Single-molecule dissection of the high-affinity cohesin–dockerin complex

Ståhl, S.; Nash, M.; Fried, Daniel B.; Slutzki, Michal; Barak, Yoav; Bayer, Edward A.; Gaub, Hermann E.

doi:10.1073/pnas.1211929109

Cited by 114 publications

(140 citation statements)

References 41 publications

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“…In such experiments, it is many times the case that the distributions of fingerprint domain unfolding and complex rupture have a significant overlap (a few exemplary cases can be found in Refs. [3,9,10,26]). In this case biasing effects will occur and should be considered in the analysis procedure.…”

Section: Discussionmentioning

confidence: 99%

“…Refolding of individual titin domains is believed to assist in muscle contraction [1], stretching forces expose cryptic binding sites involved in focal adhesions [2], and mechanically stable receptor-ligand pairs govern the assembly of large extracellular machineries and adhesion of bacterial cells to their cellulosic carbon sources [3,4]. Single-molecule pulling experiments with atomic force microscopes [5], optical tweezers [6], or magnetic tweezers [7] have become widely used techniques to study such phenomena at the singlemolecule level.…”

Section: Introductionmentioning

confidence: 99%

“…This improves curve yields and data fidelity by providing a specific molecular interaction handle to "grab" the protein of interest. Thereby, the unfolding of individual domains and the dissociation of a receptor-ligand complex can be studied in a single experiment [3,4,[8][9][10]. Also, using a known protein domain in the fusion construct provides a unique unfolding pattern that can be used to identify specific traces, when receptor-ligand unbinding itself is studied.…”

Section: Introductionmentioning

confidence: 99%

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Biasing effects of receptor-ligand complexes on protein-unfolding statistics

et al. 2016

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Protein receptor-ligand pairs are increasingly used as specific molecular handles in single-molecule proteinunfolding experiments. Further, known marker domains, also referred to as fingerprints, provide unique unfolding signatures to identify specific single-molecule interactions, when receptor-ligand pairs themselves are investigated. We show here that in cases where there is an overlap between the probability distribution associated with fingerprint domain unfolding and that associated with receptor-ligand dissociation, the experimentally measured force distributions are mutually biased. This biasing effect masks the true parameters of the underlying free energy landscape. To address this, we present a model-free theoretical framework that corrects for the biasing effect caused by such overlapping distributions.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Biasing effects of receptor-ligand complexes on protein-unfolding statistics

et al. 2016

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“…These complexes are based on a noncatalytic subunit called scaffoldin that binds to the insoluble substrate via a cellulose-specific carbohydrate-binding module (CBM). The scaffoldin also contains a set of subunitbinding modules termed cohesins that mediate the specific incorporation and organization of catalytic subunits through the dockerin, a complementary binding module carried by each enzymatic subunit (27,28). The existence of cellulosome complexes in anaerobic fungi, which differ by the absence of true cohesins and dockerins, has also been reported (29,30).…”

Section: Significancementioning

confidence: 99%

Toward combined delignification and saccharification of wheat straw by a laccase-containing designer cellulosome

Davidi

Moraïs

Artzi

et al. 2016

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

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Efficient breakdown of lignocellulose polymers into simple molecules is a key technological bottleneck limiting the production of plant-derived biofuels and chemicals. In nature, plant biomass degradation is achieved by the action of a wide range of microbial enzymes. In aerobic microorganisms, these enzymes are secreted as discrete elements in contrast to certain anaerobic bacteria, where they are assembled into large multienzyme complexes termed cellulosomes. These complexes allow for very efficient hydrolysis of cellulose and hemicellulose due to the spatial proximity of synergistically acting enzymes and to the limited diffusion of the enzymes and their products. Recently, designer cellulosomes have been developed to incorporate foreign enzymatic activities in cellulosomes so as to enhance lignocellulose hydrolysis further. In this study, we complemented a cellulosome active on cellulose and hemicellulose by addition of an enzyme active on lignin. To do so, we designed a dockerin-fused variant of a recently characterized laccase from the aerobic bacterium Thermobifida fusca. The resultant chimera exhibited activity levels similar to the wild-type enzyme and properly integrated into the designer cellulosome. The resulting complex yielded a twofold increase in the amount of reducing sugars released from wheat straw compared with the same system lacking the laccase. The unorthodox use of aerobic enzymes in designer cellulosome machinery effects simultaneous degradation of the three major components of the plant cell wall (cellulose, hemicellulose, and lignin), paving the way for more efficient lignocellulose conversion into soluble sugars en route to alternative fuels production.

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“…In addition, recruitment of cellulosomes to the bacterial cell surface via divergent type II Coh-Doc interactions allows the immediate uptake of released sugars, which are used by microbes as an energy source (1,4). The protein:protein interaction established between the Coh and Doc modules exhibit one of the strongest binding affinities found in nature, close to that of a covalent bond, and play a crucial role for both cellulosome assembly and cell-surface attachment (3,5,6). In addition, the organization and structural architecture of cellulosomes are defined by the specificity of the different Coh and Doc modules (7).…”

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confidence: 99%

Structure–function analyses generate novel specificities to assemble the components of multienzyme bacterial cellulosome complexes

Bule¹,

Cameron²,

Prates³

et al. 2018

Journal of Biological Chemistry

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Data deposition: Coordinates and observed structure factor amplitudes have been deposited in the Protein Data Bank with the wwPDB entry codes 5NRK (AcCohScaB6-DocCel5 M1) and 5NRM (AcCohScaB6-DocCel5 M2). ABSTRACTThe cellulosome is a remarkably intricate multienzyme nanomachine produced by anaerobic bacteria to degrade plant cell wall polysaccharides. Cellulosome assembly is mediated through binding of enzyme-borne dockerin modules to cohesin modules of the primary scaffoldin subunit. The anaerobic bacterium Acetivibrio cellulolyticus produces a highly intricate cellulosome comprising an adaptor scaffoldin, ScaB, whose cohesins interact with the dockerin of the primary scaffoldin (ScaA) that integrates the cellulosomal enzymes. The ScaB dockerin selectively binds to cohesin modules in ScaC that anchors the cellulosome onto the cell surface. Correct cellulosome assembly requires distinct specificities displayed by structurally related type I cohesin-dockerin pairs that mediate ScaC-ScaB and ScaAenzyme assemblies. To explore the mechanism by which these two critical protein interactions display their required specificities, we determined the crystal structure of the dockerin of a cellulosomal enzyme in complex with a ScaA cohesin. The data revealed that the enzyme-borne dockerin binds to the ScaA cohesin in two orientations, indicating two identical cohesin-binding sites. Combined mutagenesis experiments served to identify amino acid residues that modulate type I cohesin-dockerin specificity in A. cellulolyticus. Rational design was used to test the hypothesis that the ligand-binding surfaces of ScaA-and ScaB-associated dockerins mediate cohesin recognition, independent of the structural scaffold. Novel specificities could thus be engineered into one, but not both of the ligand-binding sites of ScaB, while attempts at manipulating the specificity of the enzyme-associated dockerin were unsuccessful. These data indicate that dockerin specificity requires critical interplay between the ligand-binding surface and the structural scaffold of these modules.Plant cell wall polysaccharides, primarily cellulose and hemicelluloses, are a major reservoir of carbon and energy (1), and the recycling of these complex carbohydrates by microorganisms is integral to the carbon cycle. Furthermore, as the demand for renewable sources of energy and novel molecules for the http://www.jbc.org/cgi

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Single-molecule dissection of the high-affinity cohesin–dockerin complex

Cited by 114 publications

References 41 publications

Biasing effects of receptor-ligand complexes on protein-unfolding statistics

Biasing effects of receptor-ligand complexes on protein-unfolding statistics

Toward combined delignification and saccharification of wheat straw by a laccase-containing designer cellulosome

Structure–function analyses generate novel specificities to assemble the components of multienzyme bacterial cellulosome complexes

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