The Energetics of Streptococcal Enolase Octamer Formation: The Quantitative Contributions of the Last Eight Amino Acids at the Carboxy-Terminus

RESEARCH ARTICLE The Energetics of Streptococcal Enolase Octamer Formation: The Quantitative Contributions of the Last Eight Amino Acids at the Carboxy-Terminus Jack A. Kornblatt1*, Veronica Quiros2, M. Judith Kornblatt3 1 Centre for Structural and Functional Genomics, Department of Biology, Concordia University, Montréal, Canada, 2 Department of Biology, Concordia University, Montréal, Canada, 3 Department of Chemistry and Biochemistry, Concordia University, Montréal, Canada * Abstract OPEN ACCESS Citation: Kornblatt JA, Quiros V, Kornblatt MJ (2015) The Energetics of Streptococcal Enolase Octamer Formation: The Quantitative Contributions of the Last Eight Amino Acids at the Carboxy-Terminus. PLoS ONE 10(8): e0135754. doi:10.1371/journal. pone.0135754 Editor: Peter Butko, Nagoya University, JAPAN Received: April 30, 2015 Accepted: July 25, 2015 Published: August 19, 2015 Copyright: © 2015 Kornblatt et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. All of the raw data are fully from the first author as  RA files (AUC),  txt files (CD) or as  JNB files (activity). Funding: Funding was provided by Natural Sciences and Engineering Research Council (Canada) Grant Number 9988 jak, http://www.nserc-crsng.gc.ca/. Competing Interests: The authors have declared that no competing interests exist. The enolase produced by Streptococcus pyogenes is a homo-octamer whose overall shape resembles that of a donut. The octamer is best described as a tetramer of dimers. As such, it contains two types of interfaces. The first is common to almost all enolases as most enolases that have been studied are dimers. The second is unique to the octamers and includes residues near the carboxy-terminus. The primary sequence of the enolase contains 435 residues with an added 19 as an N-terminal hexahistine tag. We have systematically truncated the carboxy-terminus, individually removing the first 8 residues. This gave rise to a series of eight structures containing respectively, 435, 434, 433, 432, 431, 430, 429 and 427 residues. The truncations cause the protein to gradually dissociate from octamers to enzymatically inactive monomers with very small amounts of intermediate tetramers and dimers. We have evaluated the contributions of the missing residues to the monomer/octamer equilibrium using a combination of analytical ultracentrifugation and activity assays. For the dissociation reaction, octamer ( ) 8 monomer truncation of all eight C-terminal residues resulted in a diminution in the standard Gibbs energy of dissociation of about 59 kJ/mole of octamer relative to the full length protein. Considering that this change is spread over eight subunits, this translates to a change in standard Gibbs interaction energy of less than 8 kJ/mole of monomer distributed over the eight monomers. The resulting proteins, containing 434, 433, 432, 431, 430, 429 and 427 residues per monomer, showed intermediate free energies of dissociation. Finally, three other mutations were introduced into our reference protein to establish how they influenced the equilibrium. The main importance of this work is it shows that for homo-multimeric proteins a small change in the standard Gibbs interaction energy between subunits can have major physiological effects. PLOS ONE | DOI:10.1371/journal.pone.0135754 August 19, 2015 1 / 12 Energetics of Octamer Formation Introduction Streptococcus pyogenes is a known pathogen responsible for several diseases (see [1] for a review). The bacterium has a full complement of glycolytic enzymes and obtains much of its energetic requirements from glycolysis[2]. Interestingly, some of the glycolytic proteins are found not only intracellularly where they function in glycolysis but are also found on the surface of the bacterium[3,4]. Amongst those on the surface is Streptococcal enolase (Str enolase) which can, in an infected host, do two things: (1) bind a host’s plasminogen and (2) assist in the spread of infections [5–7]. It does the latter via its interaction with the plasminogen/plasmin system of the host. The native Streptococcus pyogenes enolase (E.C. 4.2.1.11) catalyzes the reversible interconversion of 2-phosphoglycerate and phosphoenolpyruvate. It is a homo-octamer (Fig 1) protein containing 435 amino acid residues in each monomer. As can be seen in the Fig, while all the subunits are identical, the arrangement of the subunits is such that the protein is a tetramer of dimers. One of our laboratories (MJK) has been studying the dissociation/association reactions of different enolases, with a focus on the relationship between primary structure, quaternary structure and activity[8–14]. We asked the question: Can we destabilize the dimer-dimer interface and, if so, what is the net influence on the other properties of the protein? In this study we have focused on the removal of the carboxy-terminal amino acids, found at or close to the dimer-dimer interface. We found that their removal destabilizes the octameric structure and leads to both monomers and oligomers. The extent to which the native octamer/monomer equilibrium has been displaced by the introduction of voids in the place of amino acid residues has been quantified. The data reveal that removal of the individual amino acid residues destabilizes the dimer-dimer interface and that this in turn destabilizes the monomer/monomer interface. The energetic contribution of each of the last eight residues of each monomer to the overall octamer-monomer equilibrium is very small. Materials and Methods The routine laboratory chemicals were ACS purity or higher. 2-phosphoglyceric acid was synthesized and purified as described[15]. The buffer used throughout the study was TME-SO4. It consisted of 50 mM Tris, 1 mM MgSO4, 0.1 mM EDTA, 44 mN H2SO4, pH 7.4. The buffer used during enzymatic assays was 50 mM HEPES, 1 mM MgSO4, 1 mM 2-phosphoglyceric acid, pH 7.4. The reference protein was the enolase from Streptococcus pyogenes F137L/E363G (abbreviated: Str enolase 137/363). It differs from the sequence in the protein data bank (accession number NP_268959.1, abbreviated here: Str enolase DB) at the two indicated positions. All aspects of dealing with the DNA clone, plasmid maintenance, site directed mutagenesis, growth of the bacteria and expression and purification of the proteins have been described[8]; E. coli containing the truncations from -2 to -8 were grown at 18°C instead of 37°C. The oligonucleotides used to construct the truncations were as follows: Str enolase 137/363–1 (ΔK435) 5’- CAA ATC ATT CTA TAA CTT AAA ATA GTA GTA GGA TCC GGC TGC -3’ 5’- GCA GCC GGA TCC TAC TAC TAT TTT AAG TTA TAG AAT GAT TTG -3’ Str enolase 137/363–2 (ΔK434-K435) (...truncated)