Residue proximity information and protein model discrimination using saturation-suppressor mutagenesis

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Abstract

Identification of residue-residue contacts from primary sequence can be used to guide protein structure prediction. Using Escherichia coli CcdB as the test case, we describe an experimental method termed saturation-suppressor mutagenesis to acquire residue contact information. In this methodology, for each of five inactive CcdB mutants, exhaustive screens for suppressors were performed. Proximal suppressors were accurately discriminated from distal suppressors based on their phenotypes when present as single mutants. Experimentally identified putative proximal pairs formed spatial constraints to recover >98% of native-like models of CcdB from a decoy dataset. Suppressor methodology was also applied to the integral membrane protein, diacylglycerol kinase A where the structures determined by X-ray crystallography and NMR were significantly different. Suppressor as well as sequence co-variation data clearly point to the X-ray structure being the functional one adopted in-vivo. The methodology is applicable to any macromolecular system for which a convenient phenotypic assay exists.

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Anusmita Sahoo

Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India

Competing interests
Anusmita Sahoo, is an author on a patent application filed on behalf of the Indian Institute of Science, involving saturation suppressor mutagenesis methodology.
Shruti Khare

Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India

Competing interests
Shruti Khare, is an author on a patent application filed on behalf of the Indian Institute of Science, involving saturation suppressor mutagenesis methodology.
Sivasankar Devanarayanan

Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India

Competing interests
No competing interests declared.
Pankaj Jain

Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India

Competing interests
Pankaj Jain, is an author on a patent application filed on behalf of the Indian Institute of Science, involving saturation suppressor mutagenesis methodology.
Raghavan Varadarajan

Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India

For correspondence
varadar@mbu.iisc.ernet.in

Competing interests
Raghavan Varadarajan, is an author on a patent application filed on behalf of the Indian Institute of Science, involving saturation suppressor mutagenesis methodology.

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