Resolving dual binding conformations of cellulosome cohesin-dockerin complexes using single-molecule force spectroscopy.

Markus A Jobst,Constantin Schoeler,Edward A Bayer,Hermann E Gaub,Wolfgang Ott,Daniel B Fried,Michael A Nash,Lukas F Milles

doi:10.7554/elife.10319

Markus A Jobst, Constantin Schoeler + Show 6 more

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https://doi.org/10.7554/elife.10319

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Abstract

Receptor-ligand pairs are ordinarily thought to interact through a lock and key mechanism, where a unique molecular conformation is formed upon binding. Contrary to this paradigm, cellulosomal cohesin-dockerin (Coh-Doc) pairs are believed to interact through redundant dual binding modes consisting of two distinct conformations. Here, we combined site-directed mutagenesis and single-molecule force spectroscopy (SMFS) to study the unbinding of Coh:Doc complexes under force. We designed Doc mutations to knock out each binding mode, and compared their single-molecule unfolding patterns as they were dissociated from Coh using an atomic force microscope (AFM) cantilever. Although average bulk measurements were unable to resolve the differences in Doc binding modes due to the similarity of the interactions, with a single-molecule method we were able to discriminate the two modes based on distinct differences in their mechanical properties. We conclude that under native conditions wild-type Doc from Clostridium thermocellum exocellulase Cel48S populates both binding modes with similar probabilities. Given the vast number of Doc domains with predicted dual binding modes across multiple bacterial species, our approach opens up new possibilities for understanding assembly and catalytic properties of a broad range of multi-enzyme complexes.

Highlights

Cellulosomes are hierarchically branching protein networks developed by nature for efficient deconstruction of lignocellulosic biomass
Among Clostridium thermocellum (Ct)-Doc domains, a Ser-Thr pair located at positions 11 and 12 of F-hand motif 1 (N-terminal helix 1) is highly conserved (Figure 1A)
In order to study this remarkable plasticity in molecular recognition in greater detail, we prepared a series of mutants (Figure 2) designed to either knock out a specific binding mode or add length to the molecule at a specific position

Summary

Introduction

Cellulosomes are hierarchically branching protein networks developed by nature for efficient deconstruction of lignocellulosic biomass These enzyme complexes incorporate catalytic domains, carbohydrate binding modules (CBMs), cohesin:dockerin (Coh:Doc) pairs, and other conserved features (Demain et al, 2005; Bayer et al, 2004; Schwarz, 2001; Beguin and Aubert, 1994; Smith and Bayer, 2013; Fontes and Gilbert, 2010). Dockerins guide attachment of enzymes into the networks by binding strongly to conserved Coh domains organized within non-catalytic poly (Coh) scaffolds In addition to their nanomolar binding affinities, many archetypal Coh:Doc pairs are thought to exhibit dual binding modes (Carvalho et al, 2007; Pinheiro et al, 2008; Currie et al, 2012). The duplicated F-hand motifs resemble EF-hands found in eukaryotic calcium binding proteins (e.g., calmodulin), and provide

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