Nathan K. Karpowich

LeuT-Desipramine Structure Reveals How Antidepressants Block Neurotransmitter Reuptake

Tricyclic antidepressants exert their pharmacological effect—inhibiting the reuptake of serotonin, norepinephrine, and dopamine—by directly blocking neurotransmitter transporters (SERT, NET, and DAT, respectively) in the presynaptic membrane. The drug-binding site and the mechanism of this inhibition are poorly understood. We determined the crystal structure at 2.9 angstroms of the bacterial leucine transporter (LeuT), a homolog of SERT, NET, and DAT, in complex with leucine and the antidepressant desipramine. Desipramine binds at the inner end of the extracellular cavity of the transporter and is held in place by a hairpin loop and by a salt bridge. This binding site is separated from the leucine-binding site by the extracellular gate of the transporter. By directly locking the gate, desipramine prevents conformational changes and blocks substrate transport. Mutagenesis experiments on human SERT and DAT indicate that both the desipramine-binding site and its inhibition mechanism are probably conserved in the human neurotransmitter transporters.

Physiological Response to Membrane Protein Overexpression in E. coli

Overexpression represents a principal bottleneck in structural and functional studies of integral membrane proteins (IMPs). Although E. coli remains the leading organism for convenient and economical protein overexpression, many IMPs exhibit toxicity on induction in this host and give low yields of properly folded protein. Different mechanisms related to membrane biogenesis and IMP folding have been proposed to contribute to these problems, but there is limited understanding of the physical and physiological constraints on IMP overexpression and folding in vivo. Therefore, we used a variety of genetic, genomic, and microscopy techniques to characterize the physiological responses of Escherichia coli MG1655 cells to overexpression of a set of soluble proteins and IMPs, including constructs exhibiting different levels of toxicity and producing different levels of properly folded versus misfolded product on induction. Genetic marker studies coupled with transcriptomic results indicate only minor perturbations in many of the physiological systems implicated in previous studies of IMP biogenesis. Overexpression of either IMPs or soluble proteins tends to block execution of the standard stationary-phase transcriptional program, although these effects are consistently stronger for the IMPs included in our study. However, these perturbations are not an impediment to successful protein overexpression. We present evidence that, at least for the target proteins included in our study, there is no inherent obstacle to IMP overexpression in E. coli at moderate levels suitable for structural studies and that the biochemical and conformational properties of the proteins themselves are the major obstacles to success. Toxicity associated with target protein activity produces selective pressure leading to preferential growth of cells harboring expression-reducing and inactivating mutations, which can produce chemical heterogeneity in the target protein population, potentially contributing to the difficulties encountered in IMP crystallization.

Assembly and mechanism of a group II ECF transporter

Energy-coupling factor (ECF) transporters are a recently discovered family of primary active transporters for micronutrients and vitamins, such as biotin, thiamine, and riboflavin. Found exclusively in archaea and bacteria, including the human pathogens Listeria, Streptococcus, and Staphylococcus, ECF transporters may be the only means of vitamin acquisition in these organisms. The subunit composition of ECF transporters is similar to that of ATP binding cassette (ABC) importers, whereby both systems share two homologous ATPase subunits (A and A′), a high affinity substrate-binding subunit (S), and a transmembrane coupling subunit (T). However, the S subunit of ECF transporters is an integral membrane protein, and the transmembrane coupling subunits do not share an obvious sequence homology between the two transporter families. Moreover, the subunit stoichiometry of ECF transporters is controversial, and the detailed molecular interactions between subunits and the conformational changes during substrate translocation are unknown. We have characterized the ECF transporters from Thermotoga maritima and Streptococcus thermophilus. Our data suggests a subunit stoichiometry of 2S:2T:1A:1A′ and that S subunits for different substrates can be incorporated into the same transporter complex simultaneously. In the first crystal structure of the A–A′ heterodimer, each subunit contains a novel motif called the Q-helix that plays a key role in subunit coupling with the T subunits. Taken together, these findings suggest a mechanism for coupling ATP binding and hydrolysis to transmembrane transport by ECF transporters.

Substrate and drug binding sites in LeuT

LeuT is a member of the neurotransmitter/sodium symporter family, which includes the neuronal transporters for serotonin, norepinephrine, and dopamine. The original crystal structure of LeuT shows a primary leucine-binding site at the center of the protein. LeuT is inhibited by different classes of antidepressants that act as potent inhibitors of the serotonin transporter. The newly determined crystal structures of LeuT-antidepressant complexes provide opportunities to probe drug binding in the serotonin transporter, of which the exact position remains controversial. Structure of a LeuT-tryptophan complex shows an overlapping binding site with the primary substrate site. A secondary substrate binding site was recently identified, where the binding of a leucine triggers the cytoplasmic release of the primary substrate. This two binding site model presents opportunities for a better understanding of drug binding and the mechanism of inhibition for mammalian transporters.

Simple screening method for improving membrane protein thermostability

Biochemical and biophysical analysis on integral membrane proteins often requires monodisperse and stable protein samples. Here we describe a method to characterize protein thermostability by measuring its melting temperature in detergent using analytical size-exclusion chromatography. This quantitative method can be used to screen for compounds and conditions that stabilize the protein. With this technique we were able to assess and improve the thermostability of several membrane proteins. These conditions were in turn used to assist purification, to identify protein ligand and to improve crystal quality.

Symmetric Transporters for Asymmetric Transport

The crystal structure of a membrane transporter protein sheds light on the molecular mechanism by which glucose is absorbed by the intestine and the kidneys.

ATP binding drives substrate capture in an ECF transporter by a release-and-catch mechanism

ECF transporters are a family of active transporters for vitamins. They are composed of four subunits: a membrane-embedded substrate-binding subunit (EcfS), a transmembrane coupling subunit (EcfT) and two ATP-binding-cassette ATPases (EcfA and EcfA′). We have investigated the mechanism of the ECF transporter for riboflavin from the pathogen Listeria monocytogenes, LmECF–RibU. Using structural and biochemical approaches, we found that ATP binding to the EcfAA′ ATPases drives a conformational change that dissociates the S subunit from the EcfAA′T ECF module. Upon release from the ECF module, the RibU S subunit then binds the riboflavin transport substrate. We also find that S subunits for distinct substrates compete for the ATP-bound state of the ECF module. Our results explain how ECF transporters capture the transport substrate and reproduce the in vivo observations on S-subunit competition for which the family was named.

Rapid Bioinformatic Identification of Thermostabilizing Mutations

Ex vivo stability is a valuable protein characteristic but is laborious to improve experimentally. In addition to biopharmaceutical and industrial applications, stable protein is important for biochemical and structural studies. Taking advantage of the large number of available genomic sequences and growth temperature data, we present two bioinformatic methods to identify a limited set of amino acids or positions that likely underlie thermostability. Because these methods allow thousands of homologs to be examined in silico, they have the advantage of providing both speed and statistical power. Using these methods, we introduced, via mutation, amino acids from thermoadapted homologs into an exemplar mesophilic membrane protein, and demonstrated significantly increased thermostability while preserving protein activity.

Biophysics: Transporter in the spotlight

Membrane transporter proteins switch between conformational states to move substrates across membranes. The transition between these states can now be studied using single-molecule experiments.