Two-dimensional structural representations of protein complexes were generated, derived from structural or structure-associated data in the literature. A representation of the 20S core particle (CP) of the proteasome was derived from its X-ray crystal structure 20 As the structure of the yeast 19S regulatory particle (RP) has not been elucidated, a model of the 19S RP was adapted from Ferrell and coworkers 21, based on genetic and biochemical studies, and Sharon and colleagues 22 based on high range mass-spectrometry. A structural representation of the mediator complex was derived from models proposed using electron microscopy 23 and an interaction network based on genetic and biochemical data 24. Med19 (Rox3) was recently shown to be part of the middle module (instead of the head module) 25, and this was taken into account in our representation. A structural representation of the SAGA complex was adapted from a model derived using electron microscopy, immunolabelling, and mutant complexes 26 This study proposed that the SAGA complex contained five modules (Domains I-V), and others have proposed that additional proteins are likely to be part of these modules 27 SAGA-associated proteins were obtained from Daniel and Grant 28.

Experimental data for protein-protein interactions were from several sources. Data on protein complexes was from Gavin et al. 12. Complexes 2 and 75 described the 20S and 19S proteasomal subcomplexes respectively while Complexes 81 and 445 described the SAGA and mediator complexes respectively. Filtered yeast interactome (FYI) data was sourced from Han et al 11, and domain-domain interaction (DDI) data was extracted from iPfam release 20.0 29 using custom Perl scripts, version 5.8.7, as described previously 14.

To understand how data from different high-throughput analytical techniques can help accurately define the protein membership of complexes and elucidate topology, protein-protein interaction data was mapped onto our 2-D representations of the structures of complexes. Experimental pairwise interactions from the FYI dataset and DDI were represented by lines between protein nodes whilst membership of complexes, according to Gavin et al 12, was represented by node shading.

Our investigation involved analysing the proteasome and two transcriptional coactivator complexes. These were chosen as they are large, multisubunit complexes that have well-characterised structures. The proteasome complex, responsible for the degradation of most proteins in the cell, is composed of a 20S cylindrical core particle (CP) flanked by two 19S regulatory particles (RPs) each which contain a base and a lid 30. The mediator complex passes information from gene-specific activators and repressors to core transcriptional machinery via its interaction with TFIIH and RNA polymerase II (RNAP II) 3132. It is comprised of 25 subunits, found in the head, middle, tail, and CDK modules 2333. The SAGA complex regulates transcription of stress-induced and highly-regulated genes via histone acetylation and direct interaction with the TATA-binding protein and other transcription factors 3435 It has three distinct functional modules 26283536 along with other associated proteins. We constructed 2-D representations of the proteasome (Figure 1A), mediator complex (Figure 2A) and the SAGA complex (Figure 2B) according to the Materials and Methods. Note that our representation of the proteasome considers just one half of the structure as the proteasome is symmetrical. We sought to understand explore two key issues for these complexes. Firstly, whether high throughput protein-protein interaction datasets could accurately determine the protein members of the complexes and secondly, whether pairwise protein interactions, those from protein complexes or those predicted from the presence of certain domains can provide clues to the topology or architecture of a complex.

The FYI dataset is an intersection of different interaction datasets (see Han et al 11 for details), and is enriched for high confidence, pairwise protein interactions. We investigated how these pairwise interactions, when mapped as lines connecting proteins on our 2-D representations, reflected the features of candidate complexes. For the proteasome, interactions in the FYI dataset clearly defined the 19S RP as one complex and the 20S CP as an independent complex (Figure 1B). Protein membership was also very clear, with 100% of subunits showing at least one interaction with other subunits in the same complex. Proteins in the 19S RP show a large number of interactions with other proteins in the same complex, but far fewer interactions were seen between members of the 20S CP. It was noted, however, that the subcomplexes in the proteasome, for instance the 19S RP lid and base, could not be discerned from this data. The SAGA and mediator complexes were also clearly defined by the FYI data. All proteins of domains I to V of the SAGA complex showed multiple interactions, with most proteins showing evidence of interaction with every other protein in the complex (Figure 2C). However, it was striking that the SAGA-associated proteins showed no interactions with proteins in the SAGA complex itself; this may be due to these interactions being transient or perhaps refractory to analysis using one or more interaction-measuring techniques. In the mediator complex, proteins showed a large range in the number of their interactions, with some proteins showing 1 interaction but others showing >10. Interestingly, the CDK module subunits and protein Med1 showed no interactions with the rest of the mediator complex (Figure 2C). The CDK module is known to be a temporary inhibitor of the mediator, thus a lack of interactions may reflect the transient nature of its interactions with the mediator 32. For the complexes examined here, it was apparent that high quality pairwise interactions, from the FYI database, could accurately indicate whether proteins are likely to form a protein complex and which proteins are the constituent subunits.

To understand if data from high throughput AP-MS of complexes could accurately define the members of protein complexes, we mapped complexes from Gavin et al. 12 onto our 2-D representations. For the proteasome, the 19S and 20S subcomplexes corresponded to Gavin et al. 12 complexes 2 and 75 respectively. Encouragingly, a total of 91% of the known proteasomal subunits were seen in the high-throughput-defined complexes (see shading, Figures 3B,C). Similarly the Gavin et al. 12 complexes 81 and 445, which correspond to the SAGA and mediator complexes respectively, showed 85% of SAGA and 80% of mediator subunits (see shading, Figures 4C,D). This indicates that AP-MS has a high true positive identification rate for the constituent proteins of these complexes. However, it was also seen that high throughput AP-MS suggested a large number of other, false positive proteins as members of these complexes [see Additional files 1, 2, 3]. An additional 10 non-proteasome proteins (29%) and 23 non-mediator/SAGA proteins were observed (49%). This highlights that high throughput AP-MS alone may not accurately define the members of a complex, and is weaker than data from the FYI database for the clear definition of complexes and membership thereof. This contrasts with expectations that AP-MS, which seeks to purify protein complexes to homogeneity, should provide unambiguous data in this regard. It also contrasts with comments elsewhere 21819 to this effect.

We next sought to understand the degree to which protein interaction data, represented as pairwise interactions in the FYI dataset, could provide clues into the architecture or topology of proteins in complexes. We examined whether FYI pairwise interactions were likely or indeed possible, by reference to our structural representations. In the proteasome 19S CP, the FYI data described interactions between many proteins that are known to be structurally associated (e.g. those that form the rings in the base such as Rpt4 and Rpt5, see Figure 1A,B). However a very large number of interactions were seen for many proteins that are unlikely to be structurally associated (e.g. the FYI data suggests Rpn12 to have >10 interactions, many of which are with proteins that are a considerable distance away in 3-D space, Figure 1B). In the proteasome 20S CP, there were fewer pairwise interactions documented, and many of these reflected structural associations (Figure 1B). For example, Pup3 is known to be adjacent to Pup1 and Pre1 in the β ring and proximal to Pre8 in the neighbouring α ring (Figure 1A) and this was seen in the FYI protein-protein interactions. However, the Pre1 protein showed 10 interactions, many of which were not consistent with the structural topology of the complex. For the SAGA and mediator complexes, a similar trend was observed. The FYI data described interactions between some proteins in the SAGA or mediator complexes that are structurally adjacent, however many proteins showed an excessively large number of interactions that were not consistent with the likely positions of protein subunits in 3-D space.

There are a number of possible explanations for FYI data being weak in reflecting the structural topology of protein complexes. Yeast two-hybrid experiments, which are a key part of the FYI dataset, are known to generate false positive interactions. These can arise due to the overexpression of proteins as part of the technique, their requirement to interact in the nucleus 18 and the possible involvement of one or more endogenous subunits that bridge the 'gap' between bait and prey proteins. This bridging could explain why, along with others, Gcn5 and Spt3 apparently interact in the SAGA complex when these subunits are spatially isolated 26 (Figure 2B). Yeast-two hybrid may also detect interactions mediated by homo-domain-domain interactions that may not normally occur (see later evaluation of domain-domain interactions); this may explain some of the unexpected interactions in the proteasome 20S CP. This is an important consideration, albeit usually ignored, in the interpretation of pairwise interactions in the FYI database.

From their high throughput screen of the complexome, Gavin et al. 12 proposed that complexes can have core proteins, present all the time, as well as module and attachment proteins which interact less strongly and/or only in certain conditions. To understand the relationship of core, module and attachment proteins to the architecture of protein complexes, we overlaid all protein types from Complexes 2, 75, 81, and 445 onto our 2-D representations (Figures 3, 4). Data is also given in tabular form in Additional file 2. In our analysis, we have ignored attachment proteins; this is because they are 'singletons' that were seen to occasionally interact with the cores of complexes 12. They thus provided no information concerning the topology of a complex.

Core proteins in the proteasome, mediator and SAGA complexes (black shading, Figures 3B,C and 4C,D) were clearly seen to be interacting proteins in these complexes, particularly for the mediator complex, the proteasome 19S RP and 20S CP. It should be noted that whilst all 20S CP core proteins were not adjacent in our 2-D representations, they do in fact interact as part of the stacked α and β rings 37 For the SAGA complex, the core proteins were seen in three groups. Interestingly, the SAGA complex is yet to be crystallised and our model was built by considering data from electron microscopy, immunolabelling studies and mutant complexes 26272838 The core protein data suggests these 3 protein groups are almost certainly physically associated in the topology of the SAGA complex. Thus, for the proteins examined here and others in the Gavin et al. 12 dataset, core proteins are likely to be spatially grouped together and define aspects of the topology of complexes.

Modules, representing two or more proteins, were defined as those that are sometimes associated with the core of a complex 12. It was expected that they reflect topological features of the complexes of interest. For the proteasome, it was clear that modules 57 and 141 were comprised of proteins that were structurally associated, being adjacent or near-adjacent in our models (see numbered proteins, Figure 3C). However module 93, comprised of proteins Rpn3 and Nas6, was not consistent with our 2-D structural representation. Note that our model of the 19S RP was adapted from Ferrell and coworkers 21 and Sharon and colleagues 22, and was built in the absence of a 3-D crystal structure. Thus, it remains possible that either Rpn3 or Nas6 may be incorrectly positioned or that module 93 is not true. Evidence that Nas6 interacts with Rpn3 in the literature 29 and FYI (see Figure 1B), and FYI evidence that Rpn3 interacts with its expected neighbours (Figure 1B), suggests the latter may be the case. Examining the SAGA complex, two modules were seen – Modules 84, and 146 (see numbered proteins, Figure 4C). The proteins in Module 146 are adjacent in our model and are likely to be structurally associated; this is consistent with their classification as a module. The proteins of Module 84 (Gcn5 and Taf10), whilst proposed to both interact with Spt7 26, are not yet known to interact directly. Their existence as a module provides some evidence that this may be the case. To summarise, modules described proteins that in many cases were physically associated in a complex. This provides useful clues to the topology of proteins in a quaternary structure.

Finally, predicted domain-domain interactions (DDIs) between proteins were mapped onto our 2-D representations of complexes. Domain-domain interactions are not high throughput data per se, but were examined to understand how this data type can help in the interpretation of high throughput protein-protein interactions. The predicted DDIs in the proteasome were numerous and reflected many aspects of the 3-D positions of proteins. All proteins in the 19S RP base showed domain-domain interactions with each other (see lines connecting proteins in Figure 1C). The proteins Rpt1-6 were predicted to interact with each other by a common AAA-ATPase domain (Pfam: PF00004). This is structurally accurate as they interact with each other in a hexameric ring in the order Rpt1/2/6/4/5/3 2139. However cross-ring DDIs were also predicted due to the presence of same domain, but are unlikely to occur as they are inconsistent with the proteaseome's 3-D structure. In the 20S CP, DDIs were seen within and between all proteins of the α and β rings (Figure 1C). Around-ring DDIs were seen in the 20S CP, as would be expected. Some incorrect cross-ring DDIs were also predicted, for example between Pre1 and Pre3 that are not adjacent proteins within or between the α and β rings 2037 (Figure 1C). The reason why these were observed is due to the presence of the proteasome domain (Pfam: PF00227) in all 20S CP subunits. This domain is a putative homomeric interaction domain and thus each subunit could theoretically bind to the other subunits in the complex. Interestingly, this might explain some of the FYI interactions that were inconsistent with the proteasome topology (see Figure 1B); this is further highlighted by the overlap of many proteasome 20S CP DDIs with interactions documented in the FYI database (see Figure 1D).

In the co-activator complexes, only 6 domain-domain interactions were predicted (Figure 2D). Many of these, for example Med7-Med9 and Spt7-Gcn5, are between proteins that we expect to be structurally adjacent. In contrast to the proteasome, hetero-domain-domain interactions were seen to feature here. For example, Med7 and Med9 were predicted to interact via the Med7 protein domain (Pfam: PF05983) and RNA polymerase II transcription mediator domain (Pfam: PF07544). Part of the reason for this difference is that the interactions were extracted from the iPfam database, which is based on structural data. The structure of the proteasome is known from X-ray crystallography and mass spectrometry 2237 whilst the structure of the SAGA and mediator complexes are mostly based on interaction studies 24 and electron microscopy 2326. Accordingly, the domain-domain interactions found for the co-activator exist only due to the observation of similar domain pairs between other proteins in crystallised complexes.

A comparison of predicted DDIs in the proteasome and the co-activator complexes suggests that where a complex contains many proteins that are paralogs or of similar domain content, such as complexes with ring structures, DDI interactions are unlikely to help understand the precise structural association of subunits within a complex. However, the accurate prediction of structural associations from DDI data may be possible in complexes that contain essentially unrelated proteins whose interactions are mediated by hetero-domain-domain interactions.