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A membrane setting for the sorting motifs present in the adenovirus E3-13.7 protein which down-regulates the
O Vinogradova1, C Carlin, F D Sonnichsen
1Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106-4970, USA.
Abstract:
The adenovirus E3-13.7 protein interferes with endosomal protein sorting to down-regulate the epidermal growth factor receptor and related tyrosine kinase receptors. The cytoplasmic C terminus of this protein contains three protein sorting motifs which are related to the function of E3-13.7. In this study, the structure of a 23-residue polypeptide corresponding to this domain was examined using solution NMR and CD spectroscopic methods. The peptide was observed to exist in a mostly random structural state in aqueous solution but underwent high affinity association with dodecylphosphocholine micelles, where it adopted an ordered structure. The affinity of this peptide for the micellar surface and the structure of the bound peptide were independent of pH variation, surface charge, or attachment of a myristoyl anchor to the N-terminal. Studies with phospholipid vesicles suggested that the micellar structural results can be extrapolated to a true lipid bilayer. On the micellar surface all three sorting motifs are closely associated with the water/apolar interface: 72-YLRH and 87-LL lie within interfacial amphipathic helices, while 76-HPQY is non-helical and dimples just above the surface. These results contribute to the development of an understanding of the basis for specificity in recognition of sorting motifs by components of the cellular protein trafficking machinery.
Insights
Adenovirus E3-13.7 protein
Area of Science:
- Molecular biology
- Structural biology
- Virology
Background:
- Adenovirus E3-13.7 protein down-regulates epidermal growth factor receptor and related tyrosine kinase receptors by interfering with endosomal protein sorting.
- The cytoplasmic C terminus of E3-13.7 contains three protein sorting motifs crucial for its function.
Purpose of the Study:
- To investigate the structure and lipid-binding properties of the E3-13.7 C-terminal domain.
- To understand how the protein's sorting motifs interact with cellular membranes.
Main Methods:
- Solution Nuclear Magnetic Resonance (NMR) spectroscopy
- Circular Dichroism (CD) spectroscopy
- Dodecylphosphocholine (DPC) micelle binding assays
- Phospholipid vesicle interaction studies
Main Results:
- The 23-residue polypeptide adopted a random coil in aqueous solution.
- High-affinity binding to DPC micelles induced an ordered structure.
- Binding affinity and structure were unaffected by pH, surface charge, or N-terminal myristoylation.
- The three sorting motifs localized to the water-apol interface within interfacial amphipathic helices or a surface-dimpling non-helical structure on the micelle.
Conclusions:
- The E3-13.7 C-terminal domain undergoes a disorder-to-order transition upon membrane association.
- Structural findings on micelles are likely applicable to lipid bilayers.
- These results provide insights into the specific recognition of sorting motifs by cellular protein trafficking machinery.