Human Gpn1 purified from bacteria binds guanine nucleotides and hydrolyzes GTP as a protein dimer stabilized by its C-terminal tail
Protein Expression and Purification(2017)
Abstract
The essential GTPase Gpn1 mediates RNA polymerase II nuclear targeting and controls microtubule dynamics in yeast and human cells by molecular mechanisms still under investigation. Here, we purified human HisGpn1 expressed as a recombinant protein in bacteria E. coli BL-21 (DE3). Affinity purified HisGpn1 eluted from a size exclusion column as a protein dimer, a state conserved after removing the hexa-histidine tail and confirmed by separating HisGpn1 in native gels, and in dynamic light scattering experiments. Human HisGpn1 purity was higher than 95%, molecularly monodisperse and could be concentrated to more than 10 mg/mL without aggregating. Circular dichroism spectra showed that human HisGpn1 was properly folded and displayed a secondary structure rich in alpha helices. HisGpn1 effectively bound GDP and the non-hydrolyzable GTP analogue GMPPCP, and hydrolyzed GTP. We next tested the importance of the C-terminal tail, present in eukaryotic Gpn1 but not in the ancestral archaeal Gpn protein, on HisGpn1 dimer formation. C-terminal deleted human HisGpn1 (HisGpn1ΔC) was also purified as a protein dimer, indicating that the N-terminal GTPase domain contains the interaction surface needed for dimer formation. In contrast to HisGpn1, however, HisGpn1ΔC dimer spontaneously dissociated into monomers. In conclusion, we have developed a method to purify properly folded and functionally active human HisGpn1 from bacteria, and showed that the C-terminal tail, universally conserved in all eukaryotic Gpn1 orthologues, stabilizes the GTPase domain-mediated Gpn1 protein dimer. The availability of recombinant human Gpn1 will open new research avenues to unveil the molecular and pharmacological properties of this essential GTPase.
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Key words
Human Gpn1,GTPase,C-terminal tail,Protein dimer,E. coli BL-21 (DE3)
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