Evaluating the Conformation and Binding Interface of Cap-Binding Proteins and Complexes via Ultraviolet Photodissociation Mass Spectrometry

Abstract

We report the structural analysis of cap-binding proteins using a chemical probe/ultraviolet photodissociation (UVPD) mass spectrometry strategy for evaluating solvent accessibility of proteins. Our methodology utilized a chromogenic probe (NN) to probe the exposed amine residues of wheat eukaryotic translation initiation factor 4E (eIF4E), eIF4E in complex with a fragment of eIF4G ("mini-eIF4F"), eIF4E in complex with full length eIF4G, and the plant specific cap-binding protein, eIFiso4E. Structural changes of eIF4E in the absence and presence of excess dithiothreitol and in complex with a fragment of eIF4G or full-length eIF4G are mapped. The results indicate that there are particular lysine residues whose environment changes in the presence of dithiothreitol or eIF4G, suggesting that changes in the structure of eIF4E are occurring. On the basis of the crystal structure of wheat eIF4E and a constructed homology model of the structure for eIFiso4E, the reactivities of lysines in each protein are rationalized. Our results suggest that chemical probe/UVPD mass spectrometry can successfully predict dynamic structural changes in solution that are consistent with known crystal structures. Our findings reveal that the binding of m(7)GTP to eIF4E and eIFiso4E appears to be dependent on the redox state of a pair of cysteines near the m(7)GTP binding site. In addition, tertiary structural changes of eIF4E initiated by the formation of a complex containing a fragment of eIF4G and eIF4E were observed.

Mechanism of Cytoplasmic mRNA Translation

“…A constitutively reduced mutant (C113S) and oxidized forms of eIF4E bound m 7 GTP with a modest 1.5x difference in k off values in NMR (nuclear magnetic resonance) solution studies (Monzingo et al, 2007). Further studies using mass spectrometry and a lysine-specific chemical probe indicated structural changes occurred upon altering the redox state and support the hypothesis of a redox sensor or switch for eIF4E (O'Brien et al, 2013). It remains a tantalizing possibility that the oxidation state of eIF4E (and/or eIFiso4E) may modulate cap binding in a redox-sensing manner in plants during retrograde signaling from the chloroplast to nucleus or other processes that could regulate the level of translation in response to the redox state of the cell.…”

Characterization of Native Protein Complexes Using Ultraviolet Photodissociation Mass Spectrometry

“…There is also experimental evidence that the looped region around the Lys165 and Arg166 residues interacts with the mRNA cap. 53 UVPD of the holo -eIF4E (Figure 2) generated a wide variety of fragment ions that retained m 7 GTP (a subset of m 7 GTP-bound fragment ions is shown in Table S2). The UVPD fragmentation map of the eIF4E/m 7 GTP complex (8+) displays the backbone cleavage sites that lead to fragment ions that retain m 7 GTP (red cleavage marks in Figure 2C).…”

Structure of eIF4E in Complex with an eIF4G Peptide Supports a Universal Bipartite Binding Mode for Protein Translation

“…Despite that the two cysteines involved are strictly conserved in plant eIF4Es, suggesting that they may have some important biological role, affinity-binding assays under both reducing and oxidizing conditions showed that in both cases wheat eIF4E retained the ability to bind the cap (Monzingo et al, 2007). However, further studies using mass spectrometry and a Lys-specific chemical probe indicated structural changes upon altering the redox state, supporting the hypothesis of a redox sensor or switch for eIF4E (O'Brien et al, 2013). Whether the oxidation state of eIF4E may modulate cap binding in a redox-sensing manner in plants and regulate the level of translation in response to the redox state of the cell remains uncertain.…”