Production of heterologous and chimeric scaffoldins by Clostridium acetobutylicum ATCC 824

Clostridium acetobutylicum ATCC 824 converts sugars and various polysaccharides into acids and solvents. This bacterium, however, is unable to utilize cellulosic substrates, since it is able to secrete very small amounts of cellulosomes. To promote the utilization of crystalline cellulose, the strategy we chose aims at producing heterologous minicellulosomes, containing two different cellulases bound to a miniscaffoldin, in C. acetobutylicum. A first step toward this goal describes the production of miniCipC1, a truncated form of CipC from Clostridium cellulolyticum, and the hybrid scaffoldin Scaf 3, which bears an additional cohesin domain derived from CipA from Clostridium thermocellum. Both proteins were correctly matured and secreted in the medium, and their various domains were found to be functional.     

Cohesin-dockerin interactions within and between Clostridium josui and Clostridium thermocellum: binding selectivity between cognate dockerin and cohesin domains and species specificity

The cellulosome components are assembled into the cellulosome complex by the interaction between one of the repeated cohesin domains of a scaffolding protein and the dockerin domain of an enzyme component. We prepared five recombinant cohesin polypeptides of the Clostridium thermocellum scaffolding protein CipA, two dockerin polypeptides of C. thermocellum Xyn11A and Xyn10C, four cohesin polypeptides of Clostridium josui CipA, and two dockerin polypeptides of C. josui Aga27A and Cel8A, and qualitatively and quantitatively examined the cohesin-dockerin interactions within C. thermocellum and C. josui, respectively, and the species specificity of the cohesin-dockerin interactions between these two bacteria. Surface plasmon resonance (SPR) analysis indicated that there was a certain selectivity, with a maximal 34-fold difference in the K(D) values, in the cohesin-dockerin interactions within a combination of C. josui, although this was not detected by qualitative analysis. Affinity blotting analysis suggested that there was at least one exception to the species specificity in the cohesin-dockerin interactions, although species specificity was generally conserved among the cohesin and dockerin polypeptides from C. thermocellum and C. josui, i.e. the dockerin polypeptides of C. thermocellum Xyn11A exceptionally bound to the cohesin polypeptides from C. josui CipA. SPR analysis confirmed this exceptional binding. We discuss the relationship between the species specificity of the cohesin-dockerin binding and the conserved amino acid residues in the dockerin domains.     

The Synthesis and Biology of Fluorinated Prostacyclins

Abstract

Clostridium thermocellum produces a highly active cellulase system that consists of a high-M_r multienzyme complex termed cellulosome. Hydrolytic components of the cellulosome are organized around a large, noncatalytic glycoprotein termed CipA that acts both as a scaffolding component and a cellulose-binding factor. Catalytic subunits of the cellulosome bear conserved, noncatalytic subdomains, termed dockerin domains, which bind to receptor domains of CipA, termed cohesin domains. CipA includes nine cohesin domains, a cellulose-binding domain, and a specialized dockerin domain. Proteins of the cell envelope carrying cohesin domains that specifically bind the dockerin domain of CipA have been identified. These proteins may mediate anchoring of the cellulosomes to the cell surface. Cellulase complexes similar to the cellulosome of C. thermocellum are produced by several cellulolytic clostridia. High-Mr multienzyme complexes have also been identified in anaerobic rumen fungi. The architecture of the fungal complexes also seems to rely on the interaction of conserved, noncatalytic docking domains with a scaffolding component. However, the sequence of the fungal docking domains bears no resemblance to the clostridial dockerin domains, suggesting that the fungal and clostridial complexes arose independently.     

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. Béla?ch, C. Tardif, C. Reverbel-Leroy, C. Gaudin, and J.-P. Béla?ch, J. Bacteriol. 178:2279-2286, 1996; C. Reverbel-Leroy, A. Béla?ch, A. Bernadac, C. Gaudin, J. P. Béla?ch, 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.     

Cloning and DNA Sequencing of the Genes Encoding Clostridium josui Scaffolding Protein CipA and Cellulase CelD and Identification of Their Gene Products as Major Components of the Cellulosome

The Clostridium josui cipA and celD genes, encoding a scaffolding-like protein (CipA) and a putative cellulase (CelD), respectively, have been cloned and sequenced. CipA, with an estimated molecular weight of 120,227, consists of an N-terminal signal peptide, a cellulose-binding domain of family III, and six successive cohesin domains. The molecular architecture of C. josui CipA is similar to those of the scaffolding proteins reported so far, such as Clostridium thermocellum CipA, Clostridium cellulovorans CbpA, and Clostridium cellulolyticum CipC, but C. josui CipA is considerably smaller than the other scaffolding proteins. CelD consists of an N-terminal signal peptide, a family 48 catalytic domain of glycosyl hydrolase, and a dockerin domain. N-terminal amino acid sequence analysis of the C. josui cellulosomal proteins indicates that both CipA and CelD are major components of the cellulosome.     

Involvement of Both Dockerin Subdomains in Assembly of the Clostridium thermocellum Cellulosome

Clostridium thermocellum produces an extracellular cellulase complex termed the cellulosome. It consists of a scaffolding protein, CipA, containing nine cohesin domains and a cellulose-binding domain, and at least 14 different enzymatic subunits, each containing a conserved duplicated sequence, or dockerin domain. The cohesin-dockerin interaction is responsible for the assembly of the catalytic subunits into the cellulosome structure. Each duplicated sequence of the dockerin domain contains a region bearing homology to the EF-hand calcium-binding motif. Two subdomains, each containing a putative calcium-binding motif, were constructed from the dockerin domain of CelS, a major cellulosomal catalytic subunit. These subdomains, called DS1 and DS2, were cloned by PCR and expressed in Escherichia coli. The binding of DS1 and DS2 to R3, the third cohesin domain of CipA, was analyzed by nondenaturing gel electrophoresis. A stable complex was formed only when R3 was combined with both DS1 and DS2, indicating that the two halves of the dockerin domain interact with each other and such interaction is required for effective binding of the dockerin domain to the cohesin domain.     

A scaffoldin of the Bacteroides cellulosolvens cellulosome that contains 11 type II cohesins

A cellulosomal scaffoldin gene, termed cipBc, was identified and sequenced from the mesophilic cellulolytic anaerobe Bacteroides cellulosolvens. The gene encodes a 2,292-residue polypeptide (excluding the signal sequence) with a calculated molecular weight of 242,437. CipBc contains an N-terminal signal peptide, 11 type II cohesin domains, an internal family III cellulose-binding domain (CBD), and a C-terminal dockerin domain. Its CBD belongs to family IIIb, like that of CipV from Acetivibrio cellulolyticus but unlike the family IIIa CBDs of other clostridial scaffoldins. In contrast to all other scaffoldins thus far described, CipBc lacks a hydrophilic domain or domain X of unknown function. The singularity of CipBc, however, lies in its numerous type II cohesin domains, all of which are very similar in sequence. One of the latter cohesin domains was expressed, and the expressed protein interacted selectively with cellulosomal enzymes, one of which was identified as a family 48 glycosyl hydrolase on the basis of partial sequence alignment. By definition, the dockerins, carried by the cellulosomal enzymes of this species, would be considered to be type II. This is the first example of authentic type II cohesins that are confirmed components of a cellulosomal scaffoldin subunit rather than a cell surface anchoring component. The results attest to the emerging diversity of cellulosomes and their component sequences in nature.     

A Novel Cellulosomal Scaffoldin from Acetivibrio cellulolyticus That Contains a Family 9 Glycosyl Hydrolase

A novel cellulosomal scaffoldin gene, termed cipV, was identified and sequenced from the mesophilic cellulolytic anaerobe Acetivibrio cellulolyticus. Initial identification of the protein was based on a combination of properties, including its high molecular weight, cellulose-binding activity, glycoprotein nature, and immuno-cross-reactivity with the cellulosomal scaffoldin of Clostridium thermocellum. The cipV gene is 5,748 bp in length and encodes a 1,915-residue polypeptide with a calculated molecular weight of 199,496. CipV contains an N-terminal signal peptide, seven type I cohesin domains, an internal family III cellulose-binding domain (CBD), and an X2 module of unknown function in tandem with a type II dockerin domain at the C terminus. Surprisingly, CipV also possesses at its N terminus a catalytic module that belongs to the family 9 glycosyl hydrolases. Sequence analysis indicated the following. (i) The repeating cohesin domains are very similar to each other, ranging between 70 and 90% identity, and they also have about 30 to 40% homology with each of the other known type I scaffoldin cohesins. (ii) The internal CBD belongs to family III but differs from other known scaffoldin CBDs by the omission of a 9-residue stretch that constitutes a characteristic loop previously associated with the scaffoldins. (iii) The C-terminal type II dockerin domain is only the second such domain to have been discovered; its predicted "recognition codes" differ from those proposed for the other known dockerins. The putative calcium-binding loop includes an unusual insert, lacking in all the known type I and type II dockerins. (iv) The X2 module has about 60% sequence homology with that of C. thermocellum and appears at the same position in the scaffoldin. (v) Unlike the other known family 9 catalytic modules of bacterial origin, the CipV catalytic module is not accompanied by a flanking helper module, e.g., an adjacent family IIIc CBD or an immunoglobulin-like domain. Comparative sequence analysis of the CipV functional modules with those of the previously sequenced scaffoldins provides new insight into the structural arrangement and phylogeny of this intriguing family of microbial proteins. The modular organization of CipV is reminiscent of that of the CipA scaffoldin from C. thermocellum as opposed to the known scaffoldins from the mesophilic clostridia. The phylogenetic relationship of the different functional modules appears to indicate that the evolution of the scaffoldins reflects a collection of independent events and mechanisms whereby individual modules and other constituents are incorporated into the scaffoldin gene from different microbial sources.     

Duplicated dockerin subdomains of Clostridium thermocellum endoglucanase CelD bind to a cohesin domain of the scaffolding protein CipA with distinct thermodynamic parameters and a negative cooperativity

Mutagenized dockerin domains of endoglucanase CelD (type I) and of the cellulosome-integrating protein CipA (type II) were constructed by swapping residues 10 and 11 of the first or the second duplicated segment between the two polypeptides. These residues have been proposed to determine the specificity of cohesin-dockerin interactions. The dockerin domain of CelD still bound to the seventh cohesin domain of CipA (CohCip7), provided that mutagenesis occurred in one segment only. Binding was no longer detected by nondenaturing gel electrophoresis when both segments were mutagenized. The dockerin domain of CipA bound to the cohesin domain of SdbA as long as the second segment was intact. None of the mutated dockerins displayed detectable binding to the noncognate cohesin domain. Isothermal titration calorimetry showed that binding of the CelD dockerin to CohCip7 occurred with a high affinity [K(a) = (2.6 +/- 0.5) x 10(9) M(-1)] and a 1:1 stoichiometry. The reaction was weakly exothermic (DeltaHdegrees = -2.22 +/- 0.2 kcal x mol(-1)) and largely entropy driven (TDeltaSdegrees = 10.70 +/- 0.5 kcal x mol(-1)). The heat capacity change on complexation was negative (DeltaC(p) = -305 +/- 15 cal x mol(-1) x K(-1)). These values show that cohesin-dockerin binding is mainly hydrophobic. Mutations in the first or the second dockerin segment reduced or enhanced, respectively, the hydrophobic character of the interaction. Due to partial enthalpy-entropy compensation, these mutations induced only small changes in binding affinity. However, the binding affinity was strongly decreased when both segments were mutated, indicating strong negative cooperativity between the two mutated sites.     

Characterization and subcellular localization of the Clostridium thermocellum scaffoldin dockerin binding protein SdbA.

This article reports the characterization of the Clostridium thermocellum SdbA protein thought to anchor the cellulosome to the bacterial cell surface. The NH2-terminal region of SdbA consists of a cohesin domain which specifically binds the dockerin domain of the cellulosomal scaffolding protein CipA. The COOH-terminal region consists of a triplicated segment, termed SLH repeats, which is present in the sequence of many bacterial cell surface polypeptides. The binding parameters of the interaction between the dockerin domain of CipA and the cohesin domain of SdbA were studied by using, as a probe, the chimeric polypeptide CelC-DSCipA, which carries the dockerin domain of CipA fused to endoglucanase CelC. In the presence of Ca2+, CelC-DSCipA bound to SdbA with an affinity constant of 1.26 x 10(7) M(-1). Binding of CelC-DSCipA to SdbA as a function of Ca2+ concentration was sigmoidal, corresponding to a Hill coefficient of 2 and an affinity constant for Ca2+ of 4 x 10(6) M(-2). This suggested the presence of two cooperatively bound Ca2+ ions in the cohesin-dockerin complex. Immunoblotting of C. thermocellum subcellular fractions and electron microscopy of immunocytochemically labeled cells indicated that SdbA is located on the cell surface and is a component of the cellulosome. Together, the data confirm that SdbA could mediate anchoring of the cellulosome to the surface of C. thermocellum cells by interacting with the dockerin domain of CipA.     

Engineered proteins containing the cohesin and dockerin domains from Clostridium thermocellum provides a reversible, high affinity interaction for biotechnology applications

The cohesin-dockerin interaction, which is responsible for the formation of the cellulosome complex of cellulolytic bacteria, is a calcium-dependent, high affinity interaction. In this study, the cohesin (Cip7) and dockerin (Doc) domains of Clostridium thermocellum were fused to the cellulose-binding domain (CBD) of C. cellulovorans and the antibody-binding domain, protein LG, respectively, to form CBD-Cip7 and LG-Doc. Immobilised CBD-Cip7 was able to bind LG-Doc and subsequently antibody as determined using surface plasmon resonance. Binding was reversed by the removal of Ca2+ with EDTA. The dockerin containing fusion protein was affinity purified using an immobilised cohesin domain. Elution of the LG-Doc from the cohesin column was with EDTA. This affinity chromatography was repeated using an LG-dockerin column for the purification of cohesin fusion protein. The fusion proteins created in this report have shown that the properties of the cohesin and dockerin domains can be transferred to other protein domains and that the interaction between the cohesin and dockerin is specific, Ca2+ -dependent and reversible. We have shown that the cohesin-dockerin interaction has several properties making it suitable for use in recombinant fusion protein production and purification.     

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.     

Characterization of a cellulose binding domain from Clostridium cellulovorans endoglucanase-xylanase D and its use as a fusion partner for soluble protein expression in Escherichia coli

Different chimeric proteins combining the non-catalytic C-terminal putative cellulose binding domain of Clostridium cellulovorans endoglucanase-xylanase D (EngD) with its proline-threonine rich region PT-linker, PTCBD(EngD), cellulose binding domain of C. cellulovorans cellulose binding protein A, CBD(CbpA), cohesin domains Cip7, Coh6 and CipC1 from different clostridial species and recombinant antibody binding protein LG were constructed, expressed, purified and analyzed. The solubilities of chimeric proteins containing highly soluble domains Cip7, CipC1 and LG were not affected by fusion with PTCBD(EngD). Insoluble domain Coh6 was solubilized when fused with PTCBD(EngD). In contrast, fusion with CBD(CbpA) resulted in only a slight increase in solubility of Coh6 and even decreased solubility of CipC1 greatly. PTCBD(EngD) and Cip7-PTCBD(EngD) were shown to bind regenerated commercial amorphous cellulose Cuprophan. The purity of Cip7-PTCBD(EngD) eluted from Cuprophan was comparable to that purified by conventional ion exchange chromatography. The results demonstrated that PTCBD(EngD) can serve as a bi-functional fusion tag for solubilization of fusion partners and as a domain for the immobilization, enrichment and purification of molecules or cells on regenerated amorphous cellulose.     

Interaction between a type-II dockerin domain and a type-II cohesin domain from Clostridium thermocellum cellulosome

The interaction between the type-II dockerin domain of the scaffoldin protein CipA and the type-II cohesin domain of the outer layer protein SdbA is the fundamental mechanism for anchoring the cellulosome to the cell surface of Clostridium thermocellum. We constructed and purified a dockerin polypeptide and a cohesin polypeptide, and determined affinity constants of the interaction between them by the surface plasmon resonance method. The dissociation constant (K(D)) value was 1.8 x 10(-9) M, which is a little larger than that for the combination of a type-I dockerin and a type-I cohesin.     

Cellulosome from Clostridium cellulolyticum: molecular study of the Dockerin/Cohesin interaction.

Clostridium cellulolyticum produces cellulolytic complexes (cellulosomes) made of 10-13 cell wall degrading enzymes tightly bound to a scaffolding protein (CipC) by means of their dockerin domain. It has previously been shown that the receptor domains in CipC are the cohesin domains and that the cohesin/dockerin interaction is calcium-dependent. In the present study, surface plasmon resonance was used to demonstrate that the free cohesin1 from CipC and dockerin from CelA have the same K(D) (2.5 x 10(-)(10) M) as that of the entire CelA and a larger fragment of CipC, the latter of which contains, in addition to cohesin1, a cellulose binding domain and a hydrophilic domain of unknown function. This demonstrates that neither the catalytic domain of CelA nor the noncohesin domains of CipC have any influence on the interaction. Dockerin domains are composed of two conserved segments of 22 residues: removal of the second segment abolishes the affinity for cohesin1, whereas modified dockerins having twice the first segment, twice the second, or both segments but in a reverse order have K(D) values for cohesin1 in the same range as that observed for wild-type dockerin. These data indicate that if two segments are required for the complexation with the cohesin, segments 1 and 2 are similar enough to replace each other. Calcium overlay experiments revealed that the dockerin domain has one calcium binding site per conserved segment. Circular dichroism performed on wild-type and mutant dockerins indicates that this domain is well structured and that removal of calcium only weakly affects the secondary structure, which remains 40-45% helical.     

Molecular architecture and structural transitions of a Clostridium thermocellum mini-cellulosome

The cellulosome is a highly elaborate cell-bound multienzyme complex that efficiently orchestrates the deconstruction of cellulose and hemicellulose, two of the nature's most abundant polymers. Understanding the intricacy of these nanomachines evolved by anaerobic microbes could sustain the development of an effective process for the conversion of lignocellulosic biomass to bio-ethanol. In Clostridium thermocellum, cellulosome assembly is mediated by high-affinity protein:protein interactions (> 10⁹ M^− 1) between dockerin modules found in the catalytic subunits and cohesin modules located in a non-catalytic protein scaffold termed CipA. Whereas the atomic structures of several cellulosomal components have been elucidated, the structural organization of the complete cellulosome remains elusive. Here, we reveal that a large fragment of the cellulosome presents a mostly compact conformation in solution, by solving the three-dimensional structure of a C. thermocellum mini-cellulosome comprising three consecutive cohesin modules, each bound to one Cel8A cellulase, at 35 Å resolution by cryo-electron microscopy. Interestingly, the three cellulosomal catalytic domains are found alternately projected outward from the CipA scaffold in opposite directions, in an arrangement that could expand the area of the substrate accessible to the catalytic domains. In addition, the cellulosome can transit from this compact conformation to a multitude of diverse and flexible structures, where the linkers between cohesin modules are extended and flexible. Thus, structural transitions controlled by changes in the degree of flexibility of linkers connecting consecutive cohesin modules could regulate the efficiency of substrate recognition and hydrolysis.     

Stoichiometric Assembly of the Cellulosome Generates Maximum Synergy for the Degradation of Crystalline Cellulose,as Revealed by In Vitro Reconstitution of the Clostridium thermocellum Cellulosome

The cellulosome is a supramolecular multienzyme complex formed by species-specific interactions between the cohesin modules of scaffoldin proteins and the dockerin modules of a wide variety of polysaccharide-degrading enzymes. Cellulosomal enzymes bound to the scaffoldin protein act synergistically to degrade crystalline cellulose. However, there have been few attempts to reconstitute intact cellulosomes due to the difficulty of heterologously expressing full-length scaffoldin proteins. We describe the synthesis of a full-length scaffoldin protein containing nine cohesin modules, CipA; its deletion derivative containing two cohesin modules, ΔCipA; and three major cellulosomal cellulases, Cel48S, Cel8A, and Cel9K, of the Clostridium thermocellum cellulosome. The proteins were synthesized using a wheat germ cell-free protein synthesis system, and the purified proteins were used to reconstitute cellulosomes. Analysis of the cellulosome assembly using size exclusion chromatography suggested that the dockerin module of the enzymes stoichiometrically bound to the cohesin modules of the scaffoldin protein. The activity profile of the reconstituted cellulosomes indicated that cellulosomes assembled at a CipA/enzyme molar ratio of 1/9 (cohesin/dockerin = 1/1) and showed maximum synergy (4-fold synergy) for the degradation of crystalline substrate and ∼2.4-fold-higher synergy for its degradation than minicellulosomes assembled at a ΔCipA/enzyme molar ratio of 1/2 (cohesin/dockerin = 1/1). These results suggest that the binding of more enzyme molecules on a single scaffoldin protein results in higher synergy for the degradation of crystalline cellulose and that the stoichiometric assembly of the cellulosome, without excess or insufficient enzyme, is crucial for generating maximum synergy for the degradation of crystalline cellulose.     

A new type of cohesin domain that specifically binds the dockerin domain of the Clostridium thermocellum cellulosome-integrating protein CipA.

The cellulosome-integrating protein CipA, which serves as a scaffolding protein for the cellulolytic complex produced by Clostridium thermocellum, comprises a COOH-terminal duplicated segment termed the dockerin domain. This paper reports the cloning and sequencing of a gene, termed sdbA (for scaffoldin dockerin binding), encoding a protein which specifically binds the dockerin domain of CipA. The sequenced fragment comprises an open reading frame of 1,893 nucleotides encoding a 631-amino-acid polypeptide, termed SdbA, with a calculated molecular mass of 68,577 kDa. SAA comprises an NH2-terminal leader peptide followed by three distinct regions. The NH2-terminal region is similar to the NH2-terminal repeats of C. thermocellum OlpB and ORF2p. The central region is rich in lysine and harbors a motif present in Streptococcus M proteins. The COOH-terminal region consists of a triplicated sequence present in several bacterial cell surface proteins. The NH2-terminal region of SdbA and a fusion protein carrying the first NH2-terminal repeat of OlpB were shown to bind the dockerin domain of CipA. Thus, a new type of cohesin domain, which is present in one, two, and four copies in SdbA, ORF2p, and OlpB, respectively, can be defined. Since OlpB and most likely SdbA and ORF2p are located in the cell envelope, the three proteins probably participate in anchoring CipA (and the cellulosome) to the cell surface.     

Subcloning of a DNA fragment encoding a single cohesin domain of the Clostridium thermocellum cellulosome-integrating protein CipA: purification, crystallization, and preliminary diffraction analysis of the encoded polypeptide.

An Escherichia coli clone encoding a single cohesin domain of the cellulosome-integrating protein CipA from Clostridium thermocellum was constructed, and the corresponding polypeptide was purified, treated with papain, and crystallized from a PEG 8000 solution. Crystals exhibit orthorhombic symmetry, space group P2(1)2(1)2(1), with cell dimensions a = 37.7 A, b = 80.7 A, c = 93.3 A, and four or eight molecules in the unit cell. The crystals diffract X-rays to beyond 2 A resolution and are suitable for further crystallographic studies.     

Mapping by site-directed mutagenesis of the region responsible for cohesin-dockerin interaction on the surface of the seventh cohesin domain of Clostridium thermocellum CipA

To locate the region involved in binding dockerin domains, 15 mutations were introduced across the surface of the seventh cohesin domain of the scaffolding protein CipA, which holds together the cellulosome of Clostridium thermocellum. Mutated residues were located on both faces of the nine-stranded beta-sandwich forming the cohesin domain and on the loops connecting beta-strands 4 and 5, 6 and 7, and 8 and 9. The loop region was previously proposed, on the basis of sequence comparisons, to form a contiguous "recognition strip". Individual mutants of four residues, D39, Y74, E86, and G89, formed no complexes detectable by nondenaturing gel electrophoresis after incubation with CelD664, a shortened form of endoglucanase CelD lacking the residues linking the catalytic domain with the dockerin domain. The four sensitive residues encompass a hydrophobic region on the 5-6-3-8 face of the molecule, which overlaps partially with the recognition strip and with a hydrophobic zone involved in the formation of cohesin-cohesin dimers. Isothermal titration calorimetry showed that single cohesin mutations affecting the binding of CelD664 had significant effects on the enthalpy or entropy of binding of wild-type CelD but much lesser effects on the association constant, owing to enthalpy-entropy compensation. However, the affinity for wild-type CelD of the triple mutant affecting D39, Y74, and E86 was reduced by 2 orders of magnitude, due to negative cooperativity between mutations affecting D39 + Y74 on one hand and E86 on the other hand.