Expression, purification, crystallization and preliminary X-ray analysis of the EIIC^Glcdomain of theEscherichia coliglucose transporter

Abstract

The glucose-import system of Escherichia coli consists of a hydrophilic EIIA Glc subunit and a transmembrane EIICB Glc subunit. EIICB Glc (UniProt P69786) contains two domains: the transmembrane EIIC Glc domain (40.6 kDa) and the cytoplasmic EIIB Glc domain (8.0 kDa), which are fused by a linker that is strongly conserved among its orthologues. The EIICB Glc subunit can be split within this motif by trypsin. Here, the crystallization of the tryptic EIIC Glc domain is described. A complete data set was collected to 4.5 Å resolution at 100 K.

Structure and function of the glucose PTS transporter from Escherichia coli

“…Overexpression was carried out with freshly transformed E. coli BW25113DacrB cells (Baba et al, 2006) harboring the plasmids encoding wt-IICB (full-length; UniProtKB accession code: P69786) (Lanz and Erni, 1998), mut-IICB (full-length) (Zurbriggen et al, 2010) or mut-IIC (residues 1-394) (Zurbriggen et al, 2010). All three constructs were based on the expression vector pTSGH11 (Lanz and Erni, 1998) and had a C-terminal hexahistidine-tag (Histag) for metal affinity purification and a thrombin cleavage site for removal of the His-tag.…”

“…This feature makes the full-length protein unsuitable for 3D crystallization. Recently, the successful 3D crystallization of an E. coli IIC Glc mutant version as well as a preliminary X-ray analysis of the crystals was reported indicating diffraction to 4.5 Å resolution (Zurbriggen et al, 2010). The introduction of the three point mutations M17T, K150E and K394A into IIC Glc (herein referred to as mut-IIC, and wildtype-IIC Glc and -IICB Glc as wt-IIC and wt-IICB, respectively) was crucial for its stabilization and successful 3D crystallization (Zurbriggen et al, 2010).…”

“…Recently, the successful 3D crystallization of an E. coli IIC Glc mutant version as well as a preliminary X-ray analysis of the crystals was reported indicating diffraction to 4.5 Å resolution (Zurbriggen et al, 2010). The introduction of the three point mutations M17T, K150E and K394A into IIC Glc (herein referred to as mut-IIC, and wildtype-IIC Glc and -IICB Glc as wt-IIC and wt-IICB, respectively) was crucial for its stabilization and successful 3D crystallization (Zurbriggen et al, 2010). In contrast, 3D crystals diffracting to 3.3 Å of the diacetylchitobiose transporter from Bacillus cereus (which has no IIB domain) could successfully be grown and the structure solved (Cao et al, 2011).…”

“…This phosphoryl group originates from phosphoenolypyruvate and is sequentially transferred from enzyme I to HPr, IIA Glc , IIB Glc and finally to glucose. The IIC Glc domain has been predicted to span the membrane ten times based on topology analysis (Zurbriggen et al, 2010). Separately overexpressed and purified IIB Glc and IIC Glc domains are able to restore the carbohydrate transport and phosphorylation activities of IICB Glc (Buhr et al, 1994).…”

“…The effects of the triple mutation on the function of mut-IIC (Zurbriggen et al, 2010) and mut-IICB (full-length with M17T, K150E and K394A mutations) are unknown. Therefore, we characterized substrate binding and transport properties of mut-IIC and mut-IICB, and compared them with those of wt-IICB.…”