Factors determining intrinsic GDP release in GTPases: a structural and functional study of the ferrous iron transporter FeoB

Abstract

Factors determining intrinsic GDP release in GTPases: A structural and functional study of the ferrous iron transporter FeoB FeoB is a prokaryotic membrane protein involved in ferrous iron transport. It comprises a membrane transporter domain tethered to a soluble N-terminal domain (NFeoB) comprising two subdomains: a GTPase domain and a helical domain. The mechanism linking iron uptake to GTPase activity remains unknown. Early functional characterisation of the GTPase domain demonstrated a slow intrinsic GTP hydrolysis rate and a weak GDP affinity. Given this combination, the GTPase domain would be almost constitutively bound to GTP. This is physiologically unlikely given that FeoB would then be constitutively ‘on’ if functioning as a channel, or have too slow a hydrolysis rate if the iron transport mechanism was powered by GTP hydrolysis. In an attempt to rationalise this conundrum, the crystal structure of the Escherichia coli (E. coli) NFeoB domain was determined. However, the structure alone provided limited insight into the biochemical rationale for the weak GDP affinity and slow GTP hydrolysis. The main focus of my thesis work was thus to understand the slow GTP hydrolysis and to investigate if this reflects the in vivo rate, and to provide a molecular understanding of the weak GDP affinity in NFeoB. In this work, it was demonstrated that the intrinsically slow GTP hydrolysis rate of FeoB could be accelerated by potassium. Comparison of nucleotide free and bound E. coli NFeoB structures revealed a large conformational change in the nucleotide binding G5 motif. The importance of sequence to this region was highlighted through alanine scanning mutational analysis. In particular, mutations at positions 2 and 3 of the G5 loop had a significant impact on nucleotide affinity, and also underwent the largest conformational changes in the comparison of the nucleotide free and GTP bound structures. To determine the order of events in GDP release, a chimeric NFeoB protein combining sequence and structural characteristics of both fast and slow GDP releasing GTPases was constructed. Biochemical analysis revealed uncoupled nucleotide affinity and release rates supporting a model whereby G5 loop movement promotes nucleotide release.