Proteins act by either interacting with other proteins or small molecules such as sugars. In fact, many enzymes that interact with their low molecular weight substrates also form protein complexes, such as fatty acid synthase, a complex of 12 chains [1
]. In the past 15 years high-throughput methods have been developed to map both protein-protein interactions on a large scale, using either the yeast two-hybrid system for binary interactions [2
] or affinity purification and mass spectrometry for protein complexes [5
]. In principle, a two-hybrid screen using all the proteins of a complex should yield all the binary interactions within that complex, but this is rarely the case; in most cases only a few interactions are discovered [9
]. On the other hand, the two-hybrid system has a certain preference for transient interactions that are lost during complex purification because of the necessary washing steps [4
]. In addition, two-hybrid screening data derived from genome-scale projects is not complete or/and comprehensive. Thus, little overlap is often observed between the protein interaction datasets generated by protein-complex purification and two-hybrid studies [4
In order to infer direct interactions from complex purification data, either the matrix or spoke model has been applied to lists of co-purified proteins. More recent protein complex purification studies used the socio-affinity index
(SAI) to infer the direct interactions between complex members [5
]. A related strategy uses data from complex purification data sets to identify interacting proteins [11
]. These strategies are based on the observation that certain pairs of proteins are more frequently found in multiple purifications than others, and are thus predicted to be closer or even directly associated in the complex. Similarly, solutions have been presented to computationally identify complexes from Y2H interaction networks [12
While these experimental and computational attempts to map protein-protein interactions have produced a massive amount of data, structural analysis of complexes and interacting proteins has lagged behind. One of the goals of PPI studies is thus to identify complexes that are amenable for crystallization. Often, several strategies need to be combined to reconstruct the topology of complexes that cannot be crystallized, including proteomics, cryo-electron microscopy and others.
Recently, we have developed multiple variants of the yeast two-hybrid system and shown that different two-hybrid systems detect markedly different subsets of interactions in the same interactome [16
]. Ten different configurations of bait-prey fusions were required to detect up to 67% of a set of gold-standard interactions, whereas individual vector pairs detected only 25% on average [17
Here we describe and review a similar strategy, using yeast two-hybrid assays to map the interactions within several complexes. In addition, we analyze several well-characterized protein complexes, ranging from ribonucleotide reductase (four subunits) to spliceosomes (>200 proteins), and compare structural data to published Y2H data. Our data shows that a majority of interactions in a complex can be identitifed by systematic Y2H screening and that Y2H assays often detect subcomplexes within a larger complex that may be amenable to crystallography while the whole complex may be not crystallizable.