Publication Date

2014-04-02

Availability

Open access

Embargo Period

2014-04-02

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Biochemistry and Molecular Biology (Medicine)

Date of Defense

2014-03-25

First Committee Member

Amjad Farooq

Second Committee Member

Thomas K. Harris

Third Committee Member

Yanbin Zhang

Fourth Committee Member

Geoffrey W. Stone

Abstract

Early growth response 1 (EGR1) transcription factor orchestrates a plethora of signaling cascades in response to a wide variety of stimuli such as growth factors and hormones. Herein, using a battery of biophysical tools, I explore the molecular basis of binding of EGR1 to its cognate gene promoters. My data show that the binding of EGR1 to DNA is tightly regulated by solution pH. The binding affinity undergoes an enhancement of more than an order of magnitude with increasing pH from 5 to 8, implying that the deprotonation of an ionizable residue accounts for such behavior. This ionizable residue is identified as H382 by virtue of the fact that its substitution to nonionizable residues abolishes pH-dependence of the binding of EGR1 to DNA. Notably, H382 inserts into the major groove of DNA and stabilizes the EGR1-DNA interaction via both hydrogen bonding and van der Waals contacts. I also provide evidence that the binding of EGR1 transcription factor to DNA displays virtually zero dependence on ionic strength under physiological salt concentrations and that such feat is accomplished via favorable enthalpic contributions. Importantly, I unearth the molecular origin of such favorable enthalpy and attribute it to the ability of H382 residue to stabilize the EGR1-DNA interaction via both intermolecular hydrogen bonding and van der Waals contacts against the backdrop of salt. Consistent with this notion, the substitution of H382 residue with other amino acids faithfully restores salt-dependent binding of EGR1 to DNA in a canonical fashion. Finally, my biophysical analysis reveals that DNA sequence variations within the target gene promoters tightly modulate the energetics of binding of EGR1 and that nucleotide substitutions at certain positions are much more detrimental to EGR1-DNA interaction than others. In particular, the reduction in binding affinity poorly correlates with the loss of enthalpy and gain of entropy—a trend indicative of a complex interplay between underlying thermodynamic factors due to the differential role of water solvent upon nucleotide substitution. In sum, my findings uncover an unexpected but a key protonation-deprotonation step in the molecular recognition of EGR1 and suggest that it may act as a sensor of pH within the intracellular environment. This study reports the first example of a eukaryotic protein-DNA interaction capable of overriding the polyelectrolye effect. The work presented herein also bears important implications on understanding the molecular determinants of a key protein-DNA interaction at the cross-roads of human health and disease.

Keywords

intracellular pH; protein–DNA thermodynamics; zinc fingers; salt tolerance; polyelectrolyte effect; single nucleotide polymorphisms

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