Structural and functional characterisation of the interaction of the gene regulatory protein C.Esp1396I with its DNA recognition sites

  • Neil James Ball

Student thesis: Doctoral Thesis


The temporal regulation of restriction-modification (RM) systems is essential for the continued survival of the host cell. In many RM systems, this regulation is carried out by a controller (C) protein that is a key component of an intricate genetic switch. In the Esp136I RM system, the C-protein is a 79 amino acid protein that binds as a dimer to each of its three recognition sites (OM, OL and OR). A new purification strategy was devised to increase the yield of C.Esp1396I and two mutant proteins were expressed to either increase the solubility (Y29S) or increase the extinction coefficient (Y29W).
The monomer-dimer equilibrium constant (Kdim) was determined to be ca. 1.6 μM by sedimentation equilibrium analytical ultracentrifugation. DNA binding studies were carried out using surface plasmon resonance and the affinities of C.Esp1396I for OM, OL and OR were determined to be 0.61 nM, 5.6 nM and 121 nM respectively. Binding of a dimer of C.Esp1396I to OL increases the affinity of a second dimer binding to OR ~130-fold, and is thus highly cooperative.
The free protein structure of C.Esp1396I and an R53A mutant were determined by X-ray crystallography to 2.8 Å and 3.0 Å respectively. In both structures a flexible loop was identified between helices 3 and 4 that was able to adopt two distinct conformations. Structures of C.Esp1396I bound to either OL or OM were solved by X-ray crystallography (to 2.1 Å and 2.8 Å respectively). These nucleoprotein complex structures demonstrate that the flexible loop is involved in recognition of the C-boxes (inverted tetranucleotide repeats with the consensus: GACT), which are spaced differently in the two binding sites. R35, T36 and R46 were identified as key residues involved in sequence recognition. In the OL complex the flexible loop plays a key role in C.Esp1396I recognising two conserved elements that obey different symmetries.
Date of AwardSept 2011
Original languageEnglish
Awarding Institution
  • University of Portsmouth
SupervisorGeoff Kneale (Supervisor) & John McGeehan (Supervisor)

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