News and Publications

Structural Molecular Biology and Protein Design

J.A. Tainer, A.S. Arvai, D.P. Barondeau, S.L. Bernstein, C.M. Bruns, J. Castagnetto, S.B. Clancy, B.R. Crane, T. Cross, D. Daniels, K.T. Forest, Y. Guan, S. Han, R.A. Hallewell, M.J. Hickey, D.J. Hosfield, J. Iacovoni, T.P. Lo, T. Macke, C.D. Mol, S.S. Parikh, J.L. Pellequer, M.E. Pique, C.D. Putnam, M.M. Thayer

We focus on 4 classes of proteins at the interface between structural and cellular biology and chemistry: (1) proteins that assemble into bacterial pili needed for the attachment and motility of pathogens, (2) enzymes that regulate reactive oxygen and xenotoxin defenses, (3) enzymes that act in DNA repair and evolution, and (4) proteins that control the cell cycle. An important facet of these studies involves the design of metalloproteins to understand the activity and specificity of enzyme metal sites. Current mutational, crystallographic, and computational results are providing a fundamental understanding that should ultimately contribute to new treatments for inflammatory disorders, infectious and degenerative diseases, and cancer.

BACTERIAL PILI AND INFECTIOUS DISEASES

We seek to understand pathogenicity factors for the many bacterial pathogens that use type IV pili for attachment and motility. We have solved the atomic structure of pilin, the pilus fiber-forming protein essential for the virulence of many pathogens, including Neisseria meningitidis, Neisseria gonorrhoeae, Pseudomonas aeruginosa, Dichlobacter nodosus, Moraxella bovis, Vibrio cholerae, and enterotoxogenic Escherichia coli. A few key interactions allow a hypervariable disulfide region to undergo extreme antigenic variation to escape the host immune response.

We developed a specific assembly model for the pilus and defined the structural regions recognized by human antibodies. Our most recent structural results characterize the role of glycosylation and phosphorylation sites in pilus bundling and adhesion.

REACTIVE OXYGEN AND XENOBIOTIC CONTROL

Copper-zinc and manganese superoxide dismutases (SODs) are master regulators of intracellular reactive oxygen species, catalyzing the degradation of superoxide radicals to molecular oxygen and hydrogen peroxide. Genetic defects in the gene encoding human copper-zinc SOD cause the fatal neurodegenerative disease amyotrophic lateral sclerosis, also known as Lou Gehrig's disease. Our structural studies of human copper-zinc SOD mutants are providing insights into the molecular basis of this disease by improving our understanding of the enzyme's catalytic mechanism, electrostatic guidance, folding and stability, and metal ion binding and environment. Comparison of multiple high-resolution structures is under way to reveal common themes that link the widely distributed SOD mutations observed in patients with amyotrophic lateral sclerosis.

Our structures of manganese SOD and designed mutant enzymes provide new information on the catalytic mechanism of manganese SOD, proton-transfer pathways, and the evolutionary conservation of active-site residues in metalloenzymes. We introduced the hypothesis that extreme rather than normal substrate concentrations are a powerful constraint on residue conservation for enzyme defenses in which the ability to meet extreme conditions directly affects cell survival. Redesign of the metal site of manganese SOD and the design of a manganese SOD active site into green fluorescent protein are under way to define the requirements for the chemical activity of the manganese site in dismuting superoxide radicals.

Oxidation of lipids and cell membranes initiates a chain-reaction buildup of cytotoxic compounds implicated in aging, cancer, atherosclerosis, neurodegenerative diseases, and other illnesses. A key component in the defense against this oxidative stress is glutathione transferase A4-4. Our crystal structures of human glutathione transferase A4-4 and the results of mutagenesis studies reveal a modular basis for the evolution of the high catalytic activity of the transferase with toxic lipid peroxidation products. The mechanism of detoxification of lipid peroxidation products deduced from these recent structural and mutational results has both biochemical and pathophysiologic significance.

Nitric oxide synthases (NOSs) oxidize l-arginine to synthesize nitric oxide, a key intercellular signal and defensive cytotoxin in the nervous, cardiovascular, and immune systems. Overproduction of nitric oxide by inducible NOS (iNOS) can lead to many abnormalities, including juvenile diabetes, arthritis, aneurysms, neurodegenerative disorders, and septic shock. Therefore, specific inhibitors of iNOS have great therapeutic potential.

Our structures of monomeric murine iNOS_ox revealed that NOS has an unusual topology and heme environment, completely distinct from the cytochrome P-450s or any other known heme-containing enzyme. Most recently, we solved 3 structures of fully functional, pterin-loaded, dimeric iNOS in complex with substrate or analogs. The results provide an understanding of NOS activity that unifies biochemical findings and resolves relationships among NOS dimerization, pterin binding, recognition of l-arginine, and catalysis.

DNA REPAIR AND GENETIC EVOLUTION

Because more than 10,000 DNA bases per day are repaired in each human cell, DNA base excision repair enzymes, which protect cells from mutagenic DNA lesions, are critical to cell survival. Our structures of DNA-repair enzymes such as endonuclease III, MutY, uracil-DNA glycosylase, and AP endonuclease show in atomic detail how damaged DNA bases and abasic sites in double-stranded DNA are recognized and removed. These enzymes bind and compress the DNA backbone so that damaged DNA bases are flipped out from the double helix and into pockets specific for given damaged bases. These pockets seem ideal for the design of specific inhibitors for novel anticancer therapies.

To test our understanding of these binding pockets, we deliberately altered the specificity of uracil-DNA glycosylase by making mutants that remove cytosine or thymine from normal DNA. The endonuclease III and MutY structures represent a superfamily of DNA-repair enzymes with an important HhH motif that recognizes the DNA backbone. Our structures of the major bacterial and human DNA-repair AP endonucleases, which cut DNA at sites where bases are missing, define the active sites of the endonucleases and suggest a mechanism for recognizing missing bases and cleaving the DNA backbone for repair.

We are characterizing a number of enzymes involved in processing uracil (Fig. 1). DNA glycosylases such as uracil-DNA glycosylase act as the key initiators of the DNA base excision repair pathway by removing damaged bases such as deaminated cytosine. Uracil-DNA glycosylase is aided in the exclusion of uracil from DNA by the activity of human dUTP pyrophosphatase, which catalyzes the breakdown of uracil nucleotide triphosphates to keep uracil out of DNA. Some viruses use uracil in their DNA and have therefore evolved inhibitors, such as uracil-DNA-glycosylase inhibitor, for the cellular enzymes that remove uracil from DNA.

We recently solved high-resolution structures for uracil-DNA-glycosylase inhibitor and showed that it is an extremely effective protein mimic of the double-stranded DNA backbone (Fig. 2). Understanding the nature of this molecular mimicry may help us understand the activity of DNA base excision repair enzymes in damage recognition and also allow us to redesign uracil-DNA-glycosylase inhibitor to inhibit other DNA-repair enzymes.

CONTROL OF THE CELL CYCLE

Together with S. Reed and colleagues, Department of Molecular Biology, we are working to define the structural basis for control of the cell cycle. We solved structures of the Cks and suc1 proteins, which are essential to progression of the cycle. Our structures reveal a conformational switch that controls 2 distinct Cks folds and assemblies via a domain-swapping mechanism. The structure and mutational analyses of the human cyclin-dependent kinase in complex with human CksHs1 show how the domain swapping can regulate kinase binding. Current efforts involve structural studies of new mutants and complexes of Cks and cyclin-dependent kinase. In addition, we solved wild-type and mutant structures of the key cell-cycle and DNA-synthesis enzyme ribonucleotide reductase under conditions that retain activity of the enzyme. Metal-site redesign of ribonucleotide reductase promises to define the basis for activity of the di-iron active site in creating free radicals for the reaction chemistry.

PUBLICATIONS

Boissinot, M., Karnas, S., Lepock, J.R., Cabelli, D.E., Tainer, J.A., Getzoff, E.D., Hallewell, R.A. Function of the Greek key connection analysed using circular permutants of superoxide dismutase. EMBO J. 16:2171, 1997.

Crane, B.R., Arvai, A.S., Gachhui, R., Wu, C., Ghosh, D.K., Getzoff, E.D., Stuehr, D.J., Tainer, J.A. The structure of NO synthase oxygenase domain and inhibitor complexes. Science 278:425, 1997.

Crane, B.R., Arvai, A.S., Ghosh, D.K., Wu, C.P., Getzoff, E.D., Stuehr, D.J., Tainer, J.A. Structure of nitric oxide synthase oxygenase dimer with pterin and substrate. Science 279:2121, 1998.

Fisher, C.L., Cabelli, D.E., Hallewell, R.A., Beroza, P., Lo, T.P., Getzoff, E.D., Tainer, J.A. Computational, pulse-radiolytic and structural investigations of lysine 136 and its role in the electrostatic triad of human Cu,Zn superoxide dismutase. Proteins 29:103, 1997.

Gorman, M.A., Morera, S., Rothwell, D.G., de la Fortelle, E., Mol, C.D., Tainer, J.A., Hickson, I.D., Freemont, P.S. The crystal structure of the human DNA-repair enzyme endonuclease HAP1 suggests the recognition of extra-helical deoxyribose at DNA abasic sites. EMBO J. 16:6548, 1997.

Guan, Y., Hickey, M.J., Borgstahl, G.E.O., Hallewell, R.A., Lepock, J.R., O'Connor, D., Hsieh, Y., Nick, H.S., Silverman, D.N., Tainer, J.A. The crystal structure of Y34F mutant human mitochondrial manganese superoxide dismutase and the functional role of tyrosine 34. Biochemistry 37:4722, 1998.

Hsieh, Y., Guan, Y., Tu, C., Bratt, P.J., Angerhofer, A., Lepock, J.R., Hickey, M.J., Tainer, J.A., Nick, H.S., Silverman, D.N. Probing the active site of human manganese superoxide dismutase: The role of glutamine 143. Biochemistry 37:4731, 1998.

Marccau, M., Forest, K., Bertti, J.-L., Tainer, J.A., Nassif, X. Consequences of the loss of O-linked glycosylation of meningococcal type IV pilin on piliation and pilus-mediated adhesion. Mol. Microbiol. 27:705, 1998.

Mol, C.D., Parikh, S.S., Lo, T.P., Tainer, J.A. Structural phylogenetics of DNA base excision repair. Nucleic Acids Mol. Biol., in press.

Parikh, S.S., Mol, C.D., Slupphaug, G., Bharati, B., Krokan, H.E., Tainer, J.A. Base-excision repair initiation revealed by crystal structures and binding kinetics of human uracil-DNA glycosylase bound to DNA. EMBO J., in press.

Parikh, S.S., Mol, C.D., Tainer, J.A. Base excision repair enzyme family portrait: Integrating the structure and chemistry of an entire DNA repair pathway. Structure 5:1543, 1997.

Shen, B., Qiu, J., Hosfield, D., Tainer, J.A. Flap endonuclease homologues in Archaebacteria exist as independent proteins. Trends Biochem. Sci. 23:171, 1998.

Tong, W., Burdi, D., Riggs-Gelasco, P., Chen, S., Edmondson, D., Huynh, B.H., Stubbe, J., Hans, S., Arvai, A., Tainer, J. Characterization of Y122F R2 of Escherichia coli ribonucleotide reductase by time-resolved physical biochemical methods and x-ray crystallography. Biochemistry 37:5840, 1998.

Zu, J.S., Deng, H.-X., Lo, T.P., Mitsumoto, H., Ahmed, M.S., Hung, W.-Y., Cai, Z.-J., Tainer, J.A., Siddique, T. Exon5 encoded domain is not required for the toxic function of mutant SOD1 but essential for dismutase activity: Identification and characterization of two new SOD1 mutations associated with familial amyotrophic lateral sclerosis. Neurogenetics 1:65, 1997.