Intramolecular clasp of the cellulosomal Ruminococcus flavefaciens ScaA dockerin module confers structural stability

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Cellulosomes are essential multi-enzyme complexes for cellulose degradation.
• The ScaA dockerin (ScaADoc) from Ruminococcus flavefaciens plays a key role in integrating scaffoldin proteins within the cellulosome.
• Understanding ScaADoc structure is vital for elucidating cellulosome assembly and function.

Purpose of the Study:

• To investigate the structural and functional significance of a putative intramolecular interaction in ScaADoc.
• To determine the role of N-terminal Trp and C-terminal Pro residues in ScaADoc stability and integrity.
• To explore the prevalence of similar interactions in other dockerin modules.

Main Methods:

• Computational modeling to predict ScaADoc structure.
• Site-directed mutagenesis of key residues (Trp and Pro) to alanine.
• Circular dichroism spectroscopy to assess protein structure.
• Fluorescence spectroscopy (tryptophan and ANS) to evaluate protein environment and stability.
• NMR spectroscopy to confirm structural integrity and interactions.

Main Results:

• A computational model revealed a novel intramolecular clasp involving N-terminal Trp and C-terminal Pro residues in ScaADoc.
• Mutagenesis of these residues destabilized the Ca(2+)-bound form of ScaADoc, impacting its functional integrity.
• Spectroscopic analyses confirmed the destabilizing effects of the mutations.
• Comparative analysis of other dockerin structures indicated the presence of similar intramolecular clasp interactions.

Conclusions:

• The intramolecular clasp is a significant structural feature of the ScaADoc module, essential for its stability.
• This interaction motif appears to be a conserved characteristic across different dockerin modules.
• The findings provide new insights into the structural basis of cellulosome assembly and function.