Scaffolding Protein CipC Research Articles

Intein-mediated assembly of tunable scaffoldins for facile synthesis of designer cellulosomes.

In this study, extended artificial scaffoldins possessing multiple cohesin modules were created in vivo by employing split-intein-mediated protein ligation. Artificial scaffoldins having one Clostridium thermocellum cohesin (Coht), one carbohydrate binding module (CBM) from Clostridium cellulolyticum scaffolding protein CipC, and one to five cohesins (Cohc) derived from CipC, were assembled. These scaffoldins were used to assemble cellulosomal enzyme complexes for investigating the interplay among endoglucanase, exoglucanase, and scaffoldin-borne CBM, on the hydrolysis of a model microcrystalline cellulose substrate, Avicel. The cellulosomal complexes were assembled in vitro by incubating recombinant C. thermocellum endoglucanase (At) and C. cellulolyticum exoglucanase (Ec), with the various artificial scaffoldins. Under a fixed total cellulase concentration, improved hydrolysis is noted by recruiting both Ec and At on the same scaffoldin, for all scaffoldins tested, compared with free cellulases. The improvement is more profound with scaffoldins having a higher Cohc/Coht ratio (i.e., increased Ec/At ratio). Furthermore, among scaffoldins having the same Cohc/Coht ratio, highest rates of Avicel hydrolysis are noted when Coht, and hence an endoglucanase, is situated next to the CBM and not flanked by Cohc. These results point to the importance of using scaffoldins with sufficiently high numbers of cohesin units to achieve an optimal exo-/endo-glucanase ratio to create efficient designer cellulosomes. Furthermore, intein-trans-splicing is proven here to be an effective method for assembling complex scaffoldins and more intricate cellulosomes.

Are Cellulosome Scaffolding Protein CipC and CBM3-Containing Protein HycP, Involved in Adherence of Clostridium cellulolyticum to Cellulose?

Clostridium cellulolyticum, a mesophilic anaerobic bacterium, produces highly active enzymatic complexes called cellulosomes. This strain was already shown to bind to cellulose, however the molecular mechanism(s) involved is not known. In this context we focused on the gene named hycP, encoding a 250-kDa protein of unknown function, containing a Family-3 Carbohydrate Binding Module (CBM3) along with 23 hyaline repeat modules (HYR modules). In the microbial kingdom the gene hycP is only found in C. cellulolyticum and the very close strain recently sequenced Clostridium sp BNL1100. Its presence in C. cellulolyticum guided us to analyze its function and its putative role in adhesion of the cells to cellulose. The CBM3 of HycP was shown to bind to crystalline cellulose and was assigned to the CBM3b subfamily. No hydrolytic activity on cellulose was found with a mini-protein displaying representative domains of HycP. A C. cellulolyticum inactivated hycP mutant strain was constructed, and we found that HycP is neither involved in binding of the cells to cellulose nor that the protein has an obvious role in cell growth on cellulose. We also characterized the role of the cellulosome scaffolding protein CipC in adhesion of C. cellulolyticum to cellulose, since cellulosome scaffolding protein has been proposed to mediate binding of other cellulolytic bacteria to cellulose. A second mutant was constructed, where cipC was inactivated. We unexpectedly found that CipC is only partly involved in binding of C. cellulolyticum to cellulose. Other mechanisms for cellulose adhesion may therefore exist in C. cellulolyticum. In addition, no cellulosomal protuberances were observed at the cellular surface of C. cellulolyticum, what is in contrast to reports from several other cellulosomes producing strains. These findings may suggest that C. cellulolyticum has no dedicated molecular mechanism to aggregate the cellulosomes at the cellular surface.

Transcriptional Regulation of the Clostridium cellulolyticum cip-cel Operon: a Complex Mechanism Involving a Catabolite-Responsive Element

The cip-cel cluster of genes plays an important role in the catabolism of the substrate cellulose by Clostridium cellulolyticum. It encodes several key components of the cellulosomes, including the scaffolding protein CipC and the major cellulase Cel48F. All the genes of this cluster display linked transcription, focusing attention on the promoter upstream from the first gene, cipC. We analyzed the regulation of the cipC promoter using a transcriptional fusion approach. A single promoter is located between nucleotides -671 and -643 with respect to the ATG start codon, and the large mRNA leader sequence is processed at position -194. A catabolite-responsive element (CRE) 414 nucleotides downstream from the transcriptional start site has been shown to be involved in regulating this operon by a carbon catabolite repression mechanism. This CRE is thought to bind a CcpA-like regulator complexed with a P-Ser-Crh-like protein. Sequences surrounding the promoter sequence may also be involved in direct (sequence-dependent DNA curvature) or indirect (unknown regulator binding) regulation.

The cellulosome of Clostridium cellulolyticum

Clostridium cellulolyticum ATCC 35319 has been extensively studied in the past few years on both the enzymatic and metabolic aspects of cellulose degradation and is considered as the model of mesophilic cellulolytic clostridia. As is true of most cellulolytic clostridia, this bacterium possesses an extracellular multi-enzymatic complex, the cellulosome. Cellulosomes are highly efficient in the degradation of plant cell wall polysaccharides. In C. cellulolyticum, most of the cellulosomal genes are localised in an approximately 26 kb DNA fragment, the cel cluster, where 12 genes have been identified to date cipC- cel48F- cel8C- cel9G- cel9E- orfX- cel9H- cel9J- man5K- cel9M- rgl11Y- cel5N. The C. cellulolyticum cellulosome is organised around the scaffolding protein CipC, which permits the binding of the different cellulosomal enzymes via interactions of dockerin–cohesin domains. Twelve cellulosomal enzymes have been identified including cellulases, hemicellulase and pectinase. The state of knowledge about the structure, ultrastructure, enzymatic activity, regulation and extracellular assembly of the C. cellulolyticum cellulosome are reviewed, and potential biotechnological exploitations discussed.

Crystal structure of a cohesin module from Clostridium cellulolyticum: implications for dockerin recognition

In the assembly of the Clostridium cellulolyticum cellulosome, the multiple cohesin modules of the scaffolding protein CipC serve as receptors for cellulolytic enzymes which bear a dockerin module. The X-ray structure of a type I C. cellulolyticum cohesin module (Cc-cohesin) has been solved using molecular replacement, and refined at 2.0 Å resolution. Despite a rather low sequence identity of 32 %, this module has a fold close to those of the two Clostridium thermocellum cohesin (Ct-cohesin) modules whose 3D structures have been determined previously. Cc-cohesin forms a dimer in the crystal, as do the two Ct-cohesins. We show here that the dimer exists in solution and that addition of dockerin-containing proteins dissociates the dimer. This suggests that the dimerization interface and the cohesin/dockerin interface may overlap. The nature of the overall surface and of the dimer interface of Cc-cohesin differ notably from those of the Ct-cohesin modules, being much less polar, and this may explain the species specificity observed in the cohesin/dockerin interaction of C. cellulolyticum and C. thermocellum. We have produced a topology model of a C. cellulolyticum dockerin and of a Cc-cohesin/dockerin complex using homology modeling and available biochemical data. Our model suggests that a special residue pair, already identified in dockerin sequences, is located at the center of the cohesin surface putatively interacting with the dockerin.

Solution structure of the module X2_1 of unknown function of the cellulosomal scaffolding protein CipC of Clostridium cellulolyticum

Multidimensional, homo- and heteronuclear magnetic resonance spectroscopy combined with dynamical annealing has been used to determine the structure of a 94 residue module (X2_1) of the scaffolding protein CipC from the anaerobic bacterium Clostridium cellulolyticum. An experimental data set comprising 1647 nuclear Overhauser effect-derived restraints, 105 hydrogen bond restraints and 66 φ torsion angle restraints was used to calculate 20 converging final solutions. The calculated structures have an average rmsd about the mean structure of 0.55(±0.11) Å for backbone atoms and 1.40(±0.11) Å for all heavy atoms when fitted over the secondary structural elements. The X2_1 module has an immunoglobulin-like fold with two β-sheets packed against each other. One sheet contains three strands, the second contains four strands. An additional strand is intercalated between the β-sandwich, as well as two turns of a 3 .10 helix. X2_1 has a surprising conformational stability and may act as a conformational linker and solubility enhancer within the scaffolding protein. The fold of X2_1 is very similar to that of telokin, titin Ig domain, hemolin D2 domain, twitchin immunoglobulin domain and the first four domains of the IgSF portion of transmembrane cell adhesion molecule. As a consequence, the X2_1 module is the first prokaryotic member assigned to the I set of the immunoglobulin superfamily even though no sequence similarity with any member of this superfamily could be detected.

The extracellular xylan degradative system in Clostridium cellulolyticum cultivated on xylan: evidence for cell-free cellulosome production.

In this study, we demonstrate that the cellulosome of Clostridium cellulolyticum grown on xylan is not associated with the bacterial cell. Indeed, the large majority of the activity (about 90%) is localized in the cell-free fraction when the bacterium is grown on xylan. Furthermore, about 70% of the detected xylanase activity is associated with cell-free high-molecular-weight complexes containing avicelase activity and the cellulosomal scaffolding protein CipC. The same repartition is observed with carboxymethyl cellulase activity. The cellulose adhesion of xylan-grown cells is sharply reduced in comparison with cellulose-grown cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that cellulosomes derived from xylan- and cellulose-grown cells have different compositions. In both cases, the scaffolding protein CipC is present, but the relative proportions of the other components is dramatically changed depending on the growth substrate. We propose that, depending on the growth substrate, C. cellulolyticum is able to regulate the cell association and cellulose adhesion of cellulosomes and regulate cellulosomal composition.

Sequence analysis of scaffolding protein CipC and ORFXp, a new cohesin-containing protein in Clostridium cellulolyticum: comparison of various cohesin domains and subcellular localization of ORFXp.

The gene encoding the scaffolding protein of the cellulosome from Clostridium cellulolyticum, whose partial sequence was published earlier (S. Pagès, A. Bélaïch, C. Tardif, C. Reverbel-Leroy, C. Gaudin, and J.-P. Bélaïch, J. Bacteriol. 178:2279-2286, 1996; C. Reverbel-Leroy, A. Bélaïch, A. Bernadac, C. Gaudin, J. P. Bélaïch, and C. Tardif, Microbiology 142:1013-1023, 1996), was completely sequenced. The corresponding protein, CipC, is composed of a cellulose binding domain at the N terminus followed by one hydrophilic domain (HD1), seven highly homologous cohesin domains (cohesin domains 1 to 7), a second hydrophilic domain, and a final cohesin domain (cohesin domain 8) which is only 57 to 60% identical to the seven other cohesin domains. In addition, a second gene located 8.89 kb downstream of cipC was found to encode a three-domain protein, called ORFXp, which includes a cohesin domain. By using antiserum raised against the latter, it was observed that ORFXp is associated with the membrane of C. cellulolyticum and is not detected in the cellulosome fraction. Western blot and BIAcore experiments indicate that cohesin domains 1 and 8 from CipC recognize the same dockerins and have similar affinity for CelA (Ka = 4.8 x 10(9) M-1) whereas the cohesin from ORFXp, although it is also able to bind all cellulosome components containing a dockerin, has a 19-fold lower Ka for CelA (2.6 x 10(8) M-1). Taken together, these data suggest that ORFXp may play a role in cellulosome assembly.

Characterization of the cellulolytic complex (cellulosome) produced by Clostridium cellulolyticum.

The cellulolytic complex was isolated from Clostridium cellulolyticum grown on cellulose. Upon gel filtration, the complex was found to consist mainly of 600-kDa units, along with a 16-MDa aggregate. Its ability to degrade various substrates and its capacity to bind to the crystalline cellulose were measured. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, N-terminal sequencing, and blotting analysis showed that all of the known cellulases of this organism are present in this complex. Three major components were observed: the first component, a noncatalytic, large (160-kDa) protein, was identified based on its ability to bind to the dockerin-containing cellulases as scaffolding protein CipC. The other two components, which had molecular masses of 94 and 80.6 kDa, were identified as CelE and CelF, respectively. The identified cellulases and some other components of the cellulosome were able to bind to a miniCipC1 construct. In addition to providing an extensive description of the system, the results of the present study confirm that the dockerin-cohesin domain interaction plays an essential role in the constitution of the cellulosome.

Interaction between the endoglucanase CelA and the scaffolding protein CipC of the Clostridium cellulolyticum cellulosome

The 5' end of the cipC gene, coding for the N-terminal part of CipC, the scaffolding protein of Clostridium cellulolyticum ATCC 35319, was cloned and sequenced. It encodes a 586-amino-acid peptide, including several domains: a cellulose-binding domain, a hydrophilic domain, and two hydrophobic domains (cohesin domains). Sequence alignments showed that the N terminus of CipC and CbpA of C. cellulovorans ATCC 35296 have the same organization. The mini-CipC polypeptide, containing a cellulose-binding domain, hydrophilic domain 1, and cohesin domain 1, was overexpressed in Escherichia coli and purified. The interaction between endoglucanase CelA, with (CelA2) and without (CelA3) the characteristic clostridial C-terminal domain called the duplicated-segment or dockerin domain, and the mini-CipC polypeptide was monitored by two different methods: the interaction Western blotting (immunoblotting) method and binding assays with biotin-labeled protein. Among the various forms of CelA (CelA2, CelA3, and an intermediary form containing only part of the duplicated segment), only CelA2 was found to interact with cohesin domain 1 of CipC. The apparent equilibrium dissociation constant of the CelA2-mini-CipC complex was 7 x 10(-9)M, which indicates that there exists a high affinity between these two proteins.