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Visualizing the Unknown: Structural elucidation of CLR-3, a domain-of-unknown-function protein from Neurospora crassa

EMSL Project ID


Plant cell walls (PCW) offer a diverse range of fermentable carbohydrates that can be utilized for the production of biorefined products. Filamentous fungi are widely recognized for their efficient degradation of these materials, making them an ideal choice for all processes involving lignocellulosic feedstocks. Fungi possess intricate systems for sensing and utilizing the complex polysaccharides, enabling them to produce a wide array of PCW-degrading enzymes, including cellulases and hemicellulases. By employing targeted gene engineering, optimized strains with enhanced enzyme production can be generated. However, progress in this area is hindered by a lack of comprehensive understanding of the regulatory networks that govern fungal responses to PCW-derived sugars, particularly considering their interconnection and possibility for cross-talk.

In the past few years, cellulose and hemicellulose perception have been studied extensively in filamentous fungi, including Neurospora crassa, a filamentous ascomycete with robust growth on lignocellulosic feedstocks and reference system for fungal genetics. Importantly, cross-talk was found between mannan and cellulose signaling, severely hampering cellulase production in presence of small amounts of mannan-derived degradation products. One of the most recent discoveries was that the main activator of the cellulolytic response in N. crassa, CLR-1, is likely repressed by the protein CLR-3 in the absence of cellulose. Follow-up work (not published yet) showed that CLR-3 can indeed bind both CLR-1 (by Yeast-2-Hybrid) as well as cellulose- and mannan-derived signaling molecules (by ITC), albeit with differing kinetics. Binding of cellobiose (a cellulose-derived signaling molecule) leads to dissociation of CLR-3 from CLR-1, while this is not the case for mannobiose. Unfortunately, CLR-3 is a DUF1479 domain-containing protein and thus no structure:function information is available. However, by HDX-MS, we were able to locate the sugar-binding site within the DUF1479 domain, thus obtaining the first functional data for this domain to date and strongly indicating that CLR-3 acts as receptor for the cellulose-derived inducers, which would be completely novel. Moreover, CLR-3 might also be key to understand the mannan:cellulose cross-talk.

Unfortunately, CLR-3 has so far been resistant to crystallization trials. Using the cryo-EM resources of the PNNL – particularly in combination with the cell-free expression pipeline for higher throughput and flexibility – would therefore be our prime choice to obtain insight into the molecular mechanisms of this critical step in fungal lignocellulose perception. We thus propose to study the CLR-3:CLR-1 protein complex both alone and in combination with the relevant inducer molecules to visualize its molecular structure and dynamics. The resulting data would be directly relevant for EMSL’s Functional and Systems Biology science area, aiming to understanding proteins and pathways that connect structures and functions to phenotypic responses within cells and their environment for microbes and plants. Moreover, this project directly relates to the “1000 Fungal Proteins” project aiming to accelerate the annotation of proteins of unknown function that are highly conserved within the fungal kingdom.

Project Details

Project type
Exploratory Research
Start Date
End Date


Principal Investigator

J. Philipp Benz
Technische Universität München

Team Members

Elisabeth Tamayo
Technische Universität München

Yuxin Zhang
Technische Universität München