Michael James

Michael James

D.Phil., Oxford University

Distinguished University Professor Emeritus

Office: 780-492-4550
Lab: 780-492-2422
Fax: 780-492-0886


Understanding the biological functions of proteins, especially those of enzymes, is greatly facilitated by the knowledge of their three-dimensional structures. Our laboratory has a long-standing interest in the hydrolytic mechanisms of the serine and aspartic proteinases and their inhibitors (Maynes et al., 2005; Barrette-Ng et al., 2003a,b; Ng et al., 2000), the glycosyl hydrolases and their transition state mimics lysosomal β-hexosaminidase B (Mark et al., 2003). From these and other studies we have amassed a wealth of structural information for the interpretation of ligand binding specificity, and the chemical pathways for the hydrolysis of good substrates.  

One of the major projects in the laboratory is the structural genomics of Mycobacterium tuberculosis . In this project our laboratory has targetted ~400 ORFs from the Mtb genome; we have expressed ~200 of these proteins on a small scale and have expressed ~90 on a mg scale for purification and crystallization. We have crystallized 45 of the purified proteins and have solved the structures of 25 (for example, Maynes et al., 2003; Cherney et al., 2009). We have selected a wide range of proteins and enzymes for our studies.  Many of the first targets did not have an annotated function and our studies have contributed to the determination of their functions.  We have also concentrated on enzymes from a variety of metabolic pathways such as the enzymes of the arginine biosynthesis pathway where we have determined the structures of 6 of the key enzymes.  

Our laboratory has contributed much to the elucidation of the role of virally encoded enzymes in the maturation and infectivity of RNA viruses, such as: poliovirus, hepatitis A virus, rhinovirus 2, rabbit hemorrhagic disease virus and hepatitis C virus. We have shown that the fold of the 3C proteinases resembles that of the chymotrypsin family of serine proteinases but that the nucleophile is the sulfur atom of a cysteine residue (Bergmann et al., 1999; Bergmann et al., 2000). The 2A proteinase from Rhinovirus 2 also has a chymotrypsin-like fold and an active site cysteine but it is much smaller (140 residues) than the 3C enzymes from enteroviridae and Rhinoviridae (Petersen et al., 1999). Other targets for anti-viral compounds are the RNA dependent RNA polymerases. We have determined the structures of the RdRp from rabbit hemorrhagic disease virus (Ng et al., 2002) and from hepatitis C virus (HCV). Working in collaboration with ViroChem Pharma Inc., Laval, Quebec, we have determined the structures of ~60 inhibitors bound to HCV NS5B. These inhibitors bind to an allosteric site ~ 35 Å from the polymerase catalytic site (Wang et al., 2003; Biswal et al., 2005).  

Glycosyl hydrolases are directly linked to lysosomal storage diseases. Genetic defects in the b subunit of b hexosaminidase B are associated with Sandhoff disease whereas genetic defects in the a -subunit of Hex A are associated with Tay-Sachs disease. We have determined the structure of Hex B (the homodimer of β -subunits (Mark et al., 2003)) and recently we have determined the structure of Hex A (the heterodimer of a and β -subunits). We are collaborating with Don Mahuran of the Hospital for Sick Children on this research. Don and his group have shown that chemical chaperones (small molecule inhibitors) enhance the enzymatic activity of HexA and HexB in the lysosomes so part of our research is directed at identifying the binding sites of these molecules.  

Protein phosphatase-1 (PP-1) and protein phosphatase 2B (calcineurin) are eukaryotic serine/threonine phosphatases that share ~ 40% amino acid sequence identity in their catalytic subunits. We have determined the structures of PP-1 bound to okadaic acid (Maynes et al., 2001) and to other natural product marine toxins (Holmes et al., 2002). We have also explored the structural consequences of mutations in the β 12- β 13 loop region on the binding of okadaic acid, microcystin LR and protein phosphatase inhibitor 2 (Maynes et al., 2004). This information will assist in the design of inhibitors of calcineurin, the protein phosphatase implicated in immunosuppression for organ transplantation.  

Our laboratory has determined the 3D structure of Scytalidoglutamic peptidase (SGP), a completely new family of proteolytic enzyme, G1 from fungi (Fujinaga et al., 2004). We have shown that this family adopts a unique tertiary structure that is a new proteolytic enzyme fold. As well SGP has a unique hydrolytic mechanism for catalysis that depends on a glutamic acid carboxylate, a glutamine carboxamide and a nucleophilic water molecule. We have named this proteolytic family the EQOLISINS . From the structural work we have designed a nanomolar inhibitor of SGP and we have solved the structure of the inhibitor bound to SGP (Pillai, et al., 2007).


Selected Publications:

Pillai, B., M.M.Cherney, K. Hiraga, K Takada, K.Oda and Michael N.G.James. Crystal Structure of Scytalidoglutamic Peptidase with its First Potent Inhibitor Provides Insight into Substrate Specificity and Catalysis. J. Mol. Biol. 365:343-361(2007).
Maynes, J.T., M.M. Cherney, M.A. Qasim, M. Laskowski Jr. & M.N. James. Structure of the subtilisin Carlsberg-OMTKY3 complex reveals two different ovomucoid conformations. Acta Crystallogr D Biol Crystallogr 61:580-588 (2005).
Biswal, B.K., M.M. Cherney, M. Wang, L. Chan, C.G. Yannopoulos, D. Bilimoria, O. Nicolas, J. Bedard & M.N. James. Crystal structures of the RNA-dependent RNA polymerase genotype 2a of hepatitis C virus reveal two conformations and suggest mechanisms of inhibition by non-nucleoside inhibitors. J. Biol. Chem. 280:18202-18210 (2005).
Maynes, J.T., K.R. Perreault, M.M. Cherney, H.A. Luu, M.N.G. James & C.F.B. Holmes. Crystal structure and mutagenesis of a protein phosphatase-1:calcineurin hybrid elucidate the role of the Beta12-Beta13 loop in inhibitor binding. J. Biol. Chem. 279:43198-206 (2004).
Fujinaga, M., M.M. Cherney, H. Oyama, K. Oda & M.N.G. James. The molecular structure and catalytic mechanism of a novel carboxyl peptidase from Scytalidium lignicolum. Proc. Natl. Acad. Sci. USA 101:3364 3369 (2004).
Wang, M., K.K.S. Ng, M.M. Cherney, L. Chan, C.G. Yannopoulos, J. Bedard, N. Morin, N. Nguyen-Ba, M.H. Alaoui-Ismaili, R.C. Bethell & M.N.G. James. Non-nucleoside analogue inhibitors bind to an allosteric site on HCV NS5B polymerase. J. Biol. Chem. 278:9489-9495 (2003).
Mark, B.L., D.J. Mahuran, M.M. Cherney, D. Zhao, S. Knapp & M.N.G. James. Crystal structure of human Beta-hexosaminidase B: understanding the molecular basis of Sandhoff and Tay-Sachs disease. J. Mol. Biol. 327:1093-1109 (2003).
Barrette-Ng, I.H., K.K. Ng, M.M. Cherney, G. Pearce, C.A. Ryan & M.N. James. Structural basis of inhibition revealed by a 1:2 complex of the two-headed tomato inhibitor-II and subtilisin Carlsberg. J. Biol. Chem. 278:24062-24071 (2003).
Barrette-Ng, I.H., K.K. Ng, M.M. Cherney, G. Pearce, U. Ghani, C.A. Ryan & M.N. James. Unbound form of tomato inhibitor-II reveals interdomain flexibility and conformational variability in the reactive site loops. J. Biol. Chem. 278:31391-31400 (2003).
Ng, K.K., M.M. Cherney, A.L. Vazquez, A. Machin, J.M. Alonso, F. Parra & M.N. James. Crystal structures of active and inactive conformations of a caliciviral RNA-dependent RNA polymerase. J. Biol. Chem. 277:1381-1387 (2002).
Holmes, C.F.B., J.T. Maynes, K.R. Perreault, J.F. Dawson & M.N.G. James. Molecular enzymology underlying regulation of protein phosphatase-1 by natural toxins. Curr. Med. Chem. 9:1981-1989 (2002).
Maynes, J.T., K.S. Bateman, M.M. Cherney, A.K. Das, H.A. Luu, C.F. Holmes, M.N. James. Crystal structure of the tumor-promoter okadaic acid bound to protein phosphatase-1. J. Biol. Chem. 276:44078 44082 (2001).
Ng, K.K.S., J.F. Petersen, M.M. Cherney, C. Garen, J.J. Zalatoris, C. Rao-Naik, B.M. Dunn, M.R. Martzen, R.J. Peanasky & M.N.G. James. Structural basis for the inhibition of porcine pepsin by Ascaris pepsin inhibitor-3. Nature Structural Biology 7:653-657 (2000).
Bergmann, E.M. & M.N.G. James. The 3C Proteinases of Picornaviruses and other Positive-Sense, Single-Stranded RNA Viruses. In Proteases as Targets for Therapy (K. von der Helm, B.D. Korant & J.C. Cheronis, Eds.). Handbook of Experimental Pharmacology, Chap. 7, Vol. 140, pp. 117-143. Springer-Verlag (2000).
Bergmann, E.M., M.M. Cherney, J. McKendrick, S. Frormann, C. Luo, B.A. Malcolm, J.C. Vederas & M.N.G. James. Crystal structure of an inhibitor complex of the 3C proteinase from hepatitis A virus (HAV) and implications for the polyprotein processing in HAV. Virology 265:153-163 (1999). (PDB Accession Code: 1QA7).
Bergmann, E.M. & M.N.G. James. Proteolytic enzymes of the viruses of the family picornaviridae. In Proteases of Infectious Agents (B. Dunn, Ed.), pp. 139-163, Academic Press. (1999).
Petersen, J.F.W., M.M. Cherney, H.-D. Liebig, T. Skern, E. Kuechler & M.N.G. James. The structure of the 2A proteinase from a common cold virus: a proteinase responsible for the shut-off of host cell protein synthesis. EMBO J. 18:5463-5475 (1999).