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Research Interests

  1. Structure determination of proteins by NMR spectroscopy and X-ray crystallography
  2. Protein engineering and design
  3. Simulation and modeling of protein


Research Projects

  1. Structure basis of thermostability of proteins
  2. Structure-function studies of acidic ribosomal proteins
  3. How H. pylori urease matures?
  4. Substrate specificity and inhibitor design for 3C-like protease of Coronavirus

Structure basis of thermostability of proteins

Understanding the 'rules' of structural adaptation of how proteins remain stable and active at high temperatures is not only of great academic interests but also has potential applications in biotechnology. For example, engineering of thermostable industrial enzymes offers the benefits of increased rate of chemical reactions at higher temperatures. At high temperatures, inactivation of proteins is often caused by unfolding, which exposes the polypeptide chains to various irreversible processes (e.g. chemical modification and aggregation).  Proteins from thermophilic organisms, which adapt to grow at elevated temperatures (55-110ºC), are excellent models for this kind of studies. Although some intracellular factors are reported to stabilize protein in vivo, most thermophilic proteins are intrinsically more stable at high temperatures. Learning how these thermophilic proteins achieve thermostability will provide rules for rational design of thermostable proteins.

In our laboratory, we are currently working on two model proteins to study the structural basis of protein thermostability:
(1) Ribosomal protein L30e from Thermococcus celer
l30e.gif
(2) Acylphosphatase from Pyrococcus horikoshii
acp.gif

Collaborators

Mark Bycroft, Centre for Protein Engineering, MRC, Cambridge, UK
George Makhatdze, Penn. State University, USA
Kong-Hung Sze, University of Hong Kong

Selected Publications
  • Chan, S.H., Wilbanks, C.C., Makhatadze, G.I., Wong, K.B. (2012) Electrostatic Contribution of Surface Charge Residues to the Stability of a Thermophilic Protein: Benchmarking Experimental and Predicted pKa Values, PLoS One 7, e30296.
  • Lam, S. Y., Yeung, R. C. Y., Yu, T.-H., Sze, K.-H., and Wong, K.-B. (2011) A Rigidifying Salt-Bridge Favors the Activity of Thermophilic Enzyme at High Temperatures at the Expense of Low-Temperature Activity, PLoS Biology 9, e1001027.
  • Chan C.H., Yu, T.H., Wong, K.B. (2011) Stabilizing salt-bridge enhances protein thermostability by reducing the heat capacity change of unfolding. PLoS One 6, e21624.
  • Lee CF, Makhatadze GI, Wong KB: Effects of charge-to-alanine substitutions on the stability of ribosomal protein L30e from Thermococcus celer. Biochemistry 2005, 44:16817-16825.
  • Lee CF, Allen MD, Bycroft M, Wong KB: Electrostatic interactions contribute to reduced heat capacity change of unfolding in a thermophilic ribosomal protein L30e. J Mol Biol 2005, 348:419-431.
  • Cheung YY, Lam SY, Chu WK, Allen MD, Bycroft M, Wong KB: Crystal structure of a hyperthermophilic archaeal acylphosphatase from Pyrococcus horikoshii--structural insights into enzymatic catalysis, thermostability, and dimerization. Biochemistry 2005, 44:4601-4611.
  • Wong KB, Lee CF, Chan SH, Leung TY, Chen YW, Bycroft M: Solution structure and thermal stability of ribosomal protein L30e from hyperthermophilic archaeon Thermococcus celer. Protein Sci 2003, 12:1483-1495.
  • Chen YW, Bycroft M, Wong KB: Crystal structure of ribosomal protein L30e from the extreme thermophile Thermococcus celer: thermal stability and RNA binding. Biochemistry 2003, 42:2857-2865.
Press release

Structure-functions of ribosomal stalk proteins and its interactions with ribosomal toxin

Acidic ribosomal proteins, P0, P1, and P2, are the constituent of the P-complex that forms the stalk of eukaryotic ribosome, which involves in binding of translation factors and their activation by GTP-hydrolysis. The ribosomal stalk proteins are also involved in interacting with ribosome-inactivating proteins.  We are interested to determine the structure of acidic protein proteins, which will complement recent structural studies of prokaryotic and archaeal ribosomes, and will contribute to a better understanding of structure-function of eukaryotic ribosome.

(1) Solution structure of the N-terminal dimerization domain of P2 (Lee et al., 2010)
P2.jpg


(2) Crystal structure of C-terminal conserved region of P2 in complex with trichosanthin (Too et al., 2009)
TCS/P2 complex

Collaborators

Pang-Chui Shaw, Chinese University of Hong Kong
Guang Zhu, Hong Kong University of Science and Technology
Kong-Hung Sze, University of Hong Kong


Selected Publication
  • Lee KM, Yusa K, Chu LO, Yu, CWH, Oono M, Miyoshi T, Ito K, Shaw PC, Wong KB, Uchiumi T. (2013) Solution structure of human P1-P2 heterodimer provides insights into the role of eukaryotic stalk in recruiting ribosome-inactivating protein trichosanthin to the ribosome. Nucleic Acids Res, 41, 8776-8787
  • Lee, K.M., Yu, W.H., Chiu, Y.H., Sze, K.H., Shaw, P.C., Wong, K.B. (2012) Solution structure of the dimerization domain of the eukaryotic stalk P1/P2 complex reveals the structural organization of eukaryotic stalk complex, Nucleic Acids Res 40:3172-82
  • Lee KM, Yu CW, Chan DS, Chiu TY, Zhu G, Sze KH, Shaw PC, Wong KB: Solution structure of the dimerization domain of ribosomal protein P2 provides insights for the structural organization of eukaryotic stalk. Nucleic Acids Res 2010, 38:5206-5216.
  • Law SK, Wang RR, Mak AN, Wong KB, Zheng YT, Shaw PC: A switch-on mechanism to activate maize ribosome-inactivating protein for targeting HIV-infected cells. Nucleic Acids Res 2010, 38:6803-6812.
  • Too PH, Ma MK, Mak AN, Wong YT, Tung CK, Zhu G, Au SW, Wong KB, Shaw PC: The C-terminal fragment of the ribosomal P protein complexed to trichosanthin reveals the interaction between the ribosome-inactivating protein and the ribosome. Nucleic Acids Res 2009, 37:602-610.
  • Mak AN, Wong YT, An YJ, Cha SS, Sze KH, Au SW, Wong KB, Shaw PC: Structure-function study of maize ribosome-inactivating protein: implications for the internal inactivation region and the sole glutamate in the active site. Nucleic Acids Res 2007, 35:6259-6267.
  • Chan DS, Chu LO, Lee KM, Too PH, Ma KW, Sze KH, Zhu G, Shaw PC, Wong KB: Interaction between trichosanthin, a ribosome-inactivating protein, and the ribosomal stalk protein P2 by chemical shift perturbation and mutagenesis analyses. Nucleic Acids Res 2007, 35:1660-1672.

How H. pylori urease matures?

Helicobacter pylori is the only bacterium known to thrive in the human stomach. It damages the mucous coating of the gut, allowing stomach acid to eat away the sensitive organ lining and causing ulcers. H. pylori produces urease to spur the breakdown of urea, a naturally occurring chemical in the body, so that urea can release ammonia to neutralize the acidic environment in the gut, allowing pathogens to thrive. Unlike most other enzymes, urease does not work immediately after being produced by the bacterium. Two nickel ions have to be delivered to activate it. Our work focuses on four urease maturation factors, UreE, UreF, UreH, UreG that help the activation of urease: UreE, UreF, UreG and UreH.

Crystal structure of urease maturation factors UreF/UreH complex (Fong et al., 2011)

urefh.gif

Collaborators
Yu Wai Chen, King's College London
HZ Sun, University of Hong Kong

Selected Publication
  • Fong, Y.H., Wong, H.C., Chuck, C.P., Chen, Y.W., Sun, H., Wong, K.B. (2011) Assembly of the preactivation complex for urease maturation in Helicobacter pylori: Crystal Structure of the UreF/UreH complex, J. Biol. Chem. 286, 43241. (selected as paper of the week)
  • Fong YH, Wong HC, Yeun MH, Lau PH, Chen YW, Wong KB. (2013) Structure of UreG/UreF/UreH complex reveals how urease accessory proteins facilitate maturation of Helicobacter pylori urease. PLoS Biol, 11, e1001678.
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Press release
 

Substrate specificity and inhibitor design for 3C-like protease of Coronavirus

Coronavirus (CoV) infections cause many respiratory tract diseases like common cold, bronchiolitis and pneumonia in human. The most serious outbreak was caused by the severe acute respiraory syndrome (SARS) virus in 2003 that has resulted in a death toll of more than 700 world-wide. Although the SARS outbreak was brought under control, the high mutation and recombination rate, and a history of interspecies transmission suggest that coronavirus infections remain a potential threat to public health. As there is no approved drug currently available for CoV infections, we see there is a need to develop novel drug to combat against future outbreak of CoV infection. The 3C-like protease, which is present in all CoV and play vital role in viral replication, is an ideal drug target for this purpose. Our group is interested in profiling of the substrate specificity and in a rational design of peptidomimetic inhibitors for the 3C-like protease.

Histidine is allowed at P1 position of 3C-like protease of SARS-CoV (Chuck CP et al., 2010)

sars.jpg


Collaborators

Hak-fun Chow, Chinese University of Hong Kong
David C.C. Wan, Chinese University of Hong Kong

Selected Publication
  • Chuck CP, Chen C, Ke Z, Wan DC, Chow HF, Wong KB. (2013) Design, synthesis and crystallographic analysis of nitrile-based broad-spectrum peptidomimetic inhibitors for coronavirus 3C-like proteases. Eur J Med Chem, 59:1-6.
  • Chuck, C.P., Chow, H.F., Wan, C.C., Wong, K.B. (2011) Profiling of Substrate Specificities of 3C-Like Proteases from Group 1, 2a, 2b, and 3 Coronaviruses, PLoS One, 6, e27228
  • Chuck CP, Chong LT, Chen C, Chow HF, Wan DC, Wong KB: Profiling of substrate specificity of SARS-CoV 3CL. PLoS One 2010, 5:e13197.