Abtract
Cellulosomes are sophisticated multi-enzymatic nanomachines produced by anaerobes to effectively deconstruct plant structural carbohydrates. Cellulosome assembly involves the binding of enzyme-borne dockerins (Doc) to repeated cohesin (Coh) modules located in a non-catalytic scaffoldin. Docs appended to cellulosomal enzymes generally present two similar Coh-binding interfaces supporting a dual-binding mode, which may confer increased positional adjustment of the different complex components. Ruminococcus flavefaciens’ cellulosome is assembled from a repertoire of 223 Doc-containing proteins classified into 6 groups. Recent studies revealed that Docs of groups 3 and 6 are recruited to the cellulosome via a single-binding mode mechanism with an adaptor scaffoldin. To investigate the extent to which the single-binding mode contributes to the assembly of R. flavefaciens cellulosome, the structures of two group 1 Docs bound to Cohs of primary (ScaA) and adaptor (ScaB) scaffoldins were solved. The data revealed that group 1 Docs display a conserved mechanism of Coh recognition involving a single-binding mode. Therefore, in contrast to all cellulosomes described to date, the assembly of R. flavefaciens cellulosome involves single but not dual-binding mode Docs. Thus, this work reveals a novel mechanism of cellulosome assembly and challenges the ubiquitous implication of the dual-binding mode in the acquisition of cellulosome flexibility.
Similar content being viewed by others
Introduction
The cellulosome is one of the most intricate nanomachines Nature has evolved. Cellulosomes combine an extensive repertoire of enzymes, including glycoside hydrolases, pectate lyases and carbohydrate esterases, into a large multi-enzyme complex (molecular mass >3 MDa) that efficiently deconstructs especially recalcitrant plant structural carbohydrates, such as cellulose and hemicellulose. Highly ordered protein-protein interactions are critical to a large array of cellular and biological processes. Thus, cellulosome assembly results from the binding of enzyme borne dockerin modules (Docs) to cohesin modules (Cohs) located in macro-molecular scaffolds (scaffoldins). Integration of enzymes into the cellulosome is believed to enhance the synergistic interactions between enzymes with complementary activities while promoting enzyme stability1, 2. This process is critical to the cycling of carbon between microbes, herbivores and plants. In addition, cellulases and hemicellulases are now used in several biotechnology-based industries, such as the bio-conversion of plant biomass into renewable fuels and the development of specific molecules with biomedical applications3,4,5.
The rumen, which essentially constitutes a large fermentation chamber in the gastrointestinal tract of ruminant mammals, is a highly competitive ecological niche colonized by symbiotic microbes that have specialized in the hydrolysis of recalcitrant carbohydrates. Ruminococcus flavefaciens, a Gram-positive anaerobic bacterium of the Firmicutes phylum, is one of the major cellulolytic ruminal bacteria and the only species in this microbial ecosystem that has been shown to possess a definitive cellulosome6. Intriguingly, the rumen houses numerous subspecies of this bacterium, each with a similar set of scaffoldins but with its own spectrum of dockerin-bearing proteins (enzymes) and cellulosome architecture7, 8. The genome sequence of R. flavefaciens strain FD-1 revealed the presence of 223 dockerin-containing proteins (154 of which were identified as carbohydrate-active enzymes)8, indicating that this bacterial nanomachine is the most complex cellulosome described to date9 (Fig. 1). R. flavefaciens Docs have been organized into six groups based on primary structure homology10. This classification was recently found to be functionally relevant11, with the binding of group 1 Docs to the Cohs of scaffoldins ScaA and ScaB providing the major mechanism for cellulosome assembly in R. flavefaciens. The 96 group 1 Docs have been classified in four subgroups (a to d) although the functional significance of this subdivision remains unclear. The cellulosome is tethered to the surface of R. flavefaciens through the binding of the group 4 Doc of ScaB to the Coh of the cell surface protein ScaE. A variety of other proteins were found to contain Docs that specifically interact with cell surface Cohs rather than to the cellulosomal Cohs. These Docs were classified into group 4 and group 2. Finally, hemicellulases containing group 3 or 6 Docs bind to the adaptor scaffoldin ScaC, whose group 1 Doc locks onto the Cohs in ScaA or ScaB Cohs 1–412, 13. The ScaA Doc is the only member of group 5 and binds exclusively to ScaB Cohs 5–9. Figure 1 provides an overview of the organization of R. flavefaciens cellulosome.
In all clostridial cellulosomal systems described to date, such as Clostridium thermocellum 14, 15, C. cellulolyticum 16 and Acetivibrio cellulolyticus 17, Docs interact with their cognate Cohs through a dual-binding mode. Thus, these Docs possess the ability to bind the cognate Coh in two different orientations, by rotating ~180° with respect to its protein ligand, resulting in two different Coh-Doc conformations. The dual-binding mode results from the characteristic internal symmetry of the Doc sequence and is believed to confer additional flexibility to the macromolecular organization of cellulosomes. Recent structure/function studies, unexpectedly, showed that groups 3 and 6R. flavefaciens Docs display a single-binding mode for their target Cohs. Intriguingly, the sequence of group 1 Docs, do not seem to possess the internal symmetry required to support the dual-binding mode. This suggests that group 1 Docs may bind to their target Cohs through a single-binding mode. To test this hypothesis, we determined the X-ray crystal structure of two R. flavefaciens group 1 Docs, Doc1a and Doc1b, in complex with a ScaB (CohScaB3) and a ScaA Coh, respectively. These structures together with comprehensive biochemical analyses suggest that integration of a large repertoire of enzymes into the R. flavefaciens cellulosome operates through a single-binding mode.
Results and Discussion
Structure of R. flavefaciens ScaB cohesin 3 (RfCohScaB3)
In an initial attempt to understand the structural determinants of Coh-Doc specificity that orchestrate the correct assembly of R. flavefaciens cellulosome, the structure of the third Coh of ScaB, termed RfCohScaB3, was solved by SAD phasing. Crystals belong to space group P41212 with unit cell dimensions of a = b = 60.43 Å, c = 86.51 Å. Final data and structure-quality statistics are shown in Table S1. RfCohScaB3 displays an elliptical structure with nine β-strands, which form two β-sheets aligned in an elongated β-barrel that displays a classical “jelly-roll fold” (Figure S1A). The two sheets comprise β-strands 9, 1, 2, 7, 4 on one face and β-strands 8, 3, 6, 5 on the other face. Strands 1 and 9 align parallel to each other, thus completing the jelly-roll, while the other β-strands are antiparallel. Structural similarity search using the PDBeFold server (http://www.ebi.ac.uk/msd-srv/ssm/) revealed that the closest, functionally relevant, structural homologs of RfCohScaB3 are Cohs that bind Docs appended to enzymes, although levels of sequence similarity were relatively low. They include the Cohs from C. thermocellum ScaA (PDB code 1AOH; z score of 6.4 and root mean square deviation (rmsd) of 2.3 Å over 126 aligned residues), Pseudobacteroides cellulosolvens ScaB (PDB code 4UMS; z-score of 6.6 and rmsd of 1.97 Å over 120 aligned residues), C. cellulolyticum ScaA (PDB code 2VN5; z-score of 6.8 and rmsd of 2.3 Å over 124 aligned residues) and R. flavefaciens ScaC cohesin in complex with RfDoc3 (PDB code 5LXV; z-score of 6.9 and rmsd of 2.1 Å over 124 aligned residues). Major differences between the Coh structures were observed at β-sheet 8-3-6-5, which constitutes the protein-interacting interface (Figure S2). In particular, the ligand binding interfaces of RfCohScaB3 and RfCohScaC are dramatically different explaining differences in specificity as will be described below (Figure S1B). These observations suggest that RfCohScaB3 displays a unique mechanism of dockerin recognition not described in other Coh-Doc complexes.
Structure of novel R. flavefaciens Coh-Doc complexes
In a previous study11, ScaB Cohs 1 to 4 and ScaA Cohs were shown to bind specifically to group 1 Docs. In those studies, highly stable complexes were formed between RfCohScaB3 and a group 1a Doc, RfDoc1a, and between RfCohScaA and a group 1b Doc, RfDoc1b. RfDoc1a is a component of a family 12 carbohydrate esterase, and RfDoc1b is the C-terminal component of a family 9 glycoside hydrolase. To gain insight into the molecular mechanisms of cellulosome assembly the X-ray crystal structures of R. flavefaciens ScaA and ScaB Cohs in complex with group 1b and 1a Docs, defined as RfCohScaA-Doc1b and RfCohScaB3-Doc1a, respectively, were determined. The structure of RfCohScaB3-Doc1a was solved by molecular replacement using the RfCohScaB3 structure, described above, as the search model. The RfCohScaB3-Doc1a structure includes a single copy of the heterodimer in the asymmetric unit, as well as 323 water molecules, with RfDoc1a coordinating two calcium ions. The complex displays an elongated shape with overall dimensions of 40 × 35 × 66 Å and includes residues 5–141 of RfCohScaB3 and residues 23–96 of RfDoc1a from R. flavefaciens FD-1 (Fig. 2A). The structure of RfCohScaA-Doc1b was also solved by molecular replacement using RfCohScaB3-Doc1a as the search model. Like RfCohScaB3-Doc1a it includes a single copy of the heterodimer in the asymmetric unit, 325 water molecules and 2 calcium ions coordinated by the Doc. RfCohScaA-Doc1b is virtually identical to RfCohScaB3-Doc1a and includes residues 3–143 from RfCohScaA and residues 24–102 from RfDoc1b (Fig. 2B). Crystal parameters for the structure of the two protein complexes and data collection statistics are summarized in Table S1. In both Coh-Doc complexes the group 1 Docs bind the 8-3-5-6 sheet of the RfCohScaB3 and RfCohScaA β-sandwiches, which present a predominantly flat surface. Significantly, the structures of the RfCohScaB3-Doc1a and RfCohScaA-Doc1b complexes were found to be very similar to each other, with an average rmsd of 0.6 Å for the two chains (Fig. 2C,D). This reflects the high degree of primary structure identity (72.7% for the Cohs and 42.2% for the Docs) shown by the two complementary protein modules.
Structures of RfCohScaB3 and RfCohScaA in complex with their cognate Docs
The structures of R. flavefaciens RfCohScaB3 and RfCohScaA Cohs in complex with RfDoc1a and RfDoc1b, respectively display striking structural similarities presenting a rmsd of 0.45 Å over 136 main chain carbon atoms. As proposed above, the Doc-interacting β-sandwich face comprised β-strands 8, 3, 6 and 5 (Figure S2). No α-helices were identified in RfCohScaB3 and RfCohScaA Cohs (Fig. 2A,B; Figure S2), and they thus lack the distinctive α-helix connecting β-strands 4 and 5 in other bacterial Cohs as well as the large β-flap disrupting β-strand 8, previously observed in the R. flavefaciens ScaC group 3 Coh (ref. 12; Figure S2). The structure of RfCohScaB3, whether unbound or in complex with RfDoc1a, was essentially identical (rmsd ~0.37 Å). Thus, similar to previous descriptions15, 16, Cohs appear to be highly stable modules that do not undergo significant conformational changes upon binding to their Doc ligands.
Structures of RfDoc1a and RfDoc1b in complex with their cognate Cohs
The structures of RfDoc1a and RfDoc1b in complex with RfCohScaB3 and RfCohScaA Cohs, respectively, comprise two α-helices arranged in antiparallel orientation extending from residues (using RfDoc1a/RfDoc1b numbering) Ile-39/Ile-39 to Tyr-55/Phe-55 (helix-1) and Val-76/Asn-84 to Leu-89/Leu-97 (helix-3). The two loops connecting these structural elements, in RfDoc1a and RfDoc1b, contain a seven-residue α-helix (helix-2) extending from Asp-59/Ala-67 to Ala-65/Gly-73, respectively (Fig. 2A,B). The tertiary structures of RfDoc1a and RfDoc1b adopt a similar fold with an rmsd of 0.9 Å over 68 main chain carbon atoms. Major structural differences between RfDoc1a and RfDoc1b Docs involve the loop extending from helix-1 and helix-2, which is longer in RfDoc1b reflecting the previously identified longer linker region connecting the two duplicated repeats of group 1b Docs10. The overall tertiary structure of RfDoc1a and RfDoc1b is very similar to the enzyme-borne Docs from C. thermocellum (rmsd of ~1.4 Å, over 64 residues), A. cellulolyticus (rmsd of ~1.8 Å, over 67 residues), and R. flavefaciens group 3 Doc (Doc3) that binds the ScaC Coh (rmsd of 1.82 Å, over 59 residues). Both RfDoc1a and RfDoc1b contain two Ca2+ ions coordinated by several amino-acid residues, similar to the canonical EF-hand loop motif described in all other Docs18. The Ca2+ bound to the N-terminal repeat has a typical n, n + 2, n + 4, n + 11, plus a water molecule, pattern of coordination (Figure S3). In contrast, the second Ca2+-binding region has an atypical coordination arrangement of n, n + 6, n + 12 plus a water molecule (Figure S3).
RfCohScaB3-Doc1a and RfCohScaA-Doc1b complex interfaces - RfDoc1a and RfDoc1b helices 1 and 3 make various contacts with the surface of 8-3-6-5 β-sheets of RfCohScaB3 and RfCohScaA, respectively (Fig. 2C,D). Although the Coh-interacting platform is predominantly flat, the loop connecting β-strands 8 and 9 is elevated in relation to the 8-3-6-5 plane, thus remaining in close proximity to the N-terminus of helix-1 in the Doc structure. A slight elevation is also observed in the loop connecting β-strands 6 and 7, leading to a closer interaction with the C-terminus of helix-1. This means that the entire length of helix-1 of RfDoc1a and RfDoc1b interacts with the Coh surface, while helix-3 binds the Coh platform predominantly by the C-terminus. This contrasts with the interface of the recently described R. flavefaciens RfCohScaC-Doc3 complex where the two Doc3 helices (helix 1 and helix 3) make similar contributions to CohScaC recognition12. In RfCohScaC-Doc3, CohScaC’s α-helix located between β-strands 4 and 5, which is absent in RfCohScaB3 and RfCohScaA, is elevated in relation to the 8-3-6-5 plane allowing the entire Coh surface to be in closer proximity to both Doc α-helices. The surface electrostatic potential calculated for RfCohScaB3-Doc1a and RfCohScaA-Doc1b complexes reveal that the Coh- and Doc-interacting faces are predominantly uncharged (Figure S4). This is in contrast with C. thermocellum Coh-Doc complexes where a predominantly positive-charged Doc binds a negatively charged Coh, while the RfCohScaC-Doc3 complex interface has an intermediate charge (Figure S4).
A large network of polar (Table 1) and hydrophobic interactions (Table S2) were identified at the RfCohScaB3-Doc1a and RfCohScaA-Doc1b complex interfaces (Fig. 2C,D). Although a few differences were observed, the contacts are highly conserved between the two complexes (Fig. 2C,D). The interactions between α-helix-1 of the Docs and the R. flavefaciens Cohs are dominated by Ile-39, Ser-40, Val-43, Met-46, Gln-47 and Lys-54 of RfDoc1a and RfDoc1b and His-121/His-121, Ala-38/Ala-39, Leu-79/Leu-80 and Glu-84/Glu-85 of RfCohScaB3/RfCohScaA Cohs, respectively (Fig. 2C,D). The side chains of the Ile-39/Val-43, at positions 11 and 15 of RfDoc1a and RfDoc1b, dominate the hydrophobic recognition of the Coh by contacting with the hydrophobic platform of the Coh created by Ala-38/Ala-39 and Leu-79/Leu-80 in RfCohScaB3/RfCohScaA, respectively. The highly hydrophobic character of α-helix-1 interaction is reinforced by the contacts established by Leu-44/Ile-44, Met-46/Met-46 and Ala-50/Ser-50 of RfDoc1a/RfDoc1b with RfCohScaB3/RfCohScaA Leu-79/Leu-80 and the aliphatic region of Asn-124 side-chain. The hydrogen bond network established by α-helix 1 is dominated by the interaction of Ser-40, Gln-47 and Lys-54 with His-121/His-121, Asn-124/Asn-124 and Glu-84/Glu-85 of RfCohScaB3/RfCohScaA, respectively. The two Docs are less conserved at the C-terminus of helix-1 and this generates differences in the interaction with the Coh. Thus, RfDoc1a establishes an extra hydrogen bond between Tyr-55 Oη (Phe-55 in RfDoc1b) and Oδ2 of RfCohScaB3 Glu-84. In addition, the longer loop connecting helices 1 and 2 in RfDoc1b allows the carbonyl of His-63 to form a hydrogen bond with Asn-124 Nδ2 of RfCohScaA. In α-helix-3 the contacts are dominated by the important salt bridges established between Nε2 and Oε1 of Gln-83/Gln-91 of RfDoc1a/RfDoc1b with Oδ1 of Asn-75/Asn-76 and Nζ of Lys-77/Lys-78 of RfCohScaB3/RfCohScaA Cohs. In addition, the side chains of Leu-87/Leu-95 of RfDoc1a/RfDoc1b occupy the hydrophobic pocket created by Gly-73/Gly-74, Ile-71/Ile-72 and the aliphatic portion of Met-66/Met-67 of RfCohScaB3/RfCohScaA Cohs. The closer proximity of the two protein partners at the C-terminus of helix-3 in RfCohScaB3-Doc1a protein complex allows the formation of two extra hydrogen bonds between RfDoc1a and RfCohScaB3 that are absent in RfCohScaA-Doc1b.
RfDoc1a and RfDoc1b present a single Coh-binding interface
The binding thermodynamics of RfDoc1a and RfDoc1b to RfCohScaB3 and RfCohScaA were assessed by isothermal titration calorimetry (ITC) at 308 K, consistent with the approximate temperature of the rumen. The data, presented in Table 2 and exemplified in Figure S5, revealed a macromolecular association with a 1:1 stoichiometry and a K a of ~107–108 M−1, an affinity similar to other Coh-Doc interactions. Binding was driven by changes in enthalpy with the reduction in entropy having a negative impact on affinity. The importance of RfDoc1a and RfCohScaB3 residues for Coh-Doc recognition was initially probed through non-denaturing gel electrophoresis (NGE) (Figure S6) and then extensively explored through ITC. The data (Table 2, Fig. 3) revealed that alanine substitutions of RfDoc1a residues Ile-39 and Val-43 resulted in ~100-fold reduction in affinity of the Doc for the RfCohScaB3. Complete abolition of Coh recognition resulted from the substitution of these two non-polar residues simultaneously (Table 2, Fig. 3). The alanine substitution of RfDoc1a residues that participate in the hydrogen bond network with the Coh (namely Ser-40, Gln-47, Lys-54 and Gln-83) had little impact on affinity (Table 2, Fig. 3). In addition, a significant reduction in the affinity of RfCohScaB3 for RfDoc1a was observed following the substitution of Ala-38 with Gln and Leu-79 with Ala, the two Coh residues that create the hydrophobic environment at the RfCohScaB3 platform that binds to RfDoc1a. Again, the data suggest that RfCohScaB3 residues that hydrogen bond with RfDoc1a play a relatively small role in the binding; even when double mutants were generated the reduction in affinity was never higher than ~100 fold. Overall the data suggest that the residues that mostly influence RfCohScaB3-Doc1a interaction are Ile-39 and Val-43 at helix-1 of RfDoc1a and Ala-38 and Leu-79 located at the flat surface of RfCohScaB3 8-3-6-5 β-sheet. Thus it seems that hydrophobic interactions play a major role in RfCohScaB3-Doc1a assembly.
The observation that the Ile-39Ala/Val-43Ala Doc mutant did not bind to its target Coh suggests that R. flavefaciens group 1 Docs present a single-binding mode, in contrast to previous observations for the majority of Docs appended to enzymes in other organisms. When Docs present a dual-binding mode, mutation of a single or two closely positioned residues usually has no effect on affinity, as the other (duplicated) binding site is functional and can be accessed by its target Coh through a 180° rotation of the Doc. Inspection of the RfCohScaB3-Doc1a structure revealed that the symmetry-related residues to Ile-39 and Val-43 (amino acids that occupy the equivalent position to Ile-39 and Val-43 when the Doc has been rotated 180°) are, respectively, Val-76 and Gln-80. While the side chain of Val-76 and Ile-39 are compatible, the bulky polar side chain of Gln-80 would be incompatible with the hydrophobic pocket in the cognate Coh that interacts with Val-43. Recent data revealed that both group 3 and group 6R. flavefaciens Docs display a single-binding mode with the ScaC Coh. The internal symmetry of R. flavefaciens group 1 and group 3 Docs when compared with the well-described dual-binding mode of enzyme Docs from C. thermocellum was therefore probed by overlaying the various structures with their 2-fold related derivatives using the Matchmaker procedure from Chimera19. The superposition, displayed in Fig. 4, highlights the lack of conservation in the contacting residues when the group 1 and group 3 Docs were overlayed with their 180-rotated versions. In addition to the previously mentioned changes in group 1 Docs, Ser-40 is replaced by the non-polar Leu-77 while the critical Gln-47 is replaced by Ser-84 (Fig. 4). The lack of internal symmetry is also observed in the group 3 Docs, where both α-helices 1 and 3 are involved in Coh recognition. These data, together with the extensive mutagenesis analyses presented here, suggest that group 1 Docs display a single Coh-binding platform. In contrast, the superposition of C. thermocellum enzyme Docs revealed a well-defined internal symmetry with conservation of the Coh-interacting residues when the Doc is rotated by 180°, a property that supports a dual-binding mode (Fig. 4).
R. flavefaciens FD-1 Group 3 and Group 6 Docs present a non-dynamic binding mode to CohScaC
The 96 group 1 Docs identified in the proteome of R. flavefaciens FD-1 were previously organized in 4 subgroups, termed 1a to 1d10. RfDoc1a and RfDoc1b belong to group 1a (37 members) and group 1b (36 members), respectively, the most represented group 1 Docs. It was previously observed that group 1b Docs contain the longest linker region between the two Ca2+ repeats, although the functional significance of this remains obscure10. Recent data suggest that R. flavefaciens group 1 Docs display tight specificity for ScaA (Coh 1 and 2) and ScaB (Coh 1 to 4) Cohs. However, it remains unknown if the sub-classification of R. flavefaciens group 1 Docs has a functional significance. Thus, representative members of all R. flavefaciens Doc subgroups were expressed and purified. The capacity of the Docs to bind a range of representative Cohs from R. flavefaciens proteome was probed using a previously described cellulose microarray assay method20. The data, presented in Fig. 5 and Figure S7, revealed that all twelve Docs presented a similar binding specificity; all group 1 Docs bind tightly to CohScaA1 and CohScaB2, while not interacting with the other Cohs analyzed, including a Coh from A. cellulolyticus used as control. The primary sequences of all 13 Docs were aligned with those of group 3 Docs (Fig. 5). Initial inspection of the aligned sequences confirms, as described above, that group 1 Docs present a single-binding mode, due to a lack of internal symmetry (Figs 4 and 5). With some exceptions, strong conservation was observed in the most important residues involved in Coh recognition, namely Ile-39, Val-43, Gln-47 in helix-1 and Gln-83 and Leu-87 in helix-3 (RfDoc1a residue numbering). There are, however, a few substitutions at the Ile-39 position, but these are all to non-polar residues such as Val and Met, suggesting functional conservation at this position. Taken together, the data suggest that the subgrouping of R. flavefaciens has no functional implications.
Recent studies suggest that within the R. flavefaciens proteome six Cohs, CohScaA1 and CohScaA2 and CohScaB1–4 (Fig. 1), are able to bind the 96 group 1 Docs that recruit cellulosomal enzymes to the multi-enzyme complex11. Residues at RfCohScaB3 and RfCohScaA Cohs which make direct contacts with the Doc domains, as shown in the RfCohScaB3-Doc1a and RfCohScaA-Doc1b structures, are mostly conserved in the four other Cohs of R. flavefaciens ScaA and ScaB scaffoldins (Figure S8). Changes that might disturb the Coh-Doc interaction are observed in CohScaB4, with the replacement of the conserved Ser-37 by a Cys (β-strand 3) and the highly conserved His-121 (β-strand 8) by a Val. The His121Val substitution would remove the hydrogen bond partner for Doc Ser-40. However, this may be compensate by the Gly126Asn change observed in the loop connecting β-strands 8 and 9 of CohScaB4, which can form the required hydrogen-bonding partner for Doc Ser-40. Thus, overall conservation in the residues involved in cellulosome assembly suggests that CohScaA1, CohScaA2 and CohScaB1-4 of R. flavefaciens will be unable to discriminate between the different group 1 Docs appended to cellulosomal enzymes. In contrast, comparison of the structure of the group 1 Coh-Doc complexes with that of the group 3 RfCohScaC-Doc3 complex explains why the ScaA and ScaB Cohs cannot bind group 3 or 6 Docs, while conversely ScaC Coh is unable to recognize group 1 Docs. Other differences besides the presence of the important loop interrupting β-strand 8 in ScaC Coh, include the presence of the bulky hydrophobic side chain (usually Phe) of group 3 and 6 Docs at the critical Ser-40 position of group 1 Docs, which would make steric clashes with group 1 Cohs. Conversely, Ser-40 in group 1 Docs would not make productive interactions with the hydrophobic pocket in the ScaC Coh that is occupied by Phe side-chain in group 3 Docs.
Conclusions
Previous structure-function studies of the cellulosomes of C. thermocellum 14, 15 and C. cellulolyticum 16 revealed that Docs used to recruit the microbial enzymes to these highly intricate multi-enzyme complexes display a dual-binding mode. In addition, recent reports revealed that the attachment of cellulosomes to the P. cellulosolvens 21 and A. cellulolyticus cell surface is also mediated by Docs that display a dual-binding mode17, 22. The structure of dual-binding mode Docs presents a 2-fold internal symmetry that allow binding to the Coh partner in two 180°-related alternate positions. The fact that Docs, in general, possess two different Coh-interacting platforms displaying identical specificities suggests that the dual-binding mode could contribute to enhance the conformational flexibility of the quaternary architecture of the highly populated multi-enzyme complex. This was supported by the observation that non-cellulosomal Docs that recruit single enzymes directly to the cell surface of C. thermocellum present a single-binding mode23. In addition, the Coh-Doc interaction used by C. perfringens to assemble a two-protein toxin, which is thus also not related to cellulosome assembly, was also shown to display a single-binding mode24. In contrast, a recent analysis of the R. flavefaciens cellulosome describes a new system in which this is not observed12. In this bacterium, a large repertoire of hemicellulases is appended to group 3 and 6 Docs, which specifically bind to the Coh of the adaptor scaffoldin ScaC. ScaC contains a group 1 Doc, similar to RfDoc1a and RfDoc1b, which interacts with ScaB and ScaA Cohs. Notably, the structure of a R. flavefaciens group 3 Doc, Doc3, in complex with CohScaC, revealed the presence of a single Coh-binding interface that involves both Doc helices12. Here, we extended these studies to establish if Docs displaying a single-binding mode mechanism is a generic feature of enzyme recruitment into the R. flavefaciens cellulosome. The data revealed that, similar to previously reported group 3 and 6 Docs, lack of internal symmetry in group 1 R. flavefaciens Docs generated an unconventional single protein-binding interface. This property might be widespread among all the 96 group 1 Docs, suggesting that assembly of R. flavefaciens cellulosome involves, uniquely, single-binding mode Docs. The data presented in this report questions the widely held hypothesis that the dual-binding mode mechanism provides the conformational flexibility required to degrade plant cell walls in which the topology of these composite structures varies between plants and during the degradative process. We propose that the dual-binding mode mechanism has evolved to enable rotation of the Docs in cellulosomes with a limited scaffoldin repertoire, a requirement to minimize steric clashes between the enzyme components thus increasing the number of enzyme combinations that can populate these protein complexes. The complexity of the R. flavefaciens cellulosome primary and adaptor scaffoldins reduces the steric constraints imposed by enzyme assembly obviating the need for Docs to display a dual-binding mode.
Methods
Gene synthesis and DNA cloning
Dockerins are inherently unstable when produced in Escherichia coli. To promote dockerin stability, R. flavefaciens FD-1 group 1 dockerins from protein WP_009986495 (residues 577-649) and protein WP_009982745 (residues 783-862), termed Doc1a and Doc1b, were co-expressed in vivo with ScaB cohesin 3 (CohScaB3) and ScaA cohesin (CohScaA), respectively. The immediate binding of the expressed dockerins to the expressed cohesins confers the necessary dockerin stabilization. The genes encoding the proteins were designed considering the optimization of codon usage to maximize expression in E. coli, synthesized in vitro (NZYTech Ltd, Lisbon, Portugal) and cloned into pET28a (Merk Millipore, Germany) under the control of separate T7 promoters. The dockerin-encoding genes were positioned at the 5′ end and the cohesin-encoding genes at the 3′ end of the artificial DNA. A T7 terminator sequence (to terminate transcription of the dockerin gene) and a T7 promoter sequence (to control transcription of the cohesin gene) were incorporated between the sequences of the two genes. This construct contained NheI and NcoI recognition sites at the 5′ end and XhoI and SalI at the 3′ end specifically tailored to allow subcloning into pET-28a (Merk Millipore, Germany), such that the sequence encoding a six-residue His tag could be introduced either at the N-terminus of the dockerin (through digestion with NheI and SalI, incorporating the additional sequence MGSSHHHHHHSSGLVPRGSHMAS at the N-terminus of the polypeptide) or at the C-terminus of the cohesin (by cutting with NcoI and XhoI, which incorporates the additional sequence LEHHHHHH at the C-terminus of the polypeptide). Thus, as a result of this strategy, two pET28a plasmid derivatives were produced for each Coh-Doc pair: one leading to the expression of dockerin with an engineered hexa-histidine tag and a second derivative where the engineered tag is attached to the cohesin. The plasmids were used to express RfCohScaA-Doc1a and RfCohScaB3-Doc1b protein complexes in E. coli. Recombinant Doc1a, Doc1b, CohScaA and CohScaB3 primary sequences are presented in Table S3. To produce the recombinant cohesins and dockerins individually, two distinct cloning methods were used. Digesting the previously described cohesin-tagged version of the pET28 derivatives with BglII allowed removal of the dockerin sequence. Plasmid integrity was reconstituted by re-ligating. This strategy allowed producing two novel pET28a derivatives encoding recombinant cohesins CohScaA and CohScaB3 containing C-terminal hexa-histidine tags. Dockerin-encoding genes were cloned into the pHTP2 vector (NZYtech, Lisbon, Portugal) using NZYEasy Cloning & Expression System (NZYtech, Lisbon, Portugal), following the manufacturer’s protocol. Dockerin genes were isolated by PCR using R. flavefaciens FD-1 genomic DNA as a template and the primers shown in Table S4. Recombinant dockerins encoded by the pHTP2 derivatives contained an N-terminal thioredoxin A and an internal hexa-histidine tag for increased protein stability and solubility. Sequences of all plasmids produced were confirmed by Sanger sequencing.
To identify the residues that modulate Coh-Doc specificity, several Doc1a and CohScaB3 protein derivatives were produced by site-directed mutagenesis of the pHTP2 and pET28a derivatives encoding the two genes. Site-directed mutagenesis was performed by PCR amplification using the primers presented in Table S4, which allowed the production of nine Doc1a protein derivatives (I39A, S40A, V43A, Q47A, K54A, Q83A, L87A, I39A + V43A, V43A + Q47A) and fourteen CohScaB3 protein derivatives (A38Q, N68A, N75A, K77A, L79A, E84A, H121A, N124A, E84A + H121A, N75A + H121A, N75A + N124A, N75A + E84A, E84A + N124A, H121A + N124A). Each of the newly generated gene sequences was fully sequenced to confirm that only the desired mutation accumulated in the nucleic acid.
For the cellulose microarray experiments, a system designed to fuse the Docs with a xylanase and the Cohs to a carbohydrate-binding module was selected. This allows production of highly stable and functional Cohs that can be immobilized in a cellulose-coated glass slide and Docs that can be recognized by an α-xylanase antibody20. Thus, sequences encoding the various cohesins and selected group 1 Docs were amplified from R. flavefaciens FD-1 genomic DNA by PCR, using NZYProof polymerase (NZYTech Ltd., Portugal) and the primers shown in supplemental Table S5. After gel purification, the Doc-encoding amplicons were inserted into a xylanase-Doc cassette in the pET9d plasmid after digestion with KpnI and BamHI and ligation with T4 ligase. The resulting expressed products consist of His-tagged Docs fused to xylanase T-6 from Geobacillus stearothermophilus at the N terminus of the polyhistidine tag (XynDoc). The Coh-encoding genes were cloned into a CBM-Coh cassette in pET28a after digestion with BamHI and XhoI restriction enzymes. This resulted in His-tagged Coh recombinant derivatives fused to a CBM3a from the C. thermocellum scaffoldin ScaA (CBMCoh)25, 26.
Expression and Purification of Recombinant proteins
Preliminary expression screens revealed that when the hexa-histidine tag was located at the dockerin N-terminal end of both RfCohScaB3-Doc1a and RfCohScaA-Doc1b complexes, the expression levels of both cohesin and dockerin were higher. Tagging the cohesin resulted in the accumulation of large levels of unbound cohesin in the purification product suggesting that cohesin was expressed at higher levels than dockerins or that untagged dockerin was less stable. Thus, pET28a derivatives encoding the protein complexes formed using the tagged dockerin were subsequently used to transform E. coli BL21 (DE3) cells in order to produce RfCohScaB3-Doc1a and RfCohScaA-Doc1b protein complexes in large quantities. Recombinant E. coli were grown at 37 °C to an OD600 of 0.5. Recombinant protein expression was induced by the addition of 1 mM isopropyl β-D-1-thiogalactopyranoside followed by incubation at 19 °C for 16 hours. Cells were harvested by 15 min centrifugation at 5000 × g and resuspendend in 20 mL of immobilized-metal affinity chromatography (IMAC) binding buffer (50 mM HEPES, pH 7.5, 10 mM imidazole, 1 M NaCl, 5 mM CaCl2). Cells were then disrupted by sonication and the cell-free supernatant recovered by 30 min centrifugation at 15,000 × g. After loading the soluble fraction into a HisTrapTM nickel-charged Sepharose column (GE Healthcare, UK), initial purification was carried out by IMAC in a FPLC system (GE Healthcare, UK) using conventional protocols with a 35 mM imidazole wash and a 35–300 mM imidazole elution gradient. Fractions containing the purified cohesin–dockerin complexes were buffer exchanged into 50 mM HEPES, pH 7.5, containing 200 mM NaCl, 5 mM CaCl2 using a PD-10 Sephadex G-25M gel-filtration column (Amersham Pharmacia Biosciences, UK). A further purification step by gel-filtration chromatography was performed by loading the Coh-Doc complexes onto a HiLoad 16/60 Superdex 75 (GE Healthcare, UK) at a flow rate of 1 ml min−1. Fractions containing the purified complexes were then concentrated with Amicon Ultra-15 centrifugal devices with a 10-kDa cutoff membrane (Millipore, USA) and washed three times with molecular biology grade water (Sigma) containing 0.5 mM CaCl2. The protein concentration was estimated in a NanoDrop 2000c spectrophotometer (Thermo Scientific, USA) using a molar extinction coefficient (ε) of 9 075 M−1 cm−1 for RfCohScaB3-Doc1a and 13 075 M−1 cm−1 for RfCohScaA-Doc1b. The final protein concentrations were adjusted to 40 mg.mL−1 for the RfCohScaB3-Doc1a complex and 27 mg.mL−1 for RfCohScaA-Doc1b, and stored in molecular biology grade water containing 0.5 mM CaCl2. The purity and molecular mass of the recombinant complexes were confirmed by 14% (w/v) SDS–PAGE. A similar protocol was used to produce RfCohScaB3 used in the crystallization trials and its seleno-methionine derivative, except that in the latter the protein was expressed in the methionine auxotroph B834 strain of E. coli, using the growth conditions described by Ramakrishnan et al.27, and a reducing agent was added to all the buffers: 5 mM of 2-mercaptoethanol in affinity-chromatography buffers, 5 mM DTT in size-exclusion chromatography buffer and 1 mM TCEP in storage buffer. The final protein concentrations were adjusted to 47 mg.mL−1.
Group 1 dockerins and R. flavefaciens cohesins and their respective mutant derivatives used in native PAGE and ITC experiments were expressed as described before and purified with IMAC using nickel-charged Sepharose His GraviTrap gravity-flow columns (GE Healthcare, UK). After IMAC, the recombinant cohesin and dockerins were buffer exchanged to 50 mM HEPES pH 7.5, 0.5 mM CaCl2 and 0.5 mM TCEP using PD-10 Sephadex G-25M gel filtration columns (GE Healthcare, UK).
Nondenaturing gel electrophoresis (NGE)
For the NGE experiments, each Doc variant (30 μM) was incubated in the presence and absence of 30 μM Coh for 30 min at room temperature and separated on a 10% native (lacking SDS) polyacrylamide gel. Electrophoresis was carried out at room temperature. The gels were stained with Coomassie Blue. Complex formation was detected by the presence of an additional band, usually displaying a lower electrophoretic mobility than that of the individual modules.
Isothermal Titration Calorimetry
All ITC experiments were carried out at 308 K. The purified Doc and Coh variants were diluted to the required concentrations and filtered using a 0.45 μm syringe filter (PALL). During titrations the dockerin constructs were stirred at 307 revolutions/min in the reaction cell and titrated with 28 successive 10 μL injections of cohesin at 220 s intervals. Integrated heat effects, after correction for heats of dilution, were analyzed by nonlinear regression using a single-site model (Microcal ORIGIN version 7.0, Microcal Software, USA). The fitted data yielded the association constant (KA) and the enthalpy of binding (ΔH). Other thermodynamic parameters were calculated using the standard thermodynamic equation: ΔRTlnK A = ΔG = ΔH − TΔS.
Cellulose microarray
The cellulose microarray approach was conducted using the XynDoc/CBM-Coh fusion protein pairs, in order to evaluate cohesin-dockerin interactions by refining the method described in Barak et al.20 DNA isolation and cloning were performed as described above. The strong selective binding of the CBM to the cellulose-coated slides was used as an intrinsic purification step so that cohesins were thus applied to the glass slides as crude extracts. The dockerins were purified as described above.
Rabbit anti-XynT6 primary antibody was conjugated with fluorescent Cy3 dye and rabbit anti-CBM primary antibody with fluorescent Cy5 dye, in order to assess signal intensity and normalize with the amount of protein, respectively. Xyn-CBM fusion protein was designed, cloned and expressed in the form of crude extract, as a positive control for the Cy3- and Cy5-conjugated antibodies. For biological positive controls, pre-established interactions were included in the setup. To eliminate the possibility of any of E. coli’s background components generating a false signal, BL-21 were transformed with an empty pET28a vector, which lacks a CBM or a cohesin module. The cellulose-coated glass slides were printed with crude extracts of this negative control that were subjected to the same treatment and storage conditions.
Although protein amounts were validated on SDS-PAGE gels prior to screening, there was still printing variation resulting from the use of a hand arrayer. It was therefore necessary to estimate the ratio between the Cy3 signal intensity, which indicates the presence of XynDoc, and the Cy5 signal intensity, which stands for the amount of CBM-Coh that is present in the area of a specific spot. This was done with ‘Array Vision Evaluation 8.0’ software. Raw data were further processed in Excel to generate bar graphs.
X-ray crystallography, Structural Determination and Refinement
Crystallization conditions were set up using the sitting-drop vapor diffusion method with a robotic nanodrop dispensing system Oryx8 (Douglas Instruments, UK). Commercial kits Crystal Screen, Crystal Screen 2, PEG Ion Screen I and II from Hampton Research (California, USA), JCSG + HT96 (Molecular Dimensions, UK) and an in-house screen (80 factorial) were used for the screening. For RfCohScaB3, 1.0 μL drops of 22 and 47 mg·mL−1 of protein were mixed with 1.0 μL of reservoir solution at room temperature per well containing 50 μL of the crystallization solution. The same procedure was used for RfCohScaA-Doc1b and RfCohScaB3-Doc1a with protein drops at concentrations of 40 and 20 mg·mL−1 and 27 and 13.5 mg·mL−1, respectively. The resulting plates were then stored at 292 K.
Crystal formation from the initial screens was observed in the following 2 different conditions with the C-terminal tagged native RfCohScaB3: 0.2 M lithium sulfate, 0.1 M sodium acetate pH 4.5, 30% w/v PEG 8000; and 0.17 M ammonium sulfate, 25.5% w/v PEG 4000, 15% v/v glycerol. SeMet-derivative plates were immediately set up for structure determination, should molecular replacement methods fail. For the SeMet-RfCohScaB3 an optimization screen was set up around the range 0.1–0.5 M lithium sulfate, 0.1 M sodium acetate pH 4.5, 10–32% w/v PEG 8000 for the first condition; and 0.1–0.5 M ammonium sulfate, 10–32% w/v PEG 4000, 15% v/v glycerol for the second. The glycerol concentration was maintained at 15%, which acted as a cryoprotectant. Diffracting crystals were obtained in 12 of the 96 wells of the second optimization screen. These crystals grew to a maximum dimension of ~500 × 80 × 803 μm, within two weeks. In addition, diffracting N-tagged RfCohScaB3-Doc1a crystals were obtained in a 0.2 M ammonium nitrate and 20% w/v PEG 3550 solution while RfScaSCoh-Doc1b crystalized in a 0.2 M calcium acetate, 0.1 M sodium cacodylate trihydrate pH 6.5 and 18% PEG 8000 solution. All crystals were cryoprotected with mother solution containing 15–30% glycerol or with 100% Paratone-N (Hampton Research, USA) and flash-cooled in liquid nitrogen.
Data collection, processing, structure determination and refinement
Data for the SeMet RfCohScaB3 derivatives were collected on beamline ID23-2 at the European Synchrotron Radiation Facility (ESRF), Grenoble, France. 360° of data were collected with a ∆φ of 0.1° and an exposure of 0.04 sec. The data were collected at the wavelength of 0.8726 Å for a single-wavelength anomalous diffraction experiment. The crystal was cooled to 100 K using a gaseous nitrogen cryostream (Oxford Cryosystems) and data collected using the CCD MARMOSAIC 225 detector. The data sets were processed using iMOSFLM28 or XDS29 and AIMLESS30 from the CCP4 suite (Collaborative Computational Project, Number 4, 199431). Data collection statistics are given in Table S1. The crystals belong to the tetragonal space group (P41212), with a single molecule in the asymmetric unit, a solvent content of ~51% and a Matthews coefficient of ~2.49 Å3 Da−1 32. The SeMet-RfCohScaB3 structure was determined by single wavelength anomalous dispersion experiment with AUTOSOL33 from the PHENIX suite34). AUTOBUILD was used for building the initial structure35. Refmac536 interspersed with model adjustment in COOT37 were used for structure refinement and rebuilding. PDB_REDO was used in the penultimate round of refinement for validation purposes38. The root mean square deviation of bond lengths, bond angles, torsion angles and other indicators were continuously monitored using validation tools in COOT and MOLPROBITY. Final coordinates and structure factors were deposited in PDB under accession codes 5AOZ and R5AOZSF, respectively.
Data for the Coh-Doc complexes were collected on beamline I04-1 at the Diamond Light Source, Harwell, England (RfCohScaB3-Doc1a) and at the ESRF beamline ID-23, Grenoble, France (RfCohScaA-Doc1b) using a PILATUS 6 M detector (Dectris Ltd). Data collection and processing was done as described above. Data collection statistics are given in Table S1. The best diffracting RfCohScaB3-Doc1a crystals diffracted to a resolution of 1.26 Å and belonged to the orthorhombic space group P212121 with a single cohesin-dockerin complex in the asymmetric unit, a solvent content of ~43% and a Matthews coefficient of ~2.15 Å3 Da−1. PHASER39 was used to carry out molecular replacement using RfCohScaB3 (5AOZ) and BUCCANEER40 helped building the initial dockerin model. Refinement and model rebuilding were carried out as described for RfCohScaB3. The final round of refinement was performed using the TLS/restrained refinement procedure using each module as a single group. The best diffracting RfCohScaA-Doc1b crystals diffracted to 1.70 Å and belonged to the orthorhombic spacegroup P212121 with a single cohesin-dockerin complex in the asymmetric unit, a solvent content of ~47% and a Matthews coefficient of ~2.33 Å3 Da−1. PHASER was used to carry out molecular replacement using the RfCohScaB3-Doc1a model. Refinement occurred has described for RfCohScaB3-Doc1a. A summary of the refinement statistics is shown in Table S1. Molecular representation figures were prepared with UCSF Chimera19. Final coordinates and structure factors were deposited in PDB under accession codes 5M2O and SF5M2O for RfCohScaB3-Doc1a, and 5M2S and SF5M2S for RfCohScaA-Doc1b, respectively.
Data deposition
Coordinates and observed structure factor amplitudes have been deposited in the Protein Data Bank with the wwPDB entry codes 5AOZ (RfCohScaB3), 5M2O (RfCohScaB3-Doc1a) and 5M2S (RfCohScaA-Doc1b).
References
Fontes, C. M. G. A. & Gilbert, H. J. Cellulosomes: Highly Efficient Nanomachines Designed to Deconstruct Plant Cell Wall Complex Carbohydrates. Annu. Rev. Biochem. 79, 655–681, doi:10.1146/annurev-biochem-091208-085603 (2010).
Bayer, E. A., Belaich, J. Pede, Shoham, Y. & Lamed, D. The cellulosomes: Multienzyme Machines for Degradation of Plant Cell Wall Polysaccharides. Annu. Rev. Microbiol. 58, 521–554, doi:10.1146/annurev.micro.57.030502.091022 (2004).
Bayer, E. A., Lamed, R. & Himmel, Md. The potential of cellulases and cellulosomes for cellulosic waste management. Curr. Opin. Biotechnol. 18, 237–245, doi:10.1016/j.copbio.2007.04.004 (2007).
Demain, A. L., Newcomb, M. & Wu, J. H. D. Cellulase, Clostridia, and Ethanol. Microbiol Mol Biol Rev 69, 124–154, doi:10.1128/MMBR.69.1.124-154.2005 (2005).
Bayer, E. A., Morag, E. & Lamed, R. The cellulosome–a treasure-trove for biotechnology. Trends Biotechnol. 12, 379–386, doi:10.1016/0167-7799(94)90039-6 (1994).
Ding, S.-Y. et al. Cellulosomal Scaffoldin-Like Proteins from Ruminococcus flavefaciens. J. Bacteriol. 183, 1945–1953, doi:10.1128/JB.183.6.1945-1953.2001 (2001).
Jindou, S. et al. Cellulosome gene cluster analysis for gauging the diversity of the ruminal cellulolytic bacterium Ruminococcus flavefaciens. FEMS Microbiol. Lett. 285, 188–194, doi:10.1111/j.1574-6968.2008.01234.x (2008).
Dassa, B. et al. Rumen cellulosomics: divergent fiber-degrading strategies revealed by comparative genome-wide analysis of six ruminococcal strains. PloS One 9, e99221, doi:10.1371/journal.pone.0099221 (2014).
Berg Miller, M. E. et al. Diversity and strain specificity of plant cell wall degrading enzymes revealed by the draft genome of Ruminococcus flavefaciens FD-1. PloS One 4, e6650, doi:10.1371/journal.pone.0006650 (2009).
Rincon, M. T. et al. Abundance and diversity of dockerin-containing proteins in the fiber-degrading rumen bacterium, Ruminococcus flavefaciens FD-1. PloS One 5, e12476, doi:10.1371/journal.pone.0012476 (2010).
Ruimy, V. Organization of the Ruminococcus flavefaciens FD-1 cellulosome and features dictating recognition of its cohesin-dockerin interaction (The Weizmann Institute of Science, 2013).
Bule, P. et al. Single Binding Mode Integration of Hemicellulose-degrading Enzymes via Adaptor Scaffoldins in Ruminococcus flavefaciens Cellulosome. J. Biol. Chem. 291, 26658–26669, doi:10.1074/jbc.M116.761643 (2016).
Rincon, M. T. et al. ScaC, an Adaptor Protein Carrying a Novel Cohesin That Expands the Dockerin-Binding Repertoire of the Ruminococcus flavefaciens 17 Cellulosome. J. Bacteriol. 186, 2576–2585, doi:10.1128/JB.186.9.2576-2585.2004 (2004).
Carvalho, A. L. et al. Cellulosome assembly revealed by the crystal structure of the cohesin-dockerin complex. Proc. Natl. Acad. Sci. USA 100, 13809–13814, doi:10.1073/pnas.1936124100 (2003).
Carvalho, A. L. et al. Evidence for a dual binding mode of dockerin modules to cohesins. Proc. Natl. Acad. Sci. USA 104, 3089–3094, doi:10.1073/pnas.0611173104 (2007).
Pinheiro, B. A. et al. The Clostridium cellulolyticum dockerin displays a dual binding mode for its cohesin partner. J. Biol. Chem. 283, 18422–18430, doi:10.1074/jbc.M801533200 (2008).
Cameron, K. et al. Cell-surface Attachment of Bacterial Multienzyme Complexes Involves Highly Dynamic Protein-Protein Anchors. J. Biol. Chem. 290, 13578–13590, doi:10.1074/jbc.M114.633339 (2015).
Kretsinger, R. H. & Nockolds, C. E. Carp Muscle Calcium-binding Protein: II. Structure Determination and General Description. J. Biol. Chem. 248, 3313–3326 (1973).
Pettersen, E. F. et al. UCSF Chimera–a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612, doi:10.1002/(ISSN)1096-987X (2004).
Haimovitz, R. et al. Cohesin-dockerin microarray: Diverse specificities between two complementary families of interacting protein modules. PROTEOMICS 8, 968–979, doi:10.1002/pmic.200700486 (2008).
Cameron, K. et al. Combined Crystal Structure of a Type I Cohesin: Mutation and Affinity Binding Studies Reveal Structural Determinants of Cohesin-Dockerin Specificities. J. Biol. Chem. 290, 16215–16225, doi:10.1074/jbc.M115.653303 (2015).
Brás, J. L. et al. Diverse specificity of cellulosome attachment to the bacterial cell surface. Sci. Rep. 6, 38292, doi:10.1038/srep38292 (2016).
Brás, J. L. et al. Novel Clostridium thermocellum type I cohesin-dockerin complexes reveal a single binding mode. J. Biol. Chem. 287, 44394–44405, doi:10.1074/jbc.M112.407700 (2012).
Adams, J. J., Gregg, K., Bayer, E. A., Boraston, A. B. & Smith, S. P. Structural basis of Clostridium perfringens toxin complex formation. Proc. Natl. Acad. Sci. 105, 12194–12199, doi:10.1073/pnas.0803154105 (2008).
Handelsman, T. et al. Cohesin-dockerin interaction in cellulosome assembly: a single Asp-to-Asn mutation disrupts high-affinity cohesin-dockerin binding. FEBS Lett. 572, 195–200, doi:10.1016/j.febslet.2004.07.040 (2004).
Barak, Y. et al. Matching fusion protein systems for affinity analysis of two interacting families of proteins: the cohesin-dockerin interaction. J. Mol. Recognit. 18, 491–501, doi:10.1002/jmr.749 (2005).
Ramakrishnan, V., Finch, J. T., Graziano, V., Lee, P. L. & Sweet, R. M. Crystal structure of globular domain of histone H5 and its implications for nucleosome binding. Nature 362, 219–223, doi:10.1038/362219a0 (1993).
Battye, T. G. G., Kontogiannis, L., Johnson, O., Powell, H. R. & Leslie, A. G. W. iMOSFLM: a new graphical interface for diffraction-image processing with MOSFLM. Acta Crystallogr. D Biol. Crystallogr. 67, 271–281, doi:10.1107/S0907444910048675 (2011).
Kabsch, W. XDS. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132, doi:10.1107/S0907444909047337 (2010).
Evans, P. Scaling and assessment of data quality. Acta Crystallogr. D Biol. Crystallogr. 62, 72–82, doi:10.1107/S0907444905036693 (2006).
Winn, M. D. et al. Overview of the CCP 4 suite and current developments. Acta Crystallogr. D Biol. Crystallogr. 67, 235–242, doi:10.1107/S0907444910045749 (2011).
Matthews, B. W. Solvent content of protein crystals. J. Mol. Biol. 33, 491–497, doi:10.1016/0022-2836(68)90205-2 (1968).
Terwilliger, T. C. et al. Decision-making in structure solution using Bayesian estimates of map quality: the PHENIX AutoSol wizard. Acta Crystallogr. D Biol. Crystallogr. 65, 582–601, doi:10.1107/S0907444909012098 (2009).
Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).
Terwilliger et al. Iterative model building, structure refinement and density modification with the PHENIX AutoBuild wizard. Acta Crystallogr. D Biol. Crystallogr. 64, 61–69, doi:10.1107/S090744490705024X (2008).
Murshudov, G. N. et al. REFMAC 5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D Biol. Crystallogr. 67, 355–367, doi:10.1107/S0907444911001314 (2011).
Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132, doi:10.1107/S0907444904019158 (2004).
Joosten, R. P., Long, F., Murshudov, G. N. & Perrakis, A. The PDB_REDO server for macromolecular structure model optimization. IUCrJ 1, 213–220, doi:10.1107/S2052252514009324 (2014).
McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674, doi:10.1107/S0021889807021206 (2007).
Cowtan, K. The Buccaneer software for automated model building. 1. Tracing protein chains. Acta Crystallogr. D Biol. Crystallogr. 62, 1002–1011, doi:10.1107/S0907444906022116 (2006).
Bond, C. S. & Schüttelkopf, A. W. ALINE : a WYSIWYG protein-sequence alignment editor for publication-quality alignments. Acta Crystallogr. D Biol. Crystallogr. 65, 510–512, doi:10.1107/S0907444909007835 (2009).
Acknowledgements
The authors acknowledge financial support from Fundação para a Ciência e a Tecnologia (Lisbon, Portugal) through grant PTDC/BIA-MIC/5947/2014 and RECI/BBB-BEP/0124/2012. PB was supported by the individual fellowship SFRH/BD/86821/2012. The authors also acknowledge Professor Maria João Romão for access to the crystallization facilities of the Protein Crystallography Group in the Faculty of Science and Technology, Universidade NOVA de Lisboa, as well as the Diamond Light Source, Oxfordshire, UK (beamline IO4-1), the European Synchrotron Radiation Facility (ESRF), Grenoble, France (beamline ID23-2) for access and technical support during data collection, and the European Community’s Seventh Framework Programme (FP7/2007-2013) under BioStruct-X (grant agreement No. 283570, proposal number: Biostruct-X_4399) for funding.
Author information
Authors and Affiliations
Contributions
P.B. conducted most of the experiments. P.B. and V.D.A. analyzed the data and prepared figures and tables. VI-R performed the cellulose microarray experiments. V.D.A., L.M.A.F. and S.P.S provided assistance during experimental analysis. P.B., V.D.A., A.L.C. and S.N. performed data processing and solve the structures. P.B. and C.M.G.A.F. analyzed the data and wrote the manuscript with contributions from all the other authors. H.J.G., E.A.B. and C.M.G.A.F. directed and supervised the research.
Corresponding author
Ethics declarations
Competing Interests
The authors declare that they have no competing interests.
Additional information
Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
About this article
Cite this article
Bule, P., Alves, V.D., Israeli-Ruimy, V. et al. Assembly of Ruminococcus flavefaciens cellulosome revealed by structures of two cohesin-dockerin complexes. Sci Rep 7, 759 (2017). https://doi.org/10.1038/s41598-017-00919-w
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/s41598-017-00919-w
- Springer Nature Limited
This article is cited by
-
Understanding the microbial fibre degrading communities & processes in the equine gut
Animal Microbiome (2023)
-
Current challenges in designer cellulosome engineering
Applied Microbiology and Biotechnology (2023)
-
Dual binding in cohesin-dockerin complexes: the energy landscape and the role of short, terminal segments of the dockerin module
Scientific Reports (2018)
-
Higher order scaffoldin assembly in Ruminococcus flavefaciens cellulosome is coordinated by a discrete cohesin-dockerin interaction
Scientific Reports (2018)