Membrane Proteins: New Approaches to Probes, Technologies, and Drug Design, Part II.

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Membrane Proteins: New Approaches to Probes, Technologies, and Drug Design

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Functional reconstitution of human equilibrative nucleoside transporter-1 into styrene maleic acid co-polymer lipid particles

The human equilibrative nucleoside transporter-1 (hENT1) is important for the entry of anti-cancer and antiviral nucleoside analog therapeutics into the cell, and thus for their efficacy. Understanding of hENT1 structure -function relationship could assist with development of nucleoside analogs with better cellular uptake properties. However, structural and biophysical studies of hENT1 remain challenging as the hydrophobic nature of the protein leads to complete aggregation upon detergent-based membrane isolation. Here we report detergent-free reconstitution of the hENT1 transporter into styrene maleic acid co-polymer lipid particles (SMALPs) that form a native lipid disc. SMALP-purified hENT1, expressed in Sf9 insect cells binds a variety of ligands with a similar affinity as the protein in native membrane, and exhibits increased thermal stability compared to detergent-solubilized hENT1. hENT1-SMALPs purified using FLAG affinity M2 resin yielded similar to 0.4 mg of active and homogenous protein per liter of culture as demonstrated by ligand binding, size-exclusion chromatography and SDS-PAGE analyses. Electrospray ionization mass spectrometry (ESI-MS) analysis showed that each hENT1 lipid disc contains 16 phosphatidylcholine (PC) and 2 phosphatidylethanolamine (PE) lipid molecules. Polyunsaturated lipids are specifically excluded from the hENT1 lipid discs, possibly reflecting a functional requirement for a dynamic lipid environment. Our work demonstrates that human nucleoside transporters can be extracted and purified without removal from their native lipid environment, opening up a wide range of possibilities for their biophysical and structural studies. (C) 2017 Elsevier B.V. All rights reserved.Peer reviewe

IMPROvER : the Integral Membrane Protein Stability Selector

Identifying stabilising variants of membrane protein targets is often required for structure determination. Our new computational pipeline, the Integral Membrane Protein Stability Selector (IMPROvER) provides a rational approach to variant selection by employing three independent approaches: deep-sequence, model-based and data-driven. In silico tests using known stability data, and in vitro tests using three membrane protein targets with 7, 11 and 16 transmembrane helices provided measures of success. In vitro, individual approaches alone all identified stabilising variants at a rate better than expected by random selection. Low numbers of overlapping predictions between approaches meant a greater success rate was achieved (fourfold better than random) when approaches were combined and selections restricted to the highest ranked sites. The mix of information IMPROvER uses can be extracted for any helical membrane protein. We have developed the first general-purpose tool for selecting stabilising variants of alpha -helical membrane proteins, increasing efficiency and reducing workload. IMPROvER can be accessed at http://improver.ddns.net/IMPROvER/.Peer reviewe

Nanodisc-Tm: Rapid functional assessment of nanodisc reconstituted membrane proteins by CPM assay

Membrane proteins are generally unstable in detergents. Therefore, biochemical and biophysical studies of membrane proteins in lipidic environments provides a near native-like environment suitable for membrane proteins. However, manipulation of proteins embedded in lipid bilayer has remained difficult. Methods such as nanodiscs and lipid cubic phase have been developed for easy manipulation of membrane proteins and have yielded significant insights into membrane proteins. Traditionally functional reconstitution of receptors in nanodiscs has been studied with radioligands. We present a simple and faster method for studying the functionality of reconstituted membrane proteins for routine characterization of protein batches after initial optimization of suitable conditions using radioligands. The benefits of the method are •Faster and generic method to assess functional reconstitution of membrane proteins. •Adaptable in high throughput format (≥96 well format). •Stability measurement in near-native lipid environment and lipid dependent melting temperatures

Chapter 1 - The structure of the adenosine receptors implications for drug discovery

Extracellular adenosine mediates most of its physiological effects via an interaction with four G protein-coupled receptors (GPCRs), the adenosine receptors (ARs). These ARs are important pharmacological targets in the treatment of a wide variety of diseases from central nervous system disorders to ischemic injury. As for other GPCRs, drug development for the ARs has been hampered by the lack of structural data for this class of membrane proteins. However, in the past 3 years, this situation has changed with the elucidation of structures for the turkey ?(1)-adrenoceptor, the human ?(2)-adrenoceptor, squid rhodopsin, the activated form of bovine (rhod)opsin, the human adenosine A(2A) receptor, and most recently the CXCR4 chemokine receptor. In this review, the structural features of the human adenosine A(2A) receptor will be discussed with a particular focus on the ligand binding site. Further, the implications of this structural information for AR ligand selectivity, drug screening, homology modeling, and virtual ligand screening will be discussed

Bridge over troubled proline: assignment of intrinsically disordered proteins using (HCA)CON(CAN)H and (HCA)N(CA)CO(N)H experiments concomitantly with HNCO and i(HCA)CO(CA)NH

NMR spectroscopy is by far the most versatile and information rich technique to study intrinsically disordered proteins (IDPs). While NMR is able to offer residue level information on structure and dynamics, assignment of chemical shift resonances in IDPs is not a straightforward process. Consequently, numerous pulse sequences and assignment protocols have been developed during past several years, targeted especially for the assignment of IDPs, including experiments that employ HN, Hα or 13C detection combined with two to six indirectly detected dimensions. Here we propose two new HN-detection based pulse sequences, (HCA)CON(CAN)H and (HCA)N(CA)CO(N)H, that provide correlations with 1HN(i - 1), 13C'(i - 1) and 15N(i), and 1HN(i + 1), 13C'(i) and 15N(i) frequencies, respectively. Most importantly, they offer sequential links across the proline bridges and enable filling the single proline gaps during the assignment. We show that the novel experiments can efficiently complement the information available from existing HNCO and intraresidual i(HCA)CO(CA)NH pulse sequences and their concomitant usage enabled >95 % assignment of backbone resonances in cytoplasmic tail of adenosine receptor A2A in comparison to 73 % complete assignment using the HNCO/i(HCA)CO(CA)NH data alone

Thermodynamics and kinetics of inhibitor binding to human equilibrative nucleoside transporter subtype-1

Many nucleoside transport inhibitors are in clinical use as anti-cancer, vasodilator and cardioprotective drugs. However, little is known about the binding energetics of these inhibitors to nucleoside transporters (NTs) due to their low endogenous expression levels and difficulties in the biophysical characterization of purified protein with ligands. Here, we present kinetics and thermodynamic analyses of inhibitor binding to the human equilibrative nucleoside transporter-1 (hENT1), also known as SLC29A1. Using a radioligand binding assay, we obtained equilibrium binding and kinetic rate constants of well-known NT inhibitors--[(3)H]nitrobenzylmercaptopurine ribonucleoside ([(3)H]NBMPR), dilazep, and dipyridamole--and the native permeant, adenosine, to hENT1. We observed that the equilibrium binding affinities for all inhibitors decreased whereas, the kinetic rate constants increased with increasing temperature. Furthermore, we found that binding is enthalpy driven and thus, an exothermic reaction, implying that the transporter does not discriminate between its inhibitors and substrates thermodynamically. This predominantly enthalpy-driven binding by four chemically distinct ligands suggests that the transporter may not tolerate diversity in the type of interactions that lead to high affinity binding. Consistent with this, the measured activation energy of [(3)H]NBMPR association was relatively large (20 kcal mol(-1)) suggesting a conformational change upon inhibitor binding. For all three inhibitors the enthalpy (ΔH°) and entropy (ΔS°) contributions to the reaction energetics were determined by van't Hoff analysis to be roughly similar (25-75% ΔG°). Gains in enthalpy with increasing polar surface area of inhibitors suggest that the binding is favored by electrostatic or polar interactions between the ligands and the transporter