrimer namePrimer sequenceRestriction siteFlank amino acidsaNumber of residues flanking the BIL domain.BIL1-Rsp5p-Nrsp-bil1GAATTCATGGCTGACCAAATCCAGATCGGEcoRI+143p-Nrsp-bil1TCTAGAGCGGACGAGGACCCTTTCCGGTXbaI+52BIL2-Rsp+flanks5p-rsph-bil2GAATTCGGTGATTCATCCTTGGGGCGAEcoRI+323p-rsph-bil2TCTAGAAAACACGGCAAGGGCGAGCGGXbaI+9BIL2-Rsp-no flanks5p-rsp2-bil-only+1GAATTCCTCTCCCTGACGGCCGGGACGEcoRI+13p-rsp2-only+1TCTAGAGGGCCGGGTCACGGGATGGAGXbaI+1BIL4-Cth5p1.BIL4 CthAAAAGGATCCTGCTTTGTTGCAGGCACGATGBamHI3p408.BIL4 CthAAAATCTAGATGCATTATGCACCAATACTTCATXbaI+11522-Rca5pBILGGATCCAACTACGATCCGACGAACCCBamHI+361522-108bp3pBILTCTAGAACCATAGCCCTCAAGGCCGTCXbaI+351522+105bpa Number of residues flanking the BIL domain. Open table in a new tab Functional Assay of Protein-splicing and Cleavage Activity—The coding sequence of different BIL domains (B) was cloned in-frame between two protein tags, the maltose-binding protein (M) upstream and the chitin-binding domain (C) downstream. The chimeric protein, M-B-C, was overexpressed and extracted in E. coli bacteria as described previously (1Amitai G. Belenkiy O. Dassa B. Shainskaya A. Pietrokovski S. Mol. Microbiol. 2003; 47: 61-73Google Scholar). Protein extraction buffer contained 20 mm Tris, pH 7.4, 200 mm NaCl, 1 mm EDTA, and 1 mm sodium azide.Purification of Tagged Protein Products—Soluble protein products containing eitheraCoraMtag were purified on affinity columns using chitin (New England Biolabs) or amylose (New England Biolabs) beads, respectively. Lysed cell supernatant in extraction buffer was applied to beads for 1 h at 4 °C with shaking. Elution of proteins from chitin beads was done by mixing the beads with SDS-PAGE sample loading buffer and boiling for 2–3 min. Extraction buffer with 10 mm maltose was used to elute proteins from amylose beads.Heat Purification of BIL4-Cth Domain—The supernatant of E. coli cell lysate overexpressing the BIL4-Cth construct was heated in extraction buffer to 37–80 °C for 20 min. Soluble proteins were separated from the denatured ones by centrifugation at 13,000 rpm for 3 min and applied on an SDS gel.In Vitro Protein Transcription/Translation—In vitro transcription/translation was carried out using E. coli S30 extract for circular DNA system (Promega, Madison WI) as described by Amitai et al. (1Amitai G. Belenkiy O. Dassa B. Shainskaya A. Pietrokovski S. Mol. Microbiol. 2003; 47: 61-73Google Scholar).Western Blot Analysis—Western blot analysis was used to identify protein products containing either the M or C tag and to identify the GroEL and DnaK protein chaperones. To identify the M tag, monoclonal mouse antibodies directed at maltose-binding protein (Novus Biologicals, Littleton, CO) were used in a 1:800 ratio. To identify the C tag, polyclonal rabbit antibodies directed at CBD (New England Biolabs) were used in a ratio of 1:5000. Antibodies for GroEL were a kind gift from Prof. Amnon Horovitz (rabbit antibodies, used in a ratio of 1:1000), and DnaK mouse antibodies (Stressgen) were used in a ratio of 1:1000. The secondary antibodies used were horseradish peroxidase-conjugated goat anti-mouse IgG or goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) in a ratio of 1:10000. Chemiluminescence detection was held using SuperSignal (Pierce) according to the manufacturer's protocol.Mass Spectrometry (MS) Methods—Intact molecular weight measurements and peptide mass mapping by matrix-assisted laser desorption/ionization (MALDI) MS were performed at the Weizmann Institute Biological Mass Spectrometry unit and at the Smolar Center for proteins (Technion, Israel). Electroelution from gel followed by in-gel digestion with trypsin, chymotrypsin, or V8 proteases was performed and analyzed as described previously (37, Mehlman, T., Benjamin, M., Merhav, D., Osman, F., Ben-Asouli, Y., Goldshleger, R., Karlish, S., and Shainskaya, A. (2002) Proceedings of the 50th Conference of American Society for Mass Spectrometry, Orlando, June 2–6, 2002, American Society for Mass Spectrometry, Santa Fe, NMGoogle Scholar).N-terminal Amino Acid Sequencing—Proteins were electrophoresed by SDS-PAGE, and selected bands were prepared as described by Amitai et al. (9Amitai G. Dassa B. Pietrokovski S. J. Biol. Chem. 2004; 279: 3121-3131Google Scholar) and subjected to Edman degradation at the Weizmann Institute Biological Mass Spectrometry Unit.Computational Sequence Analysis—Sequence searches used the BLAST programs (10Altschul S.F. Madden T.L. Schaffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Google Scholar) and the BLIMPS program for block-to-sequence searches (11Henikoff S. Henikoff J.G. Alford W.J. Pietrokovski S. Gene (Amst.). 1995; 163: 17-26Google Scholar). Block multiple sequence alignments and phylogenetic analysis were conducted as described by Amitai et al. (1Amitai G. Belenkiy O. Dassa B. Shainskaya A. Pietrokovski S. Mol. Microbiol. 2003; 47: 61-73Google Scholar). Protein motifs were detected using the InterProScan tool (www.ebi.ac.uk/interpro/scan.html).RESULTSTo characterize the proteolytic activity of new A- and B-type BIL domains, each BIL domain (B) was cloned in-frame between two protein tags, maltose-binding protein (M) upstream and chitin-binding domain (C) downstream. Protein products of each chimeric gene (M-B-C) were examined in vivo and in vitro by various methods. To characterize the BIL domain activity in its native protein context, some of the domains were cloned with their full or partial native flanks, whereas others were cloned only with single residue flanks.Protein Splicing and Cleavage of an A-type BIL Domain with Ala +1 Residue—BIL4-Cth is one of the 23 A-type BIL domains we identified in the thermophilic bacterium Cth (1Amitai G. Belenkiy O. Dassa B. Shainskaya A. Pietrokovski S. Mol. Microbiol. 2003; 47: 61-73Google Scholar). It is typical of most A-type BIL domains to have all of the intein protein-splicing active site residues with the exception of the C′-flanking nucleophile (supplemental Fig. S3). Instead of Cys, Ser, or Thr invariably present in inteins, BIL4-Cth is followed by an Ala +1 residue. This is the residue present in 18% A-type BIL domains (fraction calculated as weighted average of putative active domains).The BIL4-Cth M-B-C precursor was overexpressed in vivo as a double-tagged protein, and its products were detected and analyzed. Putative protein-splicing products, the excised BIL domain and the ligated M-C flanks, and the M-B- and M-cleavage products were detected. These products were identified by Western blotting of total cell lysates and affinity-purified proteins separated on SDS-PAGE (Fig. 1A). Relative quantities of products were calculated according to measurements taken from Coomassie Blue-stained SDS gels of amylose-purified proteins and total lysates (supplemental Fig. S1). Only trace amounts of the M-B-C precursor were detected under all of the separation procedures, indicating an efficient processing. Spliced product M-C comprised 20–25% of the final products, whereas C′-cleavage product M-B comprised ∼5% of the final products. M and B proteins comprised most of the final products, indicating that they were generated by a combination of N′- and C′-cleavages. The final amount of B protein was much larger than the amount of the M-C-splicing product. This finding implies that both protein splicing and cleavage at its N′ and C′ ends released the B protein. The C product was not identified in the gels, perhaps because of cellular degradation.To characterize the putative splicing product using MALDI MS, the M-C band was extracted from the gel and digested with proteolytic enzymes. The presence of the M and C domains was verified using MS/MS analyses. Furthermore, two peptide masses corresponding to splicing-junction peptides were detected from the chymotrypsin digestion of the M-C band. One mass corresponded to a fully cleaved (N′-GSASRVDCGGLTGL-C′) peptide, and another mass corresponded to its miscleaved form (N′-GSASRVDCGGLTGLNSGLTTNPGVSAWC′) with high mass accuracies (Table II). The ligated splicing junction is between the second and third residues (Ser-Ala) with the Ser being coded by a linker joining the M tag to the BIL domain and the Ala being the native residue downstream of the BIL domain (Ala +1).Table IIMALDI-TOF results of BIL4-Cth splicing junction chymotryptic peptidesSequence position[M+H]+ calculated massaMass calculated with carboxyamidomethyl cysteine modification.[M + H]+ observed massMass accuracySequenceDappm392-4051349.651349.7144.45GSASRVDCGGLTGL392-4182634.262634.80205GSASRVDCGGLTGLNSGLTTNPGVSAWa Mass calculated with carboxyamidomethyl cysteine modification. Open table in a new tab Spliced BIL domain was purified, and its identity was verified by MS. We were able to purify the BIL domain by heat treatment, probably because it originated from a thermophilic bacterium. Incubation of total cell lysate at 80 °C left only the putative BIL domain in the soluble fraction (Fig. 1B). Intact mass MS analysis of this 15-kDa band identified the expected mass of the BIL domain, and its sequence was verified by MS/MS analysis (see Table IV and data not shown). The exact C′ end of the BIL domain was identified by MS analysis as Asn as expected (Table III).Table IVMALDI-TOF results of BIL domains splicing and cleavage products intact massCloneProbable product[M+H]+ calculated mass[M+H]+ observed massMass accuracykDa%BIL4-CthM-B58.03858.5520.89M43.09243.9832.06B14.96315.0500.58BIL2-RSP-onlyM-B56.78256.0711.25DnaK69.11869.2550.19M-B-C64.14165.4302.00BIL1-RSPGroEL/MC57.332/57.35558.3171.72RCA-1522GroEL57.33257.8100.83 Open table in a new tab Table IIIElectrospray Ionization TOF results of BIL4-Cth C-terminal tryptic peptidesSequence positionCalculated massObserved massaObserved masses are an average of [M+2H]2+, [M+3H]3+, and [M+4H]4+ masses for the first peptide and of [M+3H]3+ and [M+4H]4+ masses for the second peptide.Mass accuracySequenceDappm118-1352108.95452109.053346.8VDDFHTYHVGDNEVLVHN113-1352760.29272760.326112.1VYNFKVDDFHTYHVGDNEVLVHNa Observed masses are an average of [M+2H]2+, [M+3H]3+, and [M+4H]4+ masses for the first peptide and of [M+3H]3+ and [M+4H]4+ masses for the second peptide. Open table in a new tab A putative C′-cleavage product, M-B, was affinity-purified and identified by anti-M antibodies (Fig. 1A). Its intact mass analysis corresponded to the expected mass of a C′-cleavage product (Table IV). Other masses obtained from this sample corresponded to the M tag and to other smaller masses that could result from a cross-contamination of the M-B band by traces of smaller proteins on the gel.A protein band corresponding to the M tag was identified by Coomassie Blue staining and by Western blotting using anti-M antibodies (Fig. 1A and supplemental Fig. S1). This putative N′-cleavage product was observed in total lysates and in elutions of both chitin and amylose affinity columns.To examine whether the Tris cell extraction and protein purification buffer promoted cleavage and splicing of the M-B-C precursor, the extraction and purification procedures were repeated using different buffers (Bis-Tris propane, HEPES, sodium phosphate, and borate). Same products and relative amounts were observed with all of these control buffers (data not shown).In Vivo and in Vitro Cleavage Activities of B-type BIL Domains—B-type BIL domains are more heterogeneous in sequence than A-type domains (1Amitai G. Belenkiy O. Dassa B. Shainskaya A. Pietrokovski S. Mol. Microbiol. 2003; 47: 61-73Google Scholar). To characterize their activity, we cloned three different B-type BIL domains into the double-tagged system (described above): the two BIL domains present in R. sphaeroides termed BIL1-Rsp and BIL2-Rsp and one of the 14 BIL domains present in R. capsulatus termed 1522-Rca. The conserved C′ sequence motif of B-type BIL domains is distinct from those motifs in other known HINT domains (1Amitai G. Belenkiy O. Dassa B. Shainskaya A. Pietrokovski S. Mol. Microbiol. 2003; 47: 61-73Google Scholar). The C′ end of the cloned BIL1-Rsp and 1522-Rca is typical of B-type BIL domains, whereas BIL2-Rsp has an atypical C′ end (supplemental Fig. S3).N′-cleavage of B-type BIL1-Rsp—BIL1-Rsp, a B-type BIL domain from R. sphaeroides, was cloned between M and C tags with its native N′-14 residue and C′-51 residue flanks and overexpressed in E. coli cells. M-B-C precursor M and B-C N′-cleavage products were identified by Coomassie Blue staining and Western blotting of total lysate and affinity-purified protein samples (Fig. 2). To verify the nature of the N′-cleavage product, B-C, the band was micro-sequenced. The resulting sequence (XFTPGT) corresponded to the predicted N′ end of the BIL domain, which also includes Cys-1, which usually cannot be detected by this method (supplemental Table S-I).Fig. 2N′-cleavage activity of BIL1-Rsp. Protein products from E. coli overexpression of M-B-C construct with B-type BIL1-Rsp were eluted from amylose (A), chitin (C), or both (A+C) affinity columns or analyzed in total cell lysate (T). Proteins were separated on SDS-PAGE and either stained with Coomassie Blue or detected by anti-M, anti-C, or anti-GroEL (Anti-G) antibodies. See "Results" for discussion of GroEL cross-detection by anti-C antibodies.View Large Image Figure ViewerDownload (PPT)An additional 58-kDa band was co-purified with the M-B-C precursor. Its analysis suggests that the band might include more than a single protein species. Both anti-M and anti-C antibodies reacted with this band. However, the peptide mapping of the band identified peptides from both the M tag and the E. coli GroEL chaperone protein. Additionally, no peptides from the B and C domains were identified (data not shown). Intact mass of the band identified a mass of 58.317 kDa corresponding to GroEL and an additional unidentified protein mass of 65.175 kDa (Table IV). As a control, we checked a cross-reaction of anti-C antibodies with purified GroEL protein (supplemental Fig. S2B). Anti-C antibodies showed reactivity toward GroEL, probably because of their polyclonal nature.GroEL chaperone was detected in protein samples purified the following affinity columns: on amylose; chitin; and amylose followed by chitin. This indicates a tight and specific binding of GroEL with the precursor and/or protein products. The association of GroEL with unfolded proteins is reversible to some extent upon incubation with ATP-Mg-K (12Bukau B. Horwich A. Cell. 1998; 92: 351-366Google Scholar). Such incubation of washed protein samples bound on chitin reduced but did not eliminate the amount of GroEL eluted from chitin (supplemental Fig. S2B).C′-cleavage of B-type BIL2-Rsp in Vivo, in Vitro, and in Cell-free Systems—BIL2-Rsp was cloned between M and C tags with one native flanking residue at either end (N′-Leu and C′-Pro) and overexpressed in E. coli and in a cell-free system. In both systems, the main product was the M-B-C precursor with small amounts of M-B- and M-cleavage products (Fig. 3A). An additional band of ∼70 kDa appeared above the precursor band when expressed in vivo. This band was identified as the E. coli DnaK chaperone protein. It was not detected in the overexpressed control protein, M-C. Identity of the above products was verified by Western blotting, N-terminal sequencing of the M-B-C band, MALDI-MS peptide mapping of the M-B and DnaK bands, and MALDI MS intact mass analysis of the M-B band (Fig. 3A, Table IV, and data not shown). This last analysis gave a measured mass of 56.071 kDa, slightly smaller than the expected mass of the putative M-B product.Fig. 3C′-cleavage activity of BIL2-Rsp.A, left, protein products from E. coli overexpression of M-B-C construct with B-type BIL2-Rsp were eluted from either amylose (A) or chitin (C) affinity columns or analyzed in total cell lysate (T). Proteins were separated on SDS-PAGE and either stained with Coomassie Blue or detected by anti-M, anti-C, or anti DnaK antibodies. A, right, proteins translated in vitro in a cell-free system were labeled with [35S]Met. B, in vitro incubation of a purified precursor at 4 °C.View Large Image Figure ViewerDownload (PPT)To examine the in vitro activity of BIL2-Rsp, the overexpressed M-B-C precursor was isolated by sequential affinity columns (amylose followed by chitin) and was incubated in the extraction buffer in different temperatures for different time periods. Increasing amounts of the M-B product were clearly detected within 1 day at 4 °C (Fig. 3B). The presence of the M band may be attributed to the N′-cleavage of the BIL domain; however, the complementary B-C band was not detected. Alternatively, this could have resulted from protein degradation.Similar results were observed when BIL2-Rsp domain was cloned with its full native flanks (data not shown). However, this clone also underwent cleavage in an Arg-Arg dipeptide present in the N′-flank of the BIL domain as verified by N-terminal sequencing. This cleavage was also observed when the flanks were cloned without the BIL domain (data not shown). Thus, we suggest that this activity is unrelated to the BIL domain and is probably due to an E. coli protease (perhaps OmpT) that can cleave the BIL domain flank.No Activity of B-type Rca-1522 BIL—1522-Rca B-type BIL domain is natively present in a very large R. capsulatus protein. The domain is preceded by 1821 residues and is followed by 52 residues. The upstream flank of this BIL domain includes RTX (repeats-in toxin) calcium-binding repeat motifs, characteristic of secreted proteins (13Coote J. FEMS Microbiol. Rev. 1992; 8: 137-161Google Scholar). The BIL domain was cloned with 36 N′-flanking residues and 35 C′-flanking residues in the double tag expression vector. Overexpression of the vector yielded only the M-B-C precursor and E. coli GroEL protein as verified by Coomassie Blue staining, Western blotting, N-terminal sequencing, and MS analysis (Table IV and supplemental Fig. S2A).Isolated M-B-C precursor was incubated in vitro at 4 or 37 °C in two different environments of pH 7.4 and 8.5. No products of the precursor were detected under any of these conditions.Species and Protein Host Distribution of BIL Domains—BIL domains were identified originally in species from Gram-negative α, β, and γ Proteobacteria and from Gram-positive Actinobacteria and the Bacillus/Clostridium group (1Amitai G. Belenkiy O. Dassa B. Shainskaya A. Pietrokovski S. Mol. Microbiol. 2003; 47: 61-73Google Scholar). Further data base searches now broaden the taxonomic range of BIL domains to major bacterial divisions and lineages (supplemental Table S-II). A-type BIL domains were found in δ Proteobacteria, Cyanobacteria, Spirochaetes, Planctomycetes, and Verrucomicrobia. B-type BIL domains were found in α Proteobacteria, Rhizobium, and Silicibacter species.Sequence analyses of over a hundred identified BIL flanks reconfirmed our previous observation of the nature of the BIL domain hosts. BIL domains are present in homologs of known and predicted secreted proteins. This is exemplified by Streptomyces avermitilis, Verrucomicrobium, and Gloeobacter A-type BIL domains that are found downstream of long (400–5400 residues) Rhs core elements. Rhs elements are composite genetic elements, and their cores are believed to be cell-surface ligand-binding proteins (14Yong Dong W. J. Bacteriol. 1998; 180: 4102-4110Google Scholar). The BIL domains are present in the hypervariable core extension region that can be shuffled between the core and downstream open-reading frame regions.DISCUSSIONIn this study, we show that a typical A-type BIL domain is capable of protein splicing without a C′-nucleophilic +1 residue and that B-type BIL domains can cleave their N′ or C′ ends. Both types of domains are not uncommon, appearing in diverse bacterial divisions. These findings reflect the auto-processing nature of intein-like domains. We explain the N′- and C′-cleavage of B-type BIL domains by reactions occurr
Referência(s)