G protein-coupled receptors (GPCRs) are a large family of membrane proteins that enable cells to respond to hormones, neurotransmitters, and other external signals. Around one-third of all approved drugs act on GPCRs, making them one of the most important target classes in modern pharmacology. Although the overall architecture of GPCRs is well understood from numerous X-ray and cryo-electron microscopy structures, certain regions remain poorly characterized – particularly the flexible, intrinsically disordered segments that move too rapidly to be captured by conventional structural methods.
One such region is the N-terminal domain of the neuropeptide Y2 receptor (Y2R), a GPCR involved in regulating appetite, mood, and other physiological processes. Previous high-resolution structures of Y2R revealed the receptor’s transmembrane core and its interactions with peptide ligands, but the N-terminal region was unresolved.
In a study published in Nature Communications, researchers from Leipzig University and Martin Luther University Halle-Wittenberg used an integrative strategy combining photo–cross-linking mass spectrometry (XL-MS), molecular modeling, and functional assays to characterize these elusive interactions. Their approach enabled them to capture transient contacts between the Y2R N-terminus and its ligand, neuropeptide Y, on a timescale of micro- to milliseconds – which is something that has not been achievable with traditional methods.
We spoke with Andrea Sinz, Professor of Pharmaceutical Chemistry and Bioanalytics at Martin Luther University Halle-Wittenberg, who co-led the study, about the experimental challenges involved and what the findings could mean for drug discovery.
How did you study the flexible N-terminal segment of the Y2 receptor and its interaction with neuropeptide Y?
We used an integrative approach that combines various methods: chemical cross-linking mass spectrometry (XL-MS), computational modeling and molecular dynamics (MD) simulations, and a functional read-out by mutagenesis studies and bioluminescence resonance energy transfer (nanoBRET) assays.
These methods all served specific purposes. XL-MS using millisecond photo-reaction chemistry provided distinct interaction points between the flexible N-terminus of the Y2 receptor and its ligand neuropeptide Y. Computational modeling with Rosetta and MD simulations delivered further insights into the structural flexibility of the Y2 receptor and its dynamic interactions, and mutagenesis and nanoBRET assays served to determine the influence of specific regions on the activation of the receptor.
What motivated your team to take on this challenging research?
GPCRs, such as the Y2 receptor, are extremely important for signal transduction. As a result, they represent one of the most important classes of drug targets, with around 40% of all approved drugs acting on GPCRs. While transmembrane regions of GPCRs are well studied, the highly flexible disordered regions, such as the N-terminus of the Y2 receptor, remain largely underexplored. The major challenge with studying intrinsically disordered regions is that they lack a stable three-dimensional structure. Instead, they exist as ensembles of rapidly interconverting conformations, resembling a tangled “hairball.” There are only a few techniques in structural biology that can solve these conformational ensembles, with XL-MS being one of them. Combining XL-MS with computational methods and a functional read-out is extremely powerful in delineating the high dynamics of these intrinsically disordered regions, such as the N-terminus of the Y2 receptor.
What was the biggest analytical challenge your team faced – and how did you overcome it?
To study the N-terminus of the Y2 receptor, we first had to develop a specifically tailored XL-MS method. It took more than four years of optimization before we began to see meaningful results for this exceptionally challenging protein system.
First of all, we needed a very fast cross-linking reaction to capture the highly dynamic interaction between the Y2 receptor and its ligand, neuropeptide Y. To achieve this, we incorporated diazirine groups in the form of the artificial amino acid photo-leucine at specific positions within neuropeptide Y. When diazirines are irradiated with long-wavelength UV light (365 nm), they react in the micro- to millisecond range with other amino acids in spatial proximity.
The major challenge we faced concerned the low reaction yield of diazirines. We developed a dedicated mass spectrometry approach using highly sensitive instrumentation. We used instruments that combine ion mobility (IM) with mass spectrometry (MS) to detect and correctly identify the low-abundant cross-linked species. By combining this rapid cross-linking reaction with a highly sensitive, high-speed IM-MS method, we were able to pinpoint the interaction sites between the Y2 receptor and neuropeptide Y and to decipher the conformational ensembles underlying this interaction.
Were there any results in particular that surprised you?
We were intrigued to find that two clusters of acidic amino acids in the Y2 receptor interact with the central helix of the ligand neuropeptide Y – interactions that, until now, had eluded traditional structural biology approaches. These interactions contribute to the low-affinity binding site of the Y2 receptor and prolong the residence time of neuropeptide Y. Recruitment of arrestin-3 to the receptor depends on this interaction, whereas G-protein activation appears independent of the receptor’s intrinsically disordered, highly flexible N-terminus.
What are the next steps for your team?
Developing drugs against intrinsically disordered regions is an extremely challenging task as the conformational ensembles do not present a stable three-dimensional structure. Drugs can be raised in case a stable interaction interface is generated. A detailed structural knowledge of the interaction interface will allow us to decipher which conformational states are populated upon ligand binding. Based on this knowledge, specific small-molecule drugs could be designed that bind preferably to only one (or a few) conformational state(s).
In my group, we are exploring the general use of XL-MS for studying intrinsically disordered regions in proteins. As I mentioned before, there are only a few techniques in structural biology that can handle these highly flexible and dynamic systems. We are currently extending our work to investigating the Y1 receptor – a close relative to the Y2 receptor. Other intrinsically disordered proteins we are studying include the tumor suppressor p53 (cancer) and alpha-synuclein (Parkinson’s disease).
By studying these intrinsically disordered proteins, we hope to gain insights into the molecular mechanisms that underlie pathogenic states such as cancer or Parkinson’s disease.
Andrea Sinz is a Full Professor of Pharmaceutical Chemistry and Bioanalytics at Martin Luther University Halle-Wittenberg, Germany
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