Cohesin-Dockerin Interaction in Cellulosome Assembly

Abstract

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 theC. thermocellum dockerin differentiates between its nonrecognition and high affinity recognition (Ka∼ 109m−1) by a cohesin fromC. 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. 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 theC. thermocellum dockerin differentiates between its nonrecognition and high affinity recognition (Ka∼ 109m−1) by a cohesin fromC. 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. Overlap-extension polymerase chain reaction cellulose-binding domain recombinant cellulosomal Family-48 cellulase fromC. thermocellum recombinant cellulosomal Family-5 cellulase from C. cellulolyticum recombinant probe from the C. thermocellum scaffoldin subunit that contains the cohesin-2 module and the CBD recombinant probe from theC. cellulolyticum scaffoldin subunit that contains the CBD and cohesin-1 module polyacrylamide gel electrophoresis base pair(s) The cellulosome concept is one of the major paradigms of microbial degradation of plant cell-wall polysaccharides (1Bayer E.A. Shimon L.J.W. Lamed R. Shoham Y. J. Struct. Biol. 1998; 124: 221-234Crossref PubMed Scopus (277) Google Scholar, 2Shoham Y. Lamed R. Bayer E.A. Trends Microbiol. 1999; 7: 275-281Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar, 3Béguin P. Lemaire M. Crit. Rev. Biochem. Mol. Biol. 1996; 31: 201-236Crossref PubMed Scopus (169) Google Scholar, 4Felix C.R. Ljungdahl L.G. Annu. Rev. Microbiol. 1993; 47: 791-819Crossref PubMed Scopus (146) Google Scholar). Initially, such a multienzyme complex was described in the anaerobic thermophilic bacterium, Clostridium thermocellum (5Lamed R. Setter E. Bayer E.A. J. Bacteriol. 1983; 156: 828-836Crossref PubMed Google Scholar, 6Lamed R. Setter E. Kenig R. Bayer E.A. Biotechnol. Bioeng. Symp. 1983; 13: 163-181Google Scholar). Additional cellulosomes have been described subsequently for a variety of anaerobic bacteria and fungi (7Belaich J.-P. Tardif C. Belaich A. Gaudin C. J. Biotechnol. 1997; 57: 3-14Crossref PubMed Scopus (95) Google Scholar, 8Doi R.H. Goldstein M. Hashida S. Park J.S. Takagi M. Crit. Rev. Microbiol. 1994; 20: 87-93Crossref PubMed Scopus (85) Google Scholar, 9Fanutti C. Ponyi T. Black G.W. Hazlewood G.P. Gilbert H.J. J. Biol. Chem. 1995; 270: 29314-29322Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 10Li X. Chen H. Ljungdahl L. Appl. Environ. Microbiol. 1997; 63: 4721-4728Crossref PubMed Google Scholar, 11Bayer E.A. Chanzy H. Lamed R. Shoham Y. Curr. Opin. Struct. Biol. 1998; 8: 548-557Crossref PubMed Scopus (469) Google Scholar, 12Ding S.-Y. Bayer E.A. Steiner D. Shoham Y. Lamed R. J. Bacteriol. 2000; 182: 4915-4925Crossref PubMed Scopus (59) Google Scholar, 13Ding S.-Y. Bayer E.A. Steiner D. Shoham Y. Lamed R. J. Bacteriol. 1999; 181: 6720-6729Crossref PubMed Google Scholar). 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 (14Lamed R. Bayer E.A. Adv. Appl. Microbiol. 1988; 33: 1-46Crossref Scopus (177) Google Scholar, 15Wu J.H.D. Demain A.L. Aubert J.-P. Béguin P. Millet J. Biochemistry and Genetics of Cellulose Degradation.in: Academic Press, London1988: 117-131Google Scholar). 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 (16Chauvaux S. Béguin P. Aubert J.-P. Bhat K.M. Gow L.A. Wood T.M. Bairoch A. Biochem. J. 1990; 265: 261-265Crossref PubMed Scopus (83) Google Scholar). 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 (17Yaron S. Morag E. Bayer E.A. Lamed R. Shoham Y. FEBS Lett. 1995; 360: 121-124Crossref PubMed Scopus (125) Google Scholar, 18Lytle B. Myers C. Kruus K. Wu J.H.D. J. Bacteriol. 1996; 178: 1200-1203Crossref PubMed Google Scholar). It was further demonstrated that both duplicated segments of the dockerin domain are required for binding to the cohesin module (19Lytle B. Wu J.H.D. J. Bacteriol. 1998; 180: 6581-6585Crossref PubMed Google Scholar, 20Fierobe H.-P. Pagès S. Belaich A. Champ S. Lexa D. Belaich J.-P. Biochemistry. 1999; 38: 12822-12832Crossref PubMed Scopus (81) Google Scholar). 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 (21Pagès S. Belaich A. Belaich J.-P. Morag E. Lamed R. Shoham Y. Bayer E.A. Proteins. 1997; 29: 517-527Crossref PubMed Scopus (184) Google Scholar, 22Bayer E.A. Morag E. Lamed R. Yaron S. Shoham Y. Claeyssens M. Nerinckx W. Piens K. Carbohydrases from Trichoderma reesei and other microorganisms.in: The Royal Society of Chemistry, London1998: 39-67Google Scholar). This hypothesis has recently been corroborated experimentally by NMR spectroscopy (23Lytle B. Volkman B.F. Westler W.M. Wu J.H.D. Arch. Biochem. Biophys. 2000; 379: 237-244Crossref PubMed Scopus (56) Google Scholar). Despite the reported crystal structures for two different C. thermocellum cohesins (24Shimon L.J.W. Bayer E.A. Morag E. Lamed R. Yaron S. Shoham Y. Frolow F. Structure. 1997; 5: 381-390Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 25Tavares G.A. Béguin P. Alzari P.M. J. Mol. Biol. 1997; 273: 701-713Crossref PubMed Scopus (78) Google Scholar), 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 andC. cellulolyticum, is species-specific (21Pagès S. Belaich A. Belaich J.-P. Morag E. Lamed R. Shoham Y. Bayer E.A. Proteins. 1997; 29: 517-527Crossref PubMed Scopus (184) Google Scholar). 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 (26Mechaly A. Yaron S. Lamed R. Fierobe H.-P. Belaich A. Belaich J.-P. Shoham Y. Bayer E.A. Proteins. 2000; 39: 170-177Crossref PubMed Scopus (80) Google Scholar). 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. Escherichia colistrain 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(λDE3) 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 (26Mechaly A. Yaron S. Lamed R. Fierobe H.-P. Belaich A. Belaich J.-P. Shoham Y. Bayer E.A. Proteins. 2000; 39: 170-177Crossref PubMed Scopus (80) Google Scholar). The various CelS-derived mutants were expressed in the same host strain. DNA was manipulated by standard procedures (27Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar, 28Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith,J. A., and Struhl, K. (eds) (1992) Short in Molecular Scholar). DNA was the calcium for strain or by for and DNA was by the DNA at the termed was as reported previously (26Mechaly A. Yaron S. Lamed R. Fierobe H.-P. Belaich A. Belaich J.-P. Shoham Y. Bayer E.A. Proteins. 2000; 39: 170-177Crossref PubMed Scopus (80) Google Scholar), a of the Biochem. PubMed Scopus Google Scholar). the mutations were first in a dockerin construct and The DNA for the dockerin segment was to the enzyme a to and the dockerin as the was the same the dockerin segment were selected the enzyme and the segment was to the enzyme the same The both the dockerin and the was used subsequently as a for the of In this case, the and contained the which converted to of used for site-directed in to the position of the indicated residue in the the to and the that the was by the in the given are shown in to the position of the indicated residue in the the to and the that the was by the in the given in a new are shown the of and the other mutations were achieved PubMed Scopus Google Scholar, R. J. A. Microbiol. Lett. 1996; PubMed Google as described (26Mechaly A. Yaron S. Lamed R. Fierobe H.-P. Belaich A. Belaich J.-P. Shoham Y. Bayer E.A. Proteins. 2000; 39: 170-177Crossref PubMed Scopus (80) Google Scholar). was used as a for the of the mutations were in two and and and However, of the dockerin into in a high of to the in of the two DNA and in from this a was in which an by and was from the The was further at an and the was with the dockerin into pET9d-CelS The resulting was used as a for the of the and the same the of and were used with and with as in a The same were also used to construct and In the of was used as a with the for pET9d-CelS was used as a with The of the C. cellulolyticum cellulase A was described in an (26Mechaly A. Yaron S. Lamed R. Fierobe H.-P. Belaich A. Belaich J.-P. Shoham Y. Bayer E.A. Proteins. 2000; 39: 170-177Crossref PubMed Scopus (80) Google Scholar). mutants were expressed in as described previously (26Mechaly A. Yaron S. Lamed R. Fierobe H.-P. Belaich A. Belaich J.-P. Shoham Y. Bayer E.A. Proteins. 2000; 39: 170-177Crossref PubMed Scopus (80) Google Scholar). The were used for additional The were to a of and was to and The respective cohesin and were as described previously (17Yaron S. Morag E. Bayer E.A. Lamed R. Shoham Y. FEBS Lett. 1995; 360: 121-124Crossref PubMed Scopus (125) Google Scholar, S. Belaich A. Tardif C. C. Gaudin C. Belaich J.-P. J. Bacteriol. 1996; 178: PubMed Google Scholar). and its were as described H.-P. Pagès S. Belaich A. Champ S. Lexa D. Belaich J.-P. Biochemistry. 1999; 38: 12822-12832Crossref PubMed Scopus (81) Google Scholar, A. Yaron S. Lamed R. Fierobe H.-P. Belaich A. Belaich J.-P. Shoham Y. Bayer E.A. Proteins. 2000; 39: 170-177Crossref PubMed Scopus (80) Google Scholar). and were and as coli were to at in of with and The were to and was to a of the were in of and in a The was a of in the same The was with the same and the recombinant was with further the of the was with and of and and on of with the same The was a from to and were analyzed by The recombinant were and of by in were at a of to as described previously E.A. M. 1990; PubMed Scopus Google Scholar). The from both species binding to both and enzymes and were recognized by The affinity was as described previously (26Mechaly A. Yaron S. Lamed R. Fierobe H.-P. Belaich A. Belaich J.-P. Shoham Y. Bayer E.A. Proteins. 2000; 39: 170-177Crossref PubMed Scopus (80) Google Scholar). of the dockerin were by and The were with from C. thermocellum or C. and and were as described H.-P. Pagès S. Belaich A. Champ S. Lexa D. Belaich J.-P. Biochemistry. 1999; 38: 12822-12832Crossref PubMed Scopus (81) Google Scholar), as were The were in the same from to and to with the by of The cellulosome is a enzyme complex of the cellulosome from C. thermocellum into its the presence of at is required (14Lamed R. Bayer E.A. Adv. Appl. Microbiol. 1988; 33: 1-46Crossref Scopus (177) Google Scholar, J.H.D. Demain A.L. Biochemistry. 1988; Scopus Google Scholar). The observed of the cellulosome is a of the high affinity interaction between two different of the single dockerin domain of the individual enzyme subunits and the of the cohesin domain on the scaffoldin subunit K. S. Béguin P. P. Aubert J.-P. FEBS Lett. PubMed Scopus Google Scholar, K. P. Béguin P. 1993; PubMed Scopus Google Scholar). in to the specificity of the cohesin-dockerin interaction have with four predicted recognition residues of the dockerins from C. thermocellum and C. cellulolyticum (21Pagès S. Belaich A. Belaich J.-P. Morag E. Lamed R. Shoham Y. Bayer E.A. Proteins. 1997; 29: 517-527Crossref PubMed Scopus (184) Google Scholar), a new specificity was created the modified dockerin of one species now recognized the cohesin of the other (26Mechaly A. Yaron S. Lamed R. Fierobe H.-P. Belaich A. Belaich J.-P. Shoham Y. Bayer E.A. Proteins. 2000; 39: 170-177Crossref PubMed Scopus (80) Google Scholar). these studies, it was of to try to this interaction by four residues account for the specificity of binding between the two species. the other the to the cohesin-dockerin interaction within the same species additional residues as specificity and we the of other dockerin to the observed specificity. determine the minimal to create the new mutations of the four recognition residues were mutations were at and of the two duplicated segments of the dockerin domain in both species Thus, mutants of theC. thermocellum CelS dockerin single mutations at these in the first duplicated segment and mutations of both of the latter in each duplicated segment and and the in the present to as described previously (26Mechaly A. Yaron S. Lamed R. Fierobe H.-P. Belaich A. Belaich J.-P. Shoham Y. Bayer E.A. Proteins. 2000; 39: 170-177Crossref PubMed Scopus (80) Google The dockerin construct of the C. cellulolyticum enzyme was used as a and to the previously reported (26Mechaly A. Yaron S. Lamed R. Fierobe H.-P. Belaich A. Belaich J.-P. Shoham Y. Bayer E.A. Proteins. 2000; 39: 170-177Crossref PubMed Scopus (80) Google and mutations of the C. thermocellum CelS and the C. cellulolyticum segment segment residues at the given in of mutants were to or emulate those of its rival indicate mutation at the in a new The residues at the given in of mutants were to or emulate those of its rival indicate mutation at the The various C. thermocellum dockerins were and to and and the were with a cohesin probe from species. The results are in the of a single residue in the CelS dockerin was to create a new interaction with the C. cellulolyticum cohesin In this context, it was that the for interaction of a dockerin with the cohesin was the presence of a leucine of a threonine at position in the first duplicated Thus, mutants and with and to Indeed, this the of in this position of the thermocellum dockerins and residues of theC. cellulolyticum dockerins It is not mutation of the other residues in and the interconversion of to in position not to be required for recognition between these two species of of residues at critical in the dockerins of C. thermocellum and C. segment segment on dockerin from C. thermocellum on dockerin from C. cellulolyticum of the indicated residues are residues that at a given position are in on dockerin from C. thermocellum on dockerin from C. cellulolyticum in a new The of the indicated residues are residues that at a given position are in In study (26Mechaly A. Yaron S. Lamed R. Fierobe H.-P. Belaich A. Belaich J.-P. Shoham Y. Bayer E.A. Proteins. 2000; 39: 170-177Crossref PubMed Scopus (80) Google Scholar), we that the recognized the cohesin Nevertheless, the binding of its cohesin was In the present we additional that might reduce or abolish such intra-species self-recognition the created binding specificity between the two species. this and of the two duplicated segments of the CelS dockerin were as for site-directed mutagenesis. The for of these four was the of these residues is in theC. thermocellum dockerins to prediction prediction of B. C. J. Mol. Biol. 1993; PubMed Scopus Google Scholar, B. C. Proteins. 1994; PubMed Scopus Google Scholar, B. 1996; PubMed Google Scholar), these four residues are predicted to be in in the dockerin structure to the also be but the dockerins C. thermocellum and C. cellulolyticum a at this position and interconversion in this would be for dockerin to prediction B. 1996; PubMed Google Scholar), sequence of different dockerin from C. thermocellum and C. cellulolyticum. The CelS sequence is shown and of mutation are in The predicted of are also indicated (21Pagès S. Belaich A. Belaich J.-P. Morag E. Lamed R. Shoham Y. Bayer E.A. Proteins. 1997; 29: 517-527Crossref PubMed Scopus (184) Google in are by in the to a of prediction to prediction B. 1996; PubMed Google Scholar), sequence of different dockerin from C. thermocellum and C. cellulolyticum. The CelS sequence is shown and of mutation are in The predicted of are also indicated (21Pagès S. Belaich A. Belaich J.-P. Morag E. Lamed R. Shoham Y. Bayer E.A. Proteins. 1997; 29: 517-527Crossref PubMed Scopus (184) Google Scholar). in are by in the to a of prediction In of the mutations in and of the duplicated segments were to the mutations at and Thus, the in were to in and the residues at of the two duplicated segments and were to in the mutation in to the mutations at and In the latter two mutation to was for position of segment of as in the dockerin from C. cellulolyticum, because is more at this position among of the cellulosomal enzymes of this species The of these mutations are shown in In with the mutants and in a in their self-recognition of the C. with those of the and mutants the other in but not a of interaction with It follows that position is an in dockerin recognition. Nevertheless, the C. enzyme that to bind both mutants and to their cohesin as described This in theC. thermocellum other in to the eight still play a role in the recognition the of the affinity of the mutants were and to analysis by of the two species of cohesins and the The results are in In the are in with the and for the binding of the individual dockerins to species of cohesin. It is that the observed affinity for the cohesin-dockerin interaction in C. thermocellum were to be much those of C. cellulolyticum. The affinity for the C. cellulolyticum was in of a high affinity interaction and in with previously reported H.-P. Pagès S. Belaich A. Champ S. Lexa D. Belaich J.-P. Biochemistry. 1999; 38: 12822-12832Crossref PubMed Scopus (81) Google Scholar). In the thermophilic the affinity were and the of the It be noted that such a high affinity be required to binding thermophilic In case, the cohesin-dockerin complex in the C. thermocellum one of the affinity interactions in other high affinity and M. Bayer E.A. 1999; PubMed Scopus Google for binding of and enzymes to cohesin and enzyme were fromC. thermocellum and C. cellulolyticum were obtained by a in a new The cohesin and enzyme were fromC. thermocellum and C. cellulolyticum were obtained by a to the selectivity of cohesin-dockerin the critical mutation in which the threonine at position of the first duplicated segment was converted to a high affinity interaction with the C. with the dockerin-bearing this single mutation between a of binding and a cohesin-dockerin interaction, by a of It is to that of theC. thermocellum dockerin mutants that the mutation similarly high of for that of the enzyme from C. cellulolyticum of are the for the with a for their with the C. cellulolyticum cohesin. In the the four mutations in the dockerin of a similarly high affinity 109m−1) with the enzyme binding with the cohesin. to intra-species cohesin-dockerin the of a given dockerin to of the two species of cohesins could be by the of affinity to the high affinity for the C. thermocellum of the CelS-derived mutants a for self-recognition of However, as the number of mutations a toward for Indeed, a for recognition. In the the interaction with the rival cohesin. It is to that this is to in its mutation in and the in recognition between these two mutants be to the affinity of the cohesin-dockerin interaction of the thermophilic The of a single threonine for leucine in the C. thermocellum dockerin recognition with a C. cellulolyticum cohesin. This that the major between these two the of a hydroxyl group in leucine with the for the specificity. The presence or absence of this single decisive hydroxyl group at the position in the C. to be critical to the specificity of the cohesin-dockerin interaction in this species. It is remarkable that such a would between a high affinity interaction and interaction at This also a on the of the two cohesin species. it be that the C. cellulolyticum cohesin would a more at a yet which account for the observed of specificity in the is the that the dockerin could be to such a number of mutations self-recognition of its cohesin. Thus, the C. thermocellum dockerin could be at and in both duplicated a of one to eight mutations of the dockerin and the of recognized the cohesin of C. cellulolyticum as as its This suggests that the two species of cohesin-dockerin much more (21Pagès S. Belaich A. Belaich J.-P. Morag E. Lamed R. Shoham Y. Bayer E.A. Proteins. 1997; 29: 517-527Crossref PubMed Scopus (184) Google Scholar). the precise determinants that binding specificity of the cohesin-dockerin interaction be by three-dimensional structure of cohesin-dockerin from different species. both and NMR spectroscopy of such have In the such as site-directed or of the different to us with into the determinants of the cohesin-dockerin interaction. We the of mutants and by