Membrane proteins
Membrane proteins, which contribute to more than 60% drug targets, play versatile physiological functions. Our research has long focused on unraveling the mechanisms of membrane proteins, employing a range of approaches including structural biology, biochemistry, and electrophysiology. Prior to joining HKUST, we successfully determined the structures of several membrane proteins (Dang et al., 2010; Li et al., 2013; Dang et al., 2015; Dang et al., 2017; Dang et al., 2019; Feng et al., 2019) using x-ray crystallography and single-particle cryo-EM. These findings have provided valuable insights into the functional mechanisms of these proteins and have the potential to inform drug development efforts.
At HKUST, we are committed to advancing our understanding of the molecular mechanisms underlying vital membrane proteins. The TMEM63 family proteins are calcium-permeable channels found in animals that exhibit preferential activation in response to hypo-osmolality. These proteins have been implicated in various physiological functions and their deficiency can lead to diseases such as hearing loss. Our research efforts have led to significant progress in this field. Specifically, we have successfully determined the cryo-EM structure of mouse TMEM63C at a resolution of 3.56 Å. Through comparative analysis with TMEM63A, TMEM63B, and the plant orthologues OSCAs, we have identified structural differences that shed light on their functional distinctions (Qin et al., 2023). Moreover, utilizing structural-guided mutagenesis and calcium imaging, we have elucidated the critical roles of the coupling between TM0 and TM6 in channel activity. Furthermore, our investigations have revealed that TMEM63C primarily exists as a monomer under physiological conditions. In contrast, TMEM63B demonstrates a mixture of monomeric and dimeric forms within cells, suggesting that oligomerization serves as a regulatory mechanism for TMEM63 proteins. Our findings provide valuable insights into the functional properties and regulatory mechanisms of TMEM63 family proteins, contributing to our understanding of their involvement in physiological processes and potential implications for disease.
DNA Replication
DNA replication is a tightly regulated process that ensures faithful duplication of the genome during each cell division cycle. It relies on the replisome, a large protein complex consisting of DNA helicase, DNA polymerase, and various accessory factors. During replication elongation, the replisome also travels along chromatin to coordinate the transfer of nucleosome from the parental DNA strand in front of the fork to newly synthesized daughter strands, engage with transcription machinery to avoid transcription-replication conflict (TRC), and remove other types of large roadblocks.
In collaboration with the labs of Bik Tye and Yuanliang Zhai, our research focuses on elucidating the structural characteristics of replisome machines to gain a comprehensive understanding of the molecular mechanisms underlying DNA replication. Notably, we have successfully determined the structure of the human pre-replication complex (Li et al., 2023) as well as the complex structure of CMG helicase and the leading strand DNA polymerase (Xu et al., 2023). These findings have made significant and timely contributions to the field of DNA replication.
Methodology Development
Cryo-electron microscopy (cryo-EM) has revolutionized the field of structural biology, providing unprecedented insights into the atomic structures of biological macromolecules and driving groundbreaking discoveries that enhance our understanding of life itself. However, like any cutting-edge technology, cryo-EM presents its fair share of challenges. One of the primary obstacles lies in the preparation of high-quality cryo-samples. It has been observed that the majority of sample particles tends to adsorb to the air-water interface (AWI). This unfortunate occurrence leads to undesirable effects such as preferred orientations, aggregation, disassembly, and even denaturation of proteins, ultimately impeding the achievement of high-resolution structural determination. To surmount these challenges, various approaches have been explored, including the utilization of detergents, the application of graphene materials to coat grids, the implementation of rapid plunge-freezing robots, and tilted image collection. While these methods have shown promise, they often necessitate specialized equipment or intricate techniques, thereby limiting their widespread application and accessibility.
Our laboratory is actively engaged in the development of innovative methods to facilitate structural determination using single-particle cryo-EM. One of our ongoing endeavors involves the exploration of a novel and straightforward technique that utilizes Metallo-Supramolecular Branched Polymer (MSBP) during cryo-sample preparation (Xu et al, 2024). MSBP is a supramolecular polymer created by functionalizing the ends of polyethylene glycol (PEG) chains with bispyridyl ligands and incorporating palladium ions (Pd2+). We have successfully demonstrated that the incorporation of MSBP in cryo-sample preparation aids in keeping particles away from the air-water interface (AWI). As a result, it effectively mitigates issues associated with the AWI, such as the preferred orientation problem. Moreover, the simplicity and ease of implementation of MSBP make it accessible to the broader scientific community.
Structural studies of membrane proteins heavily rely on solubilization by detergents, which may not reflect their native states in the cellular context. Additionally, the process of identifying suitable detergents for individual membrane proteins is time-consuming and labor-intensive. We have recently developed a vesicle-based method that allows for the direct determination of membrane protein structures within their native lipid environment (Liu et al., 2023). By employing this approach, we successfully isolated vesicles containing a bacterial multidrug efflux transporter called AcrB and resolved its structure using cryo-electron microscopy. Our method offers a promising and straightforward approach for studying the structure and function of membrane proteins in their natural environment, eliminating the need for detergent screening and protein purification.
Other Efforts
Furthermore, we have made significant contributions to numerous collaborative projects, leveraging our expertise in the fields of structural biology and biochemistry.
Amidst the SARS-CoV-2 pandemic, we actively participated in establishing a local platform dedicated to vaccine identification and enhancement. This collaborative endeavor involved the lab of Zhiwei Chen at HKUMed. Our research focused on unraveling the molecular mechanisms underlying the interaction between neutralizing antibodies and the spike protein of SARS-CoV-2. Through our investigations, we have provided valuable insights into the neutralizing antibodies and their potential for optimizing and developing vaccines against a broad range of SARS-CoV-2 variants (Zhou et al., 2022; Luo et al., 2023).
In collaboration with the lab of Qinglu Zeng, we reported the cryo-EM structure of the cyanophage P-SCSP1u, a representative member of the MPP-C phages, in its native form at near-atomic resolution (Cai et al, 2023). Our study provides a detailed understanding of the assembly process of the capsid and the intricate molecular interactions within the portal-tail complex. By comparing the capsid proteins of P-SCSP1u with those of other podoviruses with known structures, we have gained valuable insights into the evolutionary aspects of T7-like viruses. Additionally, our study presents the near-atomic resolution structure of the portal-tail complex for T7-like viruses. Drawing upon previously reported structures of phage T7, we have identified an additional valve and gate, significantly contributing to the understanding of the DNA gating mechanism in T7-like viruses.