Crystal structure of an asymmetric trimer of a bacterial glutamate transporter homolog

Abstract

We report a structure of a trimeric glutamate transporter homolog from Pyrococcus horikoshii with two protomers in an inward facing state and the third in an intermediate conformation between the outward and inward facing states. The intermediate shows a cavity in the thinnest region of the transporter, which is potentially accessible to extracellular and cytoplasmic solutions. Our findings suggest a structural principle by which transport intermediates may mediate uncoupled permeation of polar solutes.

Figure 1

Crystal structure of the asymmetric trimer. a, b, Averaged 2fo-fc electron density maps at 2.5 σ (dark grey mesh) and anomalous difference Fourier maps at 5 σ (green mesh) for the IFS and the iOFS, respectively. c, Surface representations of the trimer viewed within the membrane plane. Trimerization and transport domains are colored wheat and blue, respectively. d, Cartoon representation of the protomers in the iOFS (left) and the IFS (right). The third protomer is omitted for clarity; HP1 and HP2 are yellow and red, respectively; and bound L-aspartate and sodium ions are shown as spheres.

Figure 2

Conformational transition in the iOFS. a, Cylinder representation of Glt_Ph viewed from the membrane plane in the OFS (left) and iOFS (right). The color scheme is as in Figure 1. TM8 is dark blue, and bound substrates are emphasized as spheres. Dashed lines, normal to the membrane plane, highlight the transport domain orientation differences. b, Superimposition of the trimerization domains in the OFS (yellow), iOFS (light orange) and IFS (orange). c, Surface representation of the protomers in the OFS (left), iOFS (center), and IFS (right) colored as in a. An arrow marks the enlarged crevice between the transport and trimerization domains.

Figure 3

Cavity at the domain interface in the iOFS. a, The interface is viewed from within the transport domain in the OFS (left), iOFS (center) and IFS (right). The trimerization domain is in surface representation and colored by atom type. Interacting transport domain elements are in ribbon representation and colored light blue (TM7), yellow (HP1) and red (HP2). The rest of the transport domain is omitted for clarity. The side chains making contact with the trimerization domain and bound L-aspartate are emphasized as sticks, and sodium ion in Na2 site is shown as a sphere. b, Thin cross-sections of the protomers approximately through Ser65 and Tyr195 and normal to the membrane plane. Residues lining the domain interface are labeled.