PSI Structural Biology Knowledgebase

PSI | Structural Biology Knowledgebase
Header Icons
E-Collection

Related Articles
Design and Evolution: Molecular Sleuthing Reveals Drug Selectivity
June 2015
Families in Gene Neighborhoods
June 2015
Ryanodine Receptor
April 2015
CCR5 and HIV Infection
January 2015
Drug Targets: Bile Acids in Motion
September 2014
Drug Targets: S1R's Ligands and Partners
September 2014
P2Y Receptors and Blood Clotting
September 2014
Bacterial CDI Toxins
June 2014
Glucagon Receptor
April 2014
Viroporins
March 2014
Microbial Pathogenesis: Targeting Drug Resistance in Mycobacterium tuberculosis
February 2014
Design and Discovery: Virtual Drug Screening
January 2014
Cancer Networks: IFI16-mediated p53 Activation
November 2013
G Proteins and Cancer
November 2013
Drug Discovery: Antidepressant Potential of 6-NQ SERT Inhibitors
October 2013
Drug Discovery: Finding Druggable Targets
October 2013
Drug Discovery: Identifying Dynamic Networks by CONTACT
October 2013
Drug Discovery: Modeling NET Interactions
October 2013
Membrane Proteome: GPCR Substrate Recognition and Functional Selectivity
August 2013
Infectious Diseases: Determining the Essential Structome
May 2013
NDM-1 and Antibiotics
May 2013
Microbial Pathogenesis: Computational Epitope Prediction
January 2013
Microbial Pathogenesis: Influenza Inhibitor Screen
January 2013
Microbial Pathogenesis: Measles Virus Attachment
January 2013
Cytochrome Oxidase
November 2012
Membrane Proteome: The ABCs of Transport
November 2012
Bacterial Phosphotransferase System
October 2012
Regulatory insights
September 2012
Solute Channels
September 2012
Pocket changes
July 2012
Receptor bias
July 2012
Anthrax Stealth Siderophores
June 2012
G Protein-Coupled Receptors
May 2012
Substrate specificity sleuths
April 2012
Reading out regioselectivity
December 2011
Superbugs and Antibiotic Resistance
December 2011
Terminal activation
December 2011
A change to resistance
November 2011
Docking and rolling
October 2011
Breaking down the defenses
September 2011
A2A Adenosine Receptor
May 2011
Cell wall recycler
May 2011
Subtly different
March 2011
CXCR4
January 2011
Subtle shifts
January 2011
ABA receptor diversity
November 2010
COX inhibition: Naproxen by proxy
November 2010
Zinc Transporter ZntB
July 2010
Peptidoglycan binding: Calcium-free killing
June 2010
Treating sleeping sickness
May 2010
Bacterial spore kinase
April 2010
Antibiotics and Ribosome Function
March 2010
Safer Alzheimer's drugs?
March 2010
Anthrax evasion tactics
September 2009
GPCR subunits: Separate but not equal
September 2009
Antibiotic target
August 2009
Salicylic Acid Binding Protein 2
August 2009
Lysostaphin
July 2009
Tackling influenza
June 2009
Bacterial Leucine Transporter, LeuT
May 2009
Anthrax stealth molecule
March 2009
Drug targets to aim for
February 2009
High-energy storage system
February 2009
Transporter mechanism in sight
February 2009
Scavenger Decapping Enzyme DcpS
November 2008
Blocking AmtB
September 2008

Research Themes Drug discovery

Transporter mechanism in sight

PSI-SGKB [doi:10.1038/fa_psisgkb.2009.4]
Featured Article - February 2009
Short description: Structural studies of the leucine transporter reveal details of the early steps in substrate transport across the membrane.Science 322, 1655-1661 (2008)

Secondary membrane transporters catalyse the movement of small molecules and ions across cellular membranes by linking substrate passage to transmembrane ion gradients. Although more than 200 different families of secondary transporters have been identified, with diverse protein sequences and a broad range of substrate and ion specificity, they probably work on common mechanistic principles.

These transporters lack a continuous pore for substrates to travel through, and so are thought to change conformation to allow substrates to access the transporter from each side of the membrane. The energy required is obtained from the potential energy stored in ion gradients across the membrane generated by primary, ATP-driven, transporters.

Several recently published structures of secondary transporters have shown that despite low protein sequence identity the core structure is very similar, and they may well share a similar mechanism. The first structure was of the leucine transporter LeuT, a prokaryotic member of the neurotransmitter sodium symporter (NSS) family.

Disruption of human NSS transporters is implicated in human disease, including depression, obsessive-compulsive disorder, epilepsy and autism. Their transport activity is inhibited by several classes of drugs, including tricyclic antidepressants, anticonvulsants and cocaine, but despite their importance, the molecular mechanism of substrate translocation and competitive inhibition has not been clear. Although earlier LeuT structures showed antidepressants binding in an extracellular pocket, or vestibule, they offered an explanation for noncompetitive inhibition only.

Singh et al. now present detailed X-ray crystallographic and functional studies on LeuT that identify a potential mechanism for substrate translocation. First, the authors identified tryptophan as a competitive inhibitor after screening a range of amino acids to find one that could displace leucine from the substrate site but could not be transported. They confirmed this observation using steady-state kinetic experiments.

Second, they examined the molecular basis of ligand specificity by co-crystallizing LeuT with six different amino acids. Five of these act as substrates, and when bound to the transporter, LeuT has the same outward-facing occluded conformation seen in the previously published structures, despite the variation in size of the substrates. As in the earlier structures, access from the extracellular side is blocked by just a few residues, and access from the intracellular side is blocked by almost 25 å of tightly packed protein.

But the tryptophan-bound complex has a different conformation. It is open to the outside of the cell, and it has a widened, solvent-accessible extracellular pocket where the substrate would normally bind. There are several differences between the open and the occluded state, including the rotation of three transmembrane helices 1b, 2a and 6a in the open state. In tryptophan's presence, the extracellular substrate pocket is unable to close, and therefore cannot form the occluded state necessary for transport.

Surprisingly, a second tryptophan-binding site is also observed. Two residues of the extracellular gate of LeuT —arginine 30 and aspartic acid 404 — form a low-affinity binding site for tryptophan. The authors suggest that this site is a temporary binding site for incoming amino acids before they move to the primary substrate site.

Singh et al. present a potential mechanism for LeuT in which incoming amino acids transiently bind to gating residues that are only accessible in the open-to-outside conformation. The substrate amino acid then moves to the primary binding site, and the gate residues then interact and close. This closure, or occlusion, seems to be required for the larger conformational change needed to produce the inward-facing conformation.

The structural similarities between unrelated secondary transporters make it possible that Singh et al. have uncovered principles of mechanism common to this group.

Maria Hodges

References

  1. S. K. Singh, C. L. Piscitelli, A. Yamashita & E. Gouaux A competitive inhibitor traps LeuT in an open-to-out conformation.
    Science 322, 1655-1661 (2008).

Structural Biology Knowledgebase ISSN: 1758-1338
Funded by a grant from the National Institute of General Medical Sciences of the National Institutes of Health