Limits...
LCP crystallization and X-ray diffraction analysis of VcmN, a MATE transporter from Vibrio cholerae.

Kusakizako T, Tanaka Y, Hipolito CJ, Kuroda T, Ishitani R, Suga H, Nureki O - Acta Crystallogr F Struct Biol Commun (2016)

Bottom Line: Multidrug and toxic compound extrusion (MATE) transporters, one of the multidrug exporter families, efflux xenobiotics towards the extracellular side of the membrane.Since MATE transporters expressed in bacterial pathogens contribute to multidrug resistance, they are important therapeutic targets.Here, a MATE-transporter homologue from Vibrio cholerae, VcmN, was overexpressed in Escherichia coli, purified and crystallized in lipidic cubic phase (LCP).

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.

ABSTRACT
Multidrug and toxic compound extrusion (MATE) transporters, one of the multidrug exporter families, efflux xenobiotics towards the extracellular side of the membrane. Since MATE transporters expressed in bacterial pathogens contribute to multidrug resistance, they are important therapeutic targets. Here, a MATE-transporter homologue from Vibrio cholerae, VcmN, was overexpressed in Escherichia coli, purified and crystallized in lipidic cubic phase (LCP). X-ray diffraction data were collected to 2.5 Å resolution from a single crystal obtained in a sandwich plate. The crystal belonged to space group P212121, with unit-cell parameters a = 52.3, b = 93.7, c = 100.2 Å. As a result of further LCP crystallization trials, crystals of larger size were obtained using sitting-drop plates. X-ray diffraction data were collected to 2.2 Å resolution from a single crystal obtained in a sitting-drop plate. The crystal belonged to space group P212121, with unit-cell parameters a = 61.9, b = 91.8, c = 100.9 Å. The present work provides valuable insights into the atomic resolution structure determination of membrane transporters.

No MeSH data available.


Related in: MedlinePlus

Crystals of VcmNΔC. (a) Schematic representation of the macrocyclic peptide. (b) Form A crystal. (c) Form B crystal. The scale bars represent 30 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4933005&req=5

fig2: Crystals of VcmNΔC. (a) Schematic representation of the macrocyclic peptide. (b) Form A crystal. (c) Form B crystal. The scale bars represent 30 µm.

Mentions: We performed an LCP crystallization screening of VcmNΔC. Initially, tiny crystals of a mixture of VcmNΔC and the macrocyclic peptide (Fig. 2 ▸a) were obtained under several conditions containing precipitants such as polyethylene glycol (PEG) 300 and PEG 400 using sandwich plates. As a result of optimization efforts, including the screening of additives using the StockOptions Salt and Additive Screen kits, rectangular prism-shaped crystals (form A) were obtained with a reservoir solution composed of 30% PEG 300, 100 mM sodium citrate pH 5.0, 100 mM ammonium fluoride (Fig. 2 ▸b). Crystals appeared in a day and grew to approximate dimensions of 10 × 10 × 25 µm in a week. We also attempted crystallization of VcmNΔC without the macrocyclic peptides. In contrast to the crystallization of PfMATE, we unfortunately found that the peptides did not affect the quality of the VcmNΔC crystals. Next, crystallization screening was performed using sitting-drop plates. In contrast to crystallization in sandwich plates, crystallization in sitting-drop plates is suitable for substrate-soaking experiments to determine the structures of complexes with substrates. Rod-shaped crystals (form B) were obtained in a reservoir composed of 30% PEG 500 dimethyl ether (DME), 100 mM Tris–HCl pH 8.0, 100 mM magnesium formate. The approximate dimensions of the form B crystals were 10 × 10 × 50 µm. The sizes of the crystals obtained in the sitting-drop plates were usually larger than those produced in the sandwich plates. Finally, as a result of optimization of the salt concentration and the pH of the condition, crystals with approximate dimensions of 10 × 10 × 100 µm were obtained in reservoir solutions composed of 28–33% PEG 500 DME, 100 mM Tris–HCl pH 7.5, 50 mM magnesium formate (Fig. 2 ▸c). For the substrate-soaking experiments, an 800 nl portion of reservoir solution containing 10 mM Hoechst 33342 was added to drops containing crystals. The protein crystals were incubated for about one month at 293 K and then cooled in liquid nitrogen.


LCP crystallization and X-ray diffraction analysis of VcmN, a MATE transporter from Vibrio cholerae.

Kusakizako T, Tanaka Y, Hipolito CJ, Kuroda T, Ishitani R, Suga H, Nureki O - Acta Crystallogr F Struct Biol Commun (2016)

Crystals of VcmNΔC. (a) Schematic representation of the macrocyclic peptide. (b) Form A crystal. (c) Form B crystal. The scale bars represent 30 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4933005&req=5

fig2: Crystals of VcmNΔC. (a) Schematic representation of the macrocyclic peptide. (b) Form A crystal. (c) Form B crystal. The scale bars represent 30 µm.
Mentions: We performed an LCP crystallization screening of VcmNΔC. Initially, tiny crystals of a mixture of VcmNΔC and the macrocyclic peptide (Fig. 2 ▸a) were obtained under several conditions containing precipitants such as polyethylene glycol (PEG) 300 and PEG 400 using sandwich plates. As a result of optimization efforts, including the screening of additives using the StockOptions Salt and Additive Screen kits, rectangular prism-shaped crystals (form A) were obtained with a reservoir solution composed of 30% PEG 300, 100 mM sodium citrate pH 5.0, 100 mM ammonium fluoride (Fig. 2 ▸b). Crystals appeared in a day and grew to approximate dimensions of 10 × 10 × 25 µm in a week. We also attempted crystallization of VcmNΔC without the macrocyclic peptides. In contrast to the crystallization of PfMATE, we unfortunately found that the peptides did not affect the quality of the VcmNΔC crystals. Next, crystallization screening was performed using sitting-drop plates. In contrast to crystallization in sandwich plates, crystallization in sitting-drop plates is suitable for substrate-soaking experiments to determine the structures of complexes with substrates. Rod-shaped crystals (form B) were obtained in a reservoir composed of 30% PEG 500 dimethyl ether (DME), 100 mM Tris–HCl pH 8.0, 100 mM magnesium formate. The approximate dimensions of the form B crystals were 10 × 10 × 50 µm. The sizes of the crystals obtained in the sitting-drop plates were usually larger than those produced in the sandwich plates. Finally, as a result of optimization of the salt concentration and the pH of the condition, crystals with approximate dimensions of 10 × 10 × 100 µm were obtained in reservoir solutions composed of 28–33% PEG 500 DME, 100 mM Tris–HCl pH 7.5, 50 mM magnesium formate (Fig. 2 ▸c). For the substrate-soaking experiments, an 800 nl portion of reservoir solution containing 10 mM Hoechst 33342 was added to drops containing crystals. The protein crystals were incubated for about one month at 293 K and then cooled in liquid nitrogen.

Bottom Line: Multidrug and toxic compound extrusion (MATE) transporters, one of the multidrug exporter families, efflux xenobiotics towards the extracellular side of the membrane.Since MATE transporters expressed in bacterial pathogens contribute to multidrug resistance, they are important therapeutic targets.Here, a MATE-transporter homologue from Vibrio cholerae, VcmN, was overexpressed in Escherichia coli, purified and crystallized in lipidic cubic phase (LCP).

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.

ABSTRACT
Multidrug and toxic compound extrusion (MATE) transporters, one of the multidrug exporter families, efflux xenobiotics towards the extracellular side of the membrane. Since MATE transporters expressed in bacterial pathogens contribute to multidrug resistance, they are important therapeutic targets. Here, a MATE-transporter homologue from Vibrio cholerae, VcmN, was overexpressed in Escherichia coli, purified and crystallized in lipidic cubic phase (LCP). X-ray diffraction data were collected to 2.5 Å resolution from a single crystal obtained in a sandwich plate. The crystal belonged to space group P212121, with unit-cell parameters a = 52.3, b = 93.7, c = 100.2 Å. As a result of further LCP crystallization trials, crystals of larger size were obtained using sitting-drop plates. X-ray diffraction data were collected to 2.2 Å resolution from a single crystal obtained in a sitting-drop plate. The crystal belonged to space group P212121, with unit-cell parameters a = 61.9, b = 91.8, c = 100.9 Å. The present work provides valuable insights into the atomic resolution structure determination of membrane transporters.

No MeSH data available.


Related in: MedlinePlus