Combining <i>in Vitro</i> and <i>in Silico</i> Single-Molecule Force Spectroscopy to Characterize and Tune Cellulosomal Scaffoldin Mechanics

Verdorfer, Tobias; Bernardi, Rafael C.; Meinhold, Aylin; Ott, Wolfgang; Luthey‐Schulten, Zaida; Nash, M.; Gaub, Hermann E.

doi:10.1021/jacs.7b07574

Cited by 53 publications

(67 citation statements)

References 49 publications

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“…Single-molecule force spectroscopy has been employed previously to distinguish the nature of proteinligand bonds (37) and hence infer multi-modality or conformational transitions involved in protein-ligand binding interactions (50). Single-molecule force spectroscopy in conjunction with steered MD simulations has been employed extensively to study superior mechanical stability (51), characterize the force-unfolding behavior (52), and resolve multiple binding modes of cohesin-dockerin complexes (53). However, the application of AFM-based force spectroscopy to study CBM-cellulose binding has revealed challenges in distinguishing specific vs non-specific interactions (54).…”

Section: Discussionmentioning

confidence: 99%

Multiscale Characterization of Complex Binding Interactions of Cellulolytic Enzymes Highlights Limitations of Classical Approaches

Chundawat

Nemmaru

Hackl

et al. 2020

Preprint

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Cellulolytic microorganisms, like Trichoderma reesei or Clostridium thermocellum, frequently have noncatalytic carbohydrate-binding modules (CBMs) associated with secreted or cell surface bound multidomain carbohydrate-active enzymes (CAZymes) like cellulases. Mostly type-A family CBMs are known to promote cellulose deconstruction by increasing the substrate-bound concentration of cognate cellulase catalytic domains. However, due to the interfacial nature of cellulose hydrolysis and the structural heterogeneity of cellulose, it has been challenging to fully understand the role of CBMs on cellulase activity using classical protein-ligand binding assays. Here, we report a single-molecule CAZyme assay for an industrially relevant processive cellulase Cel7A (from T. reesei) to reveal how subtle CBM1 binding differences can drastically impact cellulase motility/velocity and commitment to initial processive motion for deconstruction of two wellstudied crystalline cellulose allomorphs (namely cellulose I and III). We take a multifaceted approach to characterize the complex binding interactions of all major type-A family representative CBMs including CBM1, using an optical-tweezers based single-molecule CBM-cellulose bond 'rupture' assay to complement several classical bulk ensemble protein-ligand binding characterization methods. While our work provides a basis for the 'cautious' use of Langmuir-type adsorption models to characterize classical protein-ligand binding assay data, we highlight the critical limitations of using such overly simplistic models to gain a truly molecular-level understanding of interfacial protein binding interactions at heterogeneous solid-liquid interfaces. Finally, molecular dynamics simulations provided a theoretical basis for the complex binding behavior seen for CBM1 towards two distinct cellulose allomorphs reconciling experimental findings from multiscale analytical methods. Significance StatementMultimodal biomolecular binding interactions involving carbohydrate polymers (e.g., cellulose, starch, chitin, glycosaminoglycans) are fundamental molecular processes relevant to the recognition, biosynthesis, and degradation of all major terrestrial and aquatic biomass. Protein-carbohydrate binding interactions are also critical to industrial biotechnology operations such as enzymatically-catalyzed bioconversion of starch and lignocellulose into biochemicals like ethanol. However, despite the ubiquitous importance of such interfacial processes, we have a poor molecular-level understanding of protein-polysaccharide binding interactions. Here, we provide a comprehensive experimental and theoretical analysis of bulk ensemble versus singlemolecule binding interactions of enzyme motors and associated non-catalytic binding domains with cellulosic polysaccharides to highlight the critical limitations of applying classical biochemical assay techniques alone to understanding protein adsorption or biological activity at solid-liquid interfaces. Main Text

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Section: Discussionmentioning

confidence: 99%

Multiscale Characterization of Complex Binding Interactions of Cellulolytic Enzymes Highlights Limitations of Classical Approaches

Chundawat

Nemmaru

Hackl

et al. 2020

Preprint

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“…These unfolding force determined here are also comparable to previous results. 28,29 Moreover the mechanical resilience of the extremely high-affinity KD ~ fM interaction of a bacteriocin (colicin E9) and its immunity protein (Im9) was probed [30][31][32] . The E9:Im9 system is particularly suitable for the tethered complex approach: ColicineE9 is a fast endonuclease that kills bacteria rapidly by shredding their chromosome, recombinant expression in E. coli thus is challenging.…”

Section: Resultsmentioning

confidence: 99%

“…Only a one protein species can be fused reliably to the tip. Using tethered complexes, one can probe many different interactions with the same high-strength handle on the cantilever by swapping the tethered complex on the surface 29 . In addition, if the ultrastable handle is very reliable -as the CohE:Xdoc -it can serve as a proxy for non-refolding and sensitive complexes.…”

Section: Resultsmentioning

confidence: 99%

Is mechanical receptor ligand dissociation driven by unfolding or unbinding?

Milles¹,

Gaub²

2019

Preprint

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Mechanical force can play a pivotal role in biological systems. Single Molecule Force Spectroscopy, is a powerful tool to probe the mechanics of proteins and their binding partners. Yet, it remains unclear how complex dissociation of a protein-protein interaction under mechanical forces occurs. Are receptor and ligand unbinding, or are they unfolding? We utilize an approach wherein receptor and ligand are expressed as a single molecule fused by a long flexible linker. Force is applied to the complex via an ultrastable handle. Consequently, the events during and following complex dissociation can be monitored. We investigate two high-affinity systems: The cohesin-dockerin type I interaction in which we find that a binding partner unfolds upon complex dissociation, and a colicin-immunity protein complex in which both proteins unfold completely upon unbinding. Mechanical receptor ligand dissociation thus can encompass unfolding of one or both binding partners.

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“…To investigate the stability of the best structural models, we performed another set of SMD simulations using the 5 best models as initial structures in what we call an in silico force-spectroscopy approach 34 . Using a wide-sampling strategy, 200 steered molecular dynamics (SMD) replicas were carried out for a total of 4 µs for each binding mode, using the 5 different initial structures.…”

Section: Homology Modeling and Molecular Dynamics Simulationsmentioning

confidence: 99%

High Force Catch Bond Mechanism of Bacterial Adhesion in the Human Gut

Liu

Vera

et al. 2020

Preprint

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Bacterial colonization of the human intestine requires firm adhesion of bacteria to insoluble targets under hydrodynamic flow. Here we report the molecular mechanism behind an mechanostable protein complex responsible for resisting high shear forces and adhering bacteria to cellulose fibers in the human gut. Using single-molecule force spectroscopy (SMFS), single-molecule FRET (smFRET), and molecular dynamics (MD) simulations, we resolved two binding modes and three unbinding reaction pathways of a mechanically ultrastable R. champanellensis (Rc) Dockerin-Cohesin (Doc-Coh) complex. The complex assembles in two discrete binding modes with significantly different mechanical properties, with one breaking at ~500 pN and the other at ~200 pN at loading rates from 1-100 nN/sec. A neighboring X-module domain allosterically regulates the binding interaction and inhibits one of the lowforce pathways at high loading rates, giving rise to a new mechanism of catch bonding that manifests under force ramp protocols. Multi-state Monte Carlo simulations show strong agreement with experimental results, validating the proposed kinetic scheme. These results explain mechanistically how gut microbes regulate cell adhesion strength at high shear stress through intricate molecular mechanisms including dual-binding modes, mechanical allostery and catch bonds.When cells adhere to surfaces under flow, adhesion bonds at the cell-surface interface experience mechanical tension and resist hydrodynamic drag forces. Because of this mechanical selection pressure, adhesion proteins have evolved molecular mechanisms to deal with tension in different ways. Most bonds not involved in force transduction in vivo have lifetimes that decay exponentially with applied force, a behavior well described by the classical Bell-Evans slip bond model 1-3 . Less intuitive are catch bonds 4-7 , which are receptor-ligand interactions that serve as band pass filters for force perturbations, becoming stronger with applied force and weakening when force is released. When probed in constant force mode, the lifetime of a catch bond will rise as the force setpoint is increased. When probed in force ramp mode or constant speed mode, catch bonds typically give rise to bimodal rupture force distributions 8,9 .Different kinetic state models and network topologies can be used to describe catch bonds 10 . For example, mechanical allostery models such as the one-state two-pathway model 11 , or two independent sites model 8,12 have been applied to mathematically describe catch bond behavior 6,8,12,13 .The R. champanellensis (Rc) cellulosome 14,15 is a bacterial protein complex found in the human gut that adheres to and digests plant fiber. The large supramolecular complex is held together by Dockerin-Cohesin (Doc:Coh) interactions 16 which comprise a family of homologous high-affinity receptor-ligand pairs. A limited number of Doc:Coh complexes are known to exhibit dual-binding modes 17-21 where the complex populates two distinct binding conformations involving different sets o...

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Combining in Vitro and in Silico Single-Molecule Force Spectroscopy to Characterize and Tune Cellulosomal Scaffoldin Mechanics

Cited by 53 publications

References 49 publications

Multiscale Characterization of Complex Binding Interactions of Cellulolytic Enzymes Highlights Limitations of Classical Approaches

Multiscale Characterization of Complex Binding Interactions of Cellulolytic Enzymes Highlights Limitations of Classical Approaches

Is mechanical receptor ligand dissociation driven by unfolding or unbinding?

High Force Catch Bond Mechanism of Bacterial Adhesion in the Human Gut

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