Knowledge of the three-dimensional structure of a given protein is an absolutely essential prerequisite for fully understanding the chemical basis of a catalytic mechanism of an enzyme or for interpreting the way structural proteins like actin interact with each other. In my lab, we use X-ray crystallography in connection with computer graphics and refinement programs to establish the molecular architecture of proteins. We are eager to integrate our results with biochemical and molecular-biological data, either collected in our laboratory or available through local, national or international collaborations with specialists in the field. In addition to several colleagues here in Toronto , the Pai laboratory presently collaborates with laboratories in Dalhousie, Guelph , and Vancouver as well as Chicago , San Francisco , Seattle , Taipei , Tokyo , Kyoto and Bilbao . There are always several proteins in the laboratory we try to crystallize. Proteins whose three-dimensional structures we recently solved include:

Xanthine oxidoreductase from cows' milk
Milk xanthine oxidoreductase is an enzyme that has served as benchmark for complex flavoproteins and molybdo-enzymes for more than 100 years. In mammals, it is synthesized in its dehydrogenase form (XDH) but can be converted to an oxidase form (XO), either reversibly by oxidizing sulfhydryl groups or irreversibly by limited proteolytic digestion. The enzyme is a target for drugs directed against gout and hyperuricemia. After determining the structures of both forms, XDH and XO, we can now describe the changes associated with the transition, locate the binding sites of substrates and drug molecules and understand the interesting catalytic mechanism.

Orotidine 5'-phosphate decarboxylase
ODCase is one of the most proficient enzymes known. It catalyzes the conversion of orotidine-5'-mono-phosphate (OMP) to uridine 5'-monophosphate (UMP), the last step in the de novo biosynthesis of pyrimidine nucleotides enhancing the decarboxylation rate by 17 orders of mangitude without the help of any cofactors or metal ions. We have teamed up with medicinal chemists to design inhibitors and modified substrate molecules to investigate the mechanistic details of the enzyme. As part of our work we extended the structural analysis to include the enzyme from Methanobacterium thermoautotrophicum , an archaeal thermophile, from Plasmodium falciparum , the pathogen causing malaria, and from Homo sapiens . We also identified new substrates and obtained lead compounds for potential drugs against malaria and cancer, providing new avenues for collaboration with medicinal chemists, cell biologists and physicians.

CorA – a Mg ++ transporter
Recently, we determined the structure of our first membrane protein, a homopentamer that imports Mg ++ -ions into the cell. CorA from Thermotoga maritima represents the first divalent cation channel of known three-dimensional structure. We have generated about 70 mutants of the protein designed to probe the still elusive mechanism of ion transport through the 50 Å long tube. We will continue to investigate its mechanism applying a broad selection of biochemical and biophysical techniques. Especially rewarding are collaborations on electrophysiology and computational approaches.

Dehalogenating enzymes
In collaboration with colleagues in Chemical Engineering and at the SGC, we study several enzymes capable of removing halogen atoms from organic compounds. These enzymes were identified from the genomes of a wide array of microorganisms by bioinformatics means and confirmed by activity screens. Most remarkably, this collection includes a number of proteins that are able to break covalent C-F bonds, the most stable bonds in organic chemistry. We have determined the crystal structures of several of these dehalogenases, described substrate/inhibitor/product complexes and are now moving into the field of “time-resolved” crystallography.

2F5 – a monoclonal antibody that broadly neutralizes HIV
We have not only determined the crystal structure of this neutralizing antibody but also extended the definition of the epitope it recognizes on its target molecule gp41, the protein essential for the fusion of viral and host cell membranes. After testing the promiscuity of the antigen-binding site, we concentrate on complexes combining the primary epitope, secondary epitopes and lipid components. We undertake this research hoping that its results will help in the design of a vaccine component against HIV.

G. Garces, N. Wu, W. Gillon & E.F. Pai (2004) Anabaena Circadian Clock Proteins KaiA and KaiB Reveal a Potential Common Binding Site to Their Partner KaiC. EMBO J. 23:1688-1698.

K. Okamoto, K. Matsumoto, R. Hille, B.T. Eger, E.F. Pai & T. Nishino (2004) Crystal Structure of Xanthine Oxidoreductase in Catalysis: Hydroxylation Mechanism and Inhibition. Proc. Natl. Acad. Sci. USA, 101:7931-7936.

N. Wu and E. F. Pai (2004) Crystallographic Studies of Native and Mutant Orotidine 5'phosphate Decarboxylases. Top. Curr. Chem. 238:23-42.

M. Fujihashi, A.M. Bello, E. Poduch, L. Wei, S.C. Annedi, E.F. Pai & L. P. Kotra (2005) An Unprecedented Twist to ODCase Catalytic Activity. J. Amer. Chem. Soc. 127:15048-15050.

J. Payandeh, M. Fujihashi, W. Gillon & E.F. Pai (2006) The Crystal Structure of (S)-3-O-geranylgeranylglycerol-phosphate-synthase from Archaeoglobus fulgidus Reveals an Ancient Fold for an Ancient Enzyme. J. Biol. Chem. 281:6070-6078.

J. Payandeh & E.F. Pai (2006) A Structural Basis for Mg 2+ Homeostasis and the CorA Translocation Cycle. The EMBO J. 25:3762-3773.

R.G. Garces, W. Gillon & E.F. Pai (2007) Atomic Model of Human Rcd-1 Reveals an Armadillo-Like-Repeat Protein with in vitro Nucleic AcidBinding Properties. Prot. Science 16:176-188.

J. Payandeh & E.F. Pai (2007) Enzyme-driven Speciation: Crystallizing Archaea via Lipid Capture. J. Mol. Evol. 64 :364-374.

J. Payandeh, C. Li, M. Ramjeesingh, E. Poduch, C.E. Bear & E.F. Pai (2008) Probing Structure-Function Relationships and Gating Mechanisms in the CorA Mg 2+ Transport System. J. Biol. Chem. 283 :11721-11733.

S. Bryson, J.-P. Julien, D.E. Isenman, R. Kunert, H. Katinger & E.F. Pai (2008) Crystal Structure of the Complex between the F ab ' Fragment of the Cross-Neutralizing Anti-HIV-1Antibody 2F5 and the F ab Fragment of Its Anti-idiotypic Antibody 3H6. J. Mol. Biol. 382:910-919.

C. A. Thomson, S. Bryson , G.R. MacLean, A.L. Creagh, E.F. Pai & J.W. Schrader (2008) Germline V-Genes Imprint Innate Features on the Binding Site of a High-Affinity Antibody That Neutralizes Human Cytomegalovirus. EMBO J 27:2592-2602.

J.P. Julien, S. Bryson, J.L. Nieva & E.F. Pai (2008) Structural Details of HIV-1 Recognition by the Broadly Neutralizing Monoclonal Antibody 2F5: Epitope Conformation, Antigen-Recognition Loop Mobility and Anion-Binding Site. J. Mol. Biol. 384:377-392.

A.M. Bello, D. Konforte, E. Poduch, C. Furlonger, L. Wei, Y. Liu, Lewis, M., E.F. Pai, C.J. Paige & L.P. Kotra (2009) Structure-Activity Relationships of ODCase Inhibitors as Anticancer Agents. J. Med. Chem., in press.

M. Fujihashi, A.M. Bello, L.P. Kotra & E.F. Pai (2009) Structural Characterization of the Molecular Events during a Slow Substrate-Product Transition in Orotidine 5'-Monophosphate Decarboxylase. J. Mol. Biol., in press.