Evolution of New Protein Topologies through Multistep Gene Rearrangements

1. Evolution of New Protein Topologies through Multistep Gene Rearrangements Sergio G Peisajovich, LiatRockah and Dan S Tawfik Dibyayan DAS CS 502

2. Overview • The main issue • Protein fold • Circular Permutation • Sequential Gene rearrangements • Active Intermediates

3. DNA methyltransferases A 5-methylcytosine(m5C) class methyltransferase is used – M.HaeIII 3-D structure of the m5C class M.HaeIII

4. Permutation by Duplication Accounts for evolution of various classes of DNA methyltransferases P by D mechanism for producing a circular permutant

5. N- and C- Terminal N terminus(-NH2) refers to start of the protein chain C terminus(-COOH) refers to the end of the protein chain N - terminus C - terminus A tetrapeptide example

6. Codons The coding region of a gene is that portion of the gene’s DNA or RNA that codes for protein Each set of 3 that codes for an amino acid.

7. Creation of Circular Permutants • Gene duplication and in frame fusion. • Partial degeneration of 5’ coding region of the first copy and 3’ coding region of the second copy of the fused dimer (ITCHY Methodology)

8. Results • N-terminally truncated intermediates was generated by introducing start codons at random locations along the first copy • C-terminally truncated intermediates was generated by introducing stop codons at random locations along the second copy • C-terminally truncated intermediates were clustered in 3 groups (I, II & III) whereas N-terminally truncated ones in just 2 groups(IV & V)

10. DNA Methyltransferases are classified according to the linear order of their conserved motif sequences(I-X) and location of the TRD, defining 7 classes. Potential new class identified

11. Results • Intermediates from cluster I and II yieldedband z–class enzymes upon N-terminal truncation • Intermediates from cluster IV yielded z-class enzymes upon C-terminal truncation • No intermediates lead to g-class enzymes • Intermediates with divided TRD were discovered(clusters III and V), were named h

12. TruncatedIntermediates Do they lead to Circular Permutants? Yes How? By randomly truncating the N-terminally truncated intermediates at their C termini and C-terminally truncated intermediates at their N termini generating different libraries of potential Circular Permutants

13. Results Circular Permutants generated by cluster I and IV intermediates

14. Point Mutation • Isolate least active intermediate A180-B330 • Mutate it with circular permutant A44-B61 for M.HaeIII activity • Showed markedly enhanced in vitro activity compared to respective starting points

15. Directly selected circular permutants

16. What we found? • Selection either through intermediates or directly for permutants yielded similar results What it means? • In case of DNA methyltransferases, the same structural constraints apply to both the intermediates and the end products

17. Plausibility of P by D model • Foldability and Functionality of truncated intermediates • Do they fold properly? If so, how? • What role hydrophobic surfaces play? • Hypothesis supported by Power Law

18. ANP : Non polar accessible surface area • MW : Molecular Weight • Power Law : ANP ∝ MW0.73 • The theoretical dependence of ANP on MW was calculated using : • ANP=fNP6.3*MW0.73 • fNP: nonpolar fraction of the total area of the full length protein

19. Identification of natural h-class • No topology has thus far been identified with TRD halved(h-class) • Experiments indicated that this topology is functional • Bacillus stearothermophilus, E.coli & Hafniaalveiwere identified as members of h-class

20. Conclusion • New protein topology can evolve through multistep gene rearrangements • Point mutation favors one topology over others • Modularity of the methyltransferase fold dictates which intermediates will fold, posses function and eventually yield a new topology

21. Future Avenues • Duplicated modules swapped at protein level before permutation is completed at the gene level • If other classes, like the h-class, which was first identified in the laboratory, exists? • Methods other than Permutation by Duplication

22. Thank you !