FY2022 Annual Report

Protein Engineering and Evolution Unit
Associate Professor Paola Laurino

 

Abstract

In the past few decades, protein engineering allowed the generation of artificial enzymes to catalyze unnatural reactions and become important tools for synthetic biology. Our Unit is particularly interested in generating new enzymes by directed evolution and understanding how new protein functions emerge in Nature.

1. Staff

  • Prof. Paola Laurino, Associate Professor
  • Dr. Benjamin Clifton, Staff Scientist
  • Dr. Saacnicteh Toledo Patino, Postdoctoral Scholar
  • Dr. Prashant Jain, Postdoctoral Scholar
  • Dr. Yusran Abdillah Muthahari, Postdoctoral Scholar
  • Dr. Gen-ichiro Uechi, Technician
  • Dr. Mirco Dindo, Visiting Researcher
  • Mr. Stefano Pascarelli, PhD Student
  • Mr. Dan Kozome. PhD Student
  • Mr. Yoshiki Ochiai. PhD Student
  • Mr. Alessandro Bevilacqua. PhD Student
  • Ms. Amy Stanton Gooch. PhD Student
  • Ms. Lilian Magnus. PhD Student
  • Ms. Erika Fukuhara, PhD Student
  • Ms. Anjali Gupta, Rotation Student (Sep-Dec 2022)
  • Mr. Junho Lee, Rotation Student (Jan-Apr 2023)
  • Ms. Adriana Hernández González, Research Intern (Jan-Aug 2022)
  • Ms. Trang Vy Bui, Research Intern (from Oct 2022)
  • Ms. Diana Nechepurenko, Research Intern (from Feb 2023)

2. Collaborations

2.1 Topic: The functional instability of alanine:glyoxylate aminotransferase 

  • Type of collaboration: Joint research
  • Researchers:
    • Professor Barbara Cellini, Perugia University, Italy
    • Professor Giorgio Giardina, Rome University, Italy

3. Activities and Findings

3.1 Insertions and deletions mediated functional divergence of Rossmann fold enzymes

Nucleobase-containing coenzymes are hypothesized to be relics of an early RNA-based world that preceded the emergence of proteins. Despite the importance of coenzyme–protein synergisms, their emergence and evolution remain understudied. An excellent target to address this issue is the Rossmann fold, the most catalytically diverse and abundant protein architecture in nature. We investigated two main Rossmann lineages: the nicotinamide adenine dinucleotide phosphate (NAD(P)) and the S-adenosyl methionine (SAM)- binding superfamilies. To identify the evolutionary changes that lead to a coenzyme specificity switch on these superfamilies, we performed structural and sequence-based Hidden Markov model analysis to systematically search for key motifs in their coenzyme-binding pockets. Our analyses revealed that through insertions and deletions (InDels) and a residue substitution, the ancient β1−loop−α1 coenzyme-binding structure of NAD(P) could be reshaped into the SAM-binding β1−loop−α1 structure. To experimentally prove this obsevation, we removed three amino acids from the NAD(P)-binding pocket and solved the structure of the resulting mutant, revealing the characteristic loop features of the SAM-binding pocket. To confirm the binding to SAM, we performed isothermal titration calorimetry measurements. Molecular dynamics simulations also corroborated the role of InDels in abolishing NAD binding and acquiring SAM binding. Our results uncovered how nature may have utilized insertions and deletions to optimize the different coenzyme-binding pockets and the distinct functionalities observed for Rossmann superfamilies. This work also proposes a general mechanism by which protein templates could have been recycled through the course of evolution to adopt different coenzymes and confer distinct chemistries.

3.2 Structural dynamics shape the fitness window of alanine:glyoxylate aminotransferase

The conformational landscape of a protein is constantly expanded by genetic variations that have a minimal impact on the function(s) while causing subtle effects on protein structure. The wider the conformational space sampled by these variants, the higher the probabilities to adapt to changes in environmental conditions. However, the probability that a single mutation may result in a pathogenic phenotype also increases. Here we present a paradigmatic example of how protein evolution balances structural stability and dynamics to maximize protein adaptability and preserve protein fitness. We took advantage of known genetic variations of human alanine:glyoxylate aminotransferase (AGT1), which is present as a common major allelic form (AGT-Ma) and a minor polymorphic form (AGT-Mi) expressed in 20% of Caucasian population. By integrating crystallographic studies and molecular dynamics simulations, we show that AGT-Ma is endowed with structurally unstable (frustrated) regions, which become disordered in AGT-Mi. An in-depth biochemical characterization of variants from an anticonsensus library, encompassing the frustrated regions, correlates this plasticity to a fitness window defined by AGT-Ma and AGT-Mi. Finally, co-immunoprecipitation analysis suggests that structural frustration in AGT1 could favor additional functions related to protein–protein interactions. These results expand our understanding of protein structural evolution by establishing that naturally occurring genetic variations tip the balance between stability and frustration to maximize the ensemble of conformations falling within a well-defined fitness window, thus expanding the adaptability potential of the protein.

3.3 Inter-paralog amino acid inversion events in large phylogenies of duplicated proteins

Connecting protein sequence to function is becoming increasingly relevant since high-throughput sequencing studies accumulate large amounts of genomic data. In order to go beyond the existing database annotation, it is fundamental to understand the mechanisms underlying functional inheritance and divergence. If the homology relationship between proteins is known, can we determine whether the function diverged? In this work, we analyze different possibilities of protein sequence evolution after gene duplication and identify “inter-paralog inversions”, i.e., sites where the relationship between the ancestry and the functional signal is decoupled. The amino acids in these sites are masked from being recognized by other prediction tools. Still, they play a role in functional divergence and could indicate a shift in protein function. We develop a method to specifically recognize inter-paralog amino acid inversions in a phylogeny and test it on real and simulated datasets. In a dataset built from the Epidermal Growth Factor Receptor (EGFR) sequences found in 88 fish species, we identify 19 amino acid sites that went through inversion after gene duplication, mostly located at the ligand-binding extracellular domain. Our work uncovers an outcome of protein duplications with direct implications in protein functional annotation and sequence evolution. The developed method is optimized to work with large protein datasets and can be readily included in a targeted protein analysis pipeline.

4. Publications

4.1 Journals​

  1. Pascarelli S.; Laurino P.* “Inter-paralog amino acid inversion events in large phylogenies of duplicated proteins”, Plos Comp Biol 2022, 18, 4, e1010016. https://doi.org/10.1371/journal.pcbi.1010016
  2. Dindo, M.; Pascarelli, S.; Chiasserini, D.; Grotelli, S.; Constantini, C.; Uechi, G.; Giardina, G.*; Laurino, P.*, Cellini B*, “Structural dynamics shape the fitness window of alanine:glyoxylate aminotransferase”, Protein Science 2022, 31, e4303. https://onlinelibrary.wiley.com/doi/10.1002/pro.4303
  3. Jayaraman V, Toledo-Patiño S, Noda-García L, Laurino P, "Mechanisms of protein evolution", Protein Science, 2022, 31, e4362  https://doi.org/10.1002/pro.4362
  4. Toledo-Patiño S, Pascarelli S, Uechi GI, Laurino P, "Insertions and deletions mediated functional divergence of Rossmann fold enzymes", PNAS, 2022, 119, 48, e2207965119 https://doi.org/10.1073/pnas.2207965119
  5. Dindo M, Bevilacqua A, Laurino P, "Enzymes and Liquid-Liquid phase separation: a new era for the regulation of enzyme activity", Vol.63 No.1, Seibutsu Butsuri by The Biophysical Society of Japan, Feb 2023 https://www.biophys.jp/journal/journal_dl.php?fnm=63-1
  6. Van Toorn M, Gooch A, Boerner S, Kiyomitsu T, "NuMA deficiency causes micronuclei via checkpoint-insensitive k-fiber minus-end detachment from mitotic spindle poles", Current Biology, 6 Feb 2023 https://www.sciencedirect.com/science/article/pii/S0960982222019170

4.2 Books and other one-time publications

  1. Aoki S, Ochiai Y, “Five Useful English Correction and Translation Tools for Writing Research Papers”, Vol.40 No.6. Jikken Igaku, Experimental Medicine, Apr 2022 (Contributed Essay) 
  2. Aoki S, Ochiai Y, Kato Y, “Tools to Help You Write Your Paper in English”, Vol.40 No.16. Jikken Igaku, Experimental Medicine, Oct 2022 (Contributed Essay) 

4.3 Oral and Poster Presentations

Seminars
  1. Laurino P, "Ancient cofactors fingerprints of Rossmann Fold enzymes as a tool for cofactor engineering", Mario Negri Institute, Milan, Italy, 27 Apr 2022
  2. Laurino P, "The unexpected emergence of new enzyme functions", IFOM Milan, Italy, 12 Dec 2022
  3. Laurino P, "The unexpected emergence of new enzyme functions", Human Technopole, Milan, Italy, 13 Dec 2022
  4. Laurino P, "Insertions and Deletions (InDels): Hopeful monsters for protein engineering?", Institute for Protein Research, Osaka University, Japan, 15 Dec 2022
  5. Laurino P, "From so simple a beginning", OIST Provost Lecture, OIST, Japan, 24 Jan 2023
  6. Ochiai Y, "Engineering of improved non-specific RNA methyltransferase by ancestral sequence reconstruction”, Invited talk hosted by Professor Colin Jackson, Canberra, Australia, 10 Feb 2023
  7. Laurino P, "Insertions and Deletions mediate cofactor specificity and functional transition", SPEED Journal club, Japan, 24 Feb 2023

 

Conference Oral Presentations
  1. Ochiai Y, “Structural and functional characterization of DNA/RNA methyltransferase specificity”, Advances in Protein Folding, Evolution, and Design 2022 (online), 6-8 Apr 2022
  2. Laurino P, "Evolutionary-guided cofactor engineering", Enzyme Engineering XXVI conference, Dallas, USA, 26 May 2022
  3. Ochiai Y, “Protein engineering of DNA/RNA methyltransferase”, The 2nd Young Scientists' Meeting of the Protein Science Society of Japan (第2回蛋白質科学会 若手の会研究交流会), Tsukuba, Japan, 6 Jun 2022
  4. Ochiai Y, “Structural and functional characterization of DNA/RNA methyltransferase specificity”. The 22nd Annual Meeting of the Protein Science Society of Japan, Tsukuba, Japan, 7-9 Jun 2022
  5. Clifton, B.E., Laurino, P, “Functional characterization of the solute-binding protein repertoire of Candidatus Pelagibacter ubique”, the 22nd Annual Meeting of the Protein Science Society of Japan, Tsukuba, Japan, 9 Jun 2022
  6. Ochiai Y, “Investigation of Evolutionary Functional Origins of Promiscuity in DNA/RNA Methyltransferases”, The 36th Annual Symposium of the Protein Society, San Francisco, USA, 5-9 Jul 2022
  7. Ochiai Y, “祖先再構成法によるDNA/RNAアデニンメチル化酵素の機能変遷の追跡” Society of Young Scientists in Biophysics Kansai branch seminar生物物理若手の会 関西支部セミナー (online), 23 Jul 2022
  8. Laurino P, "Insertions and Deletions (InDels): Hopeful monsters for protein engineering?", GRC Enzyme coenzyme and Metabolic pathways, Waterville, USA, 25 Jul 2022
  9. Laurino P, "Insertions and Deletions (InDels): Hopeful monsters for protein engineering?", Multidisciplinary Symposium, Hokkaido University, Japan, 5 Aug 2022
  10. Ochiai Y, “祖先再構成法によるDNA/RNAアデニンメチル化酵素の機能変遷の追跡”. 第62回生命科学夏の学校 (62nd Summer School of Life Sciences), Sendai, Japan, 26-28 Aug 2022 (Best Presentation Award)
  11. Ochiai Y, “祖先再構成法によるDNA/RNAアデニンメチル化酵素の機能変遷の追跡”, 第62回生物物理夏の学校 (62nd Summer School of Biophysics), Gifu, Japan, 30 Aug- 2 Sep 2022
  12. Laurino P, "Insertions and Deletions (InDels): Hopeful monsters for protein engineering?" Departmental Seminar, Urbino University, Italy, 1 Sep 2022
  13. Ochiai Y, “Development of directed evolution method for changing sequence specificity and cofactor selectivity of RNA methyltransferases”. The 60th Annual Meeting of Biophysical Society of Japan, Hakodate, Japan, 28-30 Sep 2022
  14. Laurino P, "Insertions and Deletions (InDels): Hopeful monsters for protein engineering?" Active Enzyme Molecule 2022, Toyama, Japan, 1 Oct 2022
  15. Laurino P, "Insertions and Deletions mediate cofactor specificity and functional transition" Advances in Protein Design: From therapeutic Proteins to Synthetic Biology Workshop, Kfar Blum, Israel, 23 Oct 2022
  16. Bevilacqua A, "Shaping enzyme activity by tuning protocells'composition", Researcher Appreciation Week, OIST, Japan, 31 Oct 2022
  17. Laurino P, "Enzymes behavior in highly crowded membrane-less compartments" Challenge in Biomedical Complexity, OIST-Kyoto University Workshop, OIST, Japan, 2 Nov 2022
  18. Laurino P, "Insertions and Deletions (InDels): Hopeful monsters for protein engineering?" Molecular Engine Meeting, Okinawa Japan, 24 Nov 2022
  19. Ochiai Y, “Engineering of Enzymatic Activity of DNA/RNA Methyltransferase M.EcoGII by Ancestral Sequence Reconstruction”. The 46th Annual Meeting of the Molecular Biology Society of Japan, Chiba, Japan, 30 Nov-2 Dec 2022
  20. Pascarelli P, "Meta-Functionalization: when ancestry and functional relationships are broken”, The 46th Annual Meeting of the Molecular Biology Society of Japan, Chiba, Japan, 2 Dec 2022
  21. Ochiai Y, “Engineering of Enzymatic Activity of DNA/RNA Methyltransferase M.EcoGII by Ancestral Sequence Reconstruction”, The 48th Lorne Conference on Protein Structure and Function, Lorne, Australia, 5-9 Feb 2023 (Poster Presentation Awards)
  22. Toledo-Patiño S, "Design of protein wires for electron transmission as alternative for sustainable energy", The 10th Iwatani Foundation Research Presentation (online), 7 Mar 2023
  23. Laurino P, “Expanding the Substrate Scope of Fluoroacetate Dehalogenase Enzymes via Directed Evolution”, The 10th Iwatani Foundation Research Presentation (online), 7 Mar 2023

 

Conference Poster Presentations
  1. Kozome D, "InDels in the loop region shapes new protein function in the evolution of GH19 chitinase" Advances in Protein Folding, Evolution, and Design, Bayreuth, Germany, 6 Apr 2022
  2. Toledo-Patiño S, "An evolutionary path to engineer cofactor specificity from NAD to SAM employing InDels", Advances in Protein Folding, Evolution, and Design, Germany, 6-8 Apr 2022
  3. Kozome D, "InDels in the loop region shapes new protein function in the evolution of GH19 chitinase" The 36th Protein Society Annual Symposium, San Francisco, USA, 8 Jul 2022 (Poster Award)
  4. Jain P, “Directed evolution approach to understand complex kinase signaling” the Hybrid EMBO Workshop Chemical biology, Heidelberg, Germany, 5-8 Sep 2022
  5. Bevilacqua A, "Shaping enzyme activity by tuning protocells' composition", FISV2022 congress, Naples, Italy, 15 Sep 2022
  6. Gooch A, “The Sequence Space of Fluoroacetate Dehalogenase Defines Good Starting Points for the Design of Enzymes with Novel Defluorination Activities” Advances in Protein Design: From Therapeutic Proteins to Synthetic Biology”workshop, Kfar Blum, Israel, 23-27 Oct 2022
  7. Magnus L, "Cofactor specificity in FAD-binding Rossmann fold proteins", Advances in Protein Design: From therapeutic Proteins to Synthetic Biology Workshop, Kfar Blum, Israel, 26-27 Oct 2022
  8. Kozome D, "InDels in the loop region shapes new protein function in the evolution of GH19 chitinase", the 11th Molecular Engine Area Meeting 第十一回発動分子科学領域会議, Okinawa, Nov 25, 2022
  9. Pascarelli S, "Meta-functionalization: an ancestry-unrelated swap of residues in the Epidermal Growth Factor Receptor (EGFR) evolution after Teleost genome duplication”, The 46th Annual Meeting of the Molecular Biology Society of Japan, Chiba, Japan, 2 Dec 2022
  10. Fukuhara E, "Analysis of Bacillus subtilis Malate Dehydrogenase and Citrate Synthase Metabolon Formation in Phase Separated Protein Droplets", Workshop on Recent Trends in Microrheology and Microfluidics, OIST, January 10, 2023
  11. Ochiai Y, “Engineering of Enzymatic Activity of DNA/RNA Methyltransferase M.EcoGII by Ancestral Sequence Reconstruction”. The 48th Lorne Conference on Protein Structure and Function, Lorne, Australia, 5-9 Feb 2023 (Poster Presentation Awards)
  12. Toledo-Patiño S, "InDel puzzles. Insertions and deletions tailoring coenzyme binding", Lorne Proteins in Lorne Australia, 7 Feb 2023
  13. Toledo-Patiño S, "Design of protein wires for electron transmission as alternative for sustainable energy", Iwatani Grant meeting, Tokyo, 7 Mar 2023

 

5. Intellectual Property Rights and Other Specific Achievements

Nothing to report

6. Meetings and Events

6.1 Seminar

  • Date: June 29, 2022
  • Venue: Zoom
  • Speaker: Dr. Vijay Jayaraman (Weizmann Institute of Science)
  • Title: “Discovery of an anti-metabolon: a paradoxical way of regulating enzymes”

6.2 Seminar 

  • Date: August 17, 2022
  • Venue: OIST campus
  • Speaker: Prof. Stefan Matile (University of Geneva, Switzerland)
  • Title: “Translational Supramolecular Chemistry”

6.3 Seminar 

  • Date: August 22, 2022
  • Venue: OIST campus
  • Speakers: Prof. Liam Longo (Earth-Life Science Institute) 
  • Title: "On the continuity between ancient geochemistry and modern biochemistry"

 

7. Other

PL participated in the UN science summit promoting the inclusivity project ROZ.

https://ssunga77.sched.com/event/1AbND/roz-fosters-scientific-careers-through-inclusivity-interactivity-and-internationalism?iframe=no