Browsing by Author "Hailey, Kendra L."

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    Geometrical Frustration in Interleukin-33 Decouples the Dynamics of the Functional Element from the Folding Transition State Ensemble
    (Public Library of Science, 2015) Fisher, Kaitlin M.; Haglund, Ellinor; Noel, Jeffrey K.; Hailey, Kendra L.; Onuchic, José Nelson; Jennings, Patricia A.; Center for Theoretical Biological Physics
    Interleukin-33 (IL-33) is currently the focus of multiple investigations into targeting pernicious inflammatory disorders. This mediator of inflammation plays a prevalent role in chronic disorders such as asthma, rheumatoid arthritis, and progressive heart disease. In order to better understand the possible link between the folding free energy landscape and functional regions in IL-33, a combined experimental and theoretical approach was applied. IL-33 is a pseudo- symmetrical protein composed of three distinct structural elements that complicate the folding mechanism due to competition for nucleation on the dominant folding route. Trefoil 1 constitutes the majority of the binding interface with the receptor whereas Trefoils 2 and 3 provide the stable scaffold to anchor Trefoil 1. We identified that IL-33 folds with a three-state mechanism, leading to a rollover in the refolding arm of its chevron plots in strongly native conditions. In addition, there is a second slower refolding phase that exhibits the same rollover suggesting similar limitations in folding along parallel routes. Characterization of the intermediate state and the rate limiting steps required for folding suggests that the rollover is attributable to a moving transition state, shifting from a post- to pre-intermediate transition state as you move from strongly native conditions to the midpoint of the transition. On a structural level, we found that initially, all independent Trefoil units fold equally well until a QCA of 0.35 when Trefoil 1 will backtrack in order to allow Trefoils 2 and 3 to fold in the intermediate state, creating a stable scaffold for Trefoil 1 to fold onto during the final folding transition. The formation of this intermediate state and subsequent moving transition state is a result of balancing the difficulty in folding the functionally important Trefoil 1 onto the remainder of the protein. Taken together our results indicate that the functional element of the protein is geometrically frustrated, requiring the more stable elements to fold first, acting as a scaffold for docking of the functional element to allow productive folding to the native state.
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    Integrated strategy reveals the protein interface between cancer targets Bcl-2 and NAF-1
    (PNAS, 2014) Tamir, Sagi; Rotem-Bamberger, Shahar; Katz, Chen; Morcos, Faruck; Hailey, Kendra L.; Zuris, John A.; Wang, Charles; Conlan, Andrea R.; Lipper, Colin H.; Paddock, Mark L.; Mittler, Ron; Onuchic, José Nelson; Jennings, Patricia A.; Friedler, Assaf; Nechushtai, Rachel; Center for Theoretical Biological Physics
    Life requires orchestrated control of cell proliferation, cell maintenance, and cell death. Involved in these decisions are protein complexes that assimilate a variety of inputs that report on the status of the cell and lead to an output response. Among the proteins involved in this response are nutrient-deprivation autophagy factor-1 (NAF-1)- and Bcl-2. NAF-1 is a homodimeric member of the novel Fe-S protein NEET family, which binds two 2Fe-2S clusters. NAF-1 is an important partner for Bcl-2 at the endoplasmic reticulum to functionally antagonize Beclin 1-dependent autophagy [Chang NC, Nguyen M, Germain M, Shore GC (2010) EMBO J 29 (3):606–618]. We used an integrated approach involving peptide array, deuterium exchange mass spectrometry (DXMS), and functional studies aided by the power of sufficient constraints from direct coupling analysis (DCA) to determine the dominant docked conformation of the NAF-1–Bcl-2 complex. NAF-1 binds to both the pro- and antiapoptotic regions (BH3 and BH4) of Bcl-2, as demonstrated by a nested protein fragment analysis in a peptide array and DXMS analysis. A combination of the solution studies together with a new application of DCA to the eukaryotic proteins NAF-1 and Bcl-2 provided sufficient constraints at amino acid resolution to predict the interaction surfaces and orientation of the protein–protein interactions involved in the docked structure. The specific integrated approach described in this paper provides the first structural information, to our knowledge, for future targeting of the NAF-1–Bcl-2 complex in the regulation of apoptosis/autophagy in cancer biology.