Download Experimental Approaches to Protein–Protein Interactions

A Biochemical Society Focused Meeting held at University of Sheffield, Sheffield, U.K., 11–12 January 2010. Organized and Edited by Michael Sutcliffe
Biochemical Society Transactions
www.biochemsoctrans.org
(Manchester, U.K.) and Mike Williamson (Sheffield, U.K.).
Protein–protein interactions
Mike P. Williamson*1 and Michael J. Sutcliffe†
*Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, U.K., and †School of Chemical
Engineering and Analytical Science, The Mill, University of Manchester, Manchester M13 9PL, U.K.
Abstract
In the present article, we describe the two standard high-throughput methods for identification of protein
complexes: two-hybrid screens and TAP (tandem affinity purification) tagging. These methods have been
used to characterize the interactome of Saccharomyces cerevisiae, showing that the majority of proteins
are part of complexes, and that complexes typically consist of a core to which are bound ‘party’ and ‘dater’
proteins. Complexes typically are merely the sum of their parts. A particularly interesting type of complex
is the metabolon, containing enzymes within the same metabolic pathway. There is reasonably good
evidence that metabolons exist, but they have not been detected using high-thoughput assays, possibly
because of their fragility.
Introduction
Traditionally, protein–protein interactions have been identified and characterized using low-throughput biophysical
methods such as NMR, crystallography, and a range of
spectroscopic and calorimetric methods. There remains,
of course, a vital place for such methods, and indeed
many of the presentations at this meeting, Experimental
Approaches to Protein–Protein Interactions, demonstrate
the continued vitality of such measurements. However, more
recently, high-throughput methods have provided a new
insight into protein–protein interactions on a proteome-wide
scale, altering some of our ideas of the nature of interactions.
It is therefore appropriate in this introductory article to
consider the lessons learned from such studies.
High-throughput methods
There are two workhorses of high-throughput studies: twohybrid screens and TAP (tandem affinity purification) tagging. The two-hybrid screen (Figure 1) is a simple and rapid
method of finding the interaction partners of a target protein
Key words: high-throughput method, interactome, metabolon, tandem affinity purification tag
(TAP-tag), two-hybrid screen, yeast.
Abbreviations used: MIPS, Munich Information Center for Protein Sequences; TAP, tandem
affinity purification; TEV, tobacco etch virus; UAS, upstream activation sequence.
1
To whom correspondence should be addressed (email [email protected]).
Biochem. Soc. Trans. (2010) 38, 875–878; doi:10.1042/BST0380875
[1,2]. In particular, it finds proteins that interact as a binary
complex with the target protein. The screen uses a reporter
gene, whose activation is easily detected. A popular such gene
is the Escherichia coli lacZ gene, whose activation is detected
because, when grown in a medium containing X-Gal (5bromo-4-chloroindol-3-yl β-D-galactopyranoside), the cells
turn blue. The gene is activated only when a neighbouring
region of DNA known as the upstream activation sequence
(UAS) binds to a protein containing a transcription activation
domain. In unmodified cells, this protein consists of two domains: one whose function is to bind to DNA, and a
second which is the transcription activation domain. Protein–
protein interactions can then be identified by splitting
this protein into two parts, as shown in Figure 1. The DNAbinding domain is attached to a putative dimerization domain,
whereas the transcription activation domain is attached in
different clones to a library of putative dimerization partners.
Activation of the reporter gene is observed only when the
two dimerization domains interact and therefore position
the transcription activation domain in the correct position upstream of the reporter gene, giving rise to blue colonies.
This method has been very widely used, and has uncovered
a large number of interactions. It has, however, received a
good deal of criticism, mainly because it tends to identify
a high proportion of false positives: protein hits that are
unlikely to associate in vivo.
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Figure 1 The two-hybrid screen
A DNA-binding domain that binds to a UAS is fused to the target
or bait protein. A library is constructed of possible binding partners
(prey) fused to a transcriptional activation domain (which is normally
attached to the DNA-binding domain). In this way, binding of bait to prey
leads to transcription of the reporter gene such as lacZ.
TAP tagging is a clever way of purifying low-abundance
proteins and pulling them out of the cell still attached to
any binding partners they may have [3], and is discussed in
more detail in this issue of Biochemical Society Transactions by
Völkel et al. [4] It is an extension of the standard protein purification tag such as the hexahistidine tag, in which a His6 sequence is added to one end of the protein: this sequence binds
to metals such as nickel, whereas normal proteins do not, and
therefore the protein may be purified in one step by use of a
nickel-affinity column. In TAP tagging, two tags are used sequentially (Figure 2). Typically, the C-terminus of the protein
is tagged with a sequence consisting of a calmodulin-binding
peptide, followed by a cleavage site recognized by TEV (tobacco etch virus) protease, followed by Protein A. Protein A
binds to IgG. The tagged protein is therefore purified first on
IgG beads, after which the Protein A sequence is cleaved off
by TEV protease. The protein is then purified on calmodulin
beads in the presence of calcium, which is required for correct
folding of calmodulin. It is then eluted from the column using
EGTA, which removes the calcium. The second affinity step
serves as a second independent purification and also removes
the TEV protease, thereby leaving the desired complex as
(ideally) the only protein present. The method therefore
gives a reproducibly very high purification factor, allowing
the preparation of essentially completely pure protein
from the cell without overexpression. The method is
commonly used to isolate complexes, because the purification
treatments are relatively mild and so leave many complexes
intact. The purified complex can then be analysed, typically
by SDS/PAGE, after which the component proteins can
be identified by Western blotting, or more commonly by
protease digestion followed by MS of the resultant peptide
fragments and comparison with a sequence database to
identify the parent protein. By comparison with two-hybrid
screens, it identifies groups of proteins that assemble into
complexes with the target protein, and can therefore identify
proteins that do not necessarily interact directly, but do
belong to the same complex.
The TAP-tag method is also prone to errors. It generates
some false positives, in that abundant proteins, particularly
housekeeping (e.g. glycolytic) and ribosomal proteins,
tend to be picked up more often than they should [5]. It
also under-represents several classes of interactions, such
as membrane proteins (as does the two-hybrid screen).
Most significantly, the purification stages, even though
mild, mean that weak interactions are lost. It has been
frequently noted that the intracellular environment is very
Figure 2 TAP tagging
A two-step purification is used: the first uses Protein A, which binds to IgG beads. The Protein A is subsequently removed
by TEV protease. The second purification uses the affinity of a calmodulin-binding peptide (CaM-bp) for calmodulin, which is
removed by EGTA.
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Experimental Approaches to Protein–Protein Interactions
crowded, with protein occupying up to 40% of the total
fluid volume in cells (implying that the protein concentration
is very similar to that within protein crystals!) [6]. One
consequence of this macromolecular crowding is that
protein–protein interactions are stronger in the cell than they
would be in dilute solution, often by very large factors [7].
Therefore purification out of the intracellular medium almost
necessarily implies that the weaker interactions will be lost.
The interactome
The interactome (i.e. the set of interactions between all
proteins in the cell) of the yeast Saccharomyces cerevisiae has
been described by two independent groups, in both cases
using TAP tagging [8,9]. The results are broadly consistent,
in that they show that approx. 70% of proteins in the
cell have at least one interacting partner: in other words,
interaction with other proteins is the norm rather than
the exception. (And, as can be seen from other articles in
this issue of Biochemical Society Transactions, homotropic
interactions such as homodimerization are also the norm
rather than the exception; the majority of proteins exist not
as monomers, but as multimers.) The network of interactions
has the property of being scale-free: although most proteins
interact with only one or two other partners, a small number
of proteins form a large number of interactions. And protein
complexes make up virtually all aspects of cellular function.
The majority of complexes, containing only two or
three proteins, often form stable complexes in which the
components have obvious functional roles: for example, they
may be sequential enzymes in a pathway, or an enzyme plus a
regulatory protein, or an enzyme plus a protein that interacts
with substrate, or an enzyme plus a protein that locates it in
a particular region of the cell. The larger complexes tend to
contain a ‘core’ or ‘hub’, playing much the same role: these are
the key proteins that carry out the catalytic activity, and tend
to be genes that are essential for survival of the cell [10]. For
example, in the RNA polymerase II complex, the core is the
polymerase itself plus TFIIB (transcription factor IIB) and
TFIIF (transcription factor IIF), which function to track the
polymerase along its DNA substrate. In addition, there are
other proteins, which have been described as either ‘party’ or
‘dater’ proteins [11,12]. The binding of these proteins to the
core is often variable, being controlled by phosphorylation,
cell cycle, growth conditions, local concentrations and
many other factors. The party proteins are so called because
they are found attached to a range of core complexes,
but the party proteins themselves are always in the same
group. These proteins have again a well-defined function,
such as recognition of the promoter site or termination
of transcription. In this example, these two functions are
mutually exclusive, and therefore in any given complex
one might find one or the other, but not both. (Similar
conclusions were reached on purely structural grounds;
some proteins apparently have very large numbers of possible
partners, which is not physically possible simultaneously
[13].) The dater proteins are so called because they are found
with different partners in each complex, and they often have
more general functions than the party proteins.
In each case where we understand the workings of the
complex, the function of the complex is reassuringly more
or less just the sum of its parts, with different functions
being provided by modular groups of proteins, which
carry out similar functions in different complexes [8]. This
straightforward logic is, however, obscured by frequent
examples of redundancy or non-orthologous replacement.
This is not unexpected, given that the components of
complexes arose as a result of evolutionary processes.
There are clearly still many problems to be ironed out
in high-throughput methods. The two TAP-tag studies
described here show little overlap in detail [11]. There is
a hand-curated database of protein interactions assembled
by the Munich Information Center for Protein Sequences
(MIPS, http://mips.gsf.de/genre/proj/corum), which is often
cited as the ‘gold standard’ of interacting proteins [14].
Between half and one third of the complexes in MIPS
were not identified in the TAP-tag screens. The results
from the two TAP-tag screens have been combined [15] to
produce a set that is suggested to be as accurate as traditional
low-throughput methods, and the paper concludes that
complexes probably exist in a continuum of components:
some components are always associated, whereas others
come and go at different rates and with different affinities.
The yeast interactome has also been probed using twohybrid technology, generating a set of interactions that is
different again. This may of course reflect real differences
in the types and durations of interactions, and it has been
suggested that two-hybrid screens are more suited for
identifying transient and intercomplex interactions [16],
implying again that many proteins interact weakly and
transiently, often with more than one complex.
The metabolon
A particularly interesting type of complex is provided by
the metabolon. This is a concept first proposed by Welch
in the 1970s [17] and since promoted heavily by Srere [18]
among others. It is argued that many groups of enzymes
within common metabolic pathways are physically associated
together into metabolons: for example, glycolytic enzymes
or TCA (tricarboxylic acid) cycle enzymes. This idea makes
good theoretical sense: it facilitates channelling and therefore
speeds up multienzyme pathways and improves control and
regulation. The main problem, however, is that is not clearly
supported by experimental observations. The TAP-tag studies described above found almost no examples of metabolons:
indeed, the complexes identified are strikingly deficient in
metabolic complexes. It has been argued that metabolic
complexes may be weakly associated in vivo, because their
assembly needs to be regulated; for the reasons described
above, this could explain why they have not been observed.
And there are many observations that support the metabolon
concept. For example, there are six enzymes (three of which
are multifunctional) that catalyse purine biosynthesis in
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eukaryotes. In vitro, no co-localization is observed, and
there is no evidence that they associate. However, fluorescent
labelling has demonstrated that these enzymes co-localize
in vivo, and that this localization can be disrupted in response
to purine levels [19]. That study, in common with many
others, suggested further that the complexation occurs at a
membrane surface. An elegant study using isotopic labels
demonstrated channelling for most, if not all, of the glycolytic
enzymes in Arabidopsis and potato, the complexes being
formed at the mitochondrial outer membrane in response
to respiratory demand [20]. The evidence is still mixed, but it
seems likely that TAP tagging really does miss a large number
of weakly interacting, but important, complexes.
Where next?
Two clear conclusions can be reached from this analysis. The
first is that functionally important protein–protein complexes
can adopt a very wide range of affinities, ranging from very
stable homo- and hetero-dimers to transient and tightly
regulated complexes that fall apart as soon as the cell is
broken open, and therefore that have to be studied in
vivo. Such different complexes require very different experimental methods for their study. The second is that highthroughput methods are vital, but need validation and extension to weaker interactions. The participants at this meeting
illustrate these conclusions nicely, and provide an excellent introduction to the range of experimental techniques used in the
increasingly important study of protein–protein interactions.
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Received 20 January 2010
doi:10.1042/BST0380875