Basic Life Science Research Associate, Sarafan ChEM-H
BSc, Hong Kong University of Science and Technology, Molecular Biomedical Sciences (2013)
PhD, California Institute of Technology, Biochemistry and Molecular Biophysics (2019)
Discovery of VH domains that allosterically inhibit ENPP1.
Nature chemical biology
Ectodomain phosphatase/phosphodiesterase-1 (ENPP1) is overexpressed on cancer cells and functions as an innate immune checkpoint by hydrolyzing extracellular cyclic guanosine monophosphate adenosine monophosphate (cGAMP). Biologic inhibitors have not yet been reported and could have substantial therapeutic advantages over current small molecules because they can be recombinantly engineered into multifunctional formats and immunotherapies. Here we used phage and yeast display coupled with in cellulo evolution to generate variable heavy (VH) single-domain antibodies against ENPP1 and discovered a VH domain that allosterically inhibited the hydrolysis of cGAMP and adenosine triphosphate (ATP). We solved a 3.2 Å-resolution cryo-electron microscopy structure for the VH inhibitor complexed with ENPP1 that confirmed its new allosteric binding pose. Finally, we engineered the VH domain into multispecific formats and immunotherapies, including a bispecific fusion with an anti-PD-L1 checkpoint inhibitor that showed potent cellular activity.
View details for DOI 10.1038/s41589-023-01368-5
View details for PubMedID 37400538
View details for PubMedCentralID 7990037
Structural basis for activation of CB1 by an endocannabinoid analog.
2023; 14 (1): 2672
Endocannabinoids (eCBs) are endogenous ligands of the cannabinoid receptor 1 (CB1), a G protein-coupled receptor that regulates a number of therapeutically relevant physiological responses. Hence, understanding the structural and functional consequences of eCB-CB1 interactions has important implications for designing effective drugs targeting this receptor. To characterize the molecular details of eCB interaction with CB1, we utilized AMG315, an analog of the eCB anandamide to determine the structure of the AMG315-bound CB1 signaling complex. Compared to previous structures, the ligand binding pocket shows some differences. Using docking, molecular dynamics simulations, and signaling assays we investigated the functional consequences of ligand interactions with the "toggle switch" residues F2003.36 and W3566.48. Further, we show that ligand-TM2 interactions drive changes to residues on the intracellular side of TM2 and are a determinant of efficacy in activating G protein. These intracellular TM2 rearrangements are unique to CB1 and are exploited by a CB1-specific allosteric modulator.
View details for DOI 10.1038/s41467-023-37864-4
View details for PubMedID 37160876
View details for PubMedCentralID PMC10169858
Negative allosteric modulation of the glucagon receptor by RAMP2.
2023; 186 (7): 1465-1477.e18
Receptor activity-modifying proteins (RAMPs) modulate the activity of many Family B GPCRs. We show that RAMP2 directly interacts with the glucagon receptor (GCGR), a Family B GPCR responsible for blood sugar homeostasis, and broadly inhibits receptor-induced downstream signaling. HDX-MS experiments demonstrate that RAMP2 enhances local flexibility in select locations in and near the receptor extracellular domain (ECD) and in the 6th transmembrane helix, whereas smFRET experiments show that this ECD disorder results in the inhibition of active and intermediate states of the intracellular surface. We determined the cryo-EM structure of the GCGR-Gs complex at 2.9 Å resolution in the presence of RAMP2. RAMP2 apparently does not interact with GCGR in an ordered manner; however, the receptor ECD is indeed largely disordered along with rearrangements of several intracellular hallmarks of activation. Our studies suggest that RAMP2 acts as a negative allosteric modulator of GCGR by enhancing conformational sampling of the ECD.
View details for DOI 10.1016/j.cell.2023.02.028
View details for PubMedID 37001505
Negative allosteric modulation of the glucagon receptor by RAMP2
CELL PRESS. 2023: 161A
View details for Web of Science ID 000989629700786
- Negative allosteric modulation of the glucagon receptor by RAMP2. Biophysical journal 2023; 122 (3S1): 161a
Structural and dynamic insights into supra-physiological activation and allosteric modulation of a muscarinic acetylcholine receptor.
2023; 14 (1): 376
The M2 muscarinic receptor (M2R) is a prototypical G-protein-coupled receptor (GPCR) that serves as a model system for understanding GPCR regulation by both orthosteric and allosteric ligands. Here, we investigate the mechanisms governing M2R signaling versatility using cryo-electron microscopy (cryo-EM) and NMR spectroscopy, focusing on the physiological agonist acetylcholine and a supra-physiological agonist iperoxo, as well as a positive allosteric modulator LY2119620. These studies reveal that acetylcholine stabilizes a more heterogeneous M2R-G-protein complex than iperoxo, where two conformers with distinctive G-protein orientations were determined. We find that LY2119620 increases the affinity for both agonists, but differentially modulates agonists efficacy in G-protein and β-arrestin pathways. Structural and spectroscopic analysis suggest that LY211620 stabilizes distinct intracellular conformational ensembles from agonist-bound M2R, which may enhance β-arrestin recruitment while impairing G-protein activation. These results highlight the role of conformational dynamics in the complex signaling behavior of GPCRs, and could facilitate design of better drugs.
View details for DOI 10.1038/s41467-022-35726-z
View details for PubMedID 36690613
View details for PubMedCentralID PMC9870890
Structure-based design of bitopic ligands for the µ-opioid receptor.
Mu opioid receptor (µOR) agonists like fentanyl have long been used for pain management, but are considered a major public health concern due to their adverse side effects, including lethal overdose.1 To design safer therapeutics, we report a conceptually novel approach targeting conserved sodium (Na+) binding site2, observed in µOR3 and many other class A GPCRs, by bitopic fentanyl derivatives functionalized via a linker with a positively charged guanidino group. Cryo-EM structures of the most potent bitopic ligands in complex with µOR highlight the key interactions between the ligand's guanidine and the key Asp2.50 residue in the Na+ site. While the lead bitopics maintain nanomolar potency and high efficacy at Gi subtypes, they show strongly reduced arrestin recruitment, one also shows the lowest Gz-efficacy among the panel of µOR agonists, including partial and biased, morphinan and fentanyl analogs. In mice, the best bitopic ligand displayed µOR dependent antinociception with attenuated adverse effects supporting the µOR Na+ site as a potential target for the design of safer analgesics. In general, our study suggests that bitopic ligands engaging the Na+ pocket in class A GPCRs can be designed to control their efficacy and functional selectivity profiles for Gi/o/z subtypes and arrestins, thus modulating their in vivo pharmacology.
View details for DOI 10.1038/s41586-022-05588-y
View details for PubMedID 36450356
Insights into distinct signaling profiles of the OR activated by diverse agonists.
Nature chemical biology
Drugs targeting the mu-opioid receptor (muOR) are the most effective analgesics available but are also associated with fatal respiratory depression through a pathway that remains unclear. Here we investigated the mechanistic basis of action of lofentanil (LFT) and mitragynine pseudoindoxyl (MP), two muOR agonists with different safety profiles. LFT, one of the most lethal opioids, and MP, a kratom plant derivative with reduced respiratory depression in animal studies, exhibited markedly different efficacy profiles for G protein subtype activation and beta-arrestin recruitment. Cryo-EM structures of muOR-Gi1 complex with MP (2.5A) and LFT (3.2A) revealed that the two ligands engage distinct subpockets, and molecular dynamics simulations showed additional differences in the binding site that promote distinct active-state conformations on the intracellular side of the receptor where G proteins and beta-arrestins bind. These observations highlight how drugs engaging different parts of the muOR orthosteric pocket can lead to distinct signaling outcomes.
View details for DOI 10.1038/s41589-022-01208-y
View details for PubMedID 36411392
3,4-Bis(hydroxymethyl)hexane-1,6-diol-based maltosides (HDMs) for membrane-protein study: Importance of detergent rigidity-flexibility balance in protein stability.
Chemistry, an Asian journal
Detergents have been major contributors to membrane-protein structural study for decades. However, membrane proteins solubilized in conventional detergents tend to aggregate or denature over time. Stability of large eukaryotic membrane proteins with complex structures tends to be worse, necessitating development of novel detergents with improved properties. Here, we prepared a novel class of detergents, designated 3,4-bis(hydroxymethyl)hexane-1,6-diol-based maltosides (HDMs). When tested on three membrane proteins, including two G-protein-coupled receptors (GPCRs), the new detergents displayed significantly better behaviors compared with DDM. Moreover, the HDMs were superior or comparable to LMNG, an amphiphile widely used for GPCR structural study. An optimal balance of detergent rigidity vs. flexibility of the HDMs is likely responsible for their favorable behaviors toward membrane-protein stability. Thus, the current study not only introduces the HDMs, with significant potential for membrane-protein structural study, but also suggests a useful guideline for designing novel detergents for membrane-protein research.
View details for DOI 10.1002/asia.202200941
View details for PubMedID 36253323
Development of 1,3-acetonedicarboxylate-derived glucoside amphiphiles (ACAs) for membrane protein study.
2022; 13 (19): 5750-5759
Detergents are extensively used for membrane protein manipulation. Membrane proteins solubilized in conventional detergents are prone to denaturation and aggregation, rendering downstream characterization of these bio-macromolecules difficult. Although many amphiphiles have been developed to overcome the limited efficacy of conventional detergents for protein stabilization, only a handful of novel detergents have so far proved useful for membrane protein structural studies. Here, we introduce 1,3-acetonedicarboxylate-derived amphiphiles (ACAs) containing three glucose units and two alkyl chains as head and tail groups, respectively. The ACAs incorporate two different patterns of alkyl chain attachment to the core detergent unit, generating two sets of amphiphiles: ACA-As (asymmetrically alkylated) and ACA-Ss (symmetrically alkylated). The difference in the attachment pattern of the detergent alkyl chains resulted in minor variation in detergent properties such as micelle size, critical micelle concentration, and detergent behaviors toward membrane protein extraction and stabilization. In contrast, the impact of the detergent alkyl chain length on protein stability was marked. The two C11 variants (ACA-AC11 and ACA-SC11) were most effective at stabilizing the tested membrane proteins. The current study not only introduces new glucosides as tools for membrane protein study, but also provides detergent structure-property relationships important for future design of novel amphiphiles.
View details for DOI 10.1039/d2sc00539e
View details for PubMedID 35694361
View details for PubMedCentralID PMC9116450
- Development of 1,3-acetonedicarboxylate-derived glucoside amphiphiles (ACAs) for membrane protein study CHEMICAL SCIENCE 2022
Structure-based Evolution of G protein-biased mu-opioid Receptor Agonists.
Angewandte Chemie (International ed. in English)
The mu-opioid receptor (muOR) is the major target for opioid analgesics. Activation of muOR initiates signaling through G protein pathways as well as through beta-arrestin recruitment. muOR agonists that are biased towards G protein signaling pathways demonstrate diminished side effects. PZM21, discovered by computational docking, is a G protein biased muOR agonist. Here we report the cryoEM structure of PZM21 bound muOR in complex with G i protein. Structure-based evolution led to multiple PZM21 analogs with more pronounced G i protein bias and increased lipophilicity to improve CNS penetration. Among them, FH210 shows extremely low potency and efficacy for arrestin recruitment. We further determined the cryoEM structure of FH210 bound to muOR in complex with G i protein and confirmed its expected binding pose. The structural and pharmacological studies reveal a potential mechanism to reduce beta-arrestin recruitment by the muOR, and hold promise for developing next-generation analgesics with fewer adverse effects.
View details for DOI 10.1002/anie.202200269
View details for PubMedID 35385593
Foldable detergentsfor membrane protein study: Importance of detergent core flexibility in protein stabilization.
Chemistry (Weinheim an der Bergstrasse, Germany)
Membrane proteins are of biological and pharmaceutical significance. However, their structural study is extremely challenging mainly due to the fact that only a small number of chemical tools is suitable for stabilizing membrane proteins in solution. Detergents are widely used in membrane protein study, but conventional detergents are generally poor at stabilizing challenging membrane proteins such as G protein-coupled receptors and protein complexes. In the current study, we prepared tandem triazine-based maltosides (TZMs) with two amphiphilic triazine units connected by different diamine linkers, hydrazine (TZM-Hs) and 1,2-ethylenediamine (TZM-Es). These TZMs were consistently superior to a gold standard detergent (DDM) in terms of stabilizing a few membrane proteins. In addition, the TZM-Es containing a long linker showed more general protein stabilization efficacy with multiple membrane proteins than the TZM-Hs containing a short linker. This result indicates that introduction of the flexible1,2-ethylenediamine linker between two rigid triazine rings enables the TZM-Es to fold into favourable conformations in order to promote membrane protein stability. The novel concept of detergent foldability introduced in the current study has potential use for rational detergent design and membrane protein applications.
View details for DOI 10.1002/chem.202200116
View details for PubMedID 35238091
Glyco-steroidal amphiphiles (GSAs) for membrane protein structural study.
Chembiochem : a European journal of chemical biology
Integral membrane proteins pose considerable challenges to high resolution structural analysis. Maintaining membrane proteins in their native state during protein isolation is essential for structural study of these bio-macromolecules. Detergents are the most commonly used amphiphilic compounds for stabilizing membrane proteins in solution outside a lipid bilayer. We previously introduced a glyco-diosgenin (GDN) detergent that was shown to be highly effective at stabilizing a wide range of membrane proteins. This steroidal detergent has additionally gained attention due to its compatibility with membrane protein structure study via cryo-EM. However, synthetic inconvenience limits widespread use of GDN in membrane protein study. To improve its synthetic accessibility and to further enhance detergent efficacy for protein stabilization, we designed a new class of glyco-steroid-based detergents using three steroid units: cholestanol, cholesterol and diosgenin. These new detergents were efficiently prepared and showed marked efficacy for protein stabilization in evaluation with a few model membrane proteins including two G protein-coupled receptors. Some new agents were not only superior to a gold standard detergent, DDM, but were also more effective than the original GDN at preserving protein integrity long term. These agents represent valuable alternatives to GDN, and are likely to facilitate structural determination of challenging membrane proteins.
View details for DOI 10.1002/cbic.202200027
View details for PubMedID 35129249
- Sequential immunization of macaques elicits heterologous neutralizing antibodies targeting the V3-glycan patch of HIV-1 Env SCIENCE TRANSLATIONAL MEDICINE 2021; 13 (621): eabk1533
Maltose-bis(hydroxymethyl)phenol (MBPs) and Maltose-tris(hydroxymethyl)phenol (MTPs) Amphiphiles for Membrane Protein Stability.
ACS chemical biology
Membrane protein structures provide a fundamental understanding of their molecular actions and are of importance for drug development. Detergents are widely used to solubilize, stabilize, and crystallize membrane proteins, but membrane proteins solubilized in conventional detergents are prone to denaturation and aggregation. Thus, developing novel detergents with enhanced efficacy for protein stabilization remains important. We report herein the design and synthesis of a class of phenol-derived maltoside detergents. Using two different linkers, we prepared two sets of new detergents, designated maltose-bis(hydroxymethyl)phenol (MBPs) and maltose-tris(hydroxymethyl)phenol (MTPs). The evaluation of these detergents with three transporters and two G-protein coupled receptors allowed us to identify a couple of new detergents (MBP-C9 and MTP-C12) that consistently conferred enhanced stability to all tested proteins compared to a gold standard detergent (DDM). Furthermore, the data analysis based on the detergent structures provides key detergent features responsible for membrane protein stabilization that together will facilitate the future design of novel detergents.
View details for DOI 10.1021/acschembio.1c00578
View details for PubMedID 34445864
Conformationally flexible core-bearing detergents with a hydrophobic or hydrophilic pendant: Effect of pendant polarity on detergent conformation and membrane protein stability.
Membrane protein structures provide atomic level insight into essential biochemical processes and facilitate protein structure-based drug design. However, the inherent instability of these bio-macromolecules outside lipid bilayers hampers their structural and functional study. Detergent micelles can be used to solubilize and stabilize these membrane-inserted proteins in aqueous solution, thereby enabling their downstream characterizations. Membrane proteins encapsulated in detergent micelles tend to denature and aggregate over time, highlighting the need for development of new amphiphiles effective for protein solubility and stability. In this work, we present newly-designed maltoside detergents containing a pendant chain attached to a glycerol-decorated tris(hydroxylmethyl)methane (THM) core, designated GTMs. One set of the GTMs has a hydrophobic pendant (ethyl chain; E-GTMs), and the other set has a hydrophilic pendant (methoxyethoxylmethyl chain; M-GTMs) placed in the hydrophobic-hydrophilic interfaces. The two sets of GTMs displayed profoundly different behaviors in terms of detergent self-assembly and protein stabilization efficacy. These behaviors mainly arise from the polarity difference between two pendants (ethyl and methoxyethoxylmethyl chains) that results in a large variation in detergent conformation between these sets of GTMs in aqueous media. The resulting high hydrophobic density in the detergent micelle interior is likely responsible for enhanced efficacy of the M-GTMs for protein stabilization compared to the E-GTMs and a gold standard detergent DDM. A representative GTM, M-GTM-O12, was more effective for protein stability than some recently developed detergents including LMNG. This is the first case study investigating the effect of pendant polarity on detergent geometry that correlates with detergent efficacy for protein stabilization.
View details for DOI 10.1016/j.actbio.2021.04.043
View details for PubMedID 33933694
Construction, characterization, and immunization of nanoparticles that display a diverse array of influenza HA trimers
2021; 16 (3): e0247963
Current influenza vaccines do not elicit broadly protective immune responses against multiple strains. New strategies to focus the humoral immune response to conserved regions on influenza antigens are therefore required for recognition by broadly neutralizing antibodies. It has been suggested that B-cells with receptors that recognize conserved epitopes would be preferentially stimulated through avidity effects by mosaic particles presenting multiple forms of a variable antigen. We adapted SpyCatcher-based platforms, AP205 virus-like particles (VLPs) and mi3 nanoparticles (NPs), to covalently co-display SpyTagged hemagglutinin (HA) trimers from group 1 and group 2 influenza A strains. Here we show successful homotypic and heterotypic conjugation of up to 8 different HA trimers to both VLPs and NPs. We characterized the HA-VLPs and HA-NPs by cryo-electron tomography to derive the average number of conjugated HAs and their separation distances on particles, and compared immunizations of mosaic and homotypic particles in wild-type mice. Both types of HA particles elicited strong antibody responses, but the mosaic particles did not consistently elicit broader immune responses than mixtures of homotypic particles. We conclude that covalent attachment of HAs from currently-circulating influenza strains represents a viable alternative to current annual influenza vaccine strategies, but in the absence of further modifications, is unlikely to represent a method for making a universal influenza vaccine.
View details for DOI 10.1371/journal.pone.0247963
View details for Web of Science ID 000626604100046
View details for PubMedID 33661993
View details for PubMedCentralID PMC7932532
Diastereomeric Cyclopentane-Based Maltosides (CPMs) as Tools for Membrane Protein Study.
Journal of the American Chemical Society
Amphiphilic agents, called detergents, are invaluable tools for studying membrane proteins. However, membrane proteins encapsulated by conventional head-to-tail detergents tend to denature or aggregate, necessitating the development of structurally distinct molecules with improved efficacy. Here, a novel class of diastereomeric detergents with a cyclopentane core unit, designated cyclopentane-based maltosides (CPMs), were prepared and evaluated for their ability to solubilize and stabilize several model membrane proteins. A couple of CPMs displayed enhanced behavior compared with the benchmark conventional detergent, n-dodecyl-beta-d-maltoside (DDM), for all the tested membrane proteins including two G-protein-coupled receptors (GPCRs). Furthermore, CPM-C12 was notable for its ability to confer enhanced membrane protein stability compared with the previously developed conformationally rigid NBMs [J. Am. Chem. Soc. 2017, 139, 3072] and LMNG. The effect of the individual CPMs on protein stability varied depending on both the detergent configuration (cis/trans) and alkyl chain length, allowing us draw conclusions on the detergent structure-property-efficacy relationship. Thus, this study not only provides novel detergent tools useful for membrane protein research but also reports on structural features of the detergents critical for detergent efficacy in stabilizing membrane proteins.
View details for DOI 10.1021/jacs.0c09629
View details for PubMedID 33315387
New Malonate-Derived Tetraglucoside Detergents for Membrane Protein Stability.
ACS chemical biology
Membrane proteins are widely studied in detergent micelles, a membrane-mimetic system formed by amphiphilic compounds. However, classical detergents have serious limitations in their utility, particularly for unstable proteins such as eukaryotic membrane proteins and membrane protein complexes, and thus, there is an unmet need for novel amphiphiles with enhanced ability to stabilize membrane proteins. Here, we developed a new class of malonate-derived detergents with four glucosides, designated malonate-derived tetra-glucosides (MTGs), and compared these new detergents with previously reported octyl glucose neopentyl glycol (OGNG) and n-dodecyl-beta-d-maltoside (DDM). When tested with two G-protein coupled receptors (GPCRs) and three transporters, a couple of MTGs consistently conferred enhanced stability to all tested proteins compared to DDM and OGNG. As a result of favorable behaviors for a range of membrane proteins, these MTGs have substantial potential for membrane protein research. This study additionally provides a new detergent design principle based on the effect of a polar functional group (i.e., ether) on protein stability depending on its position in the detergent scaffold.
View details for DOI 10.1021/acschembio.0c00316
View details for PubMedID 32501004
- Publisher Correction: Asymmetric opening of HIV-1 Env bound to CD4 and a coreceptor-mimicking antibody. Nature structural & molecular biology 2020
- Publisher Correction: Asymmetric opening of HIV-1 Env bound to CD4 and a coreceptor-mimicking antibody. Nature structural & molecular biology 2019
Asymmetric opening of HIV-1 Env bound to CD4 and a coreceptor-mimicking antibody.
Nature structural & molecular biology
The human immunodeficiency virus (HIV-1) envelope (Env) glycoprotein, a (gp120-gp41)3 trimer, mediates fusion of viral and host cell membranes after gp120 binding to host receptor CD4. Receptor binding triggers conformational changes allowing coreceptor (CCR5) recognition through CCR5's tyrosine-sulfated amino (N) terminus, release of the gp41 fusion peptide and fusion. We present 3.3A and 3.5A cryo-EM structures of E51, a tyrosine-sulfated coreceptor-mimicking antibody, complexed with a CD4-bound open HIV-1 native-like Env trimer. Two classes of asymmetric Env interact with E51, revealing tyrosine-sulfated interactions with gp120 mimicking CCR5 interactions, and two conformations of gp120-gp41 protomers (A and B protomers in AAB and ABB trimers) that differ in their degree of CD4-induced trimer opening and induction of changes to the fusion peptide. By integrating the new structural information with previous closed and open envelope trimer structures, we modeled the order of conformational changes on the path to coreceptor binding site exposure and subsequent viral-host cell membrane fusion.
View details for DOI 10.1038/s41594-019-0344-5
View details for PubMedID 31792452
A functional enrichment test for molecular convergent evolution finds a clear protein-coding signal in echolocating bats and whales.
Proceedings of the National Academy of Sciences of the United States of America
Distantly related species entering similar biological niches often adapt by evolving similar morphological and physiological characters. How much genomic molecular convergence (particularly of highly constrained coding sequence) contributes to convergent phenotypic evolution, such as echolocation in bats and whales, is a long-standing fundamental question. Like others, we find that convergent amino acid substitutions are not more abundant in echolocating mammals compared to their outgroups. However, we also ask a more informative question about the genomic distribution of convergent substitutions by devising a test to determine which, if any, of more than 4,000 tissue-affecting gene sets is most statistically enriched with convergent substitutions. We find that the gene set most overrepresented (q-value = 2.2e-3) with convergent substitutions in echolocators, affecting 18 genes, regulates development of the cochlear ganglion, a structure with empirically supported relevance to echolocation. Conversely, when comparing to nonecholocating outgroups, no significant gene set enrichment exists. For aquatic and high-altitude mammals, our analysis highlights 15 and 16 genes from the gene sets most affected by molecular convergence which regulate skin and lung physiology, respectively. Importantly, our test requires that the most convergence-enriched set cannot also be enriched for divergent substitutions, such as in the pattern produced by inactivated vision genes in subterranean mammals. Showing a clear role for adaptive protein-coding molecular convergence, we discover nearly 2,600 convergent positions, highlight 77 of them in 3 organs, and provide code to investigate other clades across the tree of life.
View details for DOI 10.1073/pnas.1818532116
View details for PubMedID 31570615