Structure function studies on bacterial sialic acid transport proteins

Collaborators:

• Prof. Michael Apicella, MD. Department of Microbiology, The University of Iowa.
• Prof. Robert Munson, Ph.D, Department of Pediatrics, Ohio State University.
• Prof. Bradford Gibson, Buck Institute and University of California, San Fransico.

The long-term objective of this proposal is to understand how bacteria scavenge sialic acid isoforms for their use. Diseases caused by these bacteria continue to cause mortality and are of concern to human health.

Several pathogenic bacteria scavenge sialic acid from their host and use it for molecular mimicry to avoid the immune system, as a nutrient source, and as a signaling molecule. Recently, specific transport systems involved in the sialic acid isoform Neu5Ac uptake have been reported in Haemophilus influenza and Haemophilus ducreyi. In nontypeable H. influenza, it has been shown that inhibition of this transport process makes them non-virulent. Also, the ability of H. influenza to survive in biofilms is compromised. This project proposes that Pasturella multocida (a veterinary pathogen) and Vibrio cholera (a marine pathogen) also coat themselves with their host-specific sialic acid isoform to avoid complement-mediated immune response.

Sialic acid transport systems consist of a periplasmic binding protein and a membrane transport component. The periplasmic binding proteins bind to sialic acid tightly, and on binding to the membrane transporter release it to be moved into the cytoplasm. This project will carry out structure-function studies on these periplasmic sialic acid-binding proteins.

The project will use X-ray crystallography to determine the structures and isothermal titration calorimetry to elucidate the thermodynamics of sugar binding. A combination of studies including small angle x-ray scattering, site-directed mutagenesis, and functional sialic acid uptake assays will be used to understand the mechanism of uptake.

The availability of detailed structural and functional information will allow researchers to understand how these bacteria co-evolved with hosts to scavenge host sialic acid. It will also provide the structural and functional details for the rational development of drugs against these pathogenic bacteria.

Structure-function studies on Pro-inflammatory Death Domains

We are interested in understanding the molecular basis of how CARDs, PYR and other domains modulate and function in cells.

Structure Function Studies on Rieske Dioxygenases

Collaborators:

• Prof. David T Gibson (Retired).
• Prof. Hans Eklund and gang at Biomedical Center, Uppsala, Sweden.
• Prof. Rebecca Parales, The University of California, Davis.
• Prof. John Lipscomb, University of Minnesota.
• Prof. Edward I Solomon, Stanford University, Palo Alto, California.

Oxygen activation by iron is a ubiquitous process. Rieske oxygenases are non-heme iron oxyenases present in both prokaryotes and eukaryotes. The primary role they play in prokaryotes is the bioremediation of inert carbon compounds. They catalyze the first step in bioremediation by dihydroxylation of inert carbon compounds. In eukaryotes, they have been implicated in hormonal control of development. These enzymes carry out their function by the activation of dioxygen at a mono-iron center. The long-term goal of this proposal is to understand the mechanism of oxygen activation by iron and how nature uses this to catalyze reactions that are very difficult to carry out in a stereo- and regio-specific fashion in a chemistry laboratory. The laboratories previous work has determined X-ray crystallographic structures of a number of different Rieske oxygenases and their complexes to make structure-function correlations. These results allowed investigators to propose a concerted, radical mechanism for the reaction. Our recent efforts are focused on elucidating the mechanistic details of the dihydroxylation reaction; specifically, a) show that the conserved Lys/Arg-Glu-water pathway is the proton donor system for hydroperoxide formation, and b) demonstrate that the chemical steps of the reaction require the formation of a radical oxygen species. We would also like to understand how, why, and by how much mutation(s) in the active site affect regiospecificity, stereospecificity, and rates of product formation.

The substrates these enzymes catalyze and help bioremediate are environmental pollutants. Understanding the fundamental basis of the function these proteins will allow engineering of bacteria to biodegrade new environmental pollutants. The reactions catalyzed by these enzymes are stereo- and regio-specific. These enzymes have been used for the manufacture of chiral synthons in the pharmaceutical industry. The Rieske oxygenases in eukaryotes are implicated in the TGF-B and insulin/IGF-1 pathways and in cholesterol metabolism. A detailed understanding of these enzyme mechanisms may open new avenues for therapeutic intervention in these pathways.

The Cockroach Approach -- Understanding the role of glycosylation in protein structure and function

Collaborators:

• Prof. Barbara Stay and Dr. James Gray. University of Iowa.

Molecular recognition, protein stability in hostile environments, signaling are a few processes where protein glycosylation have been implicated. Defects in glycosylation have also been shown to cause of several disease states. In spite of its importance, heterogeneity of glycosylation has impeded a thorough study of glycosylation effects on protein structure and function. Here we propose a new approach for the study of protein glycosylation -- the cockroach approach.

In 1977, Professor Barbara Stay's group at the University of Iowa observed shiny protein crystals in the midgut of a cockroach embryo. In collaboration with the Stay group, structure-function studies have been initiated on the isolated protein crystals. The crystalline protein is highly glycosylated and the crystals diffract to 1.2 Angstrom resolution. Initial mass-spectrometry analysis has revealed that the dissolved crystalline protein is heterogeneous, with a molecular weight centered around 20,000 Da. The calculated mass of the amino acid sequence is 18,878 Da, so the glycosylation component contributes 10% to 12% of the observed mass. The diffraction quality of the crystals suggests that much of the glycosylation structure will be resolvable. Differential scanning calorimetry experiments, performed to study protein stability, show that the protein is stabilized at an acidic pH. Deglycosylation studies with alpha-mannosidase and endoglycosidase H reveal a complex glycosylation pattern with high mannose content.

Access to diffraction quality crystals of a glycosylated protein, grown in vivo, allow one to ask fundamentally important questions about the nature and effect of glycosylation on protein structure, stability, and hence function. Protein glycosylation is the most abundant form of post-translation modification. In human cells, between 50% to 70% of the proteins are glycosylated. The role of glycosylation on the function and properties of proteins is varied; yet, atomic resolution details and the molecular effects of glycosylation on proteins are still not well understood. To elucidate the molecular basis for the effect of glycosylation on protein structure and function, this proposal plans to carry out structure-function studies:

1. of the protein crystals grown in vivo (naturally glycosylated form of the protein)
2. by deglycosylation of dissolved protein under non-denaturing conditions (deglycosylated form of the protein)
3. of the protein expressed in yeast (form of the protein glycosylated differently)
4. of the protein expressed in E. coli (un-glycosylated form of the protein)

Structural studies using X-ray crystallography, glycan characterization by enzymatic deglycosylation followed with mass-spectrometry, and stability/folding studies using a combination of differential scanning calorimetry/circular dichroism spectroscopy will be carried out on all the forms of the protein. The results of these studies will reveal the effect of glycosylation on the crystalline protein. More broadly, it is expected that the results will elucidate the molecular basis for glycosylation effects on protein structure.

Tau -- Constitutively unfolded proteins and neurodegenerative diseases.

Collaborators:

• Prof. Gloria Lee, Department of Internal Medicine, The University of Iowa.
• Prof. Ernesto Fuentes, Department of Biochemistry, The University of Iowa.

Age related neurodegenerative diseases affect an increasing proportion of our population. Currently, one out of five individuals over the age of 75 suffer from Alzheimer's disease and as this age group expands, so will the number of people with Alzheimer's disease. There is no known cure for age related neurodegenerative diseases nor are there means to definitively diagnose such diseases, other than cognitive testing. The "gold standard" for diagnosis are tests based on post-mortem brain material where several neuropathological features have been noted. Common to several age related neurodegenerative diseases are abnormal lesions in the brain that comprise of tau protein, a known microtubule-associated protein. Tau lesions are found in Alzheimer's disease, frontotemporal dementia, progressive supranuclear palsy, corticobasal degeneration, Niemann-Pick Type C, argyrophilic grain disease, and a number of other diseases; all are now considered "tauopathies." Moreover, several age related neurodegenerative disorders are now known to be caused by mutations in the tau gene. The mutations either lead to the expression of a mutant tau protein or to an alteration in the alternative splicing of tau mRNA, resulting in a shifting of the ratio of wild type tau proteins expressed in the adult human brain.

The analysis of tau structure and function has revealed that microtubule binding activity and filament formation activity are both mediated by the carboxy terminal half of the protein. This domain of tau contains 3-4 copies of an imperfectly repeated microtubule binding motif; within the repeats are 1-2 copies of a motif thought to nucleate filament formation. Upstream of the microtubule binding domain is a proline rich region which contains a PXXP motif that interacts with the SH3 domains of the src family tyrosine kinases fyn and src. Phosphorylation of tau often causes its structure to elongate. Circular dichroism studies have not revealed any defined secondary or tertiary structural elements. However, assays mapping motifs involved in tau function have suggested that areas outside of the motif often modulate the level of activity, suggesting that the protein has conformation.

The goal of this project is to obtain structural information on tau. Specific phosphorylated sites are known to affect the ability of tau to associate with microtubules. Functionally, the most dramatic effect of the mutations has been seen on the ability of tau to associate with the SH3 domains, as determined by surface plasmon resonance. Our aim is to determine the 3-D structure of a complex of tau and an SH3 domain. This would indicate the molecular contacts involved in the tau-SH3 interaction. This interaction is likely to have a role in signal transduction during neuronal differentiation and may also be involved in the signal transduction processes that link Ab to abnormally phosphorylated tau during neuropathogenesis. Understanding these molecular contacts involved might lead to the design of compounds that could disrupt the tau-tau interaction and perhaps slow the development or disassemble neurofibrillary tangles.