About the Laboratory of Structural Biology

In 1996, the Tsinghua University Laboratory of Structural Biology was established in a 25 square metre room in the basement of the old Biology building. In 1997, the laboratory completed the purchase of the main scientific equipment and instruments, and at the beginning of 1998 started initial work. In June 1999, the laboratory moved into the newly completed Life Sciences building, and comprehensive research work started from then on.

 

"10 years to sharpen a sword".

 

"Begin a difficult undertaking".

 

The period 1999-2000 was the most arduous period for our research group. The group was constructed, tasks were condensed and grants were applied for. The teachers and students begin an undertaking together, and this period was the most painful with not too many research papers, since our manuscripts were time and time again rejected by top scientific journals.

 

At the end of 1999, our group joined with other related laboratories to successfully apply for a national "973" project titled "Principle research of protein three-dimensional structure, function and fold". This was the first significant national project undertaken by the laboratory, and simultaneously it poured new life into the construction and development of our country's few structural biology research groups.

 

In 2000, we first used the multi-wavelength anomalous diffraction (MAD) technique to determine the crystal structure of cephalosporin acylase. The successful determination of this structure by our laboratory indicated that our technology platforms for molecular biology, protein chemistry, X-ray diffraction and protein structure analysis were built and completed. This structure was chosen by renowned artist Mr. Wu Guanzhong as the source material for a large-scale sculpture he created entitled "Desire of Life", which was displayed at the National Science and Art Exhibition.

 

"The scientific research begins to accumulate".

 

2001 was the first year of our "15+" national science and technology program, and our country began to plan the start of a Structural Genomics initiative. Our research group began to take advantage of opportunity and obtained some preliminary payback in return for several years of hard work and struggle. In 2001, the laboratory published 11 research papers, including 10 in SCI indexed journals. While Tsinghua University joyfully celebrated its 90th anniversary, the laboratory welcomed Vice-Premier Li Lanqing, the three Nobel laureates Yang Zhenning, Li Zhengdao and Ding Zhaozhong, as well as the main contributor to the national "A bomb, H bomb and satellite" project. This was also a key year in which the work of the research group began to take course.

 

In 2002, the laboratory took a big leap in the quantity and quality of its research papers. The paper published in J. Biol. Chem. was the first of our results to be published in international journals with higher impact factors. For the whole year, a total of 28 papers were published in SCI journals and 25 protein crystal structures were deposited in the Protein Data Bank (PDB). A comparatively high-level research group began to form, and the research direction of the laboratory moved further towards a main focus on crystal structures of proteins related to human health, human disease or with important physiological functions.

 

In 2003, I applied for the position of Director of the Institute of Biophysics (IBP), Chinese Academy of Sciences, and for this reason our research work certainly has not suffered. Using the strengths and advantages of the Institute of Biophysics to supplement the Laboratory of Structural Biology, our research group further developed into the "Tsinghua-IBP Joint Research Group for Structural Biology" (hereafter referred to as Joint Research Group), to integrate superior resources and combine efforts in order to tackle key problems. 2003 was also the year of the SARS outbreak, and our research group took the lead in determining the first crystal structure of any protein from the SARS coronavirus in the world, namely the main protease (3CLpro or Mpro), together with its complex structure with an inhibitor. This work revealed the precise binding mode of inhibitors to the Mpro, and showed that similar kinds of inhibitors can have different binding modes. During the SARS outbreak, this was a significant achievement in SARS basic research as well as for SARS anti-viral drug design. The results of this work were published in Proc. Natl. Acad. Sci. USA, and were also reported in Science as a news focus. In the two years since it was published, the paper has been cited more than 80 times and has had tremendous influence among our international colleagues. In another significant research achievement for our laboratory, we determined the crystal structure of human FcalphaRI (CD89), the receptor specific for IgA, which was published in J. Biol. Chem. as a cover article. The laboratory published 35 SCI papers in 2003, in journals such as Proc. Natl. Acad. Sci. USA., J. Biol. Chem., J. Am. Chem. Soc., J. Mol. Biol. and so on, with an obvious increase in the quality of our publications. The research ability of our laboratory and our research level in the international structural biology field were clearly just emerging.

 

In 2004, although the quantity of the papers from our laboratory was reduced, we published 25 SCI papers in international academic journals and the overall quality of our papers steadily increased. The main research directions of our laboratory were unified and the research group was centered on the three following main aspects, for which we had already achieved a number of encouraging results:

 

1. The cloning and expression of human liver related genes. We analyzed a number of important protein crystal structures of liver proteins and their complexes, including CD89, Pirin, FKBP52, CLP, Dok1 and Dok5, and published the results in high level papers in international academic journals including Proc Natl Acad Sci USA., J. Biol. Chem., J Mol Biol. and so on.

 

2. The continuing development of systematic research on SARS coronavirus (SARS-CoV) protein structure and function. On the one hand, we determined the crystal structures of the SARS-CoV and MHV S protein fusion cores. The structure demonstrated that this fusion core is composed of a 6 helix bundle, with the MHV S protein fusion core unusually similar to that of SARS-CoV, indicating that the coronavirus membrane protein fusion process is highly conserved. Through a series of biochemical and biophysical experiments, we characterized the interaction between HR1 and HR2 peptides, thus enabling the design of fusion inhibitor peptides and opening an important new avenue towards the discovery of anti-SARS therapeutics. This series of results was published in 6 papers in J. Biol. Chem., Biochemistry (US), and so on. On the other hand, based on the crystal structure of the SARS-CoV main protease and its complex with an inhibitor, the group proposed a bold plan against coronavirus-related diseases, with the expected development of a wide-spectrum inhibitor targeting coronavirus Mpro. In addition, we also launched a team to work on the crystal structures of SARS-CoV non-structural proteins involved in viral replication. Our research group had already formed a unique system, and by this stage had become one of the most active research teams in the international study of SARS-CoV protein structure and function.

 

3. Our research group developed the new task of research and exploration of membrane protein structure and function, and made unprecedented progress in protein extraction and separation, purification, crystallization and so on.

 

At the same time, the laboratory opened up constant channels of cooperation and exchange, strengthening and reinforcing the cooperation and exchange with domestic and foreign scientific colleagues. The laboratory has established good relations with a succession of related domestic and foreign scientific research institutions for the mutual exchange of personnel, the continuous exchange of the newest research techniques and technologies, and the establishment of co-operative research projects.

 

After successfully hosting four sessions of the annual "Tsinghua International Conference of Protein Science (TICPS)", as well as successfully organizing the 10th International Conference on the Crystallization of Biomacromolecules (ICCBM10), the Laboratory of Structural Biology had gradually established a certain influence as a protein science research base, both in China and overseas. The research group developed a higher standard of research and was steadily becoming one of the most productive laboratories in the international field of protein science research.

 

2005 was a magnificent year for scientific research achievements by our group, and we obtained a series of major innovative research results. 23 SCI papers were published in internationally renowned academic journals including Cell, Nature Structural & Molecular Biology, PLoS Biology and so on. There was a huge leap in the level of publications and the quality of papers, with our average impact factor reaching 6.730. The significant research results are as follows:

 

1. Our research group was the first in the world to determine the crystal structure of the mitochondrial respiratory membrane protein Complex II, comprised of four protein subunits. Simultaneously, we also determined the membrane protein structure of the membrane protein in complex with the inhibitors 3-nitropropionate (NPA) and 2-thenoyltrifluoroacetone (TTFA). Based on the structure, we have detailed its function and biological mechanism as well as its role in mitochondrial diseases related to defects in Complex II, since the structure provides a bona fide model for the study of diseases such as familial pheochromocytoma, familial head and neck paraganglioma, and Leigh syndrome. These research results have filled an important gap in the fields of structural biology and cell biology, and have corrected and clarified an important question in textbooks about the distribution of Complex II in the inner membrane. The work, published in Cell, is a new milestone in research of the mitochondrial respiratory electron transfer chain. Incidentally, this was the first time since 1980 that mainland Chinese scientists based natively in China have published their research in the journal Cell.

 

2. Based on our crystal structure of the SARS-CoV main protease and its complex with an inhibitor, and after more than two years of unrelenting efforts, we adopted a structure-based drug design strategy together with X-ray crystallography to develop a wide-spectrum inhibitor targeting all coronavirus. At present, there are approximately 26 known types of coronavirus, which are responsible for human diseases such as SARS, the common cold, pneumonia and bronchiolitis, and which cause significant economic losses to the animal industry through virulent and often fatal diseases. Therefore, this achievement will have important benefits for the prevention and control of coronavirus-related diseases. This September, the related paper was published in PLoS Biology, who also issued a news article titled "Casting a wide net to fight coronaviruses" to the international media. This work was also reported and highly appraised by the journal Nature under the title "Virology: Crowning achievement" in their Research Highlights column. The successful design of a wide-spectrum anti-coronavirus inhibitor has latent clinical significance, and could be used as a strategy for research into the design of antiviral drugs against "bird flu".

 

3. In relation to SARS viral replication, we have determined the crystal structure of the hexadecameric super-complex assembly formed by the non-structural proteins nsp7 and nsp8. The super-complex assembly adopts a hollow cylinder conformation with four handles. The internal diameter of the cylinder is approximately 30 A and its external diameter is approximately 90 Angstrom (or 110 Angstrom if calculated including the handles). The complex is formed by 8 copies of nsp7 and 8 copies of nsp8, with nsp8 existing simultaneously in two different conformations. The outer surface of the super-complex is covered in negative charge while the surface of the internal channel is coated in positive charge, which is advantageous for nucleic acid binding. Mutagenesis experiments confirmed that the nsp7-nsp8 super-complex has strong nucleic acid binding activity, and the structure strongly suggests it might function as a processivity factor for the RNA-dependent RNA polymerase. Our research work is the first to reveal the interactions between SARS-CoV non-structural proteins, and has provided a first glimpse of the coronavirus replication machinery at the atomic level. This achievement was published in October as an article by Nature Structural and Molecular Biology, and has had an important influence on our international colleagues.

 

In view of the international influence of the results obtained by our laboratory in SARS research, Current Opinion in Structural Biology and Current Pharmaceutical Design separately invited me to write review articles on SARS protein structure, function and drug design.

 

2006 was a turning point for me and my research group. In May, I was appointed as President of Nankai University by the Central Party Committee and the State Council. I began to study to become a "proletariat statesman and educationist" and diligently fulfill my presidential responsibilities. My research work also became more of an extra-curricular hobby in nature. At present, our research group is focused on the structure-function study f important virus and tumor-related proteins, as well as structure-based drug design.

 

In 2006, as part of our program of research on SARS coronavirus protein structure, function and drug design, we continued to analyze the SARS coronavirus nsp5 and nsp7/nsp8 structures, as well as the SARS coronavirus S protein fusion core, and their complex crystal structures. We also successfully determined and published the crystal structure of nsp10, which adds a new type of fold to the protein structure family. Each monomer includes two zinc-fingers, and ten molecules assemble to form a spherical decamer. We also determined the crystal structure of MHV nsp15 in the active hexameric form. Our structure was the first uridylate specific endoribonuclease and has a novel protein fold. These two studies provide new structural information for the design of anti-coronavirus drugs and were published simultaneously in the Journal of Virology.

 

At present, in the structure-function studies of the 16 non-structural proteins involved in replication/transcription of the SARS coronavirus, 11 structures of full-length proteins, domains or complex structures have been determined to date. Among these, our group has determined six: nsp5 (Mpro), nsp7/nsp8, nsp10, nsp15 (MHV) as well as the SARS-CoV spike (S) protein fusion core protein. Moreover, five structures (nsp1, nsp9, nsp3 PLpro domain, nsp3 ADRP domain and the S protein RBD-receptor complex) have been determined by other research groups. In light of these achievements, I was invited by Current Opinion in Structural Biology and Current Pharmaceutical Design to contribute review articles summarizing the structure, function and drug design of SARS coronavirus proteins. I was also invited to contribute a chapter in the book "Anna Lindh Programme on Conflict Prevention: Health and Conflict Prevention" with the topic: " Combating SARS in China: Experiences and Efforts to Coordinate Future Prevention"

 

In addition:

 

We reported two crystal structures in J. Mol. Biol. First, we published the crystal structure of human pyrroline-5-carboxylate reductase in both the native state and in complex with NAD(P)H and elucidated its mechanism of oxidation. This enzyme has been implicated in the oxidation of the anti-tumor drug thioproline. Second, we reported the crystal structure of human glycerol 3-phosphate dehydrogenase 1 and proposed its catalytic mechanism. We also used the glycerol 3-phosphate dehydrogenase 1 structure to explain the inhibitory effects of sulfate, as well as revealing the inhibitory effects of zinc ions.

In Proc. Natl. Acad. Sci. USA, we reported the crystal structure of mouse testicular cytochrome c and its apoptotic activity, and revealed the evolutionary goal of T-Cc.

 

In J. Biol. Chem, we published the crystal structure of human Spindlin 1, which is involved in cell-cycle regulation and consists of three tandem Tudor-like domains. We also reported the crystal structure of the V domain of the human nectin-like protein Necl-1, a neural tissue-specific immunoglobulin-like cell-cell adhesion molecule, and used it as a basis to propose a model for homophilic adhesion of Necl-1 at synapses. We published the 1.8 ? crystal structure of the srGAP1-SH3 domain, which is involved in axon guidance and cell migration. This was the first structure to be determined from the Slit-Robo signaling pathway. Based on the structure and BIAcore experiments, we provided an important structural basis for the srGAP-Robo interaction. Finally, we reported the crystal structure of human cysteine dioxygenase and its complex structure with the cysteine substrate. Seven point mutants of human cysteine dioxygenase were constructed in order to research the enzyme activity and metal content. Our results provide an insight into a new mechanism of cysteine thiol dioxygenation catalyzed by cysteine dioxygenase, which is tightly associated with a thioether-bonded tyrosine-cysteine cofactor, and lay a foundation for drug design targeting diseases related to human cysteine dioxygenase, including neuro-degenerative and autoimmune diseases.

 

In 2006 we strengthened cooperation between domestic and foreign colleagues. Over the course of the year, we supported 23 students to travel to England, Germany, Japan, Hong Kong and Taiwan for academic exchange and cooperation. In October 22-26 of this year, I was the conference chair and our research group was one of the main organizers of the 4th International Conference on Structural Genomics (ICSG 2006), which was successfully held in Beijing and focused on Structural Genomics and structural biology. A prestigious meeting organized bythe International Structural Genomics Organization (ISGO), the number of participants at ICSG 2006 exceeded 520 with 290 of those registered coming from overseas, making this the largest ICSG meeting to date.

 

I would like to thank my colleagues in our research group for their unremitting hard work and endeavor over the last 10 years. I would like to thank the Ministry of Science and Technology, the Chinese Academy of Sciences, the Ministry of Education and the National Natural Science Foundation of China, as well as their respective leaders, for their continued vital support. I would also like to give my heartfelt thanks to our domestic and foreign collaborators for their cooperation and support.

 

Very best wishes to everybody for a successful 2007!

 

Zihe Rao

2006.12.20, Nankai University