Native Cys Residues Research Articles

Topology of the Na+/Proline Transporter of Escherichia coli

Hydropathy profile analysis of the amino acid sequence of the Na+/proline transporter of Escherichia coli (PutP) suggests that the protein consists of 12 transmembrane domains (TMs) which are connected by hydrophilic loops (Nakao, T., Yamato, I., and Anraku, Y. (1987) Mol. Gen. Genet. 208, 70-75). We have tested this prediction by applying a gene fusion approach in combination with a Cys accessibility analysis and site-specific proteolysis. Characterization of a series of PutP-alkaline phosphatase (PhoA) and PutP-beta-galactosidase (LacZ) hybrid proteins yields a reciprocal activity pattern of the reporter proteins that is in agreement with the topology of TMs III to XII of the 12-helix model. Placement of the PutP-PhoA and PutP-LacZ junction sites closer to the N terminus does not yield conclusive results. As a prerequisite for further topology studies, a functional PutP molecule devoid of all five native Cys residues (Cys-free PutP) is generated. Subsequently, amino acids in Cys-free PutP are replaced individually with Cys, and the accessibility of the sulfhydryl groups is analyzed. Surprisingly, Cys residues placed close to the N terminus of PutP (Ile-3 --> Cys, Thr-5 --> Cys) or into putative TM II (Ser-71 --> Cys, Glu-75 --> Cys) are highly accessible to membrane permeant and impermeant thiol reagents in intact cells. In contrast, Cys at the C terminus (Ser-502 --> Cys) reacts only with the membrane permeant but not with the impermeant reagent in intact cells. These results contradict the 12-helix motif and indicate a periplasmic location of the N terminus whereas the C terminus faces the cytoplasm. In addition, a transporter with Cys in place of Leu-37 (putative periplasmic loop (pL2) shows the same accessibility pattern as the Cys at the C terminus. Furthermore, PutP which has been purified and reconstituted into proteoliposomes in an inside-out orientation, is readily cleaved by the endoproteinase AspN before Asp-33 (pL2), Asp-112 (putative cytoplasmic loop (cL3), Asp-262 (cL7), and Asp-356 (cL9). These results suggest a cytosolic location of Asp-33 and Leu-37, thereby implying the formation of an additional TM formed by amino acids of pL2. Based on these observations, a new secondary structure model is proposed according to which the protein consists of 13 TMs with the N terminus on the outside and the C terminus facing the cytoplasm. The 13-helix structure is discussed as a common topological motif for all members of the Na+/solute cotransporter family.

Effect of cysteine replacements on the properties of the turgor sensor KdpD of Escherichia coli

Escherichia coli responds rapidly to K +-limitation or high osmolarity by induction of the kdpFABC operon coding for the high affinity K +-translocating Kdp-ATPase. This process is controlled by the membrane-bound histidine kinase KdpD and the response regulator KdpE. Here, it is demonstrated that replacements of the native Cys residues at positions 409, 852, and 874 influence distinct activities of KdpD, whereas replacements of Cys residues at positions 32, 256, and 402 have no effect. Replacements of Cys 409 in KdpD reveal that transmembrane domain I is important for perception and/or propagation of the stimulus. When Cys 409 is replaced with Ala, kdpFABC expression becomes constitutive regardless of the external stimuli. In contrast, when Cys 409 is replaced with Val or Tyr, induction of kdpFABC expression in response to different stimuli is drastically reduced. KdpD with Ser at position 409 supports levels of kdpFABC expression comparable to those seen in wild-type. Since neither the kinase nor phosphatase activity of these proteins is affected, it is proposed that different amino acid side-chains at position 409 alter the switch between the inactive and active forms of the kinase. When Cys 852 or Cys 874 is replaced with Ala or Ser, kinase activity is reduced to 10% of the wild-type level. However, kinetic studies reveal that the apparent ATP binding affinity is not affected. Surprisingly, introduction of Cys 852 and Cys 874 into a KdpD protein devoid of Cys residues leads to full recovery of the kinase activity. Labeling studies support the idea that a disulfide bridge forms between these two residues.