Analysis of MEMO1 Binding Specificity for ErbB2 Using Fluorescence Polarization and Molecular Dynamics Simulations

Abstract

ErbB2 signaling pathways are linked to breast cancer formation, growth, and aggression; therefore, understanding the behavior of proteins associated with these pathways as well as regulatory factors that influence ErbB2 function is essential. MEMO1 is a redox active protein that is shown to associate with phosphorylated ErbB2 and mediate cell motility. We have developed a fluorescence polarization assay to probe the interaction between MEMO1 and an ErbB2-derived peptide containing a phosphorylated tyrosine residue. This interaction is shown to be pH-dependent and stronger with longer peptides as would be expected for protein-protein interactions. We have quantitatively mapped the binding interface of MEMO1 to the peptide using the fluorescence polarization assay and molecular dynamics simulations. We have confirmed that phosphorylation of the peptide is essential for binding and through mutagenesis have identified residues that contribute to favorable interactions. Our results highlight the importance of the protein-protein interactions of MEMO1 that complement the oxidase activity. In the future, these studies will provide a method for screening for selective modulators of MEMO1, which will allow for additional biological investigations.

Finding MEMO—Emerging Evidence for MEMO1′s Function in Development and Disease

“…Through an unbiased screen, MEMO1 was shown to interact with phospho-Tyrosine (pY) 1227 on the C-terminus of ERBB2 after ligand (Heregulin, HRG) stimulation-a residue shown to be important for tumor cell migration [1]. Since its initial isolation, MEMO1 s interaction with ERBB2 is by far the most well characterized, relative to other signaling pathways [1,3,[7][8][9][10][11][12][13]. Given MEMO1 does not display any obvious SH2 or phospho-tyrosine binding domains, its interaction with pY1227 was thought to be mediated, in part, through the Src homology and collagen 1 (SHC) adaptor protein [1] ( Figure 1A).…”

“…However, follow-up studies, including elucidation of the MEMO1 crystal structure, have shown that MEMO1 can bind directly to pY1227 through a binding pocket contained within the protein, originally termed a 'vestigial active site' [3]. While quite extensive analyses, using an array of approaches, have been used to elucidate the key residues that mediate this interaction [3,8,11] (e.g., W16, H49, Y54, H81, D189, H192, R196, R198, C244) there have been some discrepancies. For example, while residues H81, H192 and C244 were previously shown to be essential for MEMO1 binding to an ERBB2 peptide [3], follow-up studies suggested these residues may be dispensable (or less important) for MEMO1 binding.…”

“…Out of eight metal ions tested, only in the presence of copper [Cu(II)] was MEMO1 capable of generating O 2 − -an important intermediate product of several redox reactions. When further characterizing the important residues in MEMO1 s 'active site' required for copper binding and redox activity, it was found that residue H192 (previously shown to be involved in ERBB2 binding [1]) was critical, as O 2 − production in the presence of Cu(II) was significantly less when this residue was mutated, relative to control [9]-although the stability and folding of the mutant variant may require further investigation [11]. The proposed mechanism by which MEMO1 could produce O 2 − with this metal ion was through its interactions with the importantly conserved histidine residues found in its active site.…”

Extracellular Control of Radial Glia Proliferation and Scaffolding During Cortical Development and Pathology

“…Importantly, ErbB2 expression is specific to RGs and its loss of function in the mouse unbalances the astrocyte/RG population ratio by reducing the number of elongated RGs in the developing cortex (Schmid et al, 2003). ErbB2 interacts specifically with a redox active protein, Memo1 (Newkirk et al, 2018). Although Memo1 has been known for some time to be important for cell migration (Marone et al, 2004), its role in the branching and the maintenance of the RG scaffold was identified relatively recently (Nakagawa et al, 2019).…”

Twenty years of exceptional success: The molecular education and research consortium in undergraduate computational chemistry (MERCURY)

“…The faculty published 2 peer‐reviewed books, [ 238,239 ] 9 peer‐reviewed book chapters, [ 240–248 ] and 115 peer‐reviewed research papers. [ 249–363 ] This comes to 1.6 peer‐reviewed products/faculty/year during the 3‐year grant period, which is 3.2 times the rate of publication for natural science faculty at PUIs. [ 46 ]…”