Human Gpn1 purified from bacteria binds guanine nucleotides and hydrolyzes GTP as a protein dimer stabilized by its C-terminal tail.
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.
Instituto de Física, Universidad Autónoma de San Luis Potosí, Av. Manuel Nava 6, Zona Universitaria, C.P. 78290, San Luis Potosí, San Luis Potosí, Mexico.
Rogelio
R
González-González
Instituto de Física, Universidad Autónoma de San Luis Potosí, Av. Manuel Nava 6, Zona Universitaria, C.P. 78290, San Luis Potosí, San Luis Potosí, Mexico.
José A
JA
Guerra-Moreno
Instituto de Física, Universidad Autónoma de San Luis Potosí, Av. Manuel Nava 6, Zona Universitaria, C.P. 78290, San Luis Potosí, San Luis Potosí, Mexico.
Gema R
GR
Cristóbal-Mondragón
Instituto de Física, Universidad Autónoma de San Luis Potosí, Av. Manuel Nava 6, Zona Universitaria, C.P. 78290, San Luis Potosí, San Luis Potosí, Mexico.
Violeta
V
Romero
Instituto de Física, Universidad Autónoma de San Luis Potosí, Av. Manuel Nava 6, Zona Universitaria, C.P. 78290, San Luis Potosí, San Luis Potosí, Mexico.
Sonia G
SG
Peña-Gómez
Instituto de Física, Universidad Autónoma de San Luis Potosí, Av. Manuel Nava 6, Zona Universitaria, C.P. 78290, San Luis Potosí, San Luis Potosí, Mexico.
Gabriela M
GM
Montero-Morán
IPICYT, División de Biología Molecular, Camino a la Presa San José 2055, C.P. 78216, San Luis Potosí, San Luis Potosí, Mexico.
Samuel
S
Lara-González
Area de Biofisicoquímica, Departamento de Química, Universidad Autónoma Metropolitana-Iztapalapa, San Rafael Atlixco 186, Iztapalapa C.P. 09340, Ciudad de México, Mexico.
Andrés
A
Hernández-Arana
Laboratorio de Fisicoquímica e Ingeniería de Proteínas, Facultad de Medicina, Universidad Nacional Autónoma de México, Ciudad Universitaria, Coyoacán C.P. 04510, Ciudad de México, Mexico.
Daniel A
DA
Fernández-Velasco
Instituto de Física, Universidad Autónoma de San Luis Potosí, Av. Manuel Nava 6, Zona Universitaria, C.P. 78290, San Luis Potosí, San Luis Potosí, Mexico. Electronic address: mcalera@ifisica.uaslp.mx.
Mónica R
MR
Calera
Instituto de Física, Universidad Autónoma de San Luis Potosí, Av. Manuel Nava 6, Zona Universitaria, C.P. 78290, San Luis Potosí, San Luis Potosí, Mexico. Electronic address: rsanchez@ifisica.uaslp.mx.
Roberto
R
Sánchez-Olea
D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D006160:Guanosine Triphosphate / Q000737:chemistry*; D006801:Humans; D006868:Hydrolysis; D000072417:Protein Domains; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D011994:Recombinant Proteins; D006160:Guanosine Triphosphate; C472669:GPN1 protein, human; D019204:GTP-Binding Proteins
C-terminal tail; E. coli BL-21 (DE3); GTPase; Human Gpn1; Protein dimer