Our research aims to understand the functional mechanisms of selected membrane proteins and large protein complexes through molecular structure determination by x-ray crystallography and functional characterization utilizing various biochemical and cellular techniques.
Some of our ongoing projects include water channels, FACE-1 protease, g-secretase complex, and tripeptidyl peptidase II (TPP II). As functional defects of these proteins have been associated with medical disorders such as nephrogenic diabetes insipidus. progeria, Alzheminer’s disease and obesity, understanding the molecular structures and functional understanding of these proteins at a molecular level of detail should help in the development of novel therapeutics.
We discovered that CD147 is a subunit of the g-secretase complex in its native state and plays a regulatory role in the production of amyloid b-peptides and in the processing of Notch - removal of CD147 from the g-secretase complex results in an increase in amyloid b-peptide production and a decrease in Notch cleavage. To elucidate the regulatory role of CD147 in g-secretase activity, we are determining the critical residues of CD147 involved in the regulation of g-secretase activity, utilizing CD147 wild-type and various mutant forms expressed in stable CD147-knockout HEK293 cells. In addition, we are continuing our efforts in the structure determination of g-sceretase complex in order to provide a structural basis for understanding the functional mechanism of this clinically important enzyme.
Structure determination of mammalian membrane proteins requires the availability of milligram quantities of purified proteins. To obtain such quantities, we have successfully developed a protocol for the overexpression of eukaryotic membrane proteins, FACE-1 protease and AQP6 water channel, in an E. coli overexpression system that allows for the purification of wild-type and mutant proteins for functional characterization, crystallization trials and structure determination by x-ray crystallography. Also, we are currently developing a mammalian cell expression system for the overexpression of g-secretase core complex by co-expression of the complex subunits for subsequent structure determination.
We have recently determined the structure of TPP II, a large protein complex of molecular weight ~6 MDa, by the combined use of x-ray crystallography and electron microscopy. The structure has identified the residues forming the active site and substrate-binding pocket as well as pathways leading to the active site; this information has begun to provide insight into how the substrate processing occurs and provides a structural framework for the design of new drugs for TPP II associated medical disorders.
Sampling of structures determined by our group:
Proteasome and AQP1 water channel (top row, from left to right),
bc1 complex and TPP II (bottom row, from left to right).
S. Zhou, H. Zhou, P.J. Walian and B.K. Jap. Regulation of g-Secretase in Alzheimer’s Disease. Biochemistry (Current Topics/Perspectives), 46, 2553-2563, 2007.
B.-G. Han, A.B. Guliaev, P.J. Walian and B.K. Jap. Water Transport in AQP0 Aquaporin: Molecular Dynamics Studies. J. Mol. Biol. 360, 285-296, 2006.
S. Zhou, H. Zhou, P.J. Walian and B.K. Jap. The Discovery and Role of CD147 as a Subunit of g-Secretase. Drug News and Perspectives 19,133-138, 2006.
S. Zhou, H. Zhou, P.J. Walian and B.K. Jap. CD147 is a Regulatory Subunit of the g-Secretase Complex in Alzheimer’s Disease Amyloid b-peptide Production. Proc. Natl. Acad. Sci. USA, 102, 7499-7504, 2005.
Y.D. Kwon, I. Nagy, P.D. Adams, W. Baumeister and B.K. Jap. Crystal Structures of the Rhodococcus Proteasomes with and without Its Pro-peptide: Implications for the Role of the Pro-peptide in Proteasome Assembly. J. Mol. Biol. 335, 233-245, 2004.
H. Sui, B.-G. Han, J.K. Lee, P.J. Walian and B.K. Jap. Structural Basis of Water Specific Transport through AQP1 Water Channel. Nature 414, 872-878., 2001.
S. Iwata, J. W. Lee, K. Okada, J.K. Lee, M. Iwata, B. Rasmussen, T. A. Link, S. Ramaswamy and B. K. Jap. Complete Structure of the 11-Subunit Bovine Mitochondrial Cytochrome bc1 Complex. Science 281, 64-71, 1998.
J. Löwe, D. Stock, B. Jap, P. Zwickl, W. Baumeister and R. Huber. The Crystal Structure of the 20S Proteasome at 3.4Å Resolution. Science 268, 533-539, 1995.