Abstract
Memo (mediator of ErbB2-driven cell motility) is a 297-amino-acid protein recently shown to co-precipitate with the C terminus of ErbB2 and be required for ErbB2-driven cell motility. Memo is not homologous to any known signaling proteins, and how it mediates ErbB2 signals is not known. To provide a molecular basis for understanding Memo function, we have determined and report here the 2.1A crystal structure of human Memo and show it be homologous to class III nonheme iron-dependent dioxygenases, a structural class that now includes a zinc-binding protein of unknown function. No metal binding or enzymatic activity can be detected for Memo, but Memo does bind directly to a specific ErbB2-derived phosphopeptide encompassing Tyr-1227 using its vestigial enzymatic active site. Memo thus represents a new class of phosphotyrosine-binding protein.
Highlights
HER2/ErbB2 is a member of the epidermal growth factor (EGF/ErbB)4 family of receptor tyrosine kinases, which in humans includes the EGF receptor (EGF receptor/ErbB1), ErbB3 (HER3), and ErbB4 (HER4) [1, 2]
Memo Structure—The crystal structure of full-length human Memo was determined by single wavelength anomalous diffraction, and the resulting atomic model was refined to 2.1 Å resolution (Table 1)
Memo was identified based on its ability to co-precipitate with a specific phosphopeptide within the substitution diminished pYD binding
Summary
Protein Expression and Purification—A cDNA encoding fulllength human Memo was subcloned into the pHT vector, which adds a cleavable N-terminal hexahistidine tag [11]. Selenomethionine-containing Memo was produced by expression in E. coli strain B834(DE3) grown in the presence of selenomethionine and purified to native protein. For the data shown, coupled beads were incubated with 500 ng of purified Memo or 50 ng of Shc in the presence of 1 mg/ml bovine serum albumin for 1 h at 4 °C. Data analysis was performed using the Origin software supplied by MicroCal. Induction-coupled Argon Plasma (ICAP) Spectrometry Metal Analysis—E. coli cells containing the Memo plasmid were grown in Terrific Broth medium supplemented with 0.1 mM ZnCl2. Superdex 75 column fractions containing Memo (ϳ3 M) were incubated with 5 M ZnCl2 for 1 h, concentrated to 42 M, and dialyzed for 3 h against 20 mM Tris, 0.15 M NaCl, pH 8.0, and 0.1% -mercaptoethanol. A 2-ml sample of this Memo preparation was sent to the Chemical Analysis Laboratory at the University of Georgia for metal analysis on the Thermo Jarrell-Ash 965 ICAP instrument
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