2q6h: Difference between revisions

Revision as of 19:36, 21 February 2008

Crystal Structure Analysis of LeuT complexed with L-leucine, sodium, and clomipramine

OverviewOverview

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.

About this StructureAbout this Structure

2Q6H is a Single protein structure of sequence from Aquifex aeolicus with , , and as ligands. Full crystallographic information is available from OCA.

ReferenceReference

Antidepressant binding site in a bacterial homologue of neurotransmitter transporters., Singh SK, Yamashita A, Gouaux E, Nature. 2007 Aug 23;448(7156):952-6. Epub 2007 Aug 8. PMID:17687333

Page seeded by OCA on Thu Feb 21 18:36:35 2008

Proteopedia Page Contributors and Editors (what is this?)Proteopedia Page Contributors and Editors (what is this?)

OCA

@@ Line 4: / Line 4: @@
 ==Overview==
-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 a serotonin transporter tricyclic, antidepressant 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 clomipramine noncompetitively inhibits substrate uptake., Cocrystal structures show that clomipramine 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 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.
+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.
 ==About this Structure==
@@ Line 10: / Line 10: @@
 ==Reference==
-Antidepressant binding site in a bacterial homologue of neurotransmitter transporters., Singh SK, Yamashita A, Gouaux E, Nature. 2007 Aug 8;. PMID:[http://ispc.weizmann.ac.il//pmbin/getpm?pmid=17687333 17687333]
+Antidepressant binding site in a bacterial homologue of neurotransmitter transporters., Singh SK, Yamashita A, Gouaux E, Nature. 2007 Aug 23;448(7156):952-6. Epub 2007 Aug 8. PMID:[http://ispc.weizmann.ac.il//pmbin/getpm?pmid=17687333 17687333]
 [[Category: Aquifex aeolicus]]
 [[Category: Single protein]]
 [[Category: Gouaux, E.]]
-[[Category: Singh, S.K.]]
+[[Category: Singh, S K.]]
 [[Category: Yamashita, A.]]
 [[Category: BOG]]
@@ Line 26: / Line 26: @@
 [[Category: tricyclic antidepressant]]
-''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Wed Jan 23 11:13:04 2008''
+''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 18:36:35 2008''