Computational Exploration of Structural Hypotheses for an Additional Sequence in a Mammalian Mitochondrial Protein

Background: Proteins involved in mammalian mitochondrial translation, when compared to analogous bacterial proteins, frequently have additional sequence regions whose structural or functional roles are not always clear. For example, an additional short insert sequence in the bovine mitochondrial initiation factor 2 (IF2mt) seems sufficient to fulfill the added role of eubacterial initiation factor IF1. Prior to our recent cryo-EM study that showed IF2mt to structurally occupy both the IF1 and IF2 binding sites, the spatial separation of these sites, and the short length of the insert sequence, posed ambiguity in whether it could perform the role of IF1 through occupation of the IF1 binding site on the ribosome. Results: The present study probes how well computational structure prediction methods can a priori address hypothesized roles of such additional sequences by creating quasi-atomic models of IF2mt using bacterial IF2 cryo-EM densities (that lack the insert sequences). How such initial IF2mt predictions differ from the observed IF2mt cryo-EM map and how they can be suitably improved using further sequence analysis and flexible fitting are analyzed. Conclusions: By hypothesizing that the insert sequence occupies the IF1 binding site, continuous IF2mt models that occupy both the IF2 and IF1 binding sites can be predicted computationally. These models can be improved by flexible fitting into the IF2mt cryo-EM map to get reasonable quasi-atomic IF2mt models, but the exact orientation of the insert structure may not be reproduced. Specific eukaryotic insert sequence conservation characteristics can be used to predict alternate IF2mt models that have minor secondary structure rearrangements but fewer unusually extended linker regions. Computational structure prediction methods can thus be combined with medium-resolution cryo-EM maps to explore structure-function hypotheses for additional sequence regions and to guide further biochemical experiments, especially in mammalian systems where high-resolution structures are difficult to determine.