Cohesin-Dockerin Interaction in Cellulosome Assembly

A SINGLE HYDROXYL GROUP OF A DOCKERIN DOMAIN DISTINGUISHES BETWEEN NONRECOGNITION AND HIGH AFFINITY RECOGNITION*

Adva MechalyDagger , Henri-Pierre Fierobe§, Anne Belaich§, Jean-Pierre Belaich§, Raphael Lamed||, Yuval Shoham**, and Edward A. BayerDagger DaggerDagger

From the Dagger  Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel, the § Bioénergétique et Ingéniérie des Protéines, Centre National de la Recherche Scientifique, IBSM-IFR1, Marseille 13402, France, the  Université de Provence, Marseille 13331, France, the || Department of Molecular Microbiology and Biotechnology, Tel Aviv University, Ramat Aviv 69978, Israel, and the ** Department of Food Engineering and Biotechnology, and Institute of Catalysis Science and Technology, Technion-Israel Institute of Technology, Haifa 32000, Israel

Received for publication, October 10, 2000, and in revised form, January 4, 2000


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

The assembly of enzyme components into the cellulosome complex is dictated by the cohesin-dockerin interaction. In a recent article (Mechaly, A., Yaron, S., Lamed, R., Fierobe, H.-P., Belaich, A., Belaich, J.-P., Shoham, Y., and Bayer, E. A. (2000) Proteins 39, 170-177), we provided experimental evidence that four previously predicted dockerin residues play a decisive role in the specificity of this high affinity interaction, although additional residues were also implicated. In the present communication, we examine further the contributing factors for the recognition of a dockerin by a cohesin domain between the respective cellulosomal systems of Clostridium thermocellum and Clostridium cellulolyticum. In this context, the four confirmed residues were analyzed for their individual effect on selectivity. In addition, other dockerin residues were discerned that could conceivably contribute to the interaction, and the suspected residues were similarly modified by site-directed mutagenesis. The results indicate that mutation of a single residue from threonine to leucine at a given position of the C. thermocellum dockerin differentiates between its nonrecognition and high affinity recognition (Ka ~ 109 M-1) by a cohesin from C. cellulolyticum. This suggests that the presence or absence of a single decisive hydroxyl group is critical to the observed biorecognition. This study further implicates additional residues as secondary determinants in the specificity of interaction, because interconversion of selected residues reduced intraspecies self-recognition by at least three orders of magnitude. Nevertheless, as the latter mutageneses served to reduce but not annul the cohesin-dockerin interaction within this species, it follows that other subtle alterations play a comparatively minor role in the recognition between these two modules.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

The cellulosome concept is one of the major paradigms of microbial degradation of plant cell-wall polysaccharides (1-4). Initially, such a multienzyme complex was described in the anaerobic thermophilic bacterium, Clostridium thermocellum (5, 6). Additional cellulosomes have been described subsequently for a variety of anaerobic bacteria and fungi (7-13). The various hydrolytic enzymes are incorporated into the cellulosome complex by virtue of a pivotal nonhydrolytic polypeptide, called scaffoldin, which bears a collection of cohesin modules for this purpose. Each cohesin binds a single dockerin domain located on the enzymes, thereby generating the fully assembled cellulosome.

In recent years, much effort has been devoted toward a better understanding of the cohesin-dockerin interaction. In early works, it was reported that the cellulosomal subunits are cohesively contained in the complex of C. thermocellum (14, 15). It was also noted that a duplicated segment within the dockerin sequence shows remarkable similarity with the EF-hand motif of calcium binding proteins, such as calmodulin and troponin C (16). Indeed, it was shown subsequently that the cohesin-dockerin interaction in C. thermocellum is calcium-dependent, but surprisingly nonspecific among the different dockerin-bearing enzymes of this species (17, 18). It was further demonstrated that both duplicated segments of the dockerin domain are required for binding to the cohesin module (19, 20). The three-dimensional structure of a dockerin is still unknown, although related secondary structure predictions indicated that the dockerin domain would assume an "F-helix variation" of the EF-hand motif (21, 22). This hypothesis has recently been corroborated experimentally by NMR spectroscopy (23). Despite the reported crystal structures for two different C. thermocellum cohesins (24, 25), structural information regarding the cohesin-dockerin complex and the precise residues involved in the interaction have yet to be discovered.

We have shown that the interaction among cohesins and dockerins between two cellulosome-producing bacteria, C. thermocellum and C. cellulolyticum, is species-specific (21). Sequence analysis of the various dockerin domains from the two species enabled us to predict four residues that could unambiguously account for the observed species specificity. Subsequent studies, in which the four suspected dockerin residues of one species were converted to emulate those of the other, provided experimental verification of this prediction (26). Thus, interconversion of the selected residues created new specificities in which the cohesins now recognized the dockerin from the rival species. However, in each case, despite the relevant mutations, the intra-species interaction was not destroyed, and the modified dockerins still recognized the cohesin from the same species. This implied that other residues might contribute more subtle secondary interactions that would also be involved in cohesin-dockerin recognition.

The purpose of the present work was 2-fold: first, to try to determine the minimal number of selectivity determinants in the dockerin domain that distinguish between the two species, and second, to try to identify additional residues that contribute secondarily to the cohesin-dockerin interaction. The first goal was achieved by mutation of a single dockerin residue from threonine to leucine in a C. thermocellum cellulosomal enzyme that sufficed to create the ability to bind a cohesin from C. cellulolyticum. The second goal was only partially achieved, in that up to eight separate dockerin mutations served only to reduce but not to abolish self-recognition of the C. thermocellum cohesin.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Bacterial Strains and Vectors-- Escherichia coli strain XL1-Blue was obtained from Stratagene (La Jolla, CA). E. coli strain MutS was obtained from CLONTECH Laboratories (Palo Alto, CA). E. coli strain BL21 pLysS(lambda DE3) was used as a host for the expression of the C. thermocellum CelS enzyme in pET9d (Novagen, Madison, WI), and the resulting construct was termed pET9d-CelS (26). The various CelS-derived mutants were expressed in the same host strain.

DNA Manipulation-- DNA was manipulated by standard procedures (27, 28). DNA was transformed using either the calcium chloride method for strain BL21(DE3)pLysS or by electroporation using GeneZapper (IBI, New Haven, CT) for strains XL-1 Blue and MutS. DNA sequencing was performed by the DNA Sequencing Unit at the Weizmann Institute (Rehovot, Israel).

Unique-site Elimination-- Mutant mS5 (previously termed mCelS-ct) was constructed as reported previously (26), using a modification of the unique-site elimination procedure (29). Briefly, the mutations were first produced in a detached enzyme-free dockerin construct using primers S688AT689F and S720AT721L (Table I). The DNA encoding for the entire mutated dockerin segment was transferred en bloc to the intact parent enzyme (CelS-ct), using selection primer I (thus converting a unique EagI site to SacII) and the entire mutated dockerin as the mutagenic primer. Mutant mS3 was constructed using the same approach, whereby clones containing the desired mutated dockerin segment (S688AT689F) were selected using the appropriate restriction enzyme (MluI), and the mutated segment was then transferred en bloc to the intact parent enzyme using the same selection primer. The resultant mutant, containing both the mutated dockerin and the intact catalytic domain, was used subsequently as a template for the construction of another mutant, mS1. In this case, the mutagenic primer comprised L689T, and selection primer II contained the reverse mutation, which converted the SacII site back to EagI.

                              
View this table:
[in this window]
[in a new window]
 
Table I
Details of mutagenic primers used for site-directed mutagenesis
Mutated bases are shown in bold and restriction sites are underlined.

Site-directed Mutagenesis by Overlap-extension PCR-- With the exception of mS1, mS3 and mS5, the other mutations were achieved using OE PCR1 (30, 31) as described earlier (26). mS5 was used as a template for the construction of mS6; the mutations were introduced in two OE PCR cycles via mutagenic primers R696K (forward) and R728K (reverse) and flanking primers, N-flank-AatII (forward) and C-flank-NheI (reverse). However, ligation of the mutated dockerin fragment (477 bp) into pET9D-CelS (5979 bp) resulted in a high background of nonmutated colonies, apparently due to the large difference in size of the two DNA fragments and consequent difficulty in distinguishing single- from double-digested plasmid DNA. To circumvent this problem, a triple-fragment ligation approach was undertaken, in which an extended insert, delimited by NcoI and NheI sites, was extracted from the plasmid. The insert was further digested at an internal AatII site, and the resultant NcoI-AatII-digested fragment (1958 bp) was ligated, together with the mutated dockerin fragment (AatII-NheI), into the NcoI-NheI-digested pET9d-CelS (4021 bp). The resulting mutant, mS6, was then used as a template for the construction of mS8, utilizing the mutagenic primers R700G (forward) and K732G (reverse) employing the same method. For the construction of mS7, mutagenic primers K696R (forward) and K728R (reverse) were used with flanking primers N-ter-ScaI (forward) and C-flank-NheI (reverse) with mS8 as template in a simple ligation reaction. The same flanking primers were also used to construct mS2 and mS4. In the case of mS2, mS3 was used as a template with the mutagenic primer A688S (forward), whereas for mS4, pET9d-CelS was used as a template with primer S720AT721L (reverse).

The mutant mCelA-cc of the C. cellulolyticum cellulase A was described in an earlier publication (26).

Expression of Recombinant Proteins-- All mutants were expressed in BL21(DE3)pLysS cells as described previously (26). The crude extracts were used for affinity-blotting studies without additional purification. The cells were centrifuged (6000 rpm, 10 min), resuspended to a final A600 of 10, and SDS-containing sample buffer was added, prior to boiling and SDS-PAGE.

The respective cohesin probes, Coh-cc and Coh-ct, were prepared as described previously (17, 32).

Purification of Recombinant Proteins-- CelA-cc and its mutant (mCelA-cc) were purified as described earlier (20, 26). CelS-ct, mS1, mS2, mS3, mS5, mS6, and mS8 were produced and purified as follows: E. coli clones were grown to A600 = 1.5 at 37 °C in 2 liters of Luria-Bertani medium, supplemented with 1.2% glycerol (w/v) and 50 µg·ml-1 kanamycin. The cultures were then cooled to 15 °C, and isopropyl-1-thio-beta -D-galactoside was added to a final concentration of 0.1 mM. After 16 h, the cells were centrifuged (10 min, 3000 × g), resuspended in 30 ml of 50 mM Tris-HCl (pH 8) containing 4 mM CaCl2, and broken in a French press. The crude extract was then loaded onto a column containing 5-6 g of Avicel (Fluka, Buchs, Switzerland), equilibrated in the same buffer. The cellulose was washed with the same buffer, and the recombinant protein was eluted with distilled water. To further purify the protein of interest, the eluted fraction was supplemented with Tris-HCl, pH 8.5, and CaCl2 at final concentrations of 25 and 2 mM, respectively, and loaded on 3 ml of DEAE-Trisacryl (Sepracor, Villeneuve-la-Garenne, France) equilibrated with the same buffer. The elution was performed using a linear gradient from 0 to 0.5 M NaCl, and fractions were analyzed by SDS-PAGE. The fractions containing pure recombinant protein were pooled and dialyzed using ultrafiltration (cutoff of 10 kDa; Millipore, Bedford, MA) by several concentration/dilution steps in 10 mM Tris-HCl (pH 8.0),

Protein Biotinylation-- Cohesin-containing constructs were biotinylated using biotinyl N-hydroxysuccinimide ester at a 15-fold molar ratio of reagent to protein as described previously (33). The cohesin-containing constructs from both species retained normal binding activity to both cellulose and dockerin-containing enzymes and were recognized by avidin probes.

Affinity Blot Analysis-- The affinity blotting procedure was carried out essentially as described previously (26). Briefly, samples of the mutated dockerin constructs were separated by 10% SDS-PAGE and transferred electrophoretically onto nitrocellulose membranes. The blots were reacted with biotinylated cohesin-containing constructs from either C. thermocellum or C. cellulolyticum (Coh-ct and Coh-cc, respectively) and developed using ExtrAvidin-horseradish peroxidase complexes (1:2000 dilution, Sigma Chemical Co., St. Louis, MO).

Surface Plasmon Resonance-- Experiments were performed as described earlier (20), using 10 mM Tris-maleate (pH 6.8), 0.15 M NaCl, 2.5 mM CaCl2, 0.005% P20 (BIAcore, Uppsala, Sweden) as running buffer (flow rate 25 µl min-1). Biotinylated cohesin-containing constructs (~100 resonance units) were immobilized onto streptavidin-bearing sensor chips. The cellulases were diluted in the same buffer (final concentrations ranging from 5 nM to 1 µM) and allowed to interact with the immobilized receptor by injections of 600 s.

    RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

The cellulosome is a cohesive enzyme complex of exceptional stability. To dissociate the cellulosome from C. thermocellum into its component parts, the presence of detergent at elevated temperatures is required (14, 34). The observed stability of the cellulosome is a function of the very high affinity interaction between two different types of module; i.e. the single dockerin domain of the individual enzyme subunits and the multiple copies of the cohesin domain on the scaffoldin subunit (35, 36). Recent attempts in our laboratory to control the specificity of the cohesin-dockerin interaction have met with partial success. By interconverting four predicted recognition residues of the dockerins from C. thermocellum and C. cellulolyticum (21), a new specificity was created whereby the modified dockerin of one species now recognized the cohesin of the other (26).

Following these studies, it was of interest to try to refine this interaction further, by pinpointing whether fewer than four residues may account for the specificity of binding between the two species. On the other hand, the inability to neutralize completely the cohesin-dockerin interaction within the same species implicated additional residues as auxiliary specificity determinants, and we thus revisited the potential contribution of other dockerin sites to the observed specificity.

Refinement of Critical Recognition Residues-- To determine the minimal changes necessary to create the new interspecies specificity, progressive mutations of the four putative recognition residues were designed. These particular mutations were carried out at positions 10 and 11 of the two duplicated segments of the dockerin domain in both species (Table II). Thus, mutants of the C. thermocellum CelS dockerin exhibited single mutations at these positions in the first duplicated segment (mutants mS1 and mS2), mutations of both of the latter positions in each duplicated segment alone (mS3 and mS4), and the four-residue mutant (mS5 in the present study, equivalent to mCelS-ct as described previously (26)). The mutant dockerin construct of the C. cellulolyticum CelA enzyme (mCelA-cc) was used as a cross-species reference and corresponded to the previously reported (26) four-residue mutation.

                              
View this table:
[in this window]
[in a new window]
 
Table II
Sites and mutations of the C. thermocellum CelS and the C. cellulolyticum CelA dockerins
The designated residues at the given positions in either series of mutants were interconverted to match or emulate those of its rival wild-type dockerin. Blank spaces indicate no mutation at the designated residue.

The various C. thermocellum mutant dockerins were overexpressed and subjected to SDS-PAGE and Western blotting, and the resultant blots were challenged with a cohesin probe derived from either species. The results are presented in Fig. 1. Strikingly, the conversion of a single amino acid residue in the CelS dockerin was enough to create a new specific interaction with the C. cellulolyticum cohesin (Fig. 1A). In this context, it was clear that the common denominator for interaction of a dockerin mutant with the cross-species cohesin was the presence of a leucine instead of a threonine at position 11 in the first duplicated segment. Thus, mutants mS2, mS3, and mS5 interacted strongly with Coh-cc, whereas mS1 and mS4 failed to do so. Indeed, this result reflects the conservation of hydroxy-amino acids in this particular position of the known C. thermocellum dockerins and hydrophobic residues of the C. cellulolyticum dockerins (Table III). It is not clear, however, why mutation of the other conserved residues in positions 10 and 11 (notably the interconversion of serine to alanine in position 10) appears not to be strictly required for interspecies recognition between these two species of dockerin.


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 1.   Affinity-blotting of C. thermocellum CelS-dockerin mutants using recombinant cohesin-containing probes. Enzyme samples were subjected to SDS-PAGE and blotted onto nitrocellulose membranes (see Table II for composition of the respective native and mutant enzymes). The separated proteins were probed using the desired biotinylated cohesin construct: Blot A, Coh-cc from C. cellulolyticum; Blot B, Coh-ct from C. thermocellum (17, 32). The blots were developed using an avidin-peroxidase conjugate.

                              
View this table:
[in this window]
[in a new window]
 
Table III
Distribution of amino acid residues at critical positions in the known dockerins of C. thermocellum and C. cellulolyticum
The percentage occurrence of the indicated consensus residues are shown. Non-consensus residues that appear at a given position are included parenthetically in lowercase letters.

Attempts to Reduce Self-recognition between Mutant Dockerins and Coh-ct-- In our previous study (26), we showed that the four-residue mutant, mS5, clearly recognized the cross-species cohesin probe. Nevertheless, the strong binding of its own cohesin was retained. In the present work, we sought additional sites that might reduce or abolish such intra-species self-recognition while maintaining the newly created binding specificity between the two species. For this purpose, positions 18 and 22 of the two duplicated segments of the CelS dockerin were chosen as targets for site-directed mutagenesis. The basis for selection of these four positions was 2-fold. First, the character of these residues is quite well conserved, especially in the C. thermocellum dockerins (Table III). Second, according to PHD prediction (Profile network prediction HeiDelberg) of solvent accessibility (37-39), these four residues are predicted to be in exposed positions in the dockerin structure (Table IV). According to the prediction, positions 17 may also be exposed (Table IV), but the dockerins of both C. thermocellum and C. cellulolyticum almost invariably show a lysine at this position (position 21), and interconversion in this case would thus be irrelevant.

                              
View this table:
[in this window]
[in a new window]
 
Table IV
Predicted solvent accessibility for dockerin residues
According to PHD: Profile network prediction HeiDelberg (39), based upon MAXHOM multiple sequence alignment of 20 different dockerin sequences from C. thermocellum and C. cellulolyticum. The CelS sequence is shown and sites of mutation are marked in boldface font. The predicted sites of alpha -helices are also indicated (21).

In view of the above, mutations in positions 18 and 22 of the duplicated segments were performed to accompany the mutations at positions 10 and 11 (Table II). Thus, the arginines in positions 18 were mutated to lysines in mutant mS6, and the basic residues at positions 22 of the two duplicated segments (arginine and lysine, respectively) were mutated to glycines in mS7. Mutant mS8 comprised the combined four-residue mutation in addition to the mutations at positions 10 and 11. In the latter two mutants, mutation to glycine was chosen for position 22 of segment 1 instead of alanine as in the CelA dockerin from C. cellulolyticum, because glycine is more prevalent at this position among all of the known cellulosomal enzymes of this species (Table III).

The consequences of these mutations are shown in Fig. 1. In accord with the above bioinformatics-based prediction, mutants mS7 and mS8 (mutated in positions 10, 11, 22, and/or 18) indeed displayed a visible reduction in their self-recognition of the C. thermocellum cohesin (Coh-ct), compared with those of the native CelS-ct and mutants mS1 through mS5 (Fig. 1B). On the other hand, mutant mS6 (mutated in positions 10, 11, 18, but not 22) retained a higher level of interaction with Coh-ct. It thus follows that position 22 is an important determinant in dockerin recognition. Nevertheless, unlike the native C. cellulolyticum-derived enzyme (CelA-cc) that failed to bind Coh-ct, both mutants mS7 and mS8 bound clearly to their own cohesin (Coh-ct) as described above. This indicates that, in the C. thermocellum system, other residues, in addition to the eight designated positions, still play a role in the recognition process.

Determination of Binding Constants-- On the basis of the affinity blotting data, several of the key mutants were overexpressed, purified, and subjected to analysis by surface plasmon resonance, using immobilized forms of the two species of cohesins and the BIAcore system. The results are presented in Table V. In general, the data are in total accord with the blots and provide numerical values for the binding of the individual mutant dockerins to either species of cohesin. It is worthwhile noting that the observed affinity characteristics for the cohesin-dockerin interaction in C. thermocellum were found to be much higher than those of C. cellulolyticum. The calculated affinity constant for the mesophilic C. cellulolyticum system (~1010 M-1) was in itself indicative of a very high affinity interaction and in accord with previously reported values (20). In the thermophilic system, the affinity constants were even higher and essentially exceeded the upper limits (>1011 M-1) of the BIAcore system. It should be noted that such a high affinity may be required to maintain tight binding under thermophilic conditions. In any case, the cohesin-dockerin complex in the C. thermocellum system represents one of the highest protein-protein affinity interactions known in nature, rivaling many other very high affinity associations, e.g. receptor-effector and enzyme-inhibitor pairs (40).

                              
View this table:
[in this window]
[in a new window]
 
Table V
Affinity constants for binding of wild-type and mutant dockerin-borne enzymes to immobilized cohesins
The immobilized cohesin receptors and enzyme ligands were derived from C. thermocellum (ct) and C. cellulolyticum (cc). Values were obtained by surface plasmon resonance using a BIAcore system.

With respect to the selectivity of cohesin-dockerin recognition, the critical mutation in mutant mS2 (in which the threonine at position 11 of the first duplicated segment was converted to leucine) generated a novel high affinity interaction with the C. cellulolyticum cohesin. Compared with the native dockerin-bearing enzyme, this single mutation differentiated between a lack of detectable binding and a tenacious cross-species cohesin-dockerin interaction, characterized by a measurable dissociation constant of 5 × 10-9 M. It is interesting to note that all of the C. thermocellum dockerin mutants that carried the Thr-11 right-arrow Leu mutation exhibited similarly high rates of association for Coh-cc (~106 M-1 s-1), resembling that of CelA-cc, i.e. the native enzyme from C. cellulolyticum (Table V). Also of note are the consistently higher koff values for the mutants, compared with CelA-cc, indicating a shorter half-life for their complexes with the C. cellulolyticum cohesin. In the complementary system, the four mutations in the dockerin of CelA-cc gained a similarly high affinity constant (3.3 × 109 M-1) with Coh-ct, whereas the native enzyme showed no measurable binding with the cross-species cohesin.

With respect to intra-species cohesin-dockerin recognition, the preference of a given dockerin mutant to either of the two species of cohesins could be quantified by comparing the ratio of affinity constants (see Fig. 2). Due to the very high affinity constants for the C. thermocellum system, most of the CelS-derived mutants showed a clear preference for self-recognition of Coh-ct. However, as the number of mutations increased, a trend toward preference for Coh-cc prevailed. Indeed, mutant mS8 exhibited a detectable preference for cross-species recognition. In the reverse direction, the CelA-derived mutant (mCelA-cc) clearly favored interaction with the rival cohesin. It is interesting to note that this mutant is counter-equivalent to mS5 in its mutation pattern (i.e. in positions 10 and 11). Again, the measurable difference in cross-species recognition between these two mutants may be attributed to the higher affinity of the cohesin-dockerin interaction of the thermophilic system.


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 2.   Relative binding of mutant dockerin-borne enzymes to cohesins. The ratio of affinity constants were calculated as Ka (C. thermocellum)/Ka (C. cellulolyticum). Values greater than 1 indicate a preferred affinity of the given dockerin for the C. thermocellum-derived cohesin, and values lower than 1 show a preference for the C. cellulolyticum cohesin. White bars indicate native and mutant constructs derived from C. thermocellum, and gray bars indicate those from C. cellulolyticum. Bars broken at the top indicate minimum values, either due to lack of detectable binding (Ka < 106) to one of the cohesins or to extremely strong binding (Ka > 1011) to Coh-ct. Note, mutants mS8 and mCelA-cc show a preference for the rival species of cohesin.

Concluding Perspectives-- The exchange of a single threonine for leucine in the C. thermocellum dockerin generated cross-species recognition with a C. cellulolyticum cohesin. This implies that the major difference between these two residues, i.e. the lack of a hydroxyl group in leucine compared with threonine, represents the molecular basis for the newly gained specificity. The presence or absence of this single decisive hydroxyl group at the specified position in the C. thermocellum dockerin thus appears to be critical to the specificity of the cohesin-dockerin interaction in this species. It is remarkable that such a small difference would differentiate between a very high affinity interaction and essentially no detectable interaction at all. This phenomenon also implies a complementary distinction on the surfaces of the two cohesin species. Specifically, it may be expected that the C. cellulolyticum cohesin would exhibit a more hydrophobic microenvironment at a given, yet unidentified locale, which may account for the observed change of specificity in the mutated dockerin.

No less remarkable, perhaps, is the fact that the dockerin could be subjected to such a broad number of mutations without abolishing self-recognition of its own cohesin. Thus, the C. thermocellum dockerin could be mutated at positions 10, 11, 18, and 22 in both duplicated segments, encompassing a series of one to eight mutations of the 67-residue dockerin domain, and the entire spectrum of mutated constructs recognized the cohesin of C. cellulolyticum as well as its own. This suggests that the two species of cohesin-dockerin pairs are, perhaps, much more similar than originally presumed (21).

Ultimately, the precise molecular determinants that dictate binding specificity of the cohesin-dockerin interaction will probably be resolved by three-dimensional structure determination of cohesin-dockerin complexes from different species. To date, however, both crystallogenesis and NMR spectroscopy of such complexes have proved elusive. In the interim, indirect approaches, such as site-directed or random mutagenesis of the different components, should continue to provide us with clues into the molecular determinants of the tenacious cohesin-dockerin interaction.

    ACKNOWLEDGEMENT

We appreciate the preparation of mutants mS3 and mS5 by Dr. Sima Yaron.

    FOOTNOTES

* This work was supported by a contract from the European Commission (Fourth Framework, Biotechnology Programme, BIO4-97-2303). Grants from the Israel Science Foundation (administered by the Israel Academy of Sciences and Humanities, Jerusalem) are gratefully acknowledged. Additional support was provided by the Otto Meyerhof Center for Biotechnology, established by the Minerva Foundation, (Munich, Germany).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Dagger To whom correspondence should be addressed: Tel.: 972-8-934-2373; Fax: 972-8-946-8256; E-mail: bfbayer@wicc.weizmann.ac.il.

Published, JBC Papers in Press, January 8, 2001, DOI 10.1074/jbc.M009237200

    ABBREVIATIONS

The abbreviations used are: OE PCR, Overlap-extension polymerase chain reaction; CBD, cellulose-binding domain; CelS-ct, recombinant cellulosomal Family-48 cellulase from C. thermocellum; CelA-cc, recombinant cellulosomal Family-5 cellulase from C. cellulolyticum; Coh-ct, recombinant probe from the C. thermocellum scaffoldin subunit that contains the cohesin-2 module and the CBD; Coh-cc, recombinant probe from the C. cellulolyticum scaffoldin subunit that contains the CBD and cohesin-1 module; PAGE, polyacrylamide gel electrophoresis; bp, base pair(s).

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

1. Bayer, E. A., Shimon, L. J. W., Lamed, R., and Shoham, Y. (1998) J. Struct. Biol. 124, 221-234[CrossRef][Medline] [Order article via Infotrieve]
2. Shoham, Y., Lamed, R., and Bayer, E. A. (1999) Trends Microbiol. 7, 275-281[CrossRef][Medline] [Order article via Infotrieve]
3. Béguin, P., and Lemaire, M. (1996) Crit. Rev. Biochem. Mol. Biol. 31, 201-236[Abstract]
4. Felix, C. R., and Ljungdahl, L. G. (1993) Annu. Rev. Microbiol. 47, 791-819[CrossRef][Medline] [Order article via Infotrieve]
5. Lamed, R., Setter, E., and Bayer, E. A. (1983) J. Bacteriol. 156, 828-836[Medline] [Order article via Infotrieve]
6. Lamed, R., Setter, E., Kenig, R., and Bayer, E. A. (1983) Biotechnol. Bioeng. Symp. 13, 163-181
7. Belaich, J.-P., Tardif, C., Belaich, A., and Gaudin, C. (1997) J. Biotechnol. 57, 3-14[CrossRef][Medline] [Order article via Infotrieve]
8. Doi, R. H., Goldstein, M., Hashida, S., Park, J. S., and Takagi, M. (1994) Crit. Rev. Microbiol. 20, 87-93[Medline] [Order article via Infotrieve]
9. Fanutti, C., Ponyi, T., Black, G. W., Hazlewood, G. P., and Gilbert, H. J. (1995) J. Biol. Chem. 270, 29314-29322[Abstract/Free Full Text]
10. Li, X., Chen, H., and Ljungdahl, L. (1997) Appl. Environ. Microbiol. 63, 4721-4728[Abstract]
11. Bayer, E. A., Chanzy, H., Lamed, R., and Shoham, Y. (1998) Curr. Opin. Struct. Biol. 8, 548-557[CrossRef][Medline] [Order article via Infotrieve]
12. Ding, S.-Y., Bayer, E. A., Steiner, D., Shoham, Y., and Lamed, R. (2000) J. Bacteriol. 182, 4915-4925[Abstract/Free Full Text]
13. Ding, S.-Y., Bayer, E. A., Steiner, D., Shoham, Y., and Lamed, R. (1999) J. Bacteriol. 181, 6720-6729[Abstract/Free Full Text]
14. Lamed, R., and Bayer, E. A. (1988) Adv. Appl. Microbiol. 33, 1-46
15. Wu, J. H. D., and Demain, A. L. (1988) in Biochemistry and Genetics of Cellulose Degradation (Aubert, J.-P. , Béguin, P. , and Millet, J., eds) , pp. 117-131, Academic Press, London
16. Chauvaux, S., Béguin, P., Aubert, J.-P., Bhat, K. M., Gow, L. A., Wood, T. M., and Bairoch, A. (1990) Biochem. J. 265, 261-265[Medline] [Order article via Infotrieve]
17. Yaron, S., Morag, E., Bayer, E. A., Lamed, R., and Shoham, Y. (1995) FEBS Lett. 360, 121-124[CrossRef][Medline] [Order article via Infotrieve]
18. Lytle, B., Myers, C., Kruus, K., and Wu, J. H. D. (1996) J. Bacteriol. 178, 1200-1203[Abstract]
19. Lytle, B., and Wu, J. H. D. (1998) J. Bacteriol. 180, 6581-6585[Abstract/Free Full Text]
20. Fierobe, H.-P., Pagès, S., Belaich, A., Champ, S., Lexa, D., and Belaich, J.-P. (1999) Biochemistry 38, 12822-12832[CrossRef][Medline] [Order article via Infotrieve]
21. Pagès, S., Belaich, A., Belaich, J.-P., Morag, E., Lamed, R., Shoham, Y., and Bayer, E. A. (1997) Proteins 29, 517-527[CrossRef][Medline] [Order article via Infotrieve]
22. Bayer, E. A., Morag, E., Lamed, R., Yaron, S., and Shoham, Y. (1998) in Carbohydrases from Trichoderma reesei and other microorganisms (Claeyssens, M. , Nerinckx, W. , and Piens, K., eds) , pp. 39-67, The Royal Society of Chemistry, London
23. Lytle, B., Volkman, B. F., Westler, W. M., and Wu, J. H. D. (2000) Arch. Biochem. Biophys. 379, 237-244[CrossRef][Medline] [Order article via Infotrieve]
24. Shimon, L. J. W., Bayer, E. A., Morag, E., Lamed, R., Yaron, S., Shoham, Y., and Frolow, F. (1997) Structure 5, 381-390[Medline] [Order article via Infotrieve]
25. Tavares, G. A., Béguin, P., and Alzari, P. M. (1997) J. Mol. Biol. 273, 701-713[CrossRef][Medline] [Order article via Infotrieve]
26. Mechaly, A., Yaron, S., Lamed, R., Fierobe, H.-P., Belaich, A., Belaich, J.-P., Shoham, Y., and Bayer, E. A. (2000) Proteins 39, 170-177[CrossRef][Medline] [Order article via Infotrieve]
27. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed. , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
28. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (eds) (1992) Short Protocols in Molecular Biology , Greene Publishing Associates/Wiley Interscience, New York
29. Deng, W. P., and Nickoloff, J. A. (1992) Anal. Biochem. 200, 81-88[Medline] [Order article via Infotrieve]
30. Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K., and Pease, L. R. (1989) Gene 77, 51-56[CrossRef][Medline] [Order article via Infotrieve]
31. Meza, R., Nufiez-Valdez, M.-E., Sanchez, J., and Bravo, A. (1996) FEMS Microbiol. Lett. 145, 333-339[CrossRef][Medline] [Order article via Infotrieve]
32. Pagès, S., Belaich, A., Tardif, C., Reverbel-Leroy, C., Gaudin, C., and Belaich, J.-P. (1996) J. Bacteriol. 178, 2279-2286[Abstract]
33. Bayer, E. A., and Wilchek, M. (1990) Methods Enzymol. 184, 138-160[Medline] [Order article via Infotrieve]
34. Wu, J. H. D., Orme-Johnson, W. H., and Demain, A. L. (1988) Biochemistry 27, 1703-1709
35. Tokatlidis, K., Salamitou, S., Béguin, P., Dhurjati, P., and Aubert, J.-P. (1991) FEBS Lett. 291, 185-188[CrossRef][Medline] [Order article via Infotrieve]
36. Tokatlidis, K., Dhurjati, P., and Béguin, P. (1993) Protein Eng. 6, 947-952[Abstract]
37. Rost, B., and Sander, C. (1993) J. Mol. Biol. 232, 584-599[CrossRef][Medline] [Order article via Infotrieve]
38. Rost, B., and Sander, C. (1994) Proteins 19, 55-72[Medline] [Order article via Infotrieve]
39. Rost, B. (1996) Methods Enzymol. 266, 525-539[CrossRef][Medline] [Order article via Infotrieve]
40. Wilchek, M., and Bayer, E. A. (1999) Biomol. Eng. 16, 1-4[CrossRef][Medline] [Order article via Infotrieve]


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.