Crystallization and Preliminary X-Ray Diffraction Analysis of a Mammal Inositol 1,3,4,5,6-Pentakisphosphate 2-Kinase

Franco-Echevarría, Elsa; Sanz‐Aparicio, Julia; Troffer-Charlier, N.; Poterszman, Arnaud; González, Beatriz

doi:10.1007/s10930-017-9717-y

Cited by 3 publications

(3 citation statements)

References 30 publications

(44 reference statements)

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“…Constructs for full-length IP 5 2-K recombinant expression either in bacteria (m ipk1 /pKLSLt plasmid) or insect cells were obtained, as described by us ( 34 ), from a m ipk1 cDNA (commercial clone bc062167). To produce a truncated m IP 5 2-K enzyme lacking the 21 C-terminal residues (ΔC- m IP 5 2-K), a stop codon was introduced at a position coding for residue 469 of m IP 5 2-K by site-directed mutagenesis and using as template the m ipk1 cDNA inserted into the pKLSLt vector ( 35 ).…”

Section: Methodsmentioning

confidence: 99%

“…Expression and purification of ΔC- m IP 5 2-K samples fused to LSL− was performed similarly to the full-length samples ( 34 ). Briefly, the protein was expressed in Escherichia coli BL21 Star (DE3) cells in 2TY medium supplemented with kanamycin (50 μg ml −1 ) at 310 K until an A 600 of 0.9 was reached.…”

Section: Methodsmentioning

confidence: 99%

“…All IP 5 2-K samples used for crystallization were obtained in the presence of 2 m m IP 6 . Best crystals obtained for the full-length m IP 5 2-K (expressed either in bacteria or in insect cells) diffracted to 4–4.3 Å ( 34 ), and they did not allow the structure solution. Finally, the ΔC- m IP 5 2-K construct allowed us to improve resolution to 3.2 Å from crystals grown in 0.2 m magnesium chloride, 0.1 m MES, pH 6.25, 10% (v/v) PEG 6000 and included 2 m m IP 6 and 2 m m ADP in the protein buffer.…”

Section: Methodsmentioning

confidence: 99%

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The crystal structure of mammalian inositol 1,3,4,5,6-pentakisphosphate 2-kinase reveals a new zinc-binding site and key features for protein function

Franco-Echevarría

Sanz‐Aparicio

Brearley

et al. 2017

Journal of Biological Chemistry

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Inositol 1,3,4,5,6-pentakisphosphate 2-kinases (IP5 2-Ks) are part of a family of enzymes in charge of synthesizing inositol hexakisphosphate (IP6) in eukaryotic cells. This protein and its product IP6 present many roles in cells, participating in mRNA export, embryonic development, and apoptosis. We reported previously that the full-length IP5 2-K from Arabidopsis thaliana is a zinc metallo-enzyme, including two separated lobes (the N- and C-lobes). We have also shown conformational changes in IP5 2-K and have identified the residues involved in substrate recognition and catalysis. However, the specific features of mammalian IP5 2-Ks remain unknown. To this end, we report here the first structure for a murine IP5 2-K in complex with ATP/IP5 or IP6. Our structural findings indicated that the general folding in N- and C-lobes is conserved with A. thaliana IP5 2-K. A helical scaffold in the C-lobe constitutes the inositol phosphate-binding site, which, along with the participation of the N-lobe, endows high specificity to this protein. However, we also noted large structural differences between the orthologues from these two eukaryotic kingdoms. These differences include a novel zinc-binding site and regions unique to the mammalian IP5 2-K, as an unexpected basic patch on the protein surface. In conclusion, our findings have uncovered distinct features of a mammalian IP5 2-K and set the stage for investigations into protein-protein or protein-RNA interactions important for IP5 2-K function and activity.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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The crystal structure of mammalian inositol 1,3,4,5,6-pentakisphosphate 2-kinase reveals a new zinc-binding site and key features for protein function

Franco-Echevarría

Sanz‐Aparicio

Brearley

et al. 2017

Journal of Biological Chemistry

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Protective effect of grape seed extract and orlistat co-treatment against stroke: Effect on oxidative stress and energy failure

Kadri

Ayed

Kadri

et al. 2021

Biomedicine & Pharmacotherapy

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Inositol Phosphates and Retroviral Assembly: A Cellular Perspective

Ricana

Dick

2021

Viruses

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Understanding the molecular mechanisms of retroviral assembly has been a decades-long endeavor. With the recent discovery of inositol hexakisphosphate (IP6) acting as an assembly co-factor for human immunodeficiency virus (HIV), great strides have been made in retroviral research. In this review, the enzymatic pathways to synthesize and metabolize inositol phosphates (IPs) relevant to retroviral assembly are discussed. The functions of these enzymes and IPs are outlined in the context of the cellular biology important for retroviruses. Lastly, the recent advances in understanding the role of IPs in retroviral biology are surveyed.

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Crystallization and Preliminary X-Ray Diffraction Analysis of a Mammal Inositol 1,3,4,5,6-Pentakisphosphate 2-Kinase

Cited by 3 publications

References 30 publications

The crystal structure of mammalian inositol 1,3,4,5,6-pentakisphosphate 2-kinase reveals a new zinc-binding site and key features for protein function

The crystal structure of mammalian inositol 1,3,4,5,6-pentakisphosphate 2-kinase reveals a new zinc-binding site and key features for protein function

Protective effect of grape seed extract and orlistat co-treatment against stroke: Effect on oxidative stress and energy failure

Inositol Phosphates and Retroviral Assembly: A Cellular Perspective

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