Glycerol-3-Phosphate Transport

Glycerol-3-Phosphate Transporter

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I. Introduction

Membrane transport in cells is a fundamental biological process that is mediated by various channels and transproter proteins. A major type of such proteins are the secondary active membrane transporters, which use a solute gradient to drive the translocation of other substrates. The glycerol-3-phosphate (G3P) transporter, GlpT, from Escherichia coli mediates G3P and inorganic phosphate exchange across the bacterial inner membrane. It possesses 12 transmembrane α-helices and is a member of the Major Facilitator Superfamily, the largest secondary transporter family known in the genomes sequenced to date. In the cell membrane, these proteins are responsible for the transport of a wide range of solutes, including sugars, amino acids, neurotransmitters, ions, and toxins.

For more information see Biochemistry Online: Chapter 6A - Passive and Facilitated Diffusion

II. General Structure

Wireframe
Backbone
Display a-helices

Color code:

H1 and H7

H2 and H8

H3 and H9

H4 and H10

H5 and H11

H6 and H12

The protein has 12 transmembrane a-helices which can be divided into two similar domains by a pseudo two-fold symmetry. This pseudo symmetry extends to all helices with H1 related to H7, H2 to H8, et cetera.

Display a-helices with fill

This space filling representation shows the helices in a space filled way. By rotating the molecule with your mouse, you can see that the molecular pore (which extends through the center of the protein) is closed on one side and open on the other.

Display Periplasmic Residues

The periplasmic side of the molecule is flat and protrudes only slightly into the periplasm and the pore is closed. This barrier is created by portions of H1 and H7. The space between H1 and H7, is filled with nine aromatic side chains which help close the pore completely.

Display H1 and H7 Closing Region
Display Cytoplasmic Residues

The cytoplasmic residues include the N- and C- termini which also means that the opening of the pore is within the cytoplasm.

Display All Helices Except H1 and H7

The pore opening can be seen by displaying all helices except H1 and H7 and rotating the molecule such that you are looking directly at the base of the molecule. A small opening can be seen.

Display Lys₃₇₈ and Lys₃₇₉

It is believed that the cytoplasmic loop from H10-H11 plays an integral role in helix alignment when the protein is inserted into the membrane. This is because it contains two lysine residues (Lys378 and Lys379) which have been shown to aid in insertion of the Glut1 protein (Sato et al. J. Biol. Chem. 274m 24721 (1999)).

Display Both Cytoplasmic and Periplasmic Regions

With this representation, it is clearly shown how the molecule inserts itself into the membrane. The periplasmic and cytoplasmic residues encompass the region of the protein which is membrane bound and the pore is closed.

Display Arg45 and Arg269

It is noted that Arg45 and Arg269 are embedded in H1 and H7 and by X-ray crystallography, the literature reports that the distance between then is 9.9A. The optimal hydrogen bond length for the phosphate and Arg residues would be 2.9A, this means that the phosphate ligand must induce a conformation change in the protein to bring the Arg-Arg distance to the optimal dimension. When this happens, the periplasmic ends of H1 and H7 separate and the cytoplasmic ends come together to close the cytoplasmic side. This allows for the inorganic phosphate to be released and G3P to be bound and brought back into the cell.

Display Arg₄₅ and Arg₂₆₉ as situated in H1 and H7

The substrate binding sites as located on H1 and H7.

Conclusions

The GlpT protein works in a "rocker-switch" fashion which exposes the binding site, Arg45 and Arg269, to the periplasm, yielding the outward opening conformation of the molecule. Cytoplasmic binding and periplasmic release of inorganic phosphate would allow its replacement in the substrate-binding site by G3P, which is then transferred into the cytoplasm. Substrate binding is proposed to lower the energy barrier between the inward- and outward-facing conformations of GlpT, facilitating their interconversion and allowing the inorganic phosphate gradient to drive G3P transport.