Abstract
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.
Highlights
Streptococcus pyogenes is a known pathogen responsible for several diseases
The reference protein was the enolase from Streptococcus pyogenes F137L/E363G
The genome of S. pyogenes was originally sequenced by Ferretti et al.[21]
Summary
Streptococcus pyogenes is a known pathogen responsible for several diseases (see [1] for a review). Some of the glycolytic proteins are found intracellularly where they function in glycolysis but are 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 [5,6,7]. 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
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