Cohesin–dockerin interaction in cellulosome assembly: a single Asp‐to‐Asn mutation disrupts high‐affinity cohesin–dockerin binding

Handelsman, Tal; Barak, Yoav; Nakar, David; Mechaly, Adva; Lamed, Raphael; Shoham, Yuval; Bayer, Edward A.

doi:10.1016/j.febslet.2004.07.040

Cited by 36 publications

(56 citation statements)

References 33 publications

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“…The equivalent residues in the WT dockerin; Asn-44B, Thr-49B, Ser-52B and Arg-23B (N 2), respectively, do not make direct interactions with the cohesin (SI Table 5). Mutagenesis studies support the importance of the polar interactions between the cohesin residues Ala-36A, Asn-37A, Asp-39A, Tyr-74A, Glu-86A, Gly-89A and Glu-131A (26,27) and type I dockerins, although the functional significance of Arg-77A, which seems to make important hydrogen bonds with Arg-23B and Arg-57B at the C-terminal end of the two dockerin binding sites, has not previously been explored.…”

Section: Resultsmentioning

confidence: 98%

“…3). Whereas the role of indirect hydrogen bonds in protein ligand recognition is controversial (28,29), mutating the cohesin residue Asp-39A, which makes solvent-mediated hydrogen bonds with the backbone carbonyl and amines of the dockerin residues Ile-43B, Ile-19B, Val-21B and Leu-55B, reduces affinity 1000-fold (26), suggesting that these interactions may be important in cohesin-dockerin binding. Furthermore, the cohesin residue Thr66A, which makes a solvent-mediated hydrogen bond with Lys18B, when the C-terminal binding site of the dockerin interacts with its protein ligand, is not invariant in the CipA cohesins.…”

Section: Resultsmentioning

confidence: 99%

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Evidence for a dual binding mode of dockerin modules to cohesins

Carvalho

Dias²,

Nagy

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

129

204

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The assembly of proteins that display complementary activities into macromolecular complexes is critical to cellular function. One such enzyme complex, of environmental significance, is the plant cell wall degrading apparatus of anaerobic bacteria, termed the cellulosome. The complex assembles through the interaction of enzyme-derived ''type I dockerin'' modules with the multiple ''cohesin'' modules of the scaffolding protein. Clostridium thermocellum type I dockerin modules contain a duplicated 22-residue sequence that comprises helix-1 and helix-3, respectively. The crystal structure of a C. thermocellum type I cohesin-dockerin complex showed that cohesin recognition was predominantly through helix-3 of the dockerin. The sequence duplication is reflected in near-perfect 2-fold structural symmetry, suggesting that both repeats could interact with cohesins by a common mechanism in wild-type (WT) proteins. Here, a helix-3 disrupted mutant dockerin is used to visualize the reverse binding in which the dockerin mutant is indeed rotated 180 o relative to the WT dockerin such that helix-1 now dominates recognition of its protein partner. The dual binding mode is predicted to impart significant plasticity into the orientation of the catalytic subunits within this supramolecular assembly, which reflects the challenges presented by the degradation of a heterogeneous, recalcitrant, insoluble substrate by a tethered macromolecular complex.cellulosome structure ͉ cellulosome assembly ͉ Clostridium thermocellum ͉ cohesin-dockerin

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Section: Resultsmentioning

confidence: 98%

Section: Resultsmentioning

confidence: 99%

Evidence for a dual binding mode of dockerin modules to cohesins

Carvalho

Dias²,

Nagy

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

129

204

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“…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 (8,13,16). Significantly the Ser-Thr dyad in the C. thermocellum dockerins is replaced with hydrophobic residues in the corresponding C. cellulolyticum protein modules.…”

mentioning

confidence: 93%

“…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 (8,13,16). 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.…”

Section: Thermodynamics and Stoichiometry Of Cohesin-dockerinmentioning

confidence: 99%

“…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 (6,7). The Cip molecules of Clostridium cellulolyticum and Clostridium thermocellum (designated CipC and CipA, respectively) can bind eight and nine enzymes, respectively (8), 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 (9). 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 (10).…”

mentioning

confidence: 99%

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The Clostridium cellulolyticum Dockerin Displays a Dual Binding Mode for Its Cohesin Partner

Pinheiro¹,

Proctor

Martinez-Fleites

et al. 2008

Journal of Biological Chemistry

120

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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 cohesindockerin 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.

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Characterization of a dockerin‐based affinity tag: application for purification of a broad variety of target proteins

Demishtein

Karpol

Barak

et al. 2010

J of Molecular Recognition

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Cellulose, a major component of plant matter, is degraded by a cell surface multiprotein complex called the cellulosome produced by several anaerobic bacteria. This complex coordinates the assembly of different glycoside hydrolases, via a high-affinity Ca 2R -dependent interaction between the enzyme-borne dockerin and the scaffoldin-borne cohesin modules. In this study, we characterized a new protein affinity tag, DDoc, a truncated version (48 residues) of the Clostridium thermocellum Cel48S dockerin. The truncated dockerin tag has a binding affinity (K A ) of 7.7 T 10 8 M S1, calculated by a competitive enzyme-linked assay system. In order to examine whether the tag can be used for general application in affinity chromatography, it was fused to a range of target proteins, including Aequorea victoria green fluorescent protein (GFP), C. thermocellum b-glucosidase, Escherichia coli thioesterase/protease I (TEP1), and the antibody-binding ZZ-domain from Staphylococcus aureus protein A. The results of this study significantly extend initial studies performed using the Geobacillus stearothermophilus xylanase T-6 as a model system. In addition, the enzymatic activity of a C. thermocellum b-glucosidase, purified using this approach, was tested and found to be similar to that of a b-glucosidase preparation (without the DDoc tag) purified using the standard His-tag. The truncated dockerin derivative functioned as an effective affinity tag through specific interaction with a cognate cohesin, and highly purified target proteins were obtained in a single step directly from crude cell extracts. The relatively inexpensive beaded cellulose-based affinity column was reusable and maintained high capacity after each cycle. This study demonstrates that deletion into the first Ca 2R -binding loop of the dockerin module results in an efficient and robust affinity tag that can be generally applied for protein purification.

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Cohesin–dockerin interaction in cellulosome assembly: a single Asp‐to‐Asn mutation disrupts high‐affinity cohesin–dockerin binding

Cited by 36 publications

References 33 publications

Evidence for a dual binding mode of dockerin modules to cohesins

Evidence for a dual binding mode of dockerin modules to cohesins

The Clostridium cellulolyticum Dockerin Displays a Dual Binding Mode for Its Cohesin Partner

Characterization of a dockerin‐based affinity tag: application for purification of a broad variety of target proteins

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