E coli BirA protein: the multi-tasking interaction surface

Adikaram, P.R., Beckett, D. (2012) Functional Versatility of a Single Protein Surface in Two Protein:Protein Interactions. Journal of Molecular Biology 419 (3-4): 223-233

 

A recently published study (Adikaram, P.R., Beckett, D. (2012)) brings together a number of topics examined in the Principles of Protein Structure course.  Section 10, Protein Interactions and Function, discusses dynamic protein-protein interactions including those of various enzymes and Section 8, The Protein Lifecycle, covers protein-DNA bonding.  This paper investigates a protein, BirA from E. coli, which uses a single interaction surface to interact as either a metabolic enzyme or a transcription repressor depending on the cellular requirement at any time and it considers the evolution of this multipurpose surface.

Both of the interactions start with the binding of the protein BirA to biotin and ATP to form an intermediate enzyme complex.  Biotin is a critical B complex vitamin which is synthesized by bacteria in the gut in humans and which has crucial roles in metabolism and the Krebs cycle.  If the organism is in growth mode the intermediate complex forms a heterodimer with acetyl-coenzyme A (CoA) carboxylase and transfers the biotin to a receptor subunit, so constructing the enzyme which catalyses the initiation of fatty acid synthesis.  Only when the CoA carboxylase is depleted is it energetically favourable for the BirA-biotin-ATP intermediate to fulfil its other function and form a homodimer.  The homodimer binds sequence specifically to DNA at the biotin biosynthetic operon and acts to repress the initiation of transcription.

Image adapted from (Adikaram, P.R., Beckett, D. (2012)).  This model was created using PDB 2EWN for the homodimer, PDB 1BDO for the biotin carboxyl carrier protein subunit and PDB 2EJG as a template for the heterodimer.

 

The interaction surface at the centre of both these complexes is a β sheet surrounded by five loops and both the heterodimer and the homodimer form by extension of this β sheet.  The differences occur in the loops.  Two of the loops have sequences which are found to be conserved in biotin ligases in organisms ranging from humans to bacteria.  This implies that these loops have preserved critical functionality in either the formation of the BirA-biotin-ATP intermediate or in the formation of the heterodimer or in both.  The other three loops have variable sequences and this was taken to indicate that degenerate evolution across species has produced different methods of the homodimerization required to form a transcription repressor.

In this study, 18 residues were selected across the constant and variable loops and were individually substituted for alanine to elucidate the effect of each one on the energetics of both reactions.  It was found that the transfer of biotin to its receptor protein was significantly affected by seven of the residues, most of which were part of the constant loops.  This was consistent with expectation given the conserved nature of these residues.  More surprisingly, 11 residues were found to impact the homodimerization and these were distributed across both the variable and the constant loops with some of the constant loop active residues being key to both reactions.  

So how do these results on the active residues fit with what is known about the surface interactions of the two dimers?  The heterodimer is maintained by two separate interaction sites; the constant loops provide the primary bonding region which is also the active site of the enzyme whilst a second bonding surface is provided by one of the variable loops.  This is consistent with the finding that not all of the seven key residues in heterodimerization were located on the constant loops.  The homodimer interface also comprises two interaction sites but these are symmetrical and each consists of a variable loop of one of the monomers in continuous contact with a constant loop from the other monomer.  This explains why the critical residues for homodimerization were found to include some of the constant loop residues which are also critical for heterodimerization.  This discovery has led to some interesting conclusions on the evolution of this multi-functional interaction surface.

The conserved nature of the constant loops demonstrates that the interaction with biotin and subsequent heterodimerization, which is critical to the formation of the metabolic enzyme, was the primary function of BirA, evolved in an ancestor common to bacteria and humans.  Substitution of these key residues leads to a serious depletion in the energetics of this reaction, showing that further evolution to incorporate a second function would have needed to accommodate these original sequences.  The variable loops have therefore evolved subsequently both to play a supporting role in the stabilisation of the heterodimer and, when cellular regulation allows it, to be complementary to the constant loop residues in formation of a homodimer for transcription repression.

The findings illustrate that a single surface can be used to perform two distinct functions, necessitating two distinct protein-protein interactions, where the structure required for one function has evolved to be complementary to that required for the other and where a regulatory switch is present to activate the appropriate mode.

The topic of protein-protein interactions is explored in much more detail in section 11 of the TSMB course.  This link takes you to the overview of TSMB but clicking the syllabus tag on the left hand side will give you the topics covered in each section.