Comparison of two scaffolding polypeptides for the integration of different proteins in synthetic complexes derived from the Clostridium thermocellum cellulosome

Comparison of two scaffolding polypeptides for the integration of different proteins in synthetic complexes derived from the Clostridium thermocellum cellulosome Emmanuelle Leibovitz and Pierre Be´guin Unite´ de Physiologie Cellulaire and URA 1300 CNRS, De´partement des Biotechnologies, Institut Pasteur, Paris, France The targeted integration of different polypeptides within artificial complexes derived from the Clostridium thermocellum cellulosome was investigated. Two scaffolding polypeptides, each containing two cohesin domains, were compared. Cip6 consisted of a tandem duplication of a type I cohesin domain. Cip20 was constructed by fusing the same type I cohesin domain to the type II cohesin domain of SdbA. Cip6 was mixed with varying proportions of CelD, carrying a type I dockerin domain, and CelC-DSCelD, in which the dockerin domain of CelD was fused to the endoglucanase CelC. Likewise, Cip20 was mixed with varying proportions of CelD and CelC-DSCipA-H, which carries the type II dockerin domain of CipA. Complex formation was analyzed by non-denaturing gel electrophoresis and densitometry and the composition of the bands observed was confirmed by denaturing gel electrophoresis in a second dimension. With optimal proportions of the components, the ternary complex formed of Cip6, CelD, and CelC-DSCelD accounted for 36% of the proteins entering the gel. In contrast, up to a 56% yield was obtained for the ternary complex formed of Cip20, CelD, and CelC-DSCipA-H. The results emphasize the advantage of using different types of cohesin-dockerin pairs in the design of cellulosome-derived complexes. © 1998 Elsevier Science Inc. Keywords: Cellulosome; cellulases; dockerin domain; cohesin domain; Clostridium thermocellum; protein complexes

Introduction The cellulosome concept was coined in 1983 to designate the high molecular weight form of cellulase produced by the thermophilic bacterium Clostridium thermocellum.1 It was first studied with a view to biotechnological applications such as treatment of cellulosic wastes and conversion of cellulosic substrates into solvents and fuels. Cellulosomes are cell-bound, multienzyme complexes containing 14 –18 different subunits most of which are endowed with cellulolytic or hemicellulolytic activity.2– 4 The association of cellulases within the cellulosome is required for efficient degradation of crystalline cellulose.1

Address reprint requests to Dr. P. Be´guin, Unite´ de Physiologie Cellulaire, De´partement des Biotechnologies, Institut Pasteur, 28 rue du Dr. Roux, 75724 Paris Cedex 15, France Received 30 July 1997; revised 3 November 1997; accepted 11 November 1997

Enzyme and Microbial Technology 22:588 –593, 1998 © 1998 Elsevier Science Inc. All rights reserved. 655 Avenue of the Americas, New York, NY 10010

The catalytic subunits of the cellulosome are bound to a noncatalytic scaffolding protein called CipA (cellulosome integrating protein).5–7 This scaffolding protein contains nine reiterated domains7 which act as receptors for the catalytic subunits8,9 and are called cohesin domains.10 Each catalytic subunit comprises a highly conserved domain, termed dockerin domain,10 composed of a duplication of 23 residues which binds to the cohesin domains of CipA.6,8,11 The interaction between dockerin domains of catalytic subunits and cohesin domains of CipA is called interaction of type I.12 A second type of cohesin-dockerin interaction with a specificity different from that of the type I was identified in C. thermocellum.12 This interaction is responsible for the binding of the cellulosome to the cell surface and involves the dockerin domain located at the COOH terminus of CipA and cohesin domains of the surface proteins SdbA, OlpB, and probably ORF2p.12–15 Once the basic organization of the cellulosome was elucidated, it became clear that it offered a remarkably

0141-0229/98/$19.00 PII S0141-0229(98)00251-2

Comparison of two scaffolding polypeptides: E. Leibovitz and P. Be´guin versatile opportunity to design multiprotein complexes integrating a variety of polypeptides.10,11 Indeed, noncellulosomal proteins can be fused to dockerin domains which allow them to bind to CipA or individual cohesin domains.11,16 Bayer et al.10 reviewed the numerous potential applications of cellulosome-derived complexes (coupled enzyme reactions, etc.); however, the general lack of binding specificity between various dockerin and cohesin domains of type I16 –18 would be expected to hamper the targeted integration of different polypeptides into complexes of defined topology and stoichiometry. As a first step toward the design of such complexes, we investigated how specific complex formation could be improved by using artificial scaffolding polypeptides composed of two different types of cohesin domains. Two scaffolding polypeptides were compared. The Cip6 polypeptide consisted of a tandem duplication of the seventh cohesin domain (type I) of CipA.19 The Cip20 polypeptide was constructed by fusing the seventh cohesin domain of CipA to the type II cohesin domain of SdbA (Figure 1). Cip6 was mixed with endoglucanase CelD carrying a dockerin domain of type I and CelC-DSCelD in which the same dockerin domain is fused to endoglucanase CelC.11 Cip20 was mixed with CelD and CelC-DSCipA-H, which harbors the type II dockerin domain of CipA. Owing to the possibility of forming Cip6/ CelC-DSCelD2 and Cip6/CelD2 complexes, yields were expected to be lower for the ternary Cip6/CelC-DSCelD/ CelD complex than for Cip20/CelC-DSCipA-H/CelD. Formation of binary and ternary complexes was analyzed by native gel electrophoresis and two-dimensional electrophoresis under native and denaturing conditions. Yields were quantified by densitometry.

Materials and methods Bacterial strains, plasmids, and culture conditions The bacterial strains and plasmids used in this work are described in Table 1. Escherichia coli TGI was used for cloning and sequencing. CelD and CelC-DSCelD were produced in E. coli JM101 and E. coli TGI, respectively. CelC-DSCipA-H, Cip6, and Cip20 were synthesized in E. coli M15(pREP4). E. coli was grown at 37°C in Luria-Bertani medium.20 Antibiotics were added depending on plasmids present in the host: ticarcillin (100 mg ml21), chloramphenicol (30 mg ml21), and kanamycin (25 mg ml21).

DNA manipulations DNA manipulations were performed as described by Ausubel et al.21 Restriction enzymes were used as recommended by the suppliers (New England Biolabs Beverly, MA, USA; Amersham, Buckinghamshire, England; Boehringer Mannheim, Germany). PCR amplifications22 were performed using 2.5 U pfu DNA polymerase (Stratagene La Jolla, CA, USA) and 100 pmol of each oligonucleotide primers in 100 ml of reaction mix. Oligonucleotide primers were supplied by Gibco BRL Cergy-Pontoise, France. Cloned PCR fragments were verified by sequencing.

Figure 1 Construction of pCT337 encoding the CelCDSCipA-H protein. The sequence encoding the dockerin domain of CipA is shown by hatched boxes. Numbers refer to the nucleotide sequence of CipA.7 Nucleotides changed by PCR amplification are shown in boldface type. pUC18 and pQE vectors are indicated by a thin line and the sequence encoding six histidines is represented by a black box not drawn to scale. Transcription is from left to right and indicated by an arrow. DraI, D; EcoRI, E; HindIII, H; KpnI, K; SacI, Sc; SmaI, Sm; XhoI, X; multiple cloning site, MCS.

XhoI and BamHI sites and encoding the dockerin domain of CipA was synthesized by PCR (Figure 1). The forward primer was 59-CAG GCT CCA ACT CGA GTG TGG GTA GGA-39, and the reverse primer was 59-TCC CAA TTT TAA TGG ATC CTG TGC GTC GTA-39. Thirty-five amplification cycles were performed with the following parameters: annealing, 1 min at 55°C; elongation, 1 min at 72°C; and denaturation, 1 min at 95°C. The PCR fragment digested with XhoI and BamHI, and a 1 kb SacI-XhoI fragment encoding CelC were inserted into the pQE-51 plasmid digested with BamHI and SacI, then the fragment coding for CelC-DSCipA was cut out from pQE-51 by digestion with BamHI and SacI and inserted between the 1 kb SacI-BglI fragment of pQE–51 and the 2.4 kb BglI-BglII fragment of pQE–17, yielding pCT337 which encodes the CelC-DSCipA-H polypeptide.

Construction of pCip20 Construction of pCT337 Six His residues were fused to the COOH terminus of the CelC-DSCipA polypeptide17 to facilitate purification and ensure integrity of the dockerin domain. A 234-bp fragment flanked by

The seventh cohesin domain of CipA (type I) was fused to the NH2 terminus of the SdbA cohesin domain (type II) and to six His residues to yield a chimeric scaffolding protein comprising two cohesin domains of different specificity. A 548-bp fragment

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Papers Table 1 Bacterial strains and plasmids Strain and plasmid

Escherichia coli strains TGI JM101 M15(pREP4) Plasmids pQE-51 pQE-17 pQE-31 pBC SK2 pBC/Bgl pCT332 pCT337 pCip3 pCT1834 pCip18 pCip20 pCip6

Relevant features

Source or reference

D(lac-pro) thi supE hsdD5/F9tra-36 proA1B1lacIqlacZDM15 D(lac-proAB)thi supE/F9 traD36 proA1B1lacIqlacZDM15

[34] [35] Qiaexpress kit (Qiagen)

Ampr, expression vector Ampr, expression vector Ampr, expression vector Camr, cloning vector Camr, cloning vector derived from pBC SK2 by insertion of a BglII site pUC18 derivative encoding CelC-DSCipA pQE-51/pQE-17 derivative encoding CelC-DSCipA-H pBC SK2 derivative containing the PCR fragment encoding the 7th cohesin domain of CipA followed by a linker pBC/Bgl derivative containing the PCR fragment encoding the cohesin domain of SdbA pBC/Bgl derivative containing a fragment encoding the fusion between the 7th cohesin domain of CipA and the cohesin domain of SdbA pQE-31 derivative encoding Cip20 fused to 6 His residues pQE-31 derivative encoding Cip6 fused to 6 His residues

Qiaexpress kit (Qiagen) Qiaexpress kit (Qiagen) Qiaexpress kit (Qiagen) Stratagene [36]

encoding the cohesin domain of SdbA was synthesised by PCR (Figure 2). The forward primer was 59-CG GCG GGA GAT CTA AGG GCA GAT AAA GCC-39 and the reverse primer was 59-TCC CGG TGT CTA GAA AGG CTC GTC ACC C-39. Thirty-five amplification cycles were performed using the following parameters: annealing, 1 min at 45°C; elongation, 3 min at 72°C; and denaturation, 2 min at 95°C. The PCR fragment was digested with BglII and XbaI, and inserted between the BglII and XbaI sites of pBC/Bgl to yield pCT1834. The fragment encoding the seventh cohesin domain of CipA followed by the adjacent linker was excised from pCip3 by digestion with SalI and BamHI, and inserted between the SalI and BglII sites of pCT1834, yielding pCip18. The BglII-SacI fragment of pCip18 was recloned in pQE-31, yielding pCip20; thus, the sequence encoding the chimeric polypeptide Cip20 was fused to six His codons.

[17] This study [19] This study This study This study [19]

Protein purifications Unless otherwise stated, all operations were performed at 4°C. Endoglucanase CelD and the chimeric protein CelC-DSCelD, comprising the dockerin domain of CelD fused to endoglucanase CelC, were purified from inclusion bodies as described by Tokatlidis et al.11 CelC-DSCipA-H, Cip6, and Cip20 which comprised six His residues, were purified as described previously on a Ni21-nitrilotriacetic acid column.12,19,23 Protein concentrations were calculated by measuring the absorbance at 280 nm with an Uvikon Spectrophotometer 930 (Kontron Instruments) and using the following molar extinction coefficients (m21 cm21):24 CelD, 123,200; CelC-DSCelD, 79,800; CelCDSCipA-H, 84,000; Cip6, 11,200; and Cip20, 19,600.

Figure 2 Construction of pCip20 encoding the Cip20 protein. The segments encoding the different modules are represented by boxes of different patterns. Numbers refer to the amino acid sequence of CipA and SdbA for the seventh cohesin domain of CipA and the cohesin domain of SdbA, respectively.7,12 Nucleotides that were changed by PCR amplifications are shown in boldface type. Vectors are indicated by a thin line. The sequence encoding six histidines is shown by a white box not drawn to scale. Transcription is from left to right and indicated by an arrow. BglII, Bg; BamHI, Bm; EcoRI, E; HindIII, H; KpnI, K; SalI, S; SacI, Sc; XbaI, Xb.

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Comparison of two scaffolding polypeptides: E. Leibovitz and P. Be´guin

Figure 3 Visualization of complexes performed with Cip6 (panel A) or Cip20 (panel B). Proteins were analyzed by electrophoresis on 8.5% (A) or 10% (B) polyacrylamide gels under nondenaturing conditions in the presence of 2 mM CaCl2. Panel A. Lane 1: Cip6 (73 pmol); 2: CelD (73 pmol); 3– 8: Cip6 (73 pmol), CelD (73 pmol) and varying amounts of CelC-DSCelD: 0 pmol (lane 3), 46 pmol (lane 4), 69 pmol (lane 5), 92 pmol (lane 6), 230 pmol (lane 7), and 350 pmol (lane 8); 9: Cip6 (73 pmol), CelC-DSCelD (140 pmol); 10: CelC-DSCelD (140 pmol). Panel B. Lane 1: Cip20 (66 pmol); 2: CelC-DSCipA-H (48 pmol); 3– 8: Cip20 (66 pmol), CelC-DSCipA-H (48 pmol) and varying amounts of CelD: 0 pmol (lane 3), 15 pmol (lane 4), 30 pmol (lane 5), 45 pmol (lane 6), 60 pmol (lane 7), and 120 pmol (lane 8); 9: Cip20 (66 pmol), CelD (30 pmol); 10: CelD (30 pmol). Positions of individual proteins and complexes are indicated

Formation and analysis of cohesin-dockerin complexes Proteins were mixed in 50 mm Tris-HCl pH 7.5 with 2 mm CaCl2 and incubated overnight at 4°C. Complexes were analyzed by polyacrylamide gel electrophoresis under nondenaturing conditions using the Laemmli buffer system containing 2 mm CaCl2, but no SDS or b-mercaptoethanol.25 Polyacrylamide gels were stained with Coomassie blue and bands were analyzed by densitometry with a MasterScan Interpretative Densitometer (Scanalytics, CSPI). The composition of complexes observed in nondenaturing electrophoresis was analyzed by two-dimensional electrophoresis. Complexes were first subjected to a nondenaturing electrophoresis. The corresponding gel lane was cut, treated for 2 min at 100°C with denaturing sample buffer and loaded on top of a second, SDS-containing polyacrylamide gel. After Coomassie blue staining of the gel, proteins composing complexes were visualized as aligned spots.

Results Cip6/CelC-DSCelD/CelD complexes Nondenaturing gel analysis of mixtures containing different proportions of Cip6, CelD, and CelC-DSCelD is shown in Figure 3A. Identification of the components present in each

Figure 4 Two-dimensional electrophoresis. The first dimension was performed as described in Figure 3 on a 8.5% (panel A) or 10% (panel B) polyacrylamide gel under nondenaturing conditions. Migration was from left to right. A stained gel lane with an identical sample run on the same gel is shown on top of each two-dimensional gel. The second dimension (vertical) was performed under denaturing conditions. Panel A. First dimension: Cip6 (140 pmol), CelD (150 pmol), CelC-DSCelD (180 pmol). Second dimension: lane 1, molecular mass markers 1 Cip6 (70 pmol); 2, CelC-DSCelD (16.5 pmol); 3, CelD (19 pmol). Panel B. First dimension: Cip20 (250 pmol), CelD (140 pmol), CelCDSCipA-H (95 pmol). Second dimension: lane 1, CelD (19 pmol); 2, CelC-DSCipA-H (17 pmol); 3, Cip20 (61 pmol); 4, molecular mass markers. Positions of Cip20, Cip6, and catalytic domains of CelD and CelC are indicated. Molecular masses are indicated in kilodaltons

of the bands was confirmed by denaturing gel electrophoresis in a second dimension (Figure 4A). When Cip6 was mixed with CelD (lane 3) or with CelC-DSCelD (lane 9), new bands were observed which migrated differently from the separated components (lanes 1, 2, and 10). As described previously,19 binding of CelD to cohesin domains duplicated in tandem is highly cooperative; thus, only one complex band corresponding to Cip6/CelD2 was seen in lane 3 and no band corresponding to occupancy of a single cohesin domain of Cip6 was detectable even though free Cip6 was in excess. Likewise, only one type of complex was observed upon mixing varying proportions of Cip6 and CelC-DSCelD (lane 9 and data not shown). By analogy with CelD, the complex most likely corresponds to the Cip6/ CelC-DSCelD2 form. To analyze the formation of the ternary complex Cip6/CelC-DSCelD/CelD, a Cip6:CelD ratio was chosen such as to saturate half of the cohesin domains provided by Cip6 (lane 3), and this ratio was held constant while increasing amounts of CelC-DSCelD were added to the mixture (lanes 4 – 8). At low CelD concentrations, the Cip6/CelD2 and Cip6/CelC-DSCelD2 complexes appeared to segregate (lanes 4 and 5) even though CelD and Enzyme Microb. Technol., 1998, vol. 22, May 15

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Papers CelC-DSCelD were added simultaneously to Cip6. A new species migrating between Cip6/CelD2 and Cip6/CelCDSCelD2 was observed when CelC-DSCelD was added in excess relative to Cip6 (lanes 6 – 8). Two-dimensional gel analysis confirmed that the intermediate band contained Cip6, CelC-DSCelD, and CelD (Figure 4A). The highest yield of the ternary complex was obtained in lane 7 in which it accounted for 36% of the material entering the gel. As expected, CelD was partially displaced from the complex at high CelC-DSCelD concentration (lane 8).

Cip20/CelC-DSCipA-H/CelD complexes Nondenaturing gel analysis of mixtures containing different proportions of Cip20, CelC-DSCipA-H, and CelD is shown in Figure 3B. Identification by two-dimensional gel electrophoresis of the bands present in native gel is shown in Figure 4B. Mixtures containing Cip20 and either CelCDSCipA-H (lane 3) or CelD (lane 9) displayed bands corresponding to the binary complexes Cip20/CelCDSCipA-H and Cip20/CelD, respectively. To analyze the formation of the ternary complex Cip20/CelC-DSCipA-H/ CelD, a Cip20:CelC-DSCipA-H ratio was chosen such as to yield a single band of Cip20/CelC-DSCipA-H complex (lane 3). This ratio was kept constant while increasing amounts of CelD were added to the mixture (lanes 4 – 8). Increasing the proportion of CelD led to the progressive disappearance of the Cip20/CelC-DSCipA-H complex and to the concomitant appearance of the ternary complex Cip20/CelC-DSCipA-H/CelD which migrated slower than either of the two binary complexes (lanes 4 – 8). The highest yield of ternary complex was obtained in lane 6 in which it accounted for 56% of the total proteins detected by densitometry. Binding of CelD interfered to some extent with the stability of the Cip20/CelC-DSCipA-H interaction. As a consequence, some non-complexed CelC-DSCipA-H is visible in lanes 6 – 8 along with some binary Cip20/CelD complex.

Discussion Previous studies have demonstrated that cohesin and dockerin domains may be used to integrate various polypeptides into artificial complexes based on the same cohesin-dockerin interactions as the cellulosome;11,16,17,19,26 –28 however, the formation of complexes involving more than one kind of polypeptide bound to the scaffolding component still needs to be investigated. In this study, we explored the mode of association of two different polypeptides with the same scaffolding protein. Catalytic domains of CelD and CelC were used to construct complexes with the scaffolding proteins Cip6 and Cip20, comprising a tandem duplication of type I cohesin domains and a fusion between a type I and a type II cohesin domains, respectively. Theoretically, when components are mixed in stoichiometric proportions, a mixture of Cip6, CelC-DSCelD, and CelD should yield 25% of Cip6/CelD2, 25% of Cip6/CelC-DSCelD2, and 50% of Cip6/CelC-DSCelD/CelD complexes. However, with Cip20, CelD, and CelC-DSCipA-H, the complex Cip20/ CelD/CelC-DSCipA-H should be formed with a 100% yield. As expected, the yield of ternary complex was higher 592

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for Cip20/CelC-DSCipA-H/CelD (56%) than for Cip6/ CelC-DSCelD/CelD (36%); moreover, the yield of the ternary complex Cip6/CelC-DSCelD/CelD was somewhat overestimated since the fraction of CelC-DSCelD which precipitated on top of the gel was not included in the densitometric scan (Figure 3A). Although better results were obtained with Cip20 than Cip6, in both cases yields of ternary complexes were below theoretical. Possibly, conditions encountered during electrophoresis (pH 8.8 in the separating gel) may lead to partial dissociation of the complexes. In addition, binding of one component to the scaffolding polypeptide may interfere with binding of the other. In the case of Cip20, some CelC-DSCipA-H was released from the Cip20 scaffolding protein when CelD was added (Figure 3B). Preferential displacement of CelCDSCipA-H is consistent with the lower affinity of the cohesin-dockerin interactions of type II as compared to type I.15 In the case of Cip6, which contains two identical cohesin domains, CelC-DSCelD and CelD tended to segregate and bind to separate Cip6 molecules; thus, the binary complexes Cip6/CelC-DSCelD2 and Cip6/CelD2 were found preferentially to the ternary complex Cip6/CelCDSCelD/CelD (Figure 3A, lanes 4 and 5). This suggests that interactions between catalytic domains may influence the order of the subunits along CipA and the topology of the cellulosome. Improving the usefulness of artificial cellulosome-derived complexes could be achieved by increasing the stability of the complexes, and particularly of type II cohesindockerin interactions. Chemical cross-linking or the isolation of mutants with a higher affinity may help the formation of more stable constructs. In addition, introducing further cohesin-dockerin types with distinct specificities would enhance the versatility of the system by allowing the targeted attachment of more than two different polypeptides along the scaffolding component. Mutagenesis or the isolation of cohesin-dockerin pairs found in other cellulosomeforming organisms29,30 may provide ligands with new specificities. Such tools may help biotechnologists to emulate nature in which multienzyme complexes often participate in sequential reactions. Such is the case, for example, for fatty acid synthesis, polyketide synthesis, and the nonribosomal synthesis of peptides.31–33

Acknowledgments We thank A. Dridi and M.-K. Chaveroche for purifications of Cip6, CelC-DSCelD, and CelD. We are grateful to J.-P. Aubert and M. Schwartz for their continuing interest and support. E. Leibovitz was the recipient of a fellowship from the Ministe`re de l’Enseignement Supe´rieur et de la Recherche.

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