Analysis of allosteric communication in a multienzyme complex by ancestral sequence reconstruction

Significance Enzyme complexes consist of several protein subunits that often catalyze sequential reactions in the cell. The activities of the individual subunits must be kept in phase, which requires a sophisticated communication mechanism that is mediated by certain residues. We wanted to elucidate the communication mechanisms between the α-subunits and β-subunits of the tryptophan synthase (TS) complex, which catalyzes the last 2 steps of the biosynthesis of the essential amino acid tryptophan. To this end, an approach involving the reconstruction of TSs from extinct species was followed. The results of our experimental and computational analyses identified 4 residues that contribute to the communication between the α-subunits and β-subunits and provide an explanation of how they act. Tryptophan synthase (TS) is a heterotetrameric αββα complex. It is characterized by the channeling of the reaction intermediate indole and the mutual activation of the α-subunit TrpA and the β-subunit TrpB via a complex allosteric network. We have analyzed this allosteric network by means of ancestral sequence reconstruction (ASR), which is an in silico method to resurrect extinct ancestors of modern proteins. Previously, the sequences of TrpA and TrpB from the last bacterial common ancestor (LBCA) have been computed by means of ASR and characterized. LBCA-TS is similar to modern TS by forming a αββα complex with indole channeling taking place. However, LBCA-TrpA allosterically decreases the activity of LBCA-TrpB, whereas, for example, the modern ncTrpA from Neptuniibacter caesariensis allosterically increases the activity of ncTrpB. To identify amino acid residues that are responsible for this inversion of the allosteric effect, all 6 evolutionary TrpA and TrpB intermediates that stepwise link LBCA-TS with ncTS were characterized. Remarkably, the switching from TrpB inhibition to TrpB activation by TrpA occurred between 2 successive TS intermediates. Sequence comparison of these 2 intermediates and iterative rounds of site-directed mutagenesis allowed us to identify 4 of 413 residues from TrpB that are crucial for its allosteric activation by TrpA. The effect of our mutational studies was rationalized by a community analysis based on molecular dynamics simulations. Our findings demonstrate that ancestral sequence reconstruction can efficiently identify residues contributing to allosteric signal propagation in multienzyme complexes.

[1]  Mario Marchese,et al.  Techniques and applications , 2003 .

[2]  Z. Luthey-Schulten,et al.  Dynamical networks in tRNA:protein complexes , 2009, Proceedings of the National Academy of Sciences.

[3]  M E J Newman,et al.  Community structure in social and biological networks , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[4]  G. Hammes,et al.  Kinetic studies of tryptophan synthetase. Interaction of substrates with the B subunit. , 1970, Biochemistry.

[5]  R. Merkl,et al.  Combining ancestral sequence reconstruction with protein design to identify an interface hotspot in a key metabolic enzyme complex , 2017, Proteins.

[6]  Adi Doron-Faigenboim,et al.  FastML: a web server for probabilistic reconstruction of ancestral sequences , 2012, Nucleic Acids Res..

[7]  Shaoyong Lu,et al.  Allosteric Methods and Their Applications: Facilitating the Discovery of Allosteric Drugs and the Investigation of Allosteric Mechanisms. , 2019, Accounts of chemical research.

[8]  Richard K. Moore,et al.  From theory to applications , 1986 .

[9]  A. Mozzarelli,et al.  pH Dependence of Tryptophan Synthase Catalytic Mechanism , 2004, Journal of Biological Chemistry.

[10]  E. Padlan,et al.  Three-dimensional structure of the tryptophan synthase alpha 2 beta 2 multienzyme complex from Salmonella typhimurium. , 1988, The Journal of biological chemistry.

[11]  Ruth Nussinov,et al.  Allostery in Its Many Disguises: From Theory to Applications. , 2019, Structure.

[12]  R. Merkl,et al.  Ancestral protein reconstruction: techniques and applications , 2016, Biological chemistry.

[13]  Sílvia Osuna,et al.  Deciphering the Allosterically-driven Conformational Ensemble in Tryptophan Synthase Evolution. , 2019, Journal of the American Chemical Society.

[14]  J. P. Loria,et al.  Allostery in enzyme catalysis. , 2017, Current opinion in structural biology.

[15]  Georg K. A. Hochberg,et al.  Reconstructing Ancient Proteins to Understand the Causes of Structure and Function. , 2017, Annual review of biophysics.

[16]  Adrian A Canutescu,et al.  Access the most recent version at doi: 10.1110/ps.03154503 References , 2003 .

[17]  Tyler N. Starr,et al.  Epistasis in protein evolution , 2016, Protein science : a publication of the Protein Society.

[18]  R. Merkl,et al.  Mapping the Allosteric Communication Network of Aminodeoxychorismate Synthase. , 2019, Journal of molecular biology.

[19]  K Schulten,et al.  VMD: visual molecular dynamics. , 1996, Journal of molecular graphics.

[20]  David,et al.  Three-dimensional Structure of the Tryptophan Synthase a & Multienzyme Complex from Salmonella typhimurium * , 2001 .

[21]  O. Mayans,et al.  Evolutionary Morphing of Tryptophan Synthase: Functional Mechanisms for the Enzymatic Channeling of Indole. , 2018, Journal of molecular biology.

[22]  Xin-Qiu Yao,et al.  Establishing a Framework of Using Residue-Residue Interactions in Protein Difference Network Analysis , 2019, J. Chem. Inf. Model..

[23]  R. Merkl,et al.  Ancestral Tryptophan Synthase Reveals Functional Sophistication of Primordial Enzyme Complexes. , 2016, Cell chemical biology.

[24]  Thomas L. Madden,et al.  Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. , 1997, Nucleic acids research.

[25]  Guang Hu,et al.  Integration of network models and evolutionary analysis into high-throughput modeling of protein dynamics and allosteric regulation: theory, tools and applications , 2020, Briefings Bioinform..

[26]  C. Sander,et al.  Errors in protein structures , 1996, Nature.

[27]  M. Wilmanns,et al.  Catalysis uncoupling in a glutamine amidotransferase bienzyme by unblocking the glutaminase active site. , 2012, Chemistry & biology.

[28]  M. Dunn.  Allosteric regulation of substrate channeling and catalysis in the tryptophan synthase bienzyme complex. , 2012, Archives of biochemistry and biophysics.

[29]  I. Schlichting,et al.  Loop closure and intersubunit communication in tryptophan synthase. , 1998, Biochemistry.

[30]  D. van der Spoel,et al.  GROMACS: A message-passing parallel molecular dynamics implementation , 1995 .

[31]  Keehyoung Joo,et al.  Improving physical realism, stereochemistry, and side‐chain accuracy in homology modeling: Four approaches that performed well in CASP8 , 2009, Proteins.

[32]  E. W. Miles.  The Tryptophan Synthase α2β2 Complex: A Model for Substrate Channeling, Allosteric Communication, and Pyridoxal Phosphate Catalysis , 2013, The Journal of Biological Chemistry.

[33]  W. Delano.  The PyMOL Molecular Graphics System , 2002 .

[34]  G. Fitzgerald,et al.  'I. , 2019, Australian journal of primary health.

[35]  F. Arnold,et al.  Directed Evolution Mimics Allosteric Activation by Stepwise Tuning of the Conformational Ensemble. , 2018, Journal of the American Chemical Society.

[36]  R. Merkl.  Modelling the evolution of the archeal tryptophan synthase , 2007, BMC Evolutionary Biology.

[37]  E. W. Miles.  Tryptophan synthase: a multienzyme complex with an intramolecular tunnel. , 2001, Chemical record.

[38]  F. Arnold,et al.  Directed evolution of the tryptophan synthase β-subunit for stand-alone function recapitulates allosteric activation , 2015, Proceedings of the National Academy of Sciences.

[39]  R D Appel,et al.  Protein identification and analysis tools in the ExPASy server. , 1999, Methods in molecular biology.

[40]  Zaida Luthey-Schulten,et al.  Exploring residue component contributions to dynamical network models of allostery. , 2012, Journal of chemical theory and computation.

[41]  Georg K. A. Hochberg,et al.  Evolution of protein specificity: insights from ancestral protein reconstruction. , 2017, Current opinion in structural biology.

[42]  R. Sterner,et al.  Standardized cloning vectors for protein production and generation of large gene libraries in Escherichia coli. , 2018, BioTechniques.

[43]  W. Wang,et al.  Two-stage PCR protocol allowing introduction of multiple mutations, deletions and insertions using QuikChange Site-Directed Mutagenesis. , 1999, BioTechniques.