Mutations to transcription factor MAX allosterically increase DNA selectivity by altering folding and binding...

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Engineering

Background:

• Protein selectivity, the ability to discriminate between preferred and non-preferred ligands, is crucial for biological function and protein engineering.
• The biophysical mechanisms governing protein selectivity are not fully understood, hindering predictive capabilities and engineering efforts.

Purpose of the Study:

• To investigate how variants of the transcription factor (TF) MAX alter DNA specificity and selectivity.
• To elucidate the biophysical mechanisms underlying allosteric modulation of ligand selectivity in proteins.

Main Methods:

• Assayed over 1700 Kds and 500 rate constants for MAX variants binding to multiple DNA sequences.
• Applied thermodynamic and kinetic modeling to experimental data from MAX and Pho4 (S. cerevisiae) TFs.
• Compared results with published structural data of MAX-DNA complexes.

Main Results:

• Twenty-two of 240 MAX point mutations enhanced selectivity, but not at residues directly contacting DNA.
• Mutations altered partitioning between or affinity within different protein conformations with varying intrinsic selectivity.
• Conformational heterogeneity plays a key role in determining DNA sequence selectivity.

Conclusions:

• Allosteric modulation of protein conformations provides a mechanistic basis for enhanced ligand selectivity.
• Protein conformational heterogeneity is critical for sequence selectivity and can be leveraged for protein engineering.
• Findings guide future efforts to engineer proteins with improved selectivity.