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NMR and crystal structures of the Pyrococcus horikoshii RadA intein guide a strategy for engineering a highly efficient and promiscuous intein.

Oeemig JS, Zhou D, Kajander T, Wlodawer A, Iwaï H
J Mol Biol 421 85-99 (2012)
Related entries: 2lqm, 4e2t

Cited: 47 times
EuropePMC logo PMID: 22560994

Abstract

In protein splicing, an intervening protein sequence (intein) in the host protein excises itself out and ligates two split host protein sequences (exteins) to produce a mature host protein. Inteins require the involvement for the splicing of the first residue of the extein that follows the intein (which is Cys, Ser, or Thr). Other extein residues near the splicing junctions could modulate splicing efficiency even when they are not directly involved in catalysis. Mutual interdependence between this molecular parasite (intein) and its host protein (exteins) is not beneficial for intein spread but could be advantageous for intein survival during evolution. Elucidating extein-intein dependency has increasingly become important since inteins are recognized as useful biotechnological tools for protein ligation. We determined the structures of one of inteins with high splicing efficiency, the RadA intein from Pyrococcus horikoshii (PhoRadA). The solution NMR structure and the crystal structures elucidated the structural basis for its high efficiency and directed our efforts of engineering that led to rational design of a functional minimized RadA intein. The crystal structure of the minimized RadA intein also revealed the precise interactions between N-extein and the intein. We systematically analyzed the effects at the -1 position of N-extein and were able to significantly improve the splicing efficiency of a less robust splicing variant by eliminating the unfavorable extein-intein interactions observed in the structure. This work provides an example of how unveiling structure-function relationships of inteins offer a promising way of improving their properties as better tools for protein engineering.

Reviews - 4e2u mentioned but not cited (1)

  1. Structural and dynamical features of inteins and implications on protein splicing. Eryilmaz E, Shah NH, Muir TW, Cowburn D. J Biol Chem 289 14506-14511 (2014)

Articles - 4e2u mentioned but not cited (3)

  1. NMR and crystal structures of the Pyrococcus horikoshii RadA intein guide a strategy for engineering a highly efficient and promiscuous intein. Oeemig JS, Zhou D, Kajander T, Wlodawer A, Iwaï H. J Mol Biol 421 85-99 (2012)
  2. An Unprecedented Combination of Serine and Cysteine Nucleophiles in a Split Intein with an Atypical Split Site. Bachmann AL, Mootz HD. J Biol Chem 290 28792-28804 (2015)
  3. Mini-Intein Structures from Extremophiles Suggest a Strategy for Finding Novel Robust Inteins. Hiltunen MK, Beyer HM, Iwaï H. Microorganisms 9 1226 (2021)


Reviews citing this publication (5)

  1. Protein splicing: how inteins escape from precursor proteins. Mills KV, Johnson MA, Perler FB. J Biol Chem 289 14498-14505 (2014)
  2. Recent progress in intein research: from mechanism to directed evolution and applications. Volkmann G, Mootz HD. Cell Mol Life Sci 70 1185-1206 (2013)
  3. Inteins in Science: Evolution to Application. Nanda A, Nasker SS, Mehra A, Panda S, Nayak S. Microorganisms 8 E2004 (2020)
  4. Inteins-mechanism of protein splicing, emerging regulatory roles, and applications in protein engineering. Wood DW, Belfort M, Lennon CW. Front Microbiol 14 1305848 (2023)
  5. Nature-inspired protein ligation and its applications. Pihl R, Zheng Q, David Y. Nat Rev Chem 7 234-255 (2023)

Articles citing this publication (38)

  1. Inteins: Nature's Gift to Protein Chemists. Shah NH, Muir TW. Chem Sci 5 446-461 (2014)
  2. A promiscuous split intein with expanded protein engineering applications. Stevens AJ, Sekar G, Shah NH, Mostafavi AZ, Cowburn D, Muir TW. Proc Natl Acad Sci U S A 114 8538-8543 (2017)
  3. Faster protein splicing with the Nostoc punctiforme DnaE intein using non-native extein residues. Cheriyan M, Pedamallu CS, Tori K, Perler F. J Biol Chem 288 6202-6211 (2013)
  4. Recent advances in in vivo applications of intein-mediated protein splicing. Topilina NI, Mills KV. Mob DNA 5 5 (2014)
  5. Post-translational environmental switch of RadA activity by extein-intein interactions in protein splicing. Topilina NI, Novikova O, Stanger M, Banavali NK, Belfort M. Nucleic Acids Res 43 6631-6648 (2015)
  6. Intermolecular domain swapping induces intein-mediated protein alternative splicing. Aranko AS, Oeemig JS, Kajander T, Iwaï H. Nat Chem Biol 9 616-622 (2013)
  7. Structure-based engineering and comparison of novel split inteins for protein ligation. Aranko AS, Oeemig JS, Zhou D, Kajander T, Wlodawer A, Iwaï H. Mol Biosyst 10 1023-1034 (2014)
  8. Protein splicing of a recombinase intein induced by ssDNA and DNA damage. Lennon CW, Stanger M, Belfort M. Genes Dev 30 2663-2668 (2016)
  9. An expanded library of orthogonal split inteins enables modular multi-peptide assemblies. Pinto F, Thornton EL, Wang B. Nat Commun 11 1529 (2020)
  10. A functional interplay between intein and extein sequences in protein splicing compensates for the essential block B histidine. Friedel K, Popp MA, Matern JCJ, Gazdag EM, Thiel IV, Volkmann G, Blankenfeldt W, Mootz HD. Chem Sci 10 239-251 (2019)
  11. Mycobacterial DnaB helicase intein as oxidative stress sensor. Kelley DS, Lennon CW, Li Z, Miller MR, Banavali NK, Li H, Belfort M. Nat Commun 9 4363 (2018)
  12. Conditional Protein Splicing Switch in Hyperthermophiles through an Intein-Extein Partnership. Lennon CW, Stanger M, Banavali NK, Belfort M. mBio 9 e02304-17 (2018)
  13. Chemical bypass of intein-catalyzed N-S acyl shift in protein splicing. Binschik J, Mootz HD. Angew Chem Int Ed Engl 52 4260-4264 (2013)
  14. Structural Basis for the Persistence of Homing Endonucleases in Transcription Factor IIB Inteins. Iwaï H, Mikula KM, Oeemig JS, Zhou D, Li M, Wlodawer A. J Mol Biol 429 3942-3956 (2017)
  15. Structure-activity studies on the upstream splice junction of a semisynthetic intein. Wasmuth A, Ludwig C, Mootz HD. Bioorg Med Chem 21 3495-3503 (2013)
  16. Structural basis for protein trans-splicing by a bacterial intein-like domain--protein ligation without nucleophilic side chains. Aranko AS, Oeemig JS, Iwaï H. FEBS J 280 3256-3269 (2013)
  17. Mechanism of Single-Stranded DNA Activation of Recombinase Intein Splicing. Lennon CW, Stanger MJ, Belfort M. Biochemistry 58 3335-3339 (2019)
  18. The crystal structure of the naturally split gp41-1 intein guides the engineering of orthogonal split inteins from cis-splicing inteins. Beyer HM, Mikula KM, Li M, Wlodawer A, Iwaï H. FEBS J 287 1886-1898 (2020)
  19. Conserved residues that modulate protein trans-splicing of Npu DnaE split intein. Wu Q, Gao Z, Wei Y, Ma G, Zheng Y, Dong Y, Liu Y. Biochem J 461 247-255 (2014)
  20. Crystal structures of CDC21-1 inteins from hyperthermophilic archaea reveal the selection mechanism for the highly conserved homing endonuclease insertion site. Beyer HM, Mikula KM, Kudling TV, Iwaï H. Extremophiles 23 669-679 (2019)
  21. Expressed Protein Ligation without Intein. Qiao Y, Yu G, Kratch KC, Wang XA, Wang WW, Leeuwon SZ, Xu S, Morse JS, Liu WR. J Am Chem Soc 142 7047-7054 (2020)
  22. ¹H, ¹³C and ¹⁵N NMR assignments of a Drosophila Hedgehog autoprocessing domain. Xie J, Du Z, Callahan B, Belfort M, Wang C. Biomol NMR Assign 8 279-281 (2014)
  23. SPLICEFINDER - a fast and easy screening method for active protein trans-splicing positions. Zettler J, Eppmann S, Busche A, Dikovskaya D, Dötsch V, Mootz HD, Sonntag T. PLoS One 8 e72925 (2013)
  24. Efficient Generation of Hydrazides in Proteins by RadA Split Intein. Liu J, Ekanayake O, Santoleri D, Walker K, Rozovsky S. Chembiochem 21 346-352 (2020)
  25. Protein Splicing Activity of the Haloferax volcanii PolB-c Intein Is Sensitive to Homing Endonuclease Domain Mutations. Robinzon S, Cawood AR, Ruiz MA, Gophna U, Altman-Price N, Mills KV. Biochemistry 59 3359-3367 (2020)
  26. Allosteric Influence of Extremophile Hairpin Motif Mutations on the Protein Splicing Activity of a Hyperthermophilic Intein. Chiarolanzio KC, Pusztay JM, Chavez A, Zhao J, Xie J, Wang C, Mills KV. Biochemistry 59 2459-2467 (2020)
  27. Backbone assignments of mini-RecA intein with short native exteins and an active N-terminal catalytic cysteine. Pearson CS, Belfort G, Belfort M, Shekhtman A. Biomol NMR Assign 9 235-238 (2015)
  28. Correlation of chemical shifts predicted by molecular dynamics simulations for partially disordered proteins. Karp JM, Eryilmaz E, Cowburn D. J Biomol NMR 61 35-45 (2015)
  29. Extein residues regulate the catalytic function of Spl DnaX intein enzyme by restricting the near-attack conformations of the active-site residues. Boral S, Sen S, Kushwaha T, Inampudi KK, De S. Protein Sci 32 e4699 (2023)
  30. The Convergence of the Hedgehog/Intein Fold in Different Protein Splicing Mechanisms. Beyer HM, Virtanen SI, Aranko AS, Mikula KM, Lountos GT, Wlodawer A, Ollila OHS, Iwaï H. Int J Mol Sci 21 E8367 (2020)
  31. A hydrolase-based reporter system to uncover the protein splicing performance of an archaeal intein. von der Heyde A, Lockhauserbäumer J, Uetrecht C, Elleuche S. Appl Microbiol Biotechnol 99 7613-7624 (2015)
  32. An alternative domain-swapped structure of the Pyrococcus horikoshii PolII mini-intein. Williams JE, Jaramillo MV, Li Z, Zhao J, Wang C, Li H, Mills KV. Sci Rep 11 11680 (2021)
  33. Letter Coordination of the third step of protein splicing in two cyanobacterial inteins. Ramsoomair CK, Yakely AE, Urbanski LM, Karanja K, Giaccone ZT, Siegart NM, Wang C, Gomez AV, Reitter JN, Mills KV. FEBS Lett 591 2147-2154 (2017)
  34. Labeling Ebola Virus with a Self-Splicing Fluorescent Reporter. Heiden B, Mühlberger E, Lennon CW, Hume AJ. Microorganisms 10 2110 (2022)
  35. Mechanistic Insights into Cyclic Peptide Generation by DnaE Split-Inteins through Quantitative and Structural Investigation. Kick LM, Harteis S, Koch MF, Schneider S. Chembiochem 18 2242-2246 (2017)
  36. Protein Synthesis via Activated Cysteine-Directed Protein Ligation. Yu G, Qiao Y, Blankenship LR, Liu WR. Methods Mol Biol 2530 159-167 (2022)
  37. Standard Intein Gene Expression Ramps (SIGER) for Protein-Independent Expression Control. Fages-Lartaud M, Mueller Y, Elie F, Courtade G, Hohmann-Marriott MF. ACS Synth Biol 12 1058-1071 (2023)
  38. SufB intein splicing in Mycobacterium tuberculosis is influenced by two remote conserved N-extein histidines. Panda S, Nanda A, Sahu N, Ojha DK, Pradhan B, Rai A, Suryawanshi AR, Banavali N, Nayak S. Biosci Rep 42 BSR20212207 (2022)


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