PDBsum entry: 2q72

Transport protein

PDB-id: 2q72

Contents

Description

Header details

Header records

References

PROCHECK

Protein chain

511 a.a. *

Ligands

BOG ×5

LEU

IXX ×2

Metal ions

_NA ×2

Waters ×146

* Residue conservation analysis

Tools

Clefts Calculation

PDB id:

2q72

Name:

Transport protein

Title:

Crystal structure analysis of leut complexed with l-leucine, sodium, and imipramine

Structure:

Transporter. Chain: a. Engineered: yes

Source:

Aquifex aeolicus vf5. Organism_taxid: 224324. Strain: vf5. Gene: snf. Expressed in: escherichia coli. Expression_system_taxid: 562.

UniProt:

O67854 (O67854_AQUAE)

Seq:

Struc:

Seq:

513 a.a.

Struc:

511 a.a.

Key:	PfamA domain
Secondary structure	CATH domain

Resolution:

1.70Å

R-factor:

0.203

R-free:

0.219

Authors:

S.K.Singh,A.Yamashita,E.Gouaux

Key ref:

S.K.Singh et al. (2007). Antidepressant binding site in a bacterial homologue of neurotransmitter transporters.. Nature, 448, 952-956. [PubMed id: 17687333] [DOI: 10.1038/nature06038]

Date:

05-Jun-07

Release date:

21-Aug-07

Related entries:

2a65
the same protein complexed with l-leucine and sodium
2q6h
the same protein complexed with l-leucine, clomipramine and
sodium
2qb4
the same protein complexed with l-leucine, desipramine and
sodium
2qei
the same protein complexed with l-alanine, clomipramine and
... plus others (see Header records)

Quick_links

Procheck

Clefts

Surface

Key reference

DOI no: 10.1038/nature06038

Nature 448:952-956 (2007)

PubMed id: 17687333

Antidepressant binding site in a bacterial homologue of neurotransmitter transporters.

S.K.Singh, A.Yamashita, E.Gouaux.

ABSTRACT

Sodium-coupled transporters are ubiquitous pumps that harness pre-existing sodium gradients to catalyse the thermodynamically unfavourable uptake of essential nutrients, neurotransmitters and inorganic ions across the lipid bilayer. Dysfunction of these integral membrane proteins has been implicated in glucose/galactose malabsorption, congenital hypothyroidism, Bartter's syndrome, epilepsy, depression, autism and obsessive-compulsive disorder. Sodium-coupled transporters are blocked by a number of therapeutically important compounds, including diuretics, anticonvulsants and antidepressants, many of which have also become indispensable tools in biochemical experiments designed to probe antagonist binding sites and to elucidate transport mechanisms. Steady-state kinetic data have revealed that both competitive and noncompetitive modes of inhibition exist. Antagonist dissociation experiments on the serotonin transporter (SERT) have also unveiled the existence of a low-affinity allosteric site that slows the dissociation of inhibitors from a separate high-affinity site. Despite these strides, atomic-level insights into inhibitor action have remained elusive. Here we screen a panel of molecules for their ability to inhibit LeuT, a prokaryotic homologue of mammalian neurotransmitter sodium symporters, and show that the tricyclic antidepressant (TCA) clomipramine noncompetitively inhibits substrate uptake. Cocrystal structures show that clomipramine, along with two other TCAs, binds in an extracellular-facing vestibule about 11 A above the substrate and two sodium ions, apparently stabilizing the extracellular gate in a closed conformation. Off-rate assays establish that clomipramine reduces the rate at which leucine dissociates from LeuT and reinforce our contention that this TCA inhibits LeuT by slowing substrate release. Our results represent a molecular view into noncompetitive inhibition of a sodium-coupled transporter and define principles for the rational design of new inhibitors.

Selected figure(s)

Figure 2.
Figure 2: TCAs bind in the putative permeation pathway of LeuT. F[o]–F[c] simulated annealing omit maps, both contoured at 3.0 , of clomipramine (a) and imipramine (b), in which the TCA was omitted from the simulated annealing run and subsequent phase calculation. The chlorine atom of clomipramine is green. c, LeuT tilted 15° from the membrane plane to illustrate the binding sites of clomipramine and leucine, both depicted in space-filling representation. Helices whose residues interact with clomipramine are coloured. d, Electrostatic properties of the LeuT extracellular-facing vestibule, with clomipramine (yellow) cradled in the negatively charged crevice. Electrostatic potential isocontours were set at +7 kT e^–1 (blue) and –7 kT e^–1 (red). EL4 is tinted green. To make clomipramine more visible, LeuT has been tilted an additional 5° towards the reader from the view in c ( 20° from the membrane plane).

Figure 3.
Figure 3: Clomipramine-binding site. a, Stereo view, with clomipramine and leucine carbon atoms depicted in yellow, sodium ions in purple and two water molecules in red. Residues whose interactions with clomipramine are hydrophobic, polar (Q34) or ionic (D401) are coloured grey, orange and pink, respectively. Atoms depicted as spheres interact through either a hydrogen bond or a salt bridge. b, F[o]–F[c] simulated annealing omit map, contoured at 3.0 , of D404 and R30, depicting the direct salt bridge between the guanidium of R30 and the carboxylate of D404, with displacement of two water molecules (compare to overlay in d). Distances (Å) are shown along dashed lines. c, F[o]–F[c] simulated annealing omit map, contoured at 3.0 , of the tip of EL4 (residues A317–N321), illustrating the movement 'up' of A319 in the LeuT–clomipramine crystal structure (compare to overlay in d). d, Same view as in a with residues from the original LeuT structure (PDB ID 2A65) overlaid (in green with two water molecules between D404 and R30 in cyan) onto those from the LeuT–clomipramine crystal structure (in pink with the two displaced waters in red and labelled as H[2]O (CMI)). e, CPK rendering of clomipramine, R30 and F253 illustrating how the positively charged guanidium group is sandwiched between the aromatic rings of clomipramine and F253.

The above figures are reprinted by permission from Macmillan Publishers Ltd: Nature (2007, 448, 952-956) copyright 2007.

Figures were selected by an automated process.