department of pharmacology

Phoebe L. Stewart, Ph.D.

Stewart

Director, Cleveland Center for Membrane and Structural Biology Professor, Department of Pharmacology Case Western Reserve University

School of Medicine
10900 Euclid Ave
Cleveland, OH 44106-4965
Phone: (216) 368-4349
Fax: 216-368-1300
Email: pls47@case.edu

Research

Cryo-electron microscopy (cryo-EM) plays a central role in hybrid methods as it can be used to determine structures of membrane proteins and large complexes in multiple conformations without the need for crystals. Docking of atomic resolution structures and computational models into cryo-EM density maps can often lead to a structural understanding at the near-atomic level. We are exploring cryo-EM steered molecular dynamics simulations and the development of cryo-EM guided de novo structure prediction methods. Hybrid structural technologies will enhance our ability to discern molecular mechanisms underlying biological processes in human health and disease.

We are currently applying cryo-EM structural methods to a variety of adenovirus/host factor complexes. Adenovirus is a common human pathogen that causes respiratory infections, gastrointestinal infections, and severe ocular infections. Modified forms of adenovirus have shown great potential for gene delivery and vector-based vaccination strategies. When adenovirus is injected intravenously, it induces potent innate immune and inflammatory responses, the molecular basis for which remains poorly characterized. We are investigating the interaction of adenovirus with two host factors, defensin and coagulation factor X. Human defensin 5 is a peptide from the innate immune system that neutralizes multiple adenovirus species by blocking viral cell entry. Factor X plays a role in the blood coagulation cascade and leads to highly efficient adenoviral infection of hepatocytes. We are also visualizing how heterologous HIV peptide epitopes incorporated into capsid proteins are displayed on the surface of adenovirus with the goal of helping to guide the design of multivalent vaccine vectors.

In addition, we are applying cryo-EM methods to protein and protein/DNA complexes involved in nonhomologous end joining (NHEJ) and in maintenance of circadian rhythm. DNA damage occurs from oxidative damage and exposure to ionizing radiation. If the damage is not repaired, or if it is repaired incorrectly, genetic instability may result leading to cancer or cell death. The human DNA-PKcs enzyme mitigates oncogenesis through NHEJ repair of double strand DNA breaks. Circadian rhythms regulate cellular and organismal physiology with an approximate 24 hour cycle. In humans, this clockwork regulates sleep cycles, as well as hormone and metabolic activities. Cyanobacteria are the simplest known organisms to contain circadian oscillations. The core molecular clock of cyanobacteria consists of only three proteins: KaiA, KaiB, and KaiC. These three proteins plus ATP are competent to reconstitute a phosphorylation/dephosphorylation cycle in vitro with a 24 hour oscillation pattern. In both the DNA repair and circadian rhythm systems, cryo-EM provides a way to study the structure of large and conformationally flexible complexes.

Applications

Our laboratory is focused on applying cryo-EM structural methods to a variety of biological complexes including viruses, viral/host factor complexes, engineered adenovirus-based vaccines, DNA double-strand break repair complexes, and circadian clock protein complexes.