Potassium-activated GTPase Reaction in the G Protein-coupled Ferrous Iron Transporter B

Miriam-Rose Ash,Amy Guilfoyle,Ronald J Clarke,J Mitchell Guss,Megan J Maher,Mika Jormakka

doi:10.1074/jbc.m110.111914

Miriam-Rose Ash, Amy Guilfoyle + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m110.111914

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Abstract

FeoB is a prokaryotic membrane protein responsible for the import of ferrous iron (Fe(2+)). A defining feature of FeoB is that it includes an N-terminal 30-kDa soluble domain with GTPase activity, which is required for iron transport. However, the low intrinsic GTP hydrolysis rate of this domain appears to be too slow for FeoB either to function as a channel or to possess an active Fe(2+) membrane transport mechanism. Here, we present crystal structures of the soluble domain of FeoB from Streptococcus thermophilus in complex with GDP and with the GTP analogue derivative 2'-(or -3')-O-(N-methylanthraniloyl)-beta,gamma-imidoguanosine 5'-triphosphate (mant-GMPPNP). Unlike recent structures of the G protein domain, the mant-GMPPNP-bound structure shows clearly resolved, active conformations of the critical Switch motifs. Importantly, biochemical analyses demonstrate that the GTPase activity of FeoB is activated by K(+), which leads to a 20-fold acceleration in its hydrolysis rate. Analysis of the structure identified a conserved asparagine residue likely to be involved in K(+) coordination, and mutation of this residue abolished K(+)-dependent activation. We suggest that this, together with a second asparagine residue that we show is critical for the structure of the Switch I loop, allows the prediction of K(+)-dependent activation in G proteins. In addition, the accelerated hydrolysis rate opens up the possibility that FeoB might indeed function as an active transporter.

Highlights

Small regulatory GTPase proteins (G proteins) are key binary switches in cellular processes such as differentiation, proliferation, and cell motility [1, 2]
GTP binding to the G protein domain (FeoGP) initiates the transport of Fe2ϩ across the membrane, which is halted by the hydrolysis of GTP to GDP
A slow intrinsic GTPase rate is not uncommon for G proteins, because their activity can be accelerated by GTPase-activating proteins (GAPs), which can stimulate GTP hydrolysis by several orders of magnitude [10]

Summary

EXPERIMENTAL PROCEDURES

Protein Preparation and Crystallization—DNA encoding residues 1–270 from FeoB was amplified from S. thermophilus genomic DNA (strain LMG 18311) and cloned into a pGEX4T-1 glutathione S-transferase fusion vector (GE Healthcare). Measurement of GTPase Activity by Intrinsic Tryptophan Fluorescence—The rate of GTP hydrolysis by wild-type and mutant NFeoBSt in NaCl and KCl were determined by intrinsic tryptophan fluorescence in single-turnover experiments. NFeoBSt was preincubated for 5 min at 37 °C in buffer containing 20 mM Tris, pH 8, and either NaCl (100 or 200 mM) or KCl (50 –200 mM) This solution was used to set the relative gain of the plate reader, which was adjusted to give a fluorescence signal of 90% of the maximum possible value. To measure the rate of mant-GDP release, protein (10 ␮M in 20 mM Tris, pH 8.0, and either 100 mM NaCl or KCl) was incubated for 10 min with 0.5 ␮M mant-GDP and rapidly mixed with 500 ␮M GTP in the corresponding buffer. All of the experiments were performed 7–10 times, and the data were averaged before calculating the final kobs or koff values

RESULTS

Solvent Ramachandran plotb

DISCUSSION