A Hybrid Structural Approach to Analyze Ligand Binding by the Serotonin Type 4 Receptor (5-HT4)

Padayatti, Pius S.; Wang, Liwen; Gupta, Sayan; Orban, Tivadar; Sun, Wenyu; Salom, David; Jordan, Steven R.; Palczewski, Krzysztof; Chance, Mark R.

doi:10.1074/mcp.m112.025536

Cited by 30 publications

(26 citation statements)

References 49 publications

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“…9), consistent with the known abilities of amphipols to stabilize MPs compared with detergents [219,220]. Ligand binding [221], conformational dynamics [222] and conformational changes [223,224] of MPs can also be studied using synchrotron radiolysis HRFP. This method also has the advantage of being able to study ordered water molecules in protein structures [225,226].…”

Section: Hydroxyl Radical Footprintingsupporting

confidence: 61%

Mass spectrometry-enabled structural biology of membrane proteins

Calabrese

Radford

2018

Methods

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a b s t r a c tThe last $25 years has seen mass spectrometry (MS) emerge as an integral method in the structural biology toolkit. In particular, MS has enabled the structural characterization of proteins and protein assemblies that have been intractable by other methods, especially those that are large, heterogeneous or transient, providing experimental evidence for their structural organization in support of, and in advance of, high resolution methods. The most recent frontier conquered in the field of MS-based structural biology has been the application of established methods for studying water soluble proteins to the more challenging targets of integral membrane proteins. The power of MS in obtaining structural information has been enabled by advances in instrumentation and the development of hyphenated mass spectrometry-based methods, such as ion mobility spectrometry-MS, chemical crosslinking-MS and other chemical labelling/footprinting-MS methods. In this review we detail the insights garnered into the structural biology of membrane proteins by applying such techniques. Application and refinement of these methods has yielded unprecedented insights in many areas, including membrane protein conformation, dynamics, lipid/ligand binding, and conformational perturbations due to ligand binding, which can be challenging to study using other methods.

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Section: Hydroxyl Radical Footprintingsupporting

confidence: 61%

Mass spectrometry-enabled structural biology of membrane proteins

Calabrese

Radford

2018

Methods

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“…The specific structural rearrangements that occur following activation by the tethered ligand and how cofactors and heterodimers influence the receptor allostery are remaining questions that will require alternative techniques such as structural mass spectrometry. 115,116 Targeting PARs for therapies A primary goal of structural studies is to gain insights on the mechanisms by which receptors operate at the molecular level and how they interact with potential therapeutics. These details can provide the tools required for rational drug design and high-throughput screening of chemical libraries that can be the basis for lead compound development.…”

Section: Protease-activated Receptors In Hemostasis 173mentioning

confidence: 99%

Protease-activated receptors in hemostasis

Nieman

2016

Blood

114

123

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Protease signaling in cells elicits multiple physiologically important responses via protease-activated receptors (PARs). There are 4 members of this family of G-protein–coupled receptors (PAR1-4). PARs are activated by proteolysis of the N terminus to reveal a tethered ligand. The rate-limiting step of PAR signaling is determined by the efficiency of proteolysis of the N terminus, which is regulated by allosteric binding sites, cofactors, membrane localization, and receptor dimerization. This ultimately controls the initiation of PAR signaling. In addition, these factors also control the cellular response by directing signaling toward G-protein or β-arrestin pathways. PAR1 signaling on endothelial cells is controlled by the activating protease and heterodimerization with PAR2 or PAR3. As a consequence, the genetic and epigenetic control of PARs and their cofactors in physiologic and pathophysiologic conditions have the potential to influence cellular behavior. Recent studies have uncovered polymorphisms that result in PAR4 sequence variants with altered reactivity that interact to influence platelet response. This further demonstrates how interactions within the plasma membrane can control the physiological output. Understanding the structural rearrangement following PAR activation and how PARs are allosterically controlled within the plasma membrane will determine how best to target this family of receptors therapeutically. The purpose of this article is to review how signaling from PARs is influenced by alternative cleavage sites and the physical interactions within the membrane. Going forward, it will be important to relate the altered signaling to the molecular arrangement of PARs in the cell membrane and to determine how these may be influenced genetically.

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“…Synchrotron based XF has been used to elucidate structural changes within several membrane proteins, including a G-protein coupled receptors 62,71,72 , potassium channel 80 , photosystem proteins 57,59,91 and ion transporters 84 during their activated or functional states. Availability of high resolution X-ray crystal structures of these proteins in the their native or ground state has allowed to expand the use XF approach to determine plausible role of transmembrane structural water for regulation of their functional states.…”

Section: Discussionmentioning

confidence: 99%

“…This allowed to XF-MS to be used to structurally validate a homology model for 5-HT4R receptor, for which no high resolution structure was available 72 , including predicted sites for internal water-side chain interaction, highlighting another important application of the XF-technology.…”

Section: Xf-ms In Study Of Gpcrsmentioning

confidence: 99%

Oxidative footprinting in the study of structure and function of membrane proteins: current state and perspectives

Bavro

Gupta

Ralston

2015

Biochemical Society Transactions

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Abstract:Membrane proteins, such as receptors, transporters and ion channels control the vast majority of cellular signaling and metabolite exchange processes and as such are increasingly becoming the key pharmacological targets. Difficulties of handling membrane proteins in high concentrations and requirements of membrane environments for their stabilization have made them difficult to study using traditional structural biology techniques, requiring the use of a hybrid, integrative approaches to study their dynamic properties and functional aspects in physiologically relevant conditions. In recent years significant progress has been made in the field of oxidative labeling techniques, and particularly X-radiolytic labeling in combination of mass spectroscopy (XF-MS) allowing these approaches to mature and provide valuable insights into structure and function of proteins, including membrane targets, which is difficult to obtain by other techniques, complementing available structural data. XF-MS has demonstrated unique capability for identification of structural waters and conformational changes in proteins at both high degree of spatial and temporal resolution. Here we provide a perspective of the place of XF-MS amongst other structural biology methods and showcase some of latest developments in usage of XF-MS in solving water meditated transmembrane signaling, ion transport, ion gating as well as ligand induced allosteric conformation changes in these membrane protein complexes.

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A Hybrid Structural Approach to Analyze Ligand Binding by the Serotonin Type 4 Receptor (5-HT4)

Cited by 30 publications

References 49 publications

Mass spectrometry-enabled structural biology of membrane proteins

Mass spectrometry-enabled structural biology of membrane proteins

Protease-activated receptors in hemostasis

Oxidative footprinting in the study of structure and function of membrane proteins: current state and perspectives

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