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• W1998408013 abstract "The plant cell wall degrading apparatus of anaerobic bacteria includes a large multienzyme complex termed the “cellulosome.” The complex assembles through the interaction of enzyme-derived dockerin modules with the multiple cohesin modules of the noncatalytic scaffolding protein. Here we report the crystal structure of the Clostridium cellulolyticum cohesin-dockerin complex in two distinct orientations. The data show that the dockerin displays structural symmetry reflected by the presence of two essentially identical cohesin binding surfaces. In one binding mode, visualized through the A16S/L17T dockerin mutant, the C-terminal helix makes extensive interactions with its cohesin partner. In the other binding mode observed through the A47S/F48T dockerin variant, the dockerin is reoriented by 180° and interacts with the cohesin primarily through the N-terminal helix. Apolar interactions dominate cohesin-dockerin recognition that is centered around a hydrophobic pocket on the surface of the cohesin, formed by Leu-87 and Leu-89, which is occupied, in the two binding modes, by the dockerin residues Phe-19 and Leu-50, respectively. Despite the structural similarity between the C. cellulolyticum and Clostridium thermocellum cohesins and dockerins, there is no cross-specificity between the protein partners from the two organisms. The crystal structure of the C. cellulolyticum complex shows that organism-specific recognition between the protomers is dictated by apolar interactions primarily between only two residues, Leu-17 in the dockerin and the cohesin amino acid Ala-129. The biological significance of the plasticity in dockerin-cohesin recognition, observed here in C. cellulolyticum and reported previously in C. thermocellum, is discussed. The plant cell wall degrading apparatus of anaerobic bacteria includes a large multienzyme complex termed the “cellulosome.” The complex assembles through the interaction of enzyme-derived dockerin modules with the multiple cohesin modules of the noncatalytic scaffolding protein. Here we report the crystal structure of the Clostridium cellulolyticum cohesin-dockerin complex in two distinct orientations. The data show that the dockerin displays structural symmetry reflected by the presence of two essentially identical cohesin binding surfaces. In one binding mode, visualized through the A16S/L17T dockerin mutant, the C-terminal helix makes extensive interactions with its cohesin partner. In the other binding mode observed through the A47S/F48T dockerin variant, the dockerin is reoriented by 180° and interacts with the cohesin primarily through the N-terminal helix. Apolar interactions dominate cohesin-dockerin recognition that is centered around a hydrophobic pocket on the surface of the cohesin, formed by Leu-87 and Leu-89, which is occupied, in the two binding modes, by the dockerin residues Phe-19 and Leu-50, respectively. Despite the structural similarity between the C. cellulolyticum and Clostridium thermocellum cohesins and dockerins, there is no cross-specificity between the protein partners from the two organisms. The crystal structure of the C. cellulolyticum complex shows that organism-specific recognition between the protomers is dictated by apolar interactions primarily between only two residues, Leu-17 in the dockerin and the cohesin amino acid Ala-129. The biological significance of the plasticity in dockerin-cohesin recognition, observed here in C. cellulolyticum and reported previously in C. thermocellum, is discussed. The microbial degradation of the plant cell wall is an important biological process that is central to the cycling of carbon between microorganisms, plants, and herbivores. Furthermore, the enzymes that catalyze the hydrolyses of plant structural polysaccharides are deployed in several biotechnological processes (for review see Ref. 1Bhat M.K. Biotechnol. Adv. 2000; 18: 355-383Crossref PubMed Scopus (1018) Google Scholar), although there is currently considerable interest in the application of these biocatalysts in the conversion of lignocellulose, an abundant renewable source of organic carbon, into biofuels such as ethanol and butanol (2Boudet A.M. Kajita S. Grima-Pettenati J. Goffner D. Trends Plant Sci. 2003; 8: 576-581Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar, 3Ragauskas A.J. Williams C.K. Davison B.H. Britovsek G. Cairney J. Eckert C.A. Frederick Jr., W.J. Hallett J.P. Leak D.J. Liotta C.L. Mielenz J.R. Murphy R. Templer R. Tschaplinski T. Science. 2006; 311: 484-489Crossref PubMed Scopus (4566) Google Scholar). The plant cell wall is an insoluble highly recalcitrant macromolecule consisting mainly of interlocking polysaccharides. Saprophytic microorganisms that utilize the plant cell wall as a major nutrient synthesize enzyme consortia in which the biocatalysts act in synergy to degrade the composite substrate. A common feature of the plant cell wall apparatus synthesized by both eukaryotic and prokaryotic anaerobic microorganisms is that the component enzymes assemble into large complexes, which are referred to as cellulosomes (for review see Ref. 4Bayer E.A. Belaich J.P. Shoham Y. Lamed R. Annu. Rev. Microbiol. 2004; 58: 521-554Crossref PubMed Scopus (717) Google Scholar). The cellulosome is assembled by the binding of the catalytic subunits, comprising glycoside hydrolases, esterases, and lyases, to a non-catalytic protein scaffold (hereafter referred to as Cip) (5Gerngross U.T. Romaniec M.P. Kobayashi T. Huskisson N.S. Demain A.L. Mol. Microbiol. 1993; 8: 325-334Crossref PubMed Scopus (177) Google Scholar). The integration of the plant cell wall hydrolases into the cellulosome has been proposed to potentiate the synergistic interactions between the enzymes and contributes to substrate targeting through the cellulose binding capacity of most Cip molecules (6Fierobe H.P. Bayer E.A. Tardif C. Czjzek M. Mechaly A. Belaich A. Lamed R. Shoham Y. Belaich J.P. J. Biol. Chem. 2002; 277: 49621-49630Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 7Fierobe H.P. Mingardon F. Mechaly A. Belaich A. Rincon M.T. Pages S. Lamed R. Tardif C. Belaich J.P. Bayer E.A. J. Biol. Chem. 2005; 280: 16325-16334Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). The Cip molecules of Clostridium cellulolyticum and Clostridium thermocellum (designated CipC and CipA, respectively) can bind eight and nine enzymes, respectively (8Handelsman T. Barak Y. Nakar D. Mechaly A. Lamed R. Shoham Y. Bayer E.A. FEBS Lett. 2004; 572: 195-200Crossref PubMed Scopus (35) Google Scholar), although the cellulosomes of other anaerobic bacteria deploy multiple Cip and adapter molecules in assembling as many as 96 catalytic subunits into a single complex (9Xu Q. Gao W. Ding S.Y. Kenig R. Shoham Y. Bayer E.A. Lamed R. J. Bacteriol. 2003; 185: 4548-4557Crossref PubMed Scopus (78) Google Scholar). In Clostridia the Cip contains multiple type I cohesin modules that bind tightly to the type I dockerins present on the catalytic subunits and thus assemble these enzymes into the cellulosome (10Salamitou S. Raynaud O. Lemaire M. Coughlan M. Beguin P. Aubert J.P. J. Bacteriol. 1994; 176: 2822-2827Crossref PubMed Google Scholar). In general, within a single organism, cohesin modules of the Cip display a very high level of sequence identity, and the type I dockerins appear to display little if any discrimination between their receptors in the cellulosome scaffold (11Ciruela A. Gilbert H.J. Ali B.R. Hazlewood G.P. FEBS Lett. 1998; 422: 221-224Crossref PubMed Scopus (25) Google Scholar, 12Yaron S. Morag E. Bayer E.A. Lamed R. Shoham Y. FEBS Lett. 1995; 360: 121-124Crossref PubMed Scopus (125) Google Scholar). Similarly, the type I dockerin modules also display extensive sequence identity consistent with the lack of specificity for the type I cohesins (4Bayer E.A. Belaich J.P. Shoham Y. Lamed R. Annu. Rev. Microbiol. 2004; 58: 521-554Crossref PubMed Scopus (717) Google Scholar, 13Pages 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 crystal structures of type I C. thermocellum cohesin-dockerin complexes have provided insight into the mechanism of cellulosome assembly (14Carvalho A.L. Dias F.M. Prates J.A. Nagy T. Gilbert H.J. Davies G.J. Ferreira L.M. Romao M.J. Fontes C.M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13809-13814Crossref PubMed Scopus (206) Google Scholar, 15Carvalho A.L. Pires V.M. Gloster T.M. Turkenburg J.P. Prates J.A. Ferreira L.M. Romao M.J. Davies G.J. Fontes C.M. Gilbert H.J. J. Mol. Biol. 2005; 349: 909-915Crossref PubMed Scopus (32) Google Scholar). Within the dockerin there is a tandem duplication of a 22-residue sequence that contributes an α-helix and an EF-hand calcium-binding motif and displays remarkable structural conservation. Indeed the structure of the first duplicated segment, which contains the N-terminal helix, can be superimposed precisely over the structure containing the second segment containing the C-terminal helix (helix-3). This symmetry, coupled with several mutagenesis studies (16Mechaly A. Fierobe H.P. Belaich A. Belaich J.P. Lamed R. Shoham Y. Bayer E.A. J. Biol. Chem. 2001; 276: 9883-9888Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 17Schaeffer F. Matuschek M. Guglielmi G. Miras I. Alzari P.M. Beguin P. Biochemistry. 2002; 41: 2106-2114Crossref PubMed Scopus (63) Google Scholar), indicates that the C. thermocellum type I dockerin contains two equivalent ligand-binding sites, which have been maintained during evolution. This view is entirely consistent with the crystal structure of the complexes. Thus in one complex helix-3 dominates cohesin recognition with Ser-45 and Thr-46 playing a central role in the polar interactions between the two protein partners (14Carvalho A.L. Dias F.M. Prates J.A. Nagy T. Gilbert H.J. Davies G.J. Ferreira L.M. Romao M.J. Fontes C.M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13809-13814Crossref PubMed Scopus (206) Google Scholar). In the second crystal structure the dockerin is rotated 180° relative to the cohesin, and helix-1, rather than helix-3, plays a central role in complex formation (15Carvalho A.L. Pires V.M. Gloster T.M. Turkenburg J.P. Prates J.A. Ferreira L.M. Romao M.J. Davies G.J. Fontes C.M. Gilbert H.J. J. Mol. Biol. 2005; 349: 909-915Crossref PubMed Scopus (32) Google Scholar). Thus, the equivalent residues to Ser-45 and Thr-46 in the N-terminal helix, Ser-11 and Thr-12, dominate the hydrogen-bonding interactions between the dockerin and its cohesin partner in this second binding mode. Although the sequences of C. cellulolyticum type I cohesins and dockerins are very similar to the corresponding C. thermocellum modules, there is no cross-specificity between the proteins derived from the two organisms (8Handelsman T. Barak Y. Nakar D. Mechaly A. Lamed R. Shoham Y. Bayer E.A. FEBS Lett. 2004; 572: 195-200Crossref PubMed Scopus (35) Google Scholar, 13Pages 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, 16Mechaly A. Fierobe H.P. Belaich A. Belaich J.P. Lamed R. Shoham Y. Bayer E.A. J. Biol. Chem. 2001; 276: 9883-9888Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Significantly the Ser-Thr dyad in the C. thermocellum dockerins is replaced with hydrophobic residues in the corresponding C. cellulolyticum protein modules. Mutagenesis studies have shown that replacing the Ser-Thr dyad with hydrophobic residues in the C. thermocellum dockerin, and similarly substituting the Ala-Leu and Ala-Phe motifs with hydroxyl amino acids in the C. cellulolyticum dockerin, extends ligand specificity (16Mechaly A. Fierobe H.P. Belaich A. Belaich J.P. Lamed R. Shoham Y. Bayer E.A. J. Biol. Chem. 2001; 276: 9883-9888Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Thus the mutant C. cellulolyticum and C. thermocellum dockerins gain the capacity to recognize the C. thermocellum and C. cellulolyticum cohesins, respectively, while retaining a diminished affinity for their original protein target (16Mechaly A. Fierobe H.P. Belaich A. Belaich J.P. Lamed R. Shoham Y. Bayer E.A. J. Biol. Chem. 2001; 276: 9883-9888Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Currently it is unclear whether the dual binding mode displayed by the C. thermocellum type I dockerins is a generic feature of cellulosome assembly, whereas the mechanistic basis for the organism-based specificity displayed by cohesin-dockerin partners remains essentially unknown. Here we report the crystal structure of the C. cellulolyticum dockerin-cohesin complex. The data show that the dockerin displays dyad symmetry and is able to interact with its cognate cohesin through a dual binding mode. Cohesin-dockerin recognition is dominated by hydrophobic interactions, which are centered around a hydrophobic pocket on the surface of the cohesin, formed by Leu-87 and Leu-89, which is occupied in the two binding modes by the dockerin residues Phe-19 and Leu-50, respectively. Intriguingly, organism specificity is dictated primarily by apolar interactions between only two residues, Leu-17 in the dockerin and the cohesin amino acid Ala-129. Cloning and Expression—DNA encoding the dockerin module of the GH5 C. cellulolyticum cellulase CcCel5A (residues 410-473; note that the proline at position 473 in the published sequence is a glutamate) from C. cellulolyticum was amplified by PCR from pJFAc (18Fierobe H.P. Gaudin C. Belaich A. Loutfi M. Faure E. Bagnara C. Baty D. Belaich J.P. J. Bacteriol. 1991; 173: 7956-7962Crossref PubMed Google Scholar), and the resulting product was ligated into NdeI/XhoI-digested pET22b (Novagen), to generate pHF1. The dockerin gene under the control of the pET22b T7 promoter and terminator was amplified by PCR from pHF1 using the forward primer T7f (5′-CACGATGCGTCCGGCGTAGAGGAT-3′) and the reverse primer T7br (5′-GGGGGGAGATCTATCCGGATATAGTTCCTCCTTTCA-3′) that incorporated 5′ and 3′ BglII sites. To express C. cellulolyticum dockerin and cohesin genes in the same plasmid, the resulting PCR product was digested with BglII and subcloned into the BglII site of plasmid pET-coh1B, which encodes the first cohesin module (residues 277-439) of C. cellulolyticum scaffoldin CipC (19Fierobe H.P. Pages S. Belaich A. Champ S. Lexa D. Belaich J.P. Biochemistry. 1999; 38: 12822-12832Crossref PubMed Scopus (81) Google Scholar). The resulting recombinant plasmid, termed pHF2, contained both genes organized in tandem and was sequenced to ensure that no mutations had occurred during PCR. In the wild type cohesin-dockerin complex, derived from the co-expression of the cohesin and dockerin genes from pHF2, only the cohesin contained a C-terminal His6 tag. The construction of the plasmid encoding the mutated dockerins of Cel5A and the first cohesin from CipC was performed by the overlap-extension PCR method. The regions encoding the N-terminal and C-terminal parts of the dockerin were amplified by PCR from pHF2 using mutagenic primer pairs. The resulting overlapping fragments were mixed, and a combined fragment was synthesized using the external primers. The fragment was subsequently cloned into BglII linearized pHF2, thereby generating pHF3. Positive clones were verified by DNA sequencing. To study cohesin-dockerin binding, the dockerin was also expressed independent of its cohesin partner but fused to thioredoxin, encoded by pET32a, to ensure higher levels of protein expression. DNA encoding the dockerin of the cellulase Cel5A (residues 410-475) was amplified by PCR from pHF2 and cloned into EcoRI- and XhoI-restricted pET32a to generate pHF5. Protein Expression and Purification—The pET22b derivatives encoding the cohesin-dockerin complexes and discrete cohesins and the pET32a plasmids encoding C. cellulolyticum dockerins were transformed into Escherichia coli strains BL21 (DE3) and Origami, respectively. To express the clostridial proteins, recombinant E. coli strains harboring the appropriate recombinant plasmids were cultured in LB containing 100 μg/ml ampicillin at 37 °C to mid-exponential phase (A550 0.6). Isopropyl β-d-thiogalactopyranoside was then added to a final concentration of 1 mm, and the cultures were incubated for a further 16 h at 19 °C. The cohesin-dockerin A16S-L17T and cohesin-dockerin A47S-F48T complexes were purified by metal-ion affinity chromatography, buffer exchanged into 20 mm Tris/HCl, pH 8.0, containing 2 mm CaCl2, and then further purified by anion exchange chromatography using a Source 30Q column and a gradient elution of 0-1 m NaCl (Amersham Biosciences) to separate the complexes from unbound cohesin. Fractions containing the protein complexes were buffer exchanged and then concentrated in 2 mm CaCl2 to a final concentration of 20 and 10 g/liter for the A16S-L17T and A47S-F48T complexes, respectively. Discrete His-tagged cohesins and dockerins were purified by metal-ion affinity chromatography as described previously (14Carvalho A.L. Dias F.M. Prates J.A. Nagy T. Gilbert H.J. Davies G.J. Ferreira L.M. Romao M.J. Fontes C.M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13809-13814Crossref PubMed Scopus (206) Google Scholar). Isothermal Titration Calorimetry—Isothermal titration calorimetry (ITC) 5The abbreviations used are: ITC, isothermal titration calorimetry; r.m.s.d., root mean square deviation; PDB, Protein Data Bank; SAXS, small angle x-ray scattering. was deployed to measure the affinity of native and mutant forms of the C. cellulolyticum cohesin for its dockerin partner essentially as described previously (14Carvalho A.L. Dias F.M. Prates J.A. Nagy T. Gilbert H.J. Davies G.J. Ferreira L.M. Romao M.J. Fontes C.M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13809-13814Crossref PubMed Scopus (206) Google Scholar). Briefly the wild type and mutant forms of the dockerin (7-119 μm), fused to thioredoxin, were stirred at 300 rpm in the reaction cell, which was injected with 25 × 10 or 48 × 5-μl aliquots of a 70-1715 μm solution of cohesin at 300-s intervals. The buffer consists of 50 mm NaHepes, pH 7.5, containing 2 mm CaCl2, and titrations were carried out at 308 K unless otherwise stated. Integrated heat effects, after correction for heats of dilution, were analyzed by nonlinear regressing using a single site model (Microcal ORIGIN version 7.0, Microcal Software, Northampton, MA). The fitted data yield the association constant (KA) and the change in enthalpy associated with binding (ΔH). Other thermodynamic parameters were calculated using the standard equation -RTlnKA =ΔG =ΔH - TΔS. The c values (product of the molar concentration of binding sites × KA) were >2.6. Crystallization of the C. cellulolyticum Cohesin-Dockerin Complexes and Structure Resolution—Crystals of the cohesin-dockerin A16S-L17T complex grew over a period of 10-12 days, in 0.2 m potassium sulfate and 20% w/v polyethylene glycol 3350, and were cryoprotected with 20% (v/v) of glycerol. Crystals of the cohesin-dockerin A47S-F48T complex grew over a period of 4-5 days, in 0.2 m lithium sulfate and 25% w/v polyethylene glycol monomethyl ether 2000, and were cryoprotected with 20% (v/v) of glycerol. The crystals were harvested in rayon fiber loops and frozen in liquid nitrogen. Data were collected, for both constructs, using single crystals at the European Synchrotron Radiation Facility on station ID14-2 at 100 K using an ADSC Q4 charged-coupled device detector and at a wavelength of 0.9330 Å. All diffraction data were indexed and integrated in MOSFLM (CCP4) or DENZO/SCALEPACK (20Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref Scopus (38253) Google Scholar). All other computing was carried out using the CCP4 suite unless otherwise stated. Crystals of the A16S-L17T complex belong to the space group P212121 with cell dimensions a = 39.3 Å, b = 60.5 Å, and c = 100.7 Å and one complex in the asymmetric unit. In contrast, crystals of the cohesin-dockerin A47S-F48T complex belong to the space group P3221 with approximate cell dimensions of a = b = 76.42 Å and c = 111.09 Å and two independent complexes in the asymmetric unit. Both cohesin-dockerin complex structures were solved by molecular replacement using PHASER (21McCoy A.J. Grosse-Kunstleve R.W. Adams P.D. Winn M.D. Storoni L.C. Read R.J. J. Appl. Crystallogr. 2007; 40: 658-674Crossref PubMed Scopus (13901) Google Scholar) with the search model being the structure of the previously reported apo-form of the type I C. cellulolyticum cohesin module (PDB accession code 1g1k (22Spinelli S. Fierobe H.P. Belaich A. Belaich J.P. Henrissat B. Cambillau C. J. Mol. Biol. 2000; 304: 189-200Crossref PubMed Scopus (51) Google Scholar)). Initial building of the dockerin subunits into the electron density was performed using ARP/wARP (23Collaborative Computational Project Number 4Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19668) Google Scholar), and the remaining residues were built by hand using COOT (24Emsley P. Cowtan K. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (22534) Google Scholar). Refinement was carried out with REFMAC (25Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13712) Google Scholar) with 5% of the data set aside for cross-validation purposes. A summary of the refinement statistics is shown in Table 1. Coordinates and observed structure factor amplitudes have been deposited at the Protein Data Bank (PDB codes 2vn5 and 2vn6). Figures were drawn in MOLSCRIPT (26Kraulis P.J. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar) and BOBSCRIPT (27Esnouf R.M. J. Mol. Graph Model. 1997; 15: 132-134Crossref PubMed Scopus (1794) Google Scholar).TABLE 1Data collection, phasing, and refinement statistics of C. cellulolyticum CohDocA16S/L17T and CohDocA47S/F48TCohDocA47S/F48TCohDocA16S/L17TData collectionSpace groupP3221P212121Cell dimensionsa, b, c (Å)76.4, 76.4, 111.139.3, 60.5, 100.7α, β, γ (°)90, 90, 12090, 90, 90Wavelength (Å)0.933000.93300Resolution (Å)aHighest resolution shell is shown in parentheses.50-1.90 (1.97-1.90)39.19-1.49 (1.57-1.49)RmergeaHighest resolution shell is shown in parentheses.0.067 (0.341)0.062 (0.404)I/σIaHighest resolution shell is shown in parentheses.26 (5.7)15.4 (2.0)Completeness (%)aHighest resolution shell is shown in parentheses.99.7 (99.2)98.9 (93.5)RedundancyaHighest resolution shell is shown in parentheses.6.4 (5.4)3.4 (2.5)RefinementResolution (Å)38.21-1.9019.77-1.49No. of reflections28,51137,165Rwork/Rfree0.181/0.2340.174/0.208No. of atomsProteinCohesin21001104Dockerin867508Ions (Ca2+)42Water286329B-factors (Å2)ProteinCohesin3315Dockerin3413Ions (Ca2+)309Water3830r.m.s.d.Bond lengths (Å)0.0160.010Bond angles (°)1.5421.327PDB codes2vn52vn6a Highest resolution shell is shown in parentheses. Open table in a new tab Thermodynamics and Stoichiometry of Cohesin-Dockerin Recognition—The binding of a CipC-derived C. cellulolyticum type I cohesin to its dockerin partner was assessed by ITC. The data show that at 308 K the KA is 6.50 × 108 m-1 with a ΔH of -19.3 kcal mol-1 and a TΔS of -6.84 kcal mol-1 (Table 2). There was a negative direct relationship between temperature and both ΔH and ΔS, although ΔG was not sensitive to changes in the experimental temperature (Fig. 1). At 284 and 299 K, respectively, ΔH and ΔS were 0. The heat capacity (ΔCp) of cohesin-dockerin binding was -822 cal-1 mol-1 K-1. The ITC data also showed that the stoichiometry of ligand binding was ∼1, the significance of which is discussed below.TABLE 2Thermodynamics of the binding between C. cellulolyticum dockerin and cobesin variantsCohesin variantsaThe cohesin wild type and mutants were titrated against wild type C. cellulolyticum dockerin.KAΔHTΔSnM−1 (× 106)kcal mol−1kcal mol−1Wild type650 ± 76.2−19.3 ± 0.06−6.840.915 ± 0.001T45A598 ± 57.6−19.8 ± 0.05−7.420.859 ± 0.001N47A135 ± 7.24−18.2 ± 0.04−6.710.816 ± 0.001Y49A120 ± 5.48−18.2 ± 0.04−6.750.851 ± 0.001S76A1160 ± 92.2−21.5 ± 0.05−8.690.852 ± 0.001S85A1020 ± 104−22.4 ± 0.06−9.640.852 ± 0.001L87A4.9 ± 0.12−14.0 ± 0.04−3.970.915 ± 0.002L89A8.2 ± 0.34−23.2 ± 0.07−12.10.792 ± 0.001N91A367 ± 22.8−22.3 ± 0.06−10.20.833 ± 0.001193A212 ± 11.5−26.7 ± 0.05−15.00.828 ± 0.001M135G73.6 ± 3.45−20.2 ± 0.07−9.120.803 ± 0.001K137G425 ± 28.0−22.1 ± 0.05−9.950.844 ± 0.001T45A/N47A63.6 ± 6.82−19.5 ± 0.09−8.500.882 ± 0.002T45A/N47A/Y49A2.12 ± 0.12−14.6 ± 0.19−5.700.918 ± 0.008T45A/N47A/N91A5.70 ± 0.06−15.4 ± 0.02−5.850.857 ± 0.001T45A/N47A/Y49A/N91A0.20 ± 0.01−15.4 ± 0.33−7.921.03 ± 0.02S76A/S78A/S85A795 ± 229−24.2 ± 0.02−11.60.683 ± 0.002L87A/L89A0.062 ± 0.00−11.2 ± 0.01−5.021.14 ± 0.007M135G/K137A19.2 ± 0.60−19.8 ± 0.55−9.550.836 ± 0.001Dockerin variantsbThe dockerin mutants were titrated against wild type C. cellulolyticum cohesin.A16S/L17T557 ± 0.48−18.0 ± 0.53−5.700.865 ± 0.001A16Q/L17Q776 ± 92.9−17.5 ± 0.52−4.960.988 ± 0.001A16S/L17T/A47S/F48T116 ± 6.34−18.6 ± 0.52−7.240.865 ± 0.001A47S/F48T1030 ± 131−20.8 ± 0.62−8.070.908 ± 0.001A47Q/F48Q205 ± 54.8−22.6 ± 0.03−10.81.01 ± 0.004F19A/L50A0.23 ± 0.002−18.1 ± 0.46−10.50.837 ± 0.001a The cohesin wild type and mutants were titrated against wild type C. cellulolyticum dockerin.b The dockerin mutants were titrated against wild type C. cellulolyticum cohesin. Open table in a new tab Protein Expression and Crystallization Strategy—To determine the crystal structure of the C. cellulolyticum cohesin-dockerin complex, the two proteins were co-expressed in E. coli. Initial attempts to crystallize the purified C. cellulolyticum dockerin-cohesin complex were unsuccessful. As with past work on C. thermocellum, we postulated that the failure to crystallize the complex likely reflected the dynamic interaction of the two potential ligand-binding sites in the dockerin with the cohesin. It has been suggested that the C. cellulolyticum dockerin has two ligand-binding sites in which residues dAla-16 and dLeu-17 at site 1 and dAla-47 and dPhe-48 in site 2 (C. cellulolyticum dockerin residues henceforth are prefaced with d and cohesin residues with c) play a key role in cohesin recognition (8Handelsman T. Barak Y. Nakar D. Mechaly A. Lamed R. Shoham Y. Bayer E.A. FEBS Lett. 2004; 572: 195-200Crossref PubMed Scopus (35) Google Scholar, 13Pages 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, 16Mechaly A. Fierobe H.P. Belaich A. Belaich J.P. Lamed R. Shoham Y. Bayer E.A. J. Biol. Chem. 2001; 276: 9883-9888Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). To encourage a single binding mode between the protein partners for the equivalent C. cellulolyticum complexes, two variants of the dockerin were constructed in which the functions of site 1 and site 2 were disrupted through the introduction of the mutations A16S/L17T and A47S/F48T, respectively. Diffracting crystals of the cohesin in complex with either mutant of the dockerin mutants were then obtained. Structure of the Type I Cohesin-Dockerin C. cellulolyticum Complex—The structure of the cohesin-dockerin A16S/L17T (Coh-DocA16S/L17T) and the cohesin-dockerin A47S/F48T (Coh-DocA47S/F48T) complexes were solved to 1.49- and 1.9-Å resolution, respectively, by molecular replacement using the crystal structure of the apo-form of the C. cellulolyticum type 1 cohesin (PDB code 1g1k (22Spinelli S. Fierobe H.P. Belaich A. Belaich J.P. Henrissat B. Cambillau C. J. Mol. Biol. 2000; 304: 189-200Crossref PubMed Scopus (51) Google Scholar)) as the search model. The two complexes in the asymmetric unit overlay with an r.m.s.d. of 0.18 Å (C-α atoms) for the cohesin residues and 1.0 Å for the dockerin amino acids. The individual components of the two protein complexes are extremely similar to each other (Fig. 2) with an r.m.s.d. of 0.5 Å for the C-α atoms of the cohesins and 1.0 Å for the C-α atoms of the dockerins (treated independently). The structure of the cohesin either unliganded or in complex with either dockerin variant was essentially identical (r.m.s.d. ∼0.8 Å). Thus, similar to the type I C. thermocellum cohesin, the corresponding C. cellulolyticum protein does not undergo significant conformational changes upon binding to its dockerin ligands. Structure of the C. cellulolyticum Type I Cohesin in Complex with Its Cognate Dockerin—The type I C. cellulolyticum cohesin in complex with its cognate dockerin has an elliptical structure comprising a 4-residue α-helix and 10 β-strands, which forms 2 β-sheets aligned in an elongated β-sandwich and displays a classical jelly roll fold (Fig. 2). The two sheets include β-strands 10, 1, 2, and 7 on one face (sheet A) and β-strands 5, 6, 3, and 9 on the other face (sheet B). The two sheets are connected by β-strand 8 that makes hydrogen bonds with both β-strand 9 on sheet A and β-strand 3 on sheet B. The cohesin also displays striking similarity to type I C. thermocellum cohesins (r.m.s.d. 0.8 Å). Structure of the C. cellulolyticum Type I Dockerin—The dockerin, in both complexes, has an identical structure that consists of two parallel helixes, comprising residues dAla-16 to dMet-27 and dAla-46 to dLeu-58, respectively, whereas the extended loop connecting these structural elements contains a 3-residue 310 helix (Fig. 2). The overall structure of the C. cellulolyticum dockerin is very similar to the C. thermocellum Xyn10B type 1 dockerin (r.m.s.d. 0.76 Å). The C. cellulolyticum dockerin, in both complexes, contains two Ca2+ ions coordinated by several amino acid residues in canonical EF-hand loop motifs. The coordination of the two calciums is identical to the metal ion" @default.
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• W1998408013 title "The Clostridium cellulolyticum Dockerin Displays a Dual Binding Mode for Its Cohesin Partner" @default.
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