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A.  As a postdoc fellow, I established a yeast-based high-yield GPCR preparation platform for bio-NMR study. It is notoriously difficult to prepare a large quantity of GPCR for structural biology study as the expression level for a huge majority of receptors is at a level of single digital ug or pg / 1L cell culture. This is even more outstanding for NMR research on GPCRs as a minimal mg protein is needed for a single NMR experiment. In addressing this challenge, I established an elegant GPCR preparation protocol in P. pastoris from scratch in a physical lab. Further, through optimizing 19F probe and selecting a judiciously 19F-labeling site on a GPCR, my research showed explicitly the simultaneous existence of multiple conformational states in a GPCR and the ability of shift that equilibrium of conformational ensemble with different ligands, a landmark accomplishment in the field. This advance enabled us to address a longstanding controversy regarding GPCR activation model. My research also interrogated the molecular mechanism of how cations regulated the receptor activation at the quantified conformational level. The results showed the public clearly how the monovalent cation Na+ negatively regulated the receptor activation by shifting the subpopulation to inactive states while the divalent cation Mg2+ positively modulated receptor activation by shifting the subpopulation to active conformational states, along with their dynamic transition processes.

1. Ye L, Orazietti AP, Pandey A, Prosser RS. High-efficiency expression of yeast-derived G-protein coupled receptors and 19F labeling for dynamical studies. Methods Mol Biol,  2018,1688:407-421. PubMed PMID: 29151220.

2. Ye L, Larda TS, Li Y, Manglik A, Prosser RS. A comparison of chemistry shift sensitivity of trifluoromethyl tags: optimizing resolution in 19F NMR studies of proteins. J Bio NMR, 2015, 62(1): 97-103. PubMed PMID: 25813845.

3. Ye L, Van Eps N, Zimmer M, Ernst OP, Prosser RS. Activation of the A2A adenosine G-protein-coupled receptor by conformational selection. Nature, 2016, 12;533(7602):265-8. PubMed PMID: 27144352.

4. Ye L, Neale C, Sljoka A, Lyda B, Pichugin D, Tsuchimura N, Larda ST, Pomès R, García AE, Ernst OP, Sunahara RK, Prosser RS. Mechanistic insights into allosteric regulation of the A2A adenosine G protein-coupled receptor by physiological cations. Nat Commun, 2018, 9(1):1372. PubMed PMID: 29636462. 

 

B. In my independent career since Nov. 2018, I proposed a novel concept of designing GPCR biased drugs based on receptor conformational preferences in response to ligand bindings in 2021. Towards this goal, I continued to upgrade an in-house yeast-based GPCR expression system that was early established by myself, allowing my lab to achieve the GPCR production of 5-10 mg/ 1L cell culture --- an unprecedent productivity for GPCRs. This success provides a chance to genetically integrate fluorinated unnatural amino acid (FUAA) into the receptor and study the conformational transitions and dynamics of GPCRs as the FUAA incorporation usually leads to 10-fold decrease of the productivity. Our data have demonstrated the feasibility of using FUAA incorporation for GPCR dynamics study, and project is being progressed. Benefiting from the high-yield expression of several GPCRs, my lab also established an in-membrane NMR approach studying in situ ligand-GPCR interactions, considering the in-cell membrane protein NMR is still impossible. As you may know, conventional approaches to study ligand-receptor interactions using NMR often involve laborious sample preparation, isotopic labeling, and receptor reconstitution. Each of these steps remains challenging for membrane proteins like GPCRs. This novel method integrates NMR and homogenized membrane nano-discs (~230 nm) together to characterize ligand-GPCR interactions. The approach has a great potential for drug screening as it benefits from minimal protein membrane preparation and minimizing non-specific binding. This advance allows us to examine more than 1,000 ligands with 250 mL cell culture with a simple preparation whereas the conventional approach leads to only a few samples from 6 L cell culture and the exhaustive and expensive preparation process. The in-membrane protein also maintains its structural heterogeneity that is essential for functional diversity, making it feasible for probing a more reliable ligand-GPCR interaction that is vital for faithful lead compounds discovery.

1. Wang X, McFarland A, Madsen JJ, Aalo E, Ye L*. The potential of 19F NMR application in GPCR biased drug discovery. Trends Pharmacy Sci, 2021, 42(1):19-30. PubMed PMID: 33250272. 

2. Zhao W, Wang X, Ye L*. Expression and purification of yeast derived GPCR, Gα and Gβγ subunits for structural and dynamic studies. Bio Protoc, 2021,11(4):e3919. PubMed Central PMCID: .

3. Wang X, Bushra N, Muschol M, Madsen JJ, Ye L*. An in-membrane NMR spectroscopic approach probing native ligand-GPCR interaction. Int J Biol Macromol, 2022, 206:911-916. PubMed PMID: .

4. Ye L, Wang X. A high-throughput NMR approach for in-membrane protein ligand screening. US17/677,249, 2022. (Patent)

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C. Understanding the roles of intermediate states in signaling is pivotal to unraveling the activation processes of GPCRs and guide drug development. However, the field is still struggling to define these conformational states with sufficient resolution to study their individual functions. With the foundation established in the section (2), in collaboration with Caltech and Los Alamos National Laboratory, my research pushed the frontier in delineating conformational states by trapping the intermediate states in particular constructs, proposing a five-state GPCR activation model, including two inactive states (S1 and S2), two intermediate states (S3 and S4), and one fully activated state (S5). The research also discovered a structurally conserved cation-p microswitch in regulating the transition between inactive ensemble (S1 and S2) and active (-like) ensemble (S3, S4 and S5). My recent research demonstrated the feasibility of enriching the populations of discrete states via conformation-biased mutants. These mutants adopt distinct distributions among five states that lie along the activation pathway of adenosine A2A receptor (A2AR), a class A GPCR. Using these conformation-biased mutants, my research revealed a structurally conserved cation-p microswitch between transmembrane helix VI (TM6) and Helix8 that regulates cytoplasmic cavity opening as a “gatekeeper” for G protein penetration, controlling the receptor activation from inactive ensemble to the active (-like) ensemble, different from the previous identified ionic lock between TM3 and TM6 regulating the conformational transitions within the inactive states. 

1. Wang X, Neale C, Kim SK, Goddard AW, Ye L*. Intermediate-state-trapped mutants pinpoint G protein-coupled receptor conformational allostery. Nat Commun, 2023, 14(1):1325. PubMed PMID: 36899002. 

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D. It has been trusted that GPCR activation at least include three complexes: (1) the pre-coupled GPCR-Gabgintermediate complex, (2) the partially activated GPCR-Gabg intermediate complex, and (3) the fully activated GPCR-Gabg end-state complex. The first X-ray structure of GPCR-G protein complex was elucidated in 2011 and the first cryo-EM structure of GPCR-G protein complex was resolved in 2017. Although more than 700 GPCR-G protein complex structures of over 150 GPCRs are available so far, these structures uniquely represent the fully activated GPCR-G protein end-state complex. Thus, we are still missing the structural information regarding how heterotrimeric G protein pre-coupled to the receptor and advances to the partially activated intermediate complex and eventually transitions to the fully activated end-state complex. The transient nature of pre-coupled- and partially activated- intermediate GPCR-protein complex makes the task difficult to pursue. The creation of the Intermediate-state-trapped mutants allowed us to study the functions of intermediate states and their structures in complexes with different downstream signaling partners. Thus, in collaboration with UCSF and UNC, we resolved the first intermediate GPCR-G protein (S4-G protein) complex in the field. This success provides us a 19F-qNMR-guided-cryo-EM tool to tackle those difficult conformations and their complexes in the biological systems. A series of continued work is on-going in resolving more structures of unexplored intermediate states of the GPCRs in complex with various transducers, including G proteins, GRKs, and b-arrestins, and uncover their roles in the GPCR signaling using and we expect this innovative method will be broadly applicable to other GPCRs, and any proteins of interest in studying difficult complexes. 

1. Bi M, Wang X, Wang J, Xu J, Sun W, Adediwura V, Miao Y*, Cheng Y*, Ye L*. Structure and function of an intermediate GPCR-G protein complex. BioRxiv, 2024, PubMed PMID: 38617296. 

 

E. In addition to mainstream research, I also co-authored a series of papers in helping my colleagues drive the frontiers in their respective field, by taking advantage of my expertise in NMR, biological, and membrane proteins, etc. I have helped to uncover an asymmetric enzymatic catalysis, evaluate a novel helical sulfonyl-γ-AApeptides in modulating Aβ oligomerization, evaluate a new Corona Discharge device in sterilizing the pathogens during the pandemic, and determine the dynamics of a metallo-helicoid with double-rims:polymerization, etc.

1. Kim T, Mehrabi P, Ren Z, Sljoka A, Ing C, Bezginov A, Ye L, Pomes R, Prosser RS, Pai EF. The role of dimer asymmetry and subunit dynamics in enzyme catalysis. Science, 2017,355(6322): DOI: 10.1126/science.aag2355. PubMed PMID: 28104837.

2. Liu H, Cui Y, Zhao X, Wei L, Wang X, Shen N, Odom T, Li X, Lawless W, Guida W, Cao C, Ye L, Cai J*. Helical sulfonyl-γ-AApeptides modulating Aβ oligomerization and cytotoxicity by recognizing Aβ helix. PNAS, 2024, 121,6, e2311733121. PubMed PMID: 38285951.

3. Narayanan SSKS, Wang X, Paul J, Paley V, Weng Z, Ye L*, Zhong Y*. Disinfection and electrostatic recovery of N95 respirators by Corona Discharge for safe reuse. Environ Sci Tech, 2021: doi.org/10.1021/acs.est.1c02649. PubMed PMID: 34570480.

4. Yin GQ,  Kandapal S, Liu C,  Wang H,  Jiang S,  Ji T, Yan Y,  Khalife S,  Ye L, Xu B,  Yang H, Nieh M, Li X*. Metallo-helicoid with double rims:polymerization followed by folding via intramolecular coordination. Angew Chem Int Ed Engl, 2021, 60(3): 1281-1289. PubMed PMID:  3009693.