FY2020 Annual Report

Protein Engineering and Evolution Unit
Assistant Professor Paola Laurino

 

Abstract

In the past few decades protein engineering allowed to generating artificial enzymes able to catalize unnatural reactions but also to become importnat tools for synthetic biology. Our Unit is particulary interested in generating new enzymes by directed evolution but also to understand how enzymes evolve in Nature.

1. Staff

  • Dr. Madhuri Gade, Researcher
  • Dr. Bhanu Chouhan, Researcher (till March)
  • Dr. Mirco Dindo, Researcher (JSPS Fellow)
  • Dr. Saacnicteh Toledo Patino, Researcher
  • Dr. Benjamin Clifton, Researcher (JSPS Fellow)
  • Dr. Gen-ichiro Uechi, Technician
  • Ms. Desirae Martinez, Technician (till August)
  • Mr. Stefano Pascarelli, Graduate Student
  • Mr. Dan Kozome, Graduate Student
  • Mr. Yoshiki Ochiai, Graduate Student
  • Ms. Samira Gmuer, Graduate Student
  • Mr. Andrea Testa, strategic JSPS fellow (From February 2020 to August)
  • Mr. Aiman Fariz, Intern Student (From February to March)
  • Ms. Sachie Matsuoka, Research Unit Administrator(till August)
  • Ms. Mika Uehara, Research Unit Administrator

2. Collaborations

2.1 Topic: Discovery of a new metabolite in human cells towards investigation of human methionine adenosyltransferases promiscuities

  • Type of collaboration: Joint research
  • Researchers:
    • Professor Colin Jackson, ANU, Australia

2.2 Topic: Enzymatically Active droplets

  • Type of collaboration: Joint research
  • Researchers:
    • Professor Eric R. Dufresne, ETHZ, Switzerland

2.3 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 Protein and substrate flexibility contribute to enzymatic specificity in human and bacterial methionine adenosyltransferases

Protein conformational change can facilitate the binding of non-cognate substrates and underlie promiscuous activities. However, the contribution of substrate conformational dynamics to this process is comparatively poorly understood. Here we analyse human (hMAT2A) and Escherichia coli (eMAT) methionine adenosyltransferases that have identical active sites but different substrate specificity. In the promiscuous hMAT2A, non-cognate substrates bind in a stable conformation to allow catalysis. In contrast, non-cognate substrates rarely sample stable productive binding modes in eMAT owing to increased mobility of an active site loop. Different cellular concentrations of substrate likely drove the evolutionary divergence of substrate specificity in these orthologs. The observation of catalytic promiscuity in hMAT2A led to the detection of a new human metabolite, methyl thioguanosine, that is produced at elevated level in a cancer cell line. This work establishes that identical active sites can result in different substrate specificity owing to the combined effects of both enzyme and substrate dynamics.

Figure 1: Structural and sequence aligment of MATs.

3.2 Consequences of the divergence of Methionine AdenosylTransferase

Methionine adenosyltransferase (MAT), which catalyzes the biosynthesis of S-adenosylmethionine from L-methionine and ATP, is an ancient, highly conserved enzyme present in all three domains of life. Although the MAT enzymes of each domain are believed to share a common ancestor, the sequences of archaeal MATs show a high degree of divergence from the sequences of bacterial and eukaryotic MATs. However, the structural and functional consequences of this sequence divergence are not well understood. Here, we use structural bioinformatics analysis and ancestral sequence reconstruction to highlight the consequences of archaeal MAT divergence. We show that the dimer interface containing the active site, which would be expected to be well conserved across all three domains, diverged considerably between the bacterial/eukaryotic MATs and archaeal MATs. Furthermore, the characterization of reconstructed ancestral archaeal MATs showed that they probably had low substrate specificity which expanded during their evolutionary trajectory hinting towards the observation that all the modern day MAT enzymes from the three-kingdom probably originated from a common specific ancestor and then archaea MATs diverged in sequence, structure and substrate specificity. Altogether, our results show that the archaea MAT is an ideal system for studying an enzyme family which evolved to display high degrees of divergence at the sequence/structural levels and yet are capable of performing the same catalytic reactions as their orthologous counterparts.

Figure 2: Schematic representation of observed trends in MAT.

3.3 Single EGF mutants unravel the mechanism for stabilization of Epidermal Growth Factor Receptor (EGFR) system

The Epidermal Growth Factor Receptor (EGFR) is a membrane-anchored tyrosine kinase that is able to respond to multiple extra-cellular stimuli in a selective way. Previous studies have pointed out that the modularity of this system could be carried out by ligand-induced differences in the stability of the dimerized receptor. Nevertheless, nobody has ever explored this hypothesis by observing the effects of single-mutant ligands so far. Herein, we identified residues responsible for inducing functional divergence among paralog ligands using a newly developed approach. Then, we mutated these positions and assessed the mutants’ effects on the receptor by employing a combination of molecular dynamics and experimental techniques. Although having comparable binding affinities for EGFR to the wild type, the EGF mutants induced different responses at both the receptor and the cellular level. This study shows for the first time that a single mutation in EGFR ligand is enough for changing the outcome of the activation pathway at the cellular level. These results also support the theory of biased signaling in the tyrosine kinase receptor system and show a promising new way to study ligand-receptor interactions.

Figure 3: Cross-conservation analysis

4. Publications

4.1 Journals

  1. Danielson, E.*; Dindo, M.; Porkovich, A. J.; Kumar, P.; Wang, Z.; Jain, P.; Mete, T.; Ziadi, Z.; Raghavendra, K.; Laurino, P.; Sowwan, M. Non-Enzymatic and Highly Sensitive Lactose Detection Utilizing Graphene Field-Effect Transistors. Biosensors and Bioelectronics. 2020, 165, 112419 https://doi.org/10.1016/j.bios.2020.112419

4.2 Books and other one-time publications

Nothing to report

4.3 Oral and Poster Presentations

  1. Benjamin Clifton, “RNA 2020 (25th Annual Meeting of the RNA Society)”, May 26-31, 2020.
  2. Stefano Pascarelli, "Remote BioExcel Winter School on Biomolecular Simulations", 30 November - 4 December 2020.
  3. Paola Laurino, “A protein evolution workshop”,  1-3 March 2021 OnLine Volkswagen symposium 

5. Intellectual Property Rights and Other Specific Achievements

Nothing to report

6. Meetings and Events

6.1 Seminar: "Harnessing Conformational Dynamics to Engineer New Enzymes"

  • Date: July 7, 2020
  • Venue: Online
  • Speaker: Prof. Lynn Kamerlin, (Uppsala University)

6.2 Seminar: "The reductive glycine pathway - a plug-and-play tool for one-carbon   assimilation"

  • Date: Aug 18, 2020
  • Venue: Online
  • Speaker: Dr. Arren Bar-Even, (Max Planck Institute of Molecular Plant Physiology)

6.3 Seminar: "Protein Allostery: Evolution and Correlated Motions"

  • Date: Oct 26, 2020
  • Venue: Online
  • Speaker: Prof. Nozomi Ando, (Cornell University)

6.4 Seminar: "Designing modular proteins"

  • Date: Dec 3, 2020
  • Venue: Online
  • Speaker: Dr. Fabio Parmeggiani, (University of Bristol)

 

7. Other

      1. Dr. Mirco Dindo presented a Science Dialogue demonstration at  Kyuyo High School on Jan 27, 2021.

      2. Yoshiki introduces OIST as distinct systems from other grad schools in Japan. Published in 実験医学 in Feb 2021.