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Repeat-swap homology modeling of secondary active transporters: updated protocol and prediction of elevator-type mechanisms.

ABSTRACT: Secondary active transporters are critical for neurotransmitter clearance and recycling during synaptic transmission and uptake of nutrients. These proteins mediate the movement of solutes against their concentration gradients, by using the energy released in the movement of ions down pre-existing concentration gradients. To achieve this, transporters conform to the so-called alternating-access hypothesis, whereby the protein adopts at least two conformations in which the substrate binding sites are exposed to one or other side of the membrane, but not both simultaneously. Structures of a bacterial homolog of neuronal glutamate transporters, GltPh, in several different conformational states have revealed that the protein structure is asymmetric in the outward- and inward-open states, and that the conformational change connecting them involves a elevator-like movement of a substrate binding domain across the membrane. The structural asymmetry is created by inverted-topology repeats, i.e., structural repeats with similar overall folds whose transmembrane topologies are related to each other by two-fold pseudo-symmetry around an axis parallel to the membrane plane. Inverted repeats have been found in around three-quarters of secondary transporter folds. Moreover, the (a)symmetry of these systems has been successfully used as a bioinformatic tool, called "repeat-swap modeling" to predict structural models of a transporter in one conformation using the known structure of the transporter in the complementary conformation as a template. Here, we describe an updated repeat-swap homology modeling protocol, and calibrate the accuracy of the method using GltPh, for which both inward- and outward-facing conformations are known. We then apply this repeat-swap homology modeling procedure to a concentrative nucleoside transporter, VcCNT, which has a three-dimensional arrangement related to that of GltPh. The repeat-swapped model of VcCNT predicts that nucleoside transport also occurs via an elevator-like mechanism.

SUBMITTER: Vergara-Jaque A

PROVIDER: S-EPMC4560100 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Repeat-swap homology modeling of secondary active transporters: updated protocol and prediction of elevator-type mechanisms.

Vergara-Jaque Ariela A Fenollar-Ferrer Cristina C Kaufmann Desirée D Forrest Lucy R LR

Frontiers in pharmacology 20150904

Secondary active transporters are critical for neurotransmitter clearance and recycling during synaptic transmission and uptake of nutrients. These proteins mediate the movement of solutes against their concentration gradients, by using the energy released in the movement of ions down pre-existing concentration gradients. To achieve this, transporters conform to the so-called alternating-access hypothesis, whereby the protein adopts at least two conformations in which the substrate binding sites ...[more]

PMID: 26388773

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Project description:The nucleoside transporter family is an emerging target for cancer, viral and cardiovascular diseases. Due to the difficulty in the expression, isolation and crystallization of membrane proteins, there is a lack of structural information on any of the mammalian and for that matter the human proteins. Thus the objective of this study was to build homology models for the three cloned concentrative nucleoside transporters hCNT1, hCNT2 and hCNT3 and validate them for screening towards the discovery of much needed inhibitors and probes. The recently reported crystal structure of the Vibrio cholerae concentrative nucleoside transporter (vcCNT), has satisfactory similarity to the human CNT orthologues and was thus used as a template to build homology models of all three hCNTs. The Schrödinger modeling suite was used for the exercise. External validation of the homology models was carried out by docking a set of recently reported known hCNT1 nucleoside class inhibitors at the putative binding site using induced fit docking (IDF) methodology with the Glide docking program. Then, the hCNT1 homology model was subsequently used to conduct a virtual screening of a 360,000 compound library, and 172 compounds were obtained and biologically evaluated for hCNT 1, 2 and 3 inhibitory potency and selectivity. Good quality homology models were obtained for all three hCNTs as indicated by interrogation for various structural parameters and also external validated by docking of known inhibitors. The IDF docking results showed good correlations between IDF scores and inhibitory activities; particularly for hCNT1. From the top 0.1% of compounds ranked by virtual screening with the hCNT1 homology model, 172 compounds selected and tested for against hCNT1, hCNT2 and hCNT3, yielded 14 new inhibitors (hits) of (i.e., 8% success rate). The most active compound exhibited an IC50 of 9.05 μM, which shows a greater than 25-fold higher potency than phlorizin the standard CNT inhibitor (IC50 of 250 μM). We successfully undertook homology modeling and validation for all human concentrative nucleoside transporters (hCNT 1, 2 and 3). The proof-of-concept that these models are promising for virtual screening to identify potent and selective inhibitors was also obtained using the hCNT1 model. Thus we identified a novel potent hCNT1 inhibitor that is more potent and more selective than the standard inhibitor phlorizin. The other hCNT1 hits also mostly exhibited selectivity. These homology models should be useful for virtual screening to identify novel hCNT inhibitors, as well as for optimization of hCNT inhibitors.

Dataset Information

Repeat-swap homology modeling of secondary active transporters: updated protocol and prediction of elevator-type mechanisms.

Publications

Repeat-swap homology modeling of secondary active transporters: updated protocol and prediction of elevator-type mechanisms.

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