Structural Characterization of DNA Binding Domain of Essential Mammalian Protein TTF 1
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Abstract
Transcription termination factor 1 (TTF1), being multifunctional in nature, is involved in a wide range of critical processes that make it essential for survival of the mammalian cells. TTF1 protein comprises three functional domains: the N-terminal (regulatory/inhibitory) domain, trans-activation domain, and C-terminal domain. The Myb domain is responsible for DNA-binding function of this protein and spans 550 to 732 amino acids (183 residues long). Despite the essential role of TTF1 in multiple cellular processes, there is no physical structure available to date. Purification of the functional full-length protein has been unsuccessful so far. Hence, we moved forward towards characterizing the Myb domain of this essential protein. We first constructed a three-dimensional model of the Myb domain using Robetta server and determined its stability through MD simulation in an explicit solvent. To validate the model, upon codon optimization we cloned this domain into a bacterial expression vector. The protein was then purified to homogeneity and its DNA-binding activity was checked by electro-mobility shift assay. We then proceeded to CD spectroscopy and Raman spectroscopy for secondary structure characterization. The results validated the computational model, concluding that this domain is predominantly helical in nature. The confidence built by this study now pushes us to move ahead in order to solve the atomic structure of this critical domain by crystallography or NMR spectroscopy, which in turn will decipher the exact mechanism by which this essential protein engages DNA to cater to various functions.
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The results validated the computational model, concluding that this domain is predominantly helical in nature. The confidence built by this study now pushes us to move ahead in order to solve the atomic structure of this critical domain by crystallography or NMR spectroscopy, which in turn will decipher the exact mechanism by which this essential protein engages DNA to cater to various functions
What a cool paper combining computational modeling of protein structure with experimental analysis to support it! I'm excited for the next steps listed here (crystallography or NMR) to see how those results line up with what was found here, but either way, I'm a big fan of the combined computational and experimental work!
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we used Raman spectroscopy. Raman spectrum of the buffer (control) is displayed in black, whereas that of the Myb domain is displayed in red
Did you do technical replicates? I'm curious about how consistent the data is between trials.
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Graphical quantification of intensity of the protein-DNA complex formed
I'm curious if you happened to do replicates of this and/or stats to determine if your quantification is significant?
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AlphaFold, SWISS-MODEL, and Robetta predicted compact and ordered structures with a very high percentage of α-helical conformations. While the model predicted by I-TASSER was less compact and ordered compared to the models generated by the above servers. All models have very similar and reliable statistics as per the overall SAVESv6.0 results. In summary, the structural integrity and statistics of the models derived from both homology and ab-initio methods showed considerable consistency. This confirms the reliability of Robetta and other models (excluding I-TASSER) for further computational analysis.
I really appreciate the direct comparison of the 4 methods on a single sample. This is totally beyond the scope of the paper, but I wonder if these observations would hold true with other proteins/protein domains.
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27%
Is this not just because you're BLASTing a single domain against a database of mostly full proteins?
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protein
It's a bit confusing to refer to the Myb domain as a "protein" when you aren't talking about the full-length protein.
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(Accession Number: Q62187)
Love that you included the accession number and a direct link to the sequence! It can be a pain when papers don't reference the exact protein that they're working with. It might be worth explicitly stating how you identified the Myb domain.
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neither an in silico nor a physically determined structure of the individual domains of TTF1 is available to date
Just curious if there's a structure available of the full protein? It might be useful context to see how your analysis of the Myb domain fits with the full structure (if it's available).
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DDB1
Do you know the domains that these different interacting proteins bind? The first sentence suggests that you do, and it might be useful for better understanding the particular domain that you focus on here (Myb) to know what interacts with it.
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ranging from 323 to 445 amino acids
This is very minor, but when I first read this (before looking at Figure 1), I thought this phrase meant that this specific region was 323-445 amino acids long. It might be less confusing to use language like "a specific region located between amino acid 323 and 445"? Otherwise, this section introducing the functional domains is very thorough and got me right up to speed on TTF1!
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