"Currently, due to the lack of published articles, my resume is relatively simple and plain, so I have attached the PDF of my thesis defense in the hope that Professor Xu can give me a chance for an interview." At the end of May 2022, Zhang Heng sent an email to Xu Huaqiang, a researcher at the Shanghai Institute of Materia Medica, Chinese Academy of Sciences (hereinafter referred to as the Shanghai Institute of Materia Medica). Pressing the send button, Zhang Heng felt a bit nervous because the work he did during his Ph.D. had not yet been organized into published papers—even though it took him 6 years to graduate for this project.
"Thank you for your interest in our lab. We should arrange a lab meeting for you to present next Tuesday at 6:30 PM." Zhang Heng quickly received a reply from Xu Huaqiang. The interview went smoothly, and Zhang Heng joined Xu Huaqiang's research group.
On May 15, 2024, Zhang Heng, as a co-first author, published his first paper in "Nature." This work, completed in collaboration with Xu Huaqiang's research group at the Shanghai Institute of Materia Medica, Yang Dehua's research group, and Jiang Yi's research group at the Lingang Laboratory, discovered for the first time at near-atomic resolution the mechanism of cholesterol- and lipid-mediated homodimer formation of membrane proteins during the study of norepinephrine transporter (NET), providing tangible evidence for the cell membrane's "lipid raft model."
A screenshot of a paper from "Nature."
Accidental evidence for a hypothesis from 27 years ago
It has long been known that the cell membrane not only provides cells with a relatively independent space but also serves as a window for communication with the outside world. The cell membrane, composed of a phospholipid bilayer, is populated with a variety of proteins that act as "couriers," maintaining communication between the cell and the external environment.
How do these "couriers" work on the highly dynamic cell membrane? In 1997, Finnish scholars Kai Simons and Elina Ikonen proposed the "lipid raft model," suggesting the existence of structurally dense, less mobile regions on the cell membrane formed by specific lipids, akin to rafts floating on water, where certain proteins capable of substance transport are concentrated.
Despite increasing evidence confirming the accuracy of the "lipid raft model," past research methods have failed to directly observe this structure, leading some to question: Do "lipid rafts" really exist?
In exploring the pharmacological significance of transmembrane protein structures, post-90s researcher Zhang Heng unexpectedly shed light on this question. While previous reports of the eukaryotic neurotransmitter-sodium symporter (NSS) protein showed them as monomers, Zhang Heng, during cryo-electron microscopy single-particle data processing, found that most NETs were "paired up," mutually dependent and offset. Initially thought to be an artificial result, further investigations through different sample preparation methods revealed that NETs existed in a dimeric form. Unlike the common multimeric membrane proteins formed by protein-protein interactions, Zhang Heng observed a dimeric structure presenting a "hamburger model" mediated by cholesterol and lipid molecules - where two NET monomers formed the "bread" of the hamburger, while different lipid molecules constituted the "fillings."
The representative structures of human NET homodimers in complex with substrates and six antidepressant compounds.
The tightly packed lipid molecules and lipid-dependent proteins fit well with the definition of the "lipid raft model." Furthermore, the team utilized single-particle cryo-electron microscopy to obtain high-resolution structures of human NET homodimers in complex with substrates, without substrates, and in complex with six antidepressant compounds, totaling eight structures.
Biochemical cell experiments confirmed that compared to monomeric structures, NET homodimer structures more efficiently transport neurotransmitters. Additionally, when bound to different antidepressant drugs, NET exhibited significant differences from other transporters. For instance, the affinity of maprotiline for NET is 520 times that of the serotonin transporter, while the affinity of nisoxetine for NET is 576 times that of the dopamine transporter. These findings provide important structural insights and drug design references for antidepressant drug development.
On Teacher's Day in 2023, they completed the paper and submitted it to the editorial board of "Nature." The paper was officially published online on May 15th this year.
"Both the editors and reviewers were very interested in the 'lipid raft'-like dimeric structures we observed," added Zhang Heng. "However, the 'lipid raft'-like structures we obtained are not large enough and tend to lean towards artificially simulated states. We are now working on creating larger, more natural state-like 'lipid raft' structures."
Candidate scientific illustration provided to the editorial department of "Nature," designed by Zihao Li, with butterfly imagery provided by Cuinana. (The butterfly orchid symbolizes the NET homodimer, with white petals representing the NET monomer. The yellow lip in the middle replaces cholesterol and lipid molecules. The butterfly landing on the flower symbolizes selective targeted drugs, with butterfly wings adorned with the chemical structure of antidepressants.)
Research on the Transporter of "Transport"
NET belongs to the monoamine transporter (MATs) family, which, along with the serotonin transporter (SERT) and dopamine transporter (DAT) in the same family, collectively regulate the concentration of neurotransmitters at neuronal synapses to maintain the balance of neurotransmitters in the body. NET, in particular, recycles excess norepinephrine and dopamine to prevent individuals from being in a state of prolonged excitement.
Due to its significant role in neurotransmitter transmission, MATs are the main targets of psychostimulants and antidepressants. Over the past nearly 70 years, the association of MATs with the pathology of depression has been confirmed from various perspectives such as pharmacology, genetics, molecular biology, and clinical research, and the monoamine hypothesis has also dominated the development of antidepressants for several decades.
Numerous studies indicate that abnormalities in NET function are associated with various mental disorders, such as depression, anxiety disorders, attention deficit hyperactivity disorder, and Parkinson's disease.
"My doctoral research topic was related to transporters, and creating a transporter structure was my personal wish. Professor Jiang asked me through Professor Xu if I was willing to work on neurotransmitter-related transporters, and we immediately agreed," Zhang Heng told "Chinese Science News."
However, the team led by Xu Huaqiang had not previously focused on transporters, making this project almost a "start from scratch" endeavor. It is worth mentioning that from selecting the topic to the publication of the paper, it took only 15 months. When asked why the experiments progressed so rapidly, Xu Huaqiang summarized three reasons.
First, Zhang Heng is "skillful and ingenious."
NET is a highly dynamic transmembrane protein with a total of 12 hydrophobic transmembrane domains, including substrate binding regions, outward channels, and inward channels. During the transport of neurotransmitters, there is alternating opening of channels inside and outside the cell membrane. Moreover, both the inner and outer regions of the protein are scarce, making it difficult to locate using conventional methods. Additionally, in the process of handling samples in the preliminary stage, there is a step involving separating the protein from the cell membrane, which can easily lead to the loss of crucial information.
To address this, Zhang Heng introduced the method of lipid nanodiscs. Nanodiscs are disc-shaped phospholipid bilayers assembled from phospholipids and membrane scaffold proteins, simulating the natural phospholipid bilayer membrane environment. In the environment of nanodiscs, the dimeric structure of NET is clearly visible.
NET Dimer Structure.
Next is the accumulation of basic research and technology in the laboratory's early stage.
Xu Huaqiang's team has long been committed to the study of the structure and function of neurotransmitter receptors. Since 2013, significant progress has been made in the structure and recognition mechanisms of various receptors such as serotonin receptors, dopamine receptors, and opioid receptors.
The laboratory has also developed new technologies accordingly. For example, in this study, the first author, jointly with the Jiang Yi research group, Yinyin, a postdoctoral fellow, used NanoBiT cross-linking technology (commonly known as "nanohooks") to further confirm that NET tends to form homologous dimer structures on the cell membrane. The inventor of this technology is Duan Jia, a doctoral student of Xu Huaqiang, who is now a researcher at the Shanghai Institute of Materia Medica.
Lastly, there are very "awesome" collaborators.
After the first submission, several reviewers suggested that the team supplement experiments on the binding affinity between transporters and drugs to more intuitively describe the differences in drug affinity for different transporters.
"We initially used an indirect method to measure binding affinity, but we didn't get satisfactory data for a whole two months," Zhang Heng recalled. "Later, we contacted Professor Yang Dehua, and they used radioactive isotope labeling technology. Within a month, we revised and organized everything."
"The competition in structural biology is fierce, so it is essential to make full use of the laboratory and institute platforms," Xu Huaqiang added. "I believe that through this experience, Zhang Heng can better understand the importance of finding 'strong partners' and it is also an essential part of scientific research."
Opening up new directions for the laboratory
Currently, Xu Huaqiang's team is conducting some work related to structural pharmacology around transporters.
"To be honest, I didn't expect Zhang Heng to be able to determine the structure of NET at the beginning; it can be said that he exceeded my expectations," Xu Huaqiang believes that Zhang Heng is a student with strong opinions who can independently choose research topics. "Our laboratory used to mainly focus on GPCR, but after Zhang Heng joined, we also started paying attention to transporters. He has opened up a very good direction for the laboratory."
The team members involved in the work, from left to right, are Zhang Chao, Dai Antao, Yang Dehua, Xu Huaqiang, Zhang Heng, Wu Canrong, and Hu Wen.
Members of Xu Huaqiang's team exhibit some common traits - a self-driven desire to do things well and a confidence that is unafraid of challenges.
"Teacher Xu gives us the freedom to explore and also helps us overcome research difficulties from our perspective," Zhang Heng remarked. Despite not having been with the research group for a very long time, influenced by the overall atmosphere in the lab, he can now focus more on the research itself. Xu Huaqiang's strong execution ability also propels everyone in the lab forward.
Unconsciously, Zhang Heng's initiative has also improved. He is currently entering a phase of "harvest," with a recently published paper in Nature, ongoing organization of results from collaborative research on small molecule compounds based on the design of monoamine transporter structures, and another paper already submitted.
"Shanghai Institute of Materia Medica has a very good foundation in the field of neuropharmacology, and our lab has also conducted work on central nervous system neurotransmitter-related receptors. After Zhang Heng and other students become independent, if they are interested in these scientific issues, I look forward to them carrying on the tradition of next-generation drug molecular research in the field of neurological diseases at the institute," Xu Huaqiang believes that a laboratory culture of mutual respect and free exploration can, like scientific exploration, be passed on.
Related paper information: https://doi.org/10.1038/s41586-024-07437-6
