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.
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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.
