Determination of binding affinity upon mutation for type I dockerin-cohesin complexes from Clostridium thermocellum and Clostridium cellulolyticum using deep sequencing.


Abstract

The comprehensive sequence determinants of binding affinity for type I cohesin toward dockerin from Clostridium thermocellum and Clostridium cellulolyticum was evaluated using deep mutational scanning coupled to yeast surface display. We measured the relative binding affinity to dockerin for 2970 and 2778 single point mutants of C. thermocellum and C. cellulolyticum, respectively, representing over 96% of all possible single point mutants. The interface ΔΔG for each variant was reconstructed from sequencing counts and compared with the three independent experimental methods. This reconstruction results in a narrow dynamic range of -0.8-0.5 kcal/mol. The computational software packages FoldX and Rosetta were used to predict mutations that disrupt binding by more than 0.4 kcal/mol. The area under the curve of receiver operator curves was 0.82 for FoldX and 0.77 for Rosetta, showing reasonable agreements between predictions and experimental results. Destabilizing mutations to core and rim positions were predicted with higher accuracy than support positions. This benchmark dataset may be useful for developing new computational prediction tools for the prediction of the mutational effect on binding affinities for protein-protein interactions. Experimental considerations to improve precision and range of the reconstruction method are discussed.

Submission Details

ID: WV8tmQRk

Submitter: Marie Ary

Submission Date: July 6, 2017, 11:55 a.m.

Version: 1

Publication Details
Kowalsky CA;Whitehead TA,Proteins (2016) Determination of binding affinity upon mutation for type I dockerin-cohesin complexes from Clostridium thermocellum and Clostridium cellulolyticum using deep sequencing. PMID:27699856
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
4B9F 2012-09-12 1.19 High resolution structure for family 3a carbohydrate binding module from the cipA scaffolding of clostridium thermocellum
1AOH 1998-07-08 1.7 SINGLE COHESIN DOMAIN FROM THE SCAFFOLDING PROTEIN CIPA OF THE CLOSTRIDIUM THERMOCELLUM CELLULOSOME
1NBC 1997-09-26 1.75 BACTERIAL TYPE 3A CELLULOSE-BINDING DOMAIN
3KCP 2010-02-09 1.94 Crystal structure of interacting Clostridium thermocellum multimodular components
2CCL 2007-02-13 2.03 THE S45A, T46A MUTANT OF THE TYPE I COHESIN-DOCKERIN COMPLEX FROM THE CELLULOSOME OF CLOSTRIDIUM THERMOCELLUM
2B59 2005-10-11 2.11 The type II cohesin dockerin complex
1ANU 1997-07-23 2.15 COHESIN-2 DOMAIN OF THE CELLULOSOME FROM CLOSTRIDIUM THERMOCELLUM
1OHZ 2003-11-20 2.2 Cohesin-Dockerin complex from the cellulosome of Clostridium thermocellum
5G5D 2017-04-05 3.0 Crystal Structure of the CohScaC2-XDocCipA type II complex from Clostridium thermocellum

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
90.5 Type I cohesin domain from C. thermocellum Q01866 CIPB_CLOTM
100.0 Type I cohesin domain from C. thermocellum Q06851 CIPA_CLOTH