Studying allosteric regulation in metal sensor proteins using computational methods

Abstract

In this chapter, we describe advances made in understanding the mechanism of allosteric regulation of DNA operator binding in the ArsR/SmtB family of metal-sensing proteins using computational methods. The paradigm, zinc-sensing transcriptional repressor Staphylococcus aureus CzrA represents an excellent model system to understand how metal sensor proteins maintain cellular metal homeostasis. Here, we discuss studies that helped to characterize a metal ion-mediated hydrogen-bonding pathway (HBP) that plays a dominant role in the allosteric mechanism of DNA operator binding in these proteins. The chapter discusses computational methods used to provide a molecular basis for the large conformational motions and allosteric coupling free energy (~6kcal/mol) associated with Zn(II) binding in CzrA. We present an accurate and convenient means by which to include metal ions in the nuclear magnetic resonance (NMR) structure determination process using molecular dynamics (MD) constrained by NMR-derived data. The method provides a realistic and physically viable description of the metal-binding site(s) and has potentially broad applicability in the structure determination of metal ion-bound proteins, protein folding, and metal template protein-design studies. Finally, our simulations provide strong support for a proposed HBP that physically connects the metal-binding residue, His97, to the DNA-binding interface through the αR helix that is present only in the Zn(II)-bound state. We find the interprotomer hydrogen bond interaction to be significantly stronger (~8kcal/mol) at functional allosteric metal-binding sites compared to the apo proteins. This interaction works to overcome the considerable disorder at these hydrogen-bonding sites in apo protein and functions as a "switch" to lock in a weak DNA-binding conformation once metal is bound. This interaction is found to be considerably weaker in nonresponsive metal-binding sites. These findings suggest a conserved functional role of metal-mediated second-shell coordination hydrogen bonds at allosterically responsive sites in zinc-sensing transcription regulators.

Keywords: CzrA; Molecular dynamics; Transcriptional repressor; amber; bonded model; hydrogen bond; metal ion mediated allostery; protein-DNA interactions; zinc ions.