Modular Arrangement of a Cellulosomal Scaffoldin Subunit Revealed from the Crystal Structure of a Cohesin Dyad

Noach, Ilit; Levy-Assaraf, Maly; Lamed, Raphael; Shimon, Linda J. W.; Frolow, Felix; Bayer, Edward A.

doi:10.1016/j.jmb.2010.04.013

Cited by 23 publications

(14 citation statements)

References 59 publications

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“…More recently [63], ultrastructural studies of a homogeneous mini-cellulosome containing three cohesin modules attached to three matching cellulases suggested that the flexibility of the linkers connecting consecutive cohesin modules could control structural transitions and thus regulate substrate recognition and degradation. In addition, the cellulases were found to be alternately projected from the scaffoldin in numerous directions, as was previously proposed based on the crystal structure of cohesins together with their adjacent linker segments [64,65]. This property of the scaffoldin can prevent steric clashes between neighboring catalytic modules.…”

Section: Discussionmentioning

confidence: 63%

A synthetic biology approach for evaluating the functional contribution of designer cellulosome components to deconstruction of cellulosic substrates

Vazana

Barak

Unger

et al. 2013

Biotechnol Biofuels

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BackgroundSelect cellulolytic bacteria produce multi-enzymatic cellulosome complexes that bind to the plant cell wall and catalyze its efficient degradation. The multi-modular interconnecting cellulosomal subunits comprise dockerin-containing enzymes that bind cohesively to cohesin-containing scaffoldins. The organization of the modules into functional polypeptides is achieved by intermodular linkers of different lengths and composition, which provide flexibility to the complex and determine its overall architecture.ResultsUsing a synthetic biology approach, we systematically investigated the spatial organization of the scaffoldin subunit and its effect on cellulose hydrolysis by designing a combinatorial library of recombinant trivalent designer scaffoldins, which contain a carbohydrate-binding module (CBM) and 3 divergent cohesin modules. The positions of the individual modules were shuffled into 24 different arrangements of chimaeric scaffoldins. This basic set was further extended into three sub-sets for each arrangement with intermodular linkers ranging from zero (no linkers), 5 (short linkers) and native linkers of 27–35 amino acids (long linkers). Of the 72 possible scaffoldins, 56 were successfully cloned and 45 of them expressed, representing 14 full sets of chimaeric scaffoldins. The resultant 42-component scaffoldin library was used to assemble designer cellulosomes, comprising three model C. thermocellum cellulases. Activities were examined using Avicel as a pure microcrystalline cellulose substrate and pretreated cellulose-enriched wheat straw as a model substrate derived from a native source. All scaffoldin combinations yielded active trivalent designer cellulosome assemblies on both substrates that exceeded the levels of the free enzyme systems. A preferred modular arrangement for the trivalent designer scaffoldin was not observed for the three enzymes used in this study, indicating that they could be integrated at any position in the designer cellulosome without significant effect on cellulose-degrading activity. Designer cellulosomes assembled with the long-linker scaffoldins achieved higher levels of activity, compared to those assembled with short-and no-linker scaffoldins.ConclusionsThe results demonstrate the robustness of the cellulosome system. Long intermodular scaffoldin linkers are preferable, thus leading to enhanced degradation of cellulosic substrates, presumably due to the increased flexibility and spatial positioning of the attached enzymes in the complex. These findings provide a general basis for improved designer cellulosome systems as a platform for bioethanol production.

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Section: Discussionmentioning

confidence: 63%

A synthetic biology approach for evaluating the functional contribution of designer cellulosome components to deconstruction of cellulosic substrates

Vazana

Barak

Unger

et al. 2013

Biotechnol Biofuels

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“…However, both the type II CohB1⅐CohB2 dyad crystal structure and the DocI⅐CohI 9 -XDocII⅐CohII ternary crystal structure presented here reveal multiple conformations. Within the type II CohB1⅐CohB2 dyad crystal structure, two different conformations of the linker connecting the two CohII modules were resolved (55). Our ternary complex reveals four unique conformations of the CohI 9 -X linker, each one distinct from the conformation shown in the CohI 9 -X-DocII⅐CohII structure.…”

Section: Discussionmentioning

confidence: 77%

Scaffoldin Conformation and Dynamics Revealed by a Ternary Complex from the Clostridium thermocellum Cellulosome

Currie¹,

Adams

Faucher

et al. 2012

Journal of Biological Chemistry

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“…The Ac CohScaB3 structure in complex with either XDocScaAN145G or XDocScaAN178G is very similar to the structures of unbound A. cellulolyticus type-II Cohs14161718, suggesting that type-II Cohs do not undergo major structural changes upon binding. Similar to the C. thermocellum ScaF and ScaC2 type-II Cohs6, Ac CohScaB3 displays an elongated jelly-roll topology in a nine-stranded flattened β-sandwich structure, defined by two β-sheets, disrupted by additional secondary structures: a unique crowning α-helix between β-strands 6 and 7 and two β-flap regions that interrupt β-strands 4 and 8 (Fig.…”

Section: Resultsmentioning

confidence: 89%

Diverse specificity of cellulosome attachment to the bacterial cell surface

Brás

Pinheiro

Cameron

et al. 2016

Sci Rep

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During the course of evolution, the cellulosome, one of Nature’s most intricate multi-enzyme complexes, has been continuously fine-tuned to efficiently deconstruct recalcitrant carbohydrates. To facilitate the uptake of released sugars, anaerobic bacteria use highly ordered protein-protein interactions to recruit these nanomachines to the cell surface. Dockerin modules located within a non-catalytic macromolecular scaffold, whose primary role is to assemble cellulosomal enzymatic subunits, bind cohesin modules of cell envelope proteins, thereby anchoring the cellulosome onto the bacterial cell. Here we have elucidated the unique molecular mechanisms used by anaerobic bacteria for cellulosome cellular attachment. The structure and biochemical analysis of five cohesin-dockerin complexes revealed that cell surface dockerins contain two cohesin-binding interfaces, which can present different or identical specificities. In contrast to the current static model, we propose that dockerins utilize multivalent modes of cohesin recognition to recruit cellulosomes to the cell surface, a mechanism that maximises substrate access while facilitating complex assembly.

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Modular Arrangement of a Cellulosomal Scaffoldin Subunit Revealed from the Crystal Structure of a Cohesin Dyad

Cited by 23 publications

References 59 publications

A synthetic biology approach for evaluating the functional contribution of designer cellulosome components to deconstruction of cellulosic substrates

A synthetic biology approach for evaluating the functional contribution of designer cellulosome components to deconstruction of cellulosic substrates

Scaffoldin Conformation and Dynamics Revealed by a Ternary Complex from the Clostridium thermocellum Cellulosome

Diverse specificity of cellulosome attachment to the bacterial cell surface

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