Electronegative clusters modulate folding status and RNA binding of unstructured RNA-binding proteins.

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Electronegative clusters (ENCs), rich in acidic residues or phosphorylation sites, are common in RNA-binding proteins.
• While ENCs typically inhibit RNA binding in structured domains, their role in unstructured domains is less understood.

Purpose of the Study:

• To elucidate the mechanism by which phosphorylated ENCs enhance RNA binding affinity in the unstructured RNA-binding domain (RBD) of histone pre-mRNA stem-loop binding protein (SLBP).
• To investigate the generalizability of ENC function by engineering an ENC onto a different unstructured RBD.

Main Methods:

• Nuclear Magnetic Resonance (NMR) spectroscopy
• Molecular Dynamics (MD) simulations
• Protein engineering (grafting ENCs and introducing charged residues)

Main Results:

• ENC phosphorylation induces a transient folded state in apo SLBP, resembling the RNA-bound conformation.
• The phosphorylated ENC allosterically interacts with a loop region, stabilizing the SLBP-RNA complex and enhancing binding affinity to the sub-picomolar range.
• Engineering an ENC onto a different unstructured RBD (DRBM1) increased its folded state and inhibited RNA binding, while introducing basic residues counteracted this effect.

Conclusions:

• ENCs can promote the folding of intrinsically disordered RNA-binding domains.
• The effect of ENCs on RNA binding is context-dependent, influenced by the domain's intrinsic properties and the presence of electropositive charges on its surface.