Infrared Multiphoton Dissociation Enables Top-Down Characterization of Membrane Protein Complexes and G Protein-Coupled Receptors.

Membrane proteins are challenging to analyze by native mass spectrometry (MS) as their hydrophobic nature typically requires stabilization in detergent micelles that are removed prior to analysis via collisional activation. There is however a practical limit to the amount of energy which can be applied, which often precludes subsequent characterization by top-down MS. To overcome this barrier, we have applied a modified Orbitrap Eclipse Tribrid mass spectrometer coupled to an infrared laser within a high-pressure linear ion trap. We show how tuning the intensity and time of incident photons enables liberation of membrane proteins from detergent micelles. Specifically, we relate the ease of micelle removal to the infrared absorption of detergents in both condensed and gas phases. Top-down MS via infrared multiphoton dissociation (IRMPD), results in good sequence coverage enabling unambiguous identification of membrane proteins and their complexes. By contrasting and comparing the fragmentation patterns of the ammonia channel with two class A GPCRs, we identify successive cleavage of adjacent amino acids within transmembrane domains. Using gas-phase molecular dynamics simulations, we show that areas prone to fragmentation maintain aspects of protein structure at increasing temperatures. Altogether, we propose a rationale to explain why and where in the protein fragment ions are generated.

Infrared Multiphoton Dissociation Enables Top-Down Characterization of Membrane Protein Complexes and G Protein-Coupled Receptors.

Membrane proteins are challenging to analyze by native mass spectrometry (MS) as their hydrophobic nature typically requires stabilization in detergent micelles that are removed prior to analysis via collisional activation. There is however a practical limit to the amount of energy which can be applied, which often precludes subsequent characterization by top-down MS. To overcome this barrier, we have applied a modified Orbitrap Eclipse Tribrid mass spectrometer coupled to an infrared laser within a high-pressure linear ion trap. We show how tuning the intensity and time of incident photons enables liberation of membrane proteins from detergent micelles. Specifically, we relate the ease of micelle removal to the infrared absorption of detergents in both condensed and gas phases. Top-down MS via infrared multiphoton dissociation (IRMPD), results in good sequence coverage enabling unambiguous identification of membrane proteins and their complexes. By contrasting and comparing the fragmentation patterns of the ammonia channel with two class A GPCRs, we identify successive cleavage of adjacent amino acids within transmembrane domains. Using gas-phase molecular dynamics simulations, we show that areas prone to fragmentation maintain aspects of protein structure at increasing temperatures. Altogether, we propose a rationale to explain why and where in the protein fragment ions are generated.

Mass spectrometry captures biased signalling and allosteric modulation of a G-protein-coupled receptor.

G-protein-coupled receptors signal through cognate G proteins. Despite the widespread importance of these receptors, their regulatory mechanisms for G-protein selectivity are not fully understood. Here we present a native mass spectrometry-based approach to interrogate both biased signalling and allosteric modulation of the ß1-adrenergic receptor in response to various ligands. By simultaneously capturing the effects of ligand binding and receptor coupling to different G proteins, we probed the relative importance of specific interactions with the receptor through systematic changes in 14 ligands, including isoprenaline derivatives, full and partial agonists, and antagonists. We observed enhanced dynamics of the intracellular loop 3 in the presence of isoprenaline, which is capable of acting as a biased agonist. We also show here that endogenous zinc ions augment the binding in receptor-Gs complexes and propose a zinc ion-binding hotspot at the TM5/TM6 intracellular interface of the receptor-Gs complex. Further interrogation led us to propose a mechanism in which zinc ions facilitate a structural transition of the intermediate complex towards the stable state.

Disruption of the Unique ABCG-Family NBD:NBD Interface Impacts Both Drug Transport and ATP Hydrolysis.

ABCG2 is one of a triumvirate of human multidrug ATP binding cassette (ABC) transporters that are implicated in the defense of cells and tissues against cytotoxic chemicals, but these transporters can also confer chemotherapy resistance states in oncology. Understanding the mechanism of ABCG2 is thus imperative if we are to be able to counter its deleterious activity. The structure of ABCG2 and its related family members (ABCG5/G8) demonstrated that there were two interfaces between the nucleotide binding domains (NBD). In addition to the canonical ATP "sandwich-dimer" interface, there was a second contact region between residues at the C-terminus of the NBD. We investigated this second interface by making mutations to a series of residues that are in close interaction with the opposite NBD. Mutated ABCG2 isoforms were expressed in human embryonic kidney (HEK) 293T cells and analysed for targeting to the membrane, drug transport, and ATPase activity. Mutations to this second interface had a number of effects on ABCG2, including altered drug specificity, altered drug transport, and, in two mutants, a loss of ATPase activity. The results demonstrate that this region is particularly sensitive to mutation and can impact not only direct, local NBD events (i.e., ATP hydrolysis) but also the allosteric communication to the transmembrane domains and drug transport.

ABCG2: does resolving its structure elucidate the mechanism?

ABCG2 is one of a few human membrane transporters which display the amazing ability to transport multiple different chemicals out of cells. These multidrug pumps, which have orthologues in all organisms, are important in humans in the context of drug pharmacokinetics, especially with respect to resistance to chemotherapy. In 2016, we presented a mini-review on ABCG2 which identified many areas of exciting research progress as well as many areas of frustrating ignorance. Just 2 years on the field has advanced, particularly with respect to structural biology as the cryo-electron microscopy revolution has brought us new insights into the structure and mechanism of ABCG2. In this update, we evaluate the degree to which new data have enhanced our understanding of the structure and mechanism of ABCG2 and whether we are now in a position to translate some of these findings into inhibitor design and development.

Polymorphisms of the Multidrug Pump ABCG2: A Systematic Review of Their Effect on Protein Expression, Function, and Drug Pharmacokinetics.

The widespread expression and polyspecificity of the multidrug ABCG2 efflux transporter make it an important determinant of the pharmacokinetics of a variety of substrate drugs. Null ABCG2 expression has been linked to the Junior blood group. Polymorphisms affecting the expression or function of ABCG2 may have clinically important roles in drug disposition and efficacy. The most well-studied single nucleotide polymorphism (SNP), Q141K (421C>A), is shown to decrease ABCG2 expression and activity, resulting in increased total drug exposure and decreased resistance to various substrates. The effect of Q141K can be rationalized by inspection of the ABCG2 structure, and the effects of this SNP on protein processing may make it a target for pharmacological intervention. The V12M SNP (34G>A) appears to improve outcomes in cancer patients treated with tyrosine kinase inhibitors, but the reasons for this are yet to be established, and this residue's role in the mechanism of the protein is unexplored by current biochemical and structural approaches. Research into the less-common polymorphisms is confined to in vitro studies, with several polymorphisms shown to decrease resistance to anticancer agents such as SN-38 and mitoxantrone. In this review, we present a systematic analysis of the effects of ABCG2 polymorphisms on ABCG2 function and drug pharmacokinetics. Where possible, we use recent structural advances to present a molecular interpretation of the effects of SNPs and indicate where we need further in vitro experiments to fully resolve how SNPs impact ABCG2 function.

Residues contributing to drug transport by ABCG2 are localised to multiple drug-binding pockets.

Multidrug binding and transport by the ATP-binding cassette transporter ABCG2 is a factor in the clinical resistance to chemotherapy in leukaemia, and a contributory factor to the pharmacokinetic profiles of many other prescribed drugs. Despite its importance, the structural basis of multidrug transport, i.e. the ability to transport multiple distinct chemicals, has remained elusive. Previous research has shown that at least two residues positioned towards the cytoplasmic end of transmembrane helix 3 (TM3) of the transporter play a role in drug transport. We hypothesised that other residues, either in the longitudinal span of TM3, or a perpendicular slice through the intracellular end of other TM helices would also contribute to drug binding and transport by ABCG2. Single-point mutant isoforms of ABCG2 were made at ∼30 positions and were analysed for effects on protein expression, localisation (western blotting, confocal microscopy) and function (flow cytometry) in a mammalian stable cell line expression system. Our data were interpreted in terms of recent structural data on the ABCG protein subfamily and enabled us to propose a surface-binding site for the drug mitoxantrone (MX) as well as a second, buried site for the same drug. Further mutational analysis of residues that spatially separate these two sites prompts us to suggest a molecular and structural pathway for MX transport by ABCG2.