Investigating Receptor-ligand Systems of the Cellulosome with AFM-based Single-molecule Force Spectroscopy

Summary

Cellulosomes are multienzyme complexes designed for digesting cellulose. AFM-based SMFS was used to study the mechanical properties and folding configuration of cellulosome-associated protein assemblies. We present a complete workflow for protein immobilization, data acquisition, and data analysis to study the interactions of individual receptor-ligand complexes involved in cellulosome assembly.

Abstract

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the enzymes onto the noncatalytic scaffold protein is directed by interactions among a family of related receptor-ligand pairs comprising interacting cohesin and dockerin modules. The extremely strong binding between cohesin and dockerin modules results in dissociation constants in the low picomolar to nanomolar range, which may hamper accurate off-rate measurements with conventional bulk methods. Single-molecule force spectroscopy (SMFS) with the atomic force microscope measures the response of individual biomolecules to force, and in contrast to other single-molecule manipulation methods (i.e. optical tweezers), is optimal for studying high-affinity receptor-ligand interactions because of its ability to probe the high-force regime (>120 pN). Here we present our complete protocol for studying cellulosomal protein assemblies at the single-molecule level. Using a protein topology derived from the native cellulosome, we worked with enzyme-dockerin and carbohydrate binding module-cohesin (CBM-cohesin) fusion proteins, each with an accessible free thiol group at an engineered cysteine residue. We present our site-specific surface immobilization protocol, along with our measurement and data analysis procedure for obtaining detailed binding parameters for the high-affinity complex. We demonstrate how to quantify single subdomain unfolding forces, complex rupture forces, kinetic off-rates, and potential widths of the binding well. The successful application of these methods in characterizing the cohesin-dockerin interaction responsible for assembly of multidomain cellulolytic complexes is further described.

Introduction

Cellulosomes are large multienzyme complexes displayed on the surface of anaerobic cellulolytic bacteria (e.g. C. thermocellum) that have evolved to efficiently depolymerize plant cell wall lignocellulose into soluble oligosaccharides¹. A central attribute of cellulosomes is the high-affinity cohesin-dockerin interaction. In the most prominent paradigm, a highly conserved 60-75 amino acid type I dockerin module is displayed at the C-terminal end of the various bacterial enzymes. The dockerin module directs assembly of synergistic combinations of enzymes onto the noncatalytic scaffold protein ('scaffoldin'), which comprises a p....

Protocol

A schematic of the pulling geometry used in this work to probe the cohesin-dockerin interaction is shown in Figure 1A. The protein immobilization protocol reported here for cantilever and cover glass functionalization is a modified version of the procedure published previously²⁷. The proteins were expressed from plasmid vectors in E. coli using conventional methods. The proteins were designed with a solvent-accessible thiol group, which was used in combination with maleimide chemistry to tether the protein via a stable thioether linkage to the cover glass surface and cantilever. The engineered cysteine residues in both the CBM-cohe....

Results

We used the described procedure to investigate a type I cohesin-dockerin pair from C. thermocellum. Upon successful binding of the cohesin-dockerin pair, the recorded force distance traces showed characteristic peak patterns. A typical trace is shown in Figure 4a. Every peak in the trace represents the unfolding of one protein subdomain with the last peak corresponding to the dissociation of the receptor-ligand complex.

For the CBM-cohesin-dockerin-xylanase complex in.......

Discussion

To obtain meaningful data from single molecule force spectroscopy experiments, it is crucial to achieve well-defined and reproducible pulling geometries. The protocol used here results in site-specific immobilization of protein complexes in a defined pulling geometry.

The cantilevers used in this study were chosen due to their force sensitivity and high resonance frequency in water. Moreover, the small tip curvature of approximately 10 nm is advantageous for single molecule experiments due to .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
3-Aminopropyl dimethyl ethoxysilane	ABCR GmbH	AB110423
5 kDa NHS-PEG-maleimide	Rapp Polymer	13 5000-65-35
TCEP Disulfide reducing gel	Thermo Scientific, Pierce	77712	www.thermoscientific.com/pierce
Tris(hydroxymethyl)aminomethane
BioLever mini silicon nitride cantilevers	Olympus	BL-AC40TS-C2	Soft batches
XYZ Piezoelectric actuators	Physik Instrumente GmbH
Infrared “broad spectrum” IR laser	Superlum
MFP-3D AFM Controller	Asylum Research
Igor Pro 6.31	Wavemetrics		Data acquisition and analysis
Sodium chloride
Calcium chloride
pH Meter
Sodium borate
Tweezers
Cover glasses	Thermo Scientific, Menzel-Gläser		24 mm diameter, 0.5 mm thickness
PTFE sample holder			custom made
Sonicator bath
Ethanol			analytical purity
Sulfuric acid (concentrated)			analytical purity
Hydrogen peroxide (30%)			analytical purity
Orbital shaker
Toluene			analytical purity
Filter paper
Glass slides
Microtubes
Micropipettes
Centrifuge			suitable for microtubes
Rotator
Petri dishes
Beakers
Optical microscope