The Laboratory of Structural Biology in Tsinghua University focuses on systematic and thorough research of the relationship between 3-D protein structure and function, based on molecular biology, protein biochemistry and X-ray crystallography. We not only study the relation between structure and function, but also try to provide a structural basis for designing potential human disease-related drugs and to find valuable mutants, complexes or drug candidates (leading compounds) using protein engineering, cell biology and other biological techniques.
Our research is mainly focused in four main areas:
1. SARS coronavirus basic research
The 2003 global outbreak of severe acute respiratory syndrome (SARS) was caused by a newly identified virus, called the SARS coronavirus (SARS-CoV). As with many other laboratories in China, Zihe Rao devoted all of his efforts to basic research of the SARS coronavirus. Adopting a “structural proteomics” approach, his group systematically expressed and purified 48 proteins or domains encoded by the SARS coronavirus in order to study their three-dimensional structures, to elucidate their structure-function relationships, and to provide essential information for the design of anti-SARS therapeutics and vaccines. Rao’s group continues to be very active in this area.
2. Influenza research
The recent emergence of highly pathogenic avian influenza H5N1 viruses poses a significant global threat to human health, with more than 400 human cases and 256 fatalities reported worldwide since 2003. Currently approved influenza drugs may have limited effectiveness in the event of an influenza pandemic due to the emergence of resistant strains of H5N1 subtype influenza viruses. Zihe Rao’s group is therefore active in elucidating the underlying mechanisms of the virus life cycle, with a view towards the development of new approaches and drugs for anti-viral therapy.
2.1 Crystal structure of the polymerase PAC-PB1N complex from an avian influenza H5N1 virus
Zihe Rao’s group determined the structure of an important component of the influenza virus polymerase heterotrimer (PA, PB1, PB2). His group was first to determine the crystal structure of the PA protein C-terminal domain (PAC) in complex with a N-terminal peptide fragment of PB1. The peptide, corresponding to the N-terminal 15 amino acids of PB1 (PB1N), binds to a highly conserved site on PAC and has been shown to inhibit the polymerase function by blocking assembly of the heterotrimer. The PAC-PB1N complex structure, reported in Nature, is therefore is a major target for the discovery of novel anti-influenza drugs (1).
2.2 Crystal structure of an avian influenza polymerase PAN reveals an endonuclease active site
Zihe Rao’s group determined the crystal structure of the N-terminal domain of the polymerase PA subunit, revealing it to have endonuclease activity and thus resolving a controversy about its location in the influenza virus polymerase complex. Inhibitors of the endonuclease activity by the polymerase complex have been shown to abolish viral replication in cell culture. This work has been published in Nature (2). His group went on to solve a series of crystal structures of PAN in complex with nucleotides (3). These structures should therefore accelerate the development of lead compounds as a prelude to novel anti-influenza drugs.
3. EV71 research: A sensor/adaptor mechanism for enterovirus uncoating from structures of EV71
Enteroviruses cause diseases in mammals and some 66 types infect humans. Enterovirus 71 (EV71), a major agent of hand-foot-and-mouth disease in children, can cause severe central nervous system disease and mortality. At present no vaccine or antiviral therapy is available. Zihe Rao’s group has determined highresolution structures for the mature virus and immature particles lacking RNA. Whilst the mature virus is mostly similar to other enteroviruses, the empty particles are dramatically expanded, with notable fissures, and resemble elusive enterovirus uncoating intermediates previously only characterized at low resolution. Hydrophobic capsid pockets are collapsed in the expanded particle, providing a detailed explanation of the molecular mechanism for receptorbinding triggered enterovirus uncoating. The results indicate that a portion of the enterovirus VP1 GH loop acts as an adaptor-sensor for the attachment of a range of receptors, converting heterologous inputs to a generic uncoating mechanism. This mechanism spotlights novel points for therapeutic intervention(4).
4. AIDS research: Crystal structure of the SIV matrix antigen and implications for virus assembly
AIDS, caused by the human immunodeficiency virus (HIV), has already claimed more than 20 million lives worldwide and is one of the greatest threats to human health in the 21st century. Structural studies of HIV and the closely related simian immunodeficiency virus (SIV) have greatly increased our understanding of the HIV life cycle and led to the development of antiretroviral drugs. In 1995, while working as a senior research scientist in Oxford University, Zihe Rao determined the first three-dimensional structure of the SIV matrix antigen (MA) (5), which shares 50% sequence identity with the HIV MA. MA is a component of Pr55Gag, the sole protein required for assembly of the virion shell. MA targets Pr55 to the plasma membrane, and facilitates incorporation of the virus envelope protein and assembly of the Pr55Gag shell. Cleavage of Pr55 by the viral protease produces the mature protein of relative molecular mass 17-18K, which underlies the host-derived membrane and is important in both virus entry and nuclear localization of the virion core. Zihe Rao’s structure, published in Nature (5), led to the first mechanism of virus assembly for HIV and other lentiviruses. This mechanism of virus assembly is now widely accepted by scientists all over the world. Rao’s group has continued to work on another important HIV protein, the Virion Infectivity Factor (Vif). Its precise function is still being studied, but naturally occurring HIV-1 strains with mutations in Vif are known to replicate at significantly lower levels compared to the wild-type virus.
5. Crystal structure of the mitochondrial respiratory membrane protein Complex II
Zihe Rao’s group was first to successfully determine the three-dimensional structure of the mitochondrial respiratory membrane protein Complex II (6), thus expanding the research fields of mitochondrial structural biology and cell biology. It is an important achievement which should have significant impacts in life science and medical research, since mutations in Complex II are implicated in a number of mitochondrial diseases including familial or non-familial head and neck paraganglioma, familial or non-familial pheochromocytoma, midgut carcinoid, Merkel cell carcinoma and Leigh syndrome Mitochondria, as cellular organelles, are the "energy factory" of the cell and are mainly responsible for cell aerobic respiration. They realize energy transformation through the oxidation-phosphorylation process and provide most of the energy for cell activity. The oxidation process in mitochondria is carried out by four respiratory membrane protein complexes inside the mitochondrial inner membrane (Complex I, II, III and IV). Since the 1990s, determining the structures of these four membrane protein complexes has been a major challenge and leading scientists from the United States, Japan, England and Germany have made great efforts in this field. Until recently, only scientists from the United States and Japan had been successful in determining the crystal structures of mitochondrial Complex III and Complex IV. No breakthroughs had been made on either mitochondrial Complex I or Complex II until the work by Rao’s group.
Zihe Rao and colleagues began their structural studies of the mitochondrial respiratory membrane protein Complex II in 2001 using new methods. They chose to extract and purify the membrane protein complex from porcine heart and finally determined the structure of this complex in 2005. Complex II, also known as succinate:ubiquinone oxidoreductase, is comprised of four different protein subunits: the flavoprotein (622 amino acids), iron-sulfur protein (252 amino acids), and two membrane-anchor proteins (CybL, 140 amino acids and CybS, 103 amino acids) with a total of six trans-membrane helices. The Complex II structure also includes prosthetic groups required for electron transfer from succinate to ubiquinone. The structure correlates the protein environments around prosthetic groups with their unique midpoint redox potentials. Two ubiquinone binding sites are discussed and elucidated by a complex structure with inhibitors 3-nitropropionate and 2-thenoyltrifluoroacetone (TTFA). The availability of the Complex II structure provides a bona fide model for the study of human mitochondrial diseases related to mutations in this complex and helps to complete our understanding of the mitochondrial respiratory electron transfer chain.
The structure of the mitochondrial respiratory Complex II was published in Cell (6). Incidentally, this was the first paper by Chinese scientists working entirely in mainland China to be published in Cell for 25 years.
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6. Structure and function of the EGF domain of human blood clotting factor IX
Various diverse extracellular proteins possess Ca(2+)-binding epidermal growth factor (EGF)-like domains, the function of which remains uncertain. In 1995, during his work in Oxford University, Zihe Rao determined, at high resolution (1.5 Å), the crystal structure of such a domain from human clotting factor IX, as a complex with Ca2+ . The Ca2+ ligands form a classic pentagonal bipyramid with six ligands contributed by one polypeptide chain and the seventh supplied by a neighboring EGF-like domain. The crystal structure identifies the role of Ca2+ in maintaining the conformation of the N-terminal region of the domain, but more importantly demonstrates that Ca2+ can directly mediate protein-protein contacts. The observed crystal packing of the domains provided the first plausible model for the association of multiple tandemly linked EGF-like domains in proteins such as fibrillin-1, Notch, and protein S. This model is consistent with the known functional data and suggests a general biological role for these domains. This work has been published in Cell (7).
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This area is still being updated and further details will be added later.
1. Yuan P, Bartlam M, Lou Z, Chen S, Zhou J, He X, Lv Z, Ge R, Li X, Deng T, Fodor E, Rao Z* & Liu Y*. 2009. Crystal structure of an avian influenza polymerase PA(N) reveals an endonuclease active site. Nature, 458(7240):909-13
2. He X, Zhou J, Bartlam M, Zhang R, Ma J, Lou Z, Li X, Li J, Joachimiak A., Zeng Z, Ge R, Rao Z* & Liu YF*. 2008. Crystal structure of the polymerase PA(C)-PB1(N) complex from an avian influenza H5N1 virus. Nature, 454(7208):1123-6.
3. Zhao, C., Z. Lou, Y. Guo, M. Ma, Y. Chen, S. Liang, L. Zhang, S. Chen, X. Li, Y. Liu, M. Bartlam, and Z. Rao, 2009. Nucleoside monophosphate complex structures of the endonuclease domain from the influenza virus polymerase PA subunit reveal the substrate binding site inside the catalytic center. J Virol 83: 9024-30.
4. Wang X, Peng W, Ren J, Hu Z, Xu Ji, Lou Z, Li X, Yin W, Shen X, Porta C, Walter T, Rowlands D, Wang J*, Stuart D*, Fry E*, and Rao Z*. A sensor/adaptor mechanism for enterovirus uncoating from structures of EV71. Nature Structural & Molecular Biology (in press)
5. Rao, Z., A. S. Belyaev, E. Fry, P. Roy, I. M. Jones, and D. I. Stuart. 1995. Crystal structure of SIV matrix antigen and implications for virus assembly. Nature 378:743.
6. Sun F, X. Huo, Y. Zhai, A. Wang, J. Xu, D. Su, M. Bartlam, and Z. Rao*. 2005. Crystal structure of mitochondrial respiratory membrane protein complex II. Cell 121(7):1043-57.
7. Rao, Z., P. Handford, M. Mayhew, V. Knott, G. G. Brownlee, and D. Stuart. 1995. The structure of a Ca(2+)-binding epidermal growth factor-like domain: its role in protein-protein interactions. Cell 82:131.