2021 Nobel Prize winner paper: How does the pain receptor TRPV1 perceive various natural stimuli?

Structural biology helps to understand the mechanism of the occurrence and development of macroscopic phenomena at the molecular level, especially due to the increasing maturity and wide application of single-particle cryo-electron microscopy

With the excellent performance of AlphaFold2 in protein structure prediction, the research direction and future of structural biology have also been heatedly discussed

It is undeniable that protein prediction will promote scientific research from other existing and potential perspectives.

On September 7, 2021, the Yifan Cheng laboratory of the University of California, San Francisco and the David Julius laboratory published a research paper entitled "Structural snapshots of TRPV1 reveal mechanism of polymodal functionality" in Cell magazine

In this study, under the conditions of applying different natural stimuli and simulating different physiological environments, with the help of single-particle cryo-electron microscopy, a series of intermediate state conformations and activation mechanisms of the capsaicin receptor TRPV1 were analyzed

The study's co-corresponding author, Professor David Julius, has just won this year's Nobel Prize in Physiology or Medicine for his research on the capsaicin receptor TRPV1

TPRV1 is a non-specific transport tetrameric ion channel, which is closely related to pain and inflammation

The natural stimulants of TPRV1 include capsaicin, the active ingredient of pepper, polypeptide toxin (DkTx) produced by poisonous spiders, and gum lipotoxin (RTX) isolated from Euphorbia

Due to the importance of signal transmission mediated by TPRV1, it has become a potential analgesic target with clinical significance
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However, due to the complexity of its signal integration, the development and application of analgesics for TPRV1 still have side effects such as impairing thermoregulation
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Therefore, under different stimuli, it is necessary to explore the diversity, uniqueness and commonalities of TRPV1 activation pathways in order to provide deeper and more targeted guidance for the development of TPRV1 targeted drugs
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The author recombined the protein TPRV1 into a lipid environment, expressed and purified the polypeptide toxin DkTx in vitro, and prepared frozen samples of the two complexes
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By optimizing the sample preparation conditions and data processing parameters, a series of intermediate structures of pre-combination, single-combination and double-combination were successfully identified when the channel was closed and opened
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These gradual conformational changes, especially the outward shift of the S1-S4 domain from the initial conformation to the full conformation, strongly explain how the peptide agonist DkTx binds to the outside of the cell and triggers the low gate opening of TPRV1
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Unlike the DkTx binding site, the vanillic acid agonist RTX binds to the region where the lower door of TPRV1 is located, thereby directly regulating the opening state of the lower door
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In the absence of ligand binding, the binding site of the TPRV1 vanillic acid agonist is occupied by the endogenous lipid molecule PI
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This means that TPRV1 activated by RTX must first competitively replace PI in the binding pocket, and then regulate ion channels
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Under low-salt conditions, the author successfully revealed the various possibilities of RTX molecules competing with PI for four binding pockets, namely, single RTX molecules, bimolecular ortho and para positions, and the binding of three and four RTX molecules
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Each time the RTX molecule completes a binding pocket competition, the corresponding subunit conformation will change, but only when all four subunits are occupied by RTX and cause sufficient conformational changes can the gate of TPRV1 be shown to open.

The accumulation of acid in local tissues of the body can also turn on the signal transduction of TPRV1
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Previous functional experiments proved that the two amino acids on the extracellular side of TRPV1 constitute the key sites for acid response
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Based on the decomposition of multiple intermediate state conformations, the author directly observed how these two amino acid positions undergo local structural changes under acidic conditions and gradually affect the conformation of TPRV1
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Although these intermediate states are not completely open, the conformational changes in the S1-S4 domains that act similarly to DkTx explain the acidic environment and promote the opening of TPRV1
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The author further explored the transport mechanism of TPRV1 to large cations (such as NMDG)
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By adding large cations to the solution and preparing frozen samples, the authors solved the multiple conformations of TPRV1
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These conformations cover how NMDG expands the pore area outside the cell and gradually enters the ion transport pathway
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These findings confirm the adaptability of TPRV1 to different physiological environments and the mechanism that mediates the transport of ions of different sizes across the membrane
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Dr.Kaihua Zhang, a postdoctoral fellow at the University of California, San Francisco, is the first author of this paper
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Professor Yifan Cheng from the Department of Biochemistry and Biophysics at the University of California, San Francisco and Professor David Julius from the Department of Physiology are the co-corresponding authors
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The David Julius laboratory discovered TRPV1 in 1997 and confirmed that it is an ion channel that can be activated by heat and capsaicin to cause pain
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Yifan Cheng's laboratory has been engaged in research on cryo-electron microscopy methods and applications
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The two laboratories have long-term cooperation
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In 2013, the first use of single-particle cryo-electron microscopy to analyze the atomic structure of TRPV1 triggered a revolution in structural biology
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In 2016, Dr.Kaihua Zhang joined the cooperative team for post-doctoral research
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He successfully used the advantages of single-particle cryo-electron microscopy to analyze the heterogeneity of the sample, and further explained the relationship between the structure and function of TRPV1 through the study of the intermediate state structure, and interpreted the protein analyzer from a biological point of view
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The importance of intermediate structure.
These findings will help to better understand the functions of TRP family ion channels and other signaling proteins, and provide new ideas for the development of TRPV1 targeted drugs.

In terms of structural biology, especially in the context of using artificial intelligence structure prediction, the use of single-particle cryo-electron microscopy to capture the intermediate state structure to explain the function of the protein may be a new research direction in the future.

Article link: https://doi.org/10.1016/j.cell.2021.08.012