BACKGROUND
in eukaryotic organisms, there exist at least three distinct multi-protein assemblies that are jointly referred to as 'pci complexes'  <cit>  and have a similar subunit architecture despite their fundamentally different function: i) the proteasome lid, a subcomplex of the 19s proteasome regulator and the 26s proteasome, ii) the cop <dig> signalosome or csn complex, and iii) the eukaryotic translation initiation factor eif <dig>  as a common feature, these complexes are composed of multiple subunits harbouring the pci domain, named after the three participating complexes  <cit> , sometimes also referred to as the pint domain  <cit> .
other subunits of these complexes are characterized by a second shared homology domain called mpn   <cit> .
among these complexes, the proteasome lid and the csn share a particular degree of analogy.
both complexes consist of eight core subunits, six of them of the pci class and two of the mpn class.
as described previously  <cit>  and summarized in table  <dig>  there is a clear 1: <dig> correspondence between the paralogous sets of pci and mpn subunits.
in addition, a similar ring-shaped structure was observed for the two complexes  <cit>  and there is evidence that in those rings paralogous subunits occupy equivalent positions  <cit> .
by contrast, the eif <dig> complex has a smaller number of pci subunits  and its two mpn subunits are absent in several unicellular eukaryotes.
unlike the proteasome lid and the csn, the eif <dig> complex contains a number of non-pci/non-mpn subunits, which are required for its function in translation.
despite the common homology domains and a similar structure, the functions of the three pci complexes are very different.
the proteasome lid, in combination with the 'base' complex containing a hexameric ring of aaa-atpases, forms the 19s regulatory particle, which in turn constitutes an essential subcomplex of the 26s proteasome  <cit> .
the lid complex contains an intrinsic deubiquitinating activity, which is encoded by the mpn subunit rpn <dig> that has the hallmarks of a metalloprotease  <cit> .
no specific function has been described for the pci subunits of the lid.
the csn complex has been first described as a regulator of photomorphogenesis in plants, but seems to regulate diverse cellular processes like signal transduction, regulation of transcription or cell proliferation  <cit> .
csn <dig>  an mpn-bearing subunit of the signalosome, which is analogous to rpn <dig>  also encodes a metalloprotease that is essential for the removal of the ubiquitin-like protein nedd <dig> from cullins  <cit>  the third pci complex, the translation initiation factor eif <dig>  promotes the formation of preinitiation complexes and works as a scaffold by binding to other initiation factors, to ribosomes and to mrna  <cit> .
both mpn subunits of eif <dig> lack the residues necessary for metal binding  <cit>  and are most likely catalytically inactive.
so far, the metal-containing mpn subunits and the non-pci/non-mpn portion of the complex constitute the only known carriers of functionality.
the pci proteins themselves seem to be the main building blocks of the complexes, a fact already suggested by their high abundance.
there are several hints that the pci subunits are crucial for proper complex assembly  <cit> .
the mpn subunits of the three complexes are rather well conserved and the detection of mpn domains and their boundaries is relatively straightforward.
by contrast, the degree of conservation between pci subunits is highly variable.
sequence similarity between the corresponding subunits of proteasome lid and csn is generally easy to spot, while the detection of similarity between other paralogous pci subunits typically requires sophisticated sequence comparison approaches, such as the generalized profile method  <cit> .
a particular challenge is the detection of the highly divergent pci domains in the budding yeast csn-like complex  <cit>  and those of the eif <dig> complex, where only three pci subunits could be detected in the initial survey  <cit> .
due to this difficulties, it is to be expected that there are still a number of highly divergent pci domain proteins in eukaryotic genomes, which have eluded detection so far.
a second issue in the bioinformatical definition of the pci domain concerns the position of its n-terminal boundary.
in general, homology domains are thought to correspond to structural domains in the sense of autonomous folding units; they are typically characterized by a pronounced loss of sequence similarity at the domain boundaries.
while this is true for the pci domain c-terminus, the n-terminal domain boundary is blurred through a gradual decay in sequence similarity instead of a sharp drop.
as a consequence, different pci domain boundaries have been used in the literature  <cit>  and in various domain databases like prosite  <cit> , pfam  <cit>  and smart  <cit> .
the corresponding accession numbers are ps <dig>  pf <dig> and sm <dig>  respectively.
during an exhaustive bioinformatical analysis of proteasome subunits and other components of the ubiquitin/proteasome system, we obtained two independent results jointly suggesting that a structure-based redefinition of the pci domain is appropriate: on one hand, we detected multiple instances of tpr-like repeats in the n-terminus of many pci proteins, which suggests that the homology between the proteasome and csn components is not restricted to the pci domain itself.
on the other hand, we detected a previously overlooked pci domain in the novel eif <dig> subunit eif3k  <cit> .
most interestingly, an x-ray structure of eif3k has been published recently  <cit> .
based on this structure and on our alignment data, we suggest a bipartite consensus model for the canonical pci proteins, consisting of a c-terminal 'winged helix' domain preceded by an extended helical repeat region.
we use this model to re-evaluate some bioinformatical and experimental findings that have been enigmatic so far.
RESULTS
tpr-like helical repeats in pci proteins
in most pci proteins, the canonical pci domain occupies a region of approximately  <dig> residues close to the carboxy-terminus of the sequence.
the n-terminal non-pci portion of the proteins is moderately conserved between species and only poorly conserved between different pci subunits – even between the analogous subunits of the lid and the csn.
upon submitting those pci proteins to profile- or hmm-based domain detection services, no significant matches were obtained for the n-termini of the proteins.
however, the prosite  <cit>  profile for the tetratrico-peptide repeat  yielded a number of closely sub-significant matches in multiple pci proteins, e.g.
rpn <dig> from s. bayanus  and csn <dig> from e. histolytica .
the tpr repeat family  <cit>  is very heterogeneous, and tpr motif descriptors such as the prosite profile are known to miss several instances of bona fide tpr repeats.
upon closer inspection, most pci proteins exhibit multiple regions of similarity to profiles derived from established tpr repeats , although the similarity scores for each of the single regions do not reach statistical significance.
no relevant similarity scores were obtained for other helical repeat motifs, such as heat or armadillo repeats.
to further investigate if a tpr-like structure should be assumed for the n-terminal portions of all pci proteins, we performed a secondary structure prediction for each of the protein families individually.
to that aim, we constructed multiple alignments for representative members of each subunit family and submitted the alignment to phd and jpred prediction servers  <cit> .
as a result, all pci subunits of lid and csn are predicted to adopt an all-helical secondary structure upstream of the pci domain.
interestingly, these helical regions merge seamlessly into the pci domain, at least if the longer pci versions of prosite and pfam are used.
this finding is in agreement with the observation of several regions with weak tpr-similarity within the n-terminal part of the pci domain itself .
further support for a tpr-like structure comes from a sequence-based fold recognition for lid and csn subunits using the superfamily-service  <cit> .
several subunits like rpn <dig> from budding yeast and human csn <dig> were found to have significant scores for the tpr fold upstream of the pci domain .
the predicted all-helical secondary structure of the non-pci portion of lid and csn subunits consists of several short helices that appear to occur in pairs.
to test whether those bi-helical segments correspond to the structural elements of a tpr-like repeat, we selected several examples starting immediately upstream of the predicted pci domains.
when multiple alignments of those bi-helical segments were used for profile construction and in subsequent database searches several bona fide tpr proteins were found to match within the tpr region, with the bi-helices being in the correct tpr register, these segments were also classified as tpr-like.
no matches to established heat- or armadillo-repeat proteins were found, demonstrating that the scores are not just caused by an arbitrary helical repeat arrangement.
it should be pointed out that none of the singular observations described above is able to prove a statistically significant sequence relationship between the n-terminal portions of pci proteins and true tpr-repeats.
taken as a whole, the results strongly suggest that there is a general tendency of pci domains to be preceded by an α-helical repeat structure that has at least some specific relationship to the tetratrico-peptide repeat.
a previously unrecognized pci domain in eif3k
in the first surveys of recognizable pci domains, only three pci subunits of the eif <dig> complex had been detected  <cit> .
more recently, a number of novel eif <dig> components have been identified: eif3j  <cit> , eif3k  <cit>  and eif3l  <cit> .
among these novel subunits, only eif3l has been reported to harbour a pci domain  <cit> , interestingly also preceded by a tpr-region.
in order to find further indications of divergent pci domains, we performed a thorough profile analysis of all uncharacterized eif <dig> subunits.
a generalized profile was constructed from the conserved portion of representative eif3k orthologs from vertebrates, invertebrates, plants and fungi.
after a scaling step, the resulting profile was run against a nonredundant protein database.
apart from the eif3k proteins already used for profile construction, the only other sequences matching with significance were selected pci subunits of the proteasome and the csn, among them rice csn <dig>  and the drosophila rpn <dig> homologue .
all of the twenty top-scoring sequences could be identified as either csn8- or rpn12-homologs.
as shown in table  <dig>  csn <dig> and rpn <dig> are the corresponding pci subunits in the csn and the lid, respectively.
csn <dig> and rpn <dig> are the most divergent pci subunits of the proteasome and the signalosome, respectively, and their pci domains appear to be shorter than that of the more typical family members.
our observations provide good bioinformatical evidence that eif3k is the fifth pci-containing subunit of the eif <dig> complex and most likely a direct analogue of csn <dig> and rpn <dig> .
a structural model for the canonical pci domain
the discovery of a pci domain in eif3k is of particular importance, as a three-dimensional structure of eif <dig> has been solved recently  <cit> .
so far, no structural information on the pci domain has been available, and a structural model for the canonical pci domain based on the alignment shown in figure  <dig> should allow interesting insights into the architecture of the pci complexes.
a detailed analysis of the eif3k structure  <cit>  reveals a bipartite structure of two subdomains that are in close contact through a large inter-domain surface patch .
the c-terminal half-domain is a globular α/β structure with an "αβααββ" arrangement.
the three β-strands are very short and form an antiparallel sheet.
the whole c-terminal part can be classified as a "winged helix" fold and thus is referred to as "wh-domain"  <cit> .
by contrast, the n-terminal half-domain is entirely helical with a core of six regularly-spaced helices that form three antiparallel helical hairpin elements.
the resulting superhelix is reminiscent of the solenoids found in helical repeats such as heat, armadillo and tpr.
somewhat unusual are the short 3– <dig> helices that connect the consecutive α-hairpins.
according to wei et al.
<cit>  the n-terminal half-domain resembles structurally mainly heat and armadillo repeats, and thus the name "ham-domain" was proposed.
the bipartite structure of eif3k is in good overall agreement with the secondary structure predictions for the single pci domain families and also with our result of tpr-like helical repeats partially overlapping the pci domain.
it was therefore of special interest to make a detailed comparison of the eif3k structure and the profile-guided alignment of the canonical pci superfamily shown in figure  <dig>
within the n-terminal subdomain, the sequence conservation between the different pci domain families is relatively poor and some aspects of the alignment shown in figure  <dig> are not very reliable.
nevertheless, there is a good correspondence between the helices that build the α-hairpins of eif3k and the uninterrupted sequence blocks in the pci alignment.
the gap-regions in the pci alignment are typically caused by insertion events in selected pci subfamilies.
in no case, a deletion of one or more of the hairpin helices is observed.
this finding suggests that the helical hairpin structure is conserved in most or all pci domains.
our alignment and the derived secondary structure predictions suggest that the short  <dig> helices that connect the helical hairpins in eif3k are absent in most other pci proteins.
as mentioned in the previous paragraphs, there are several instances of subsignificant sequence similarity to tpr repeats found also within the n-terminal subdomain of the pci domain.
by contrast, no similarity to heat or armadillo-repeats has been observed.
thus, we prefer to interpret the helical hairpin structure of the n-terminal subdomain as atypical tpr-like repeats rather than as the heat/armadillo repeats suggested by wei et al.
<cit> .
the globular c-terminal subdomain  is generally better conserved than the helical n-terminal domain and as a consequence, the part of the alignment covering this structural subdomain shown in figure  <dig> is more reliable.
the "αβααββ" arrangement of α- and β-regions is distributed over two large sequence blocks with a single major gap region between "αβα" and "αββ".
as can be seen in figure  <dig>  no important secondary structure element is interrupted by a gap found in the pci alignment.
like in the n-terminal subdomain, the wh portion shows a good concordance between the secondary structure predicted from the canonical pci families and the structural elements of the eif3k structure, apart from minor problems in predicting one of the very short β-strands.
taken together, the comparison of the pci alignment with the eif3k structure  shows that the two structures are clearly compatible and suggests that the canonical pci domains will have an analogous bipartite fold similar to that shown in figure  <dig>  the prediction of tpr-like helical repeats n-terminal of the proper pci domain suggests that they form an extension of the helical repeat region of the first pci subdomain.
the implications of this model for the overall pci structure will be discussed below.
discussion
revision of the eif <dig> complex stoichiometry
while the proteasome lid and the csn complex have an analogous architecture of six pci-subunits and two mpn-subunits, the more distantly related eif <dig> complex has so far only three readily detectable pci proteins: eif3a , eif3c  and eif3e   <cit> .
recent work by morris-desbois et al.
<cit>  has grouped eif3l  with the pci components of eif <dig>  and our work described above adds eif3k  to the ranks of pci proteins.
besides the pci subunits, vertebrate eif <dig> complexes also contain two mpn proteins: eif3f  and eif3h .
unlike the situation in the lid and csn complexes, both mpn subunits of eif <dig> have lost their metal-coordinating residues and are most likely catalytically inactive.
in addition, several unicellular eukaryotes, including budding yeasts, do not seem to have any eif3-associated mpn proteins.
comparing the stoichiometry of eif <dig> with the two better-conserved pci complexes, only one pci subunit seems to be missing.
our sequence analysis efforts have also included other known eif <dig> subunits, but no indications for further pci domains could be obtained .
given the high degree of pci sequence divergence, it cannot be fully excluded that one of the non-pci/non-mpn subunits  harbours a cryptic pci domain that has eluded our detection.
on the other hand, it is well conceivable that eif <dig> has a deviating subunit composition.
in yeast and several other organisms, not only the mpn proteins are missing but also the number of pci components is reduced, as eif3e and eif3l are absent.
at present, it is not clear whether the corresponding positions in the complex are left empty or are filled by additional copies of the remaining pci components.
in evolutionary terms, it appears likely that the eif <dig> complex is a 'degraded' copy of an ancient lid-like complex, which has lost its mpn+/jamm mediated catalytic activity and potentially some of its pci subunits.
in turn, by acquiring a group of novel non-pci/non-mpn subunits, the eif <dig> complex has gained a functionality that is different  to the proteasome lid and its cousin, the csn complex.
the bipartite structure of the pci domain
in our original discovery note  <cit> , we had defined the pci domain as a homology domain, i.e.
as a region of localized similarity found within multiple proteins that are otherwise unrelated.
the results presented here suggest that this view should be revised.
the sequence regions detected as pci domains by bioinformatical methods seems to consist of two structurally distinct domains.
the c-terminal portion, which in eif3k is referred to as the wh-domain, is much better conserved in sequence than the n-terminal portion, and the c-terminal boundary of the pci homology domain is relatively well defined by a notable loss of sequence conservation.
by contrast, the n-terminal boundary of the homology domain has always been ill-defined, as the overall sequence conservation in this region is low and different families of pci proteins appear to lose their similarity at different positions.
as a consequence, different domain databases and their associated web-servers detect pci-domains  of varying length in the order prosite > pfam > smart.
using the eif3k-derived structural model, most of these observations can be readily explained.
the c-terminal pci/pint boundary, which is agreed on by all domain databases, corresponds to the c-terminal boundary of the structural wh-like domain.
the n-terminal boundary of the pint domain, as described in the smart database, essentially corresponds to the n-terminus of the wh-like domain.
the pci domain of the pfam database corresponds to the wh-portion plus a single α-helical hairpin repeat.
finally, the pci domain as described in the prosite database covers the wh-portion and all three helical hairpin repeats found in the eif3k structure.
of the three representations, the pint domain of the smart database is structurally most correct, as it describes a true autonomously folding domain.
the observation that some pci families lose their sequence conservation at different n-terminal positions can be explained by assuming a variable number of helical repeat motifs for those proteins.
as an extreme example, only the wh-like region could be detected in eif3e by our profile searches, and the secondary structure prediction for the eif3e family suggests a β-structure instead of the usual helical-hairpin repeats upstream of the wh region.
this finding can be taken as a further hint for structural and functional independence of the n- and c-terminal sub-regions of the pci homology domain.
the nature of the n-terminal helical repeat extension
our finding of tpr-like repeats preceding many pci domains, combined with the helical repeat structure of the n-terminal portion of the pci domain itself, leads to the interesting question if these repeats are of the same type and may form a continuous solenoid structure.
the authors of the eif3k crystal structure propose a structural relationship between the eif3k n-terminus and the heat motif based on superposition calculations with dali  <cit> .
by contrast, our sequence-based analysis methods rather point to an evolutionary relationship to the tpr motif, both for the region preceding the pci domain and for the first helical hairpin of the pci domain itself.
a related finding was reported for rpn <dig> and csn <dig> elsewhere, where a homology domain termed "pam"  with tpr-like properties has been proposed  <cit> .
our findings suggest that the pam-domain is a special case of a more widespread preference of the wh-portion of the pci domain to be preceded by tpr-like repeats.
in addition, both our results and those of ciccarelli et al.
argue in favour of a continuity between the n-terminal repeats and those found within the pci domain.
due to the borderline sequence similarity between the classical tpr motif and the distinct helical hairpins of the pci proteins, a completely novel type of bi-helical repeats distinct from tpr and heat/armadillo or some kind of intermediary form cannot be ruled out.
structurally, heat and tpr repeats are relatively similar and both tend to form superhelical solenoid structures  <cit> .
without assuming a particular repeat family, we have attempted a rough estimation of what a typical pci component of the lid or the csn complex might look like.
figure 3b shows schematically a pci protein with a wh-like domain at the c-terminus , preceded by three helical-repeats assumed to lie within the pci boundaries according to prosite , which are in turn preceded by three additional helical repeats that represent the tpr-related n-terminal extension .
as we do not assume a particular repeat family with a well-known radius of solenoid curvature, we use the values derived from the first two helical hairpins of the eif <dig> structure instead.
it should be stressed that the model of figure 3b with its 'boomerang'-shaped architecture can only give a very coarse approximation of the real situation.
both the solenoid curvature and the exact number of n-terminal repeat extensions are rough estimations.
nevertheless, the model appears to be roughly compatible with the electron density maps of the lid and csn complexes  <cit> .
a structural scaffold for three multi-protein complexes
pci proteins constitute the main components of the proteasome lid and the csn complex and also form the structural core of the translation initiation factor eif <dig>  so far, no catalytic activity has been described for pci proteins.
given the lack of invariant polar residues, such a role appears unlikely.
the role of the pci domains is most likely that of a scaffold for the other complex subunits and other binding partners.
there are at least three distinct structural roles that pci proteins have to fulfil: i) maintaining the integrity of the complex by binding to other pci proteins, ii) attaching the mpn-subunits to the complex, and iii) binding to other partners such as the base-complex in the case of the proteasome lid or the rna-binding subunits of the eif <dig> complex.
the assignment of these functionalities to the different regions of the pci proteins, and equally important, the source for the subunit interaction specificity or promiscuity have been subject to several experimental studies, some of them published while others have been presented at a recent meeting on pci complexes  <cit> .
the pci model presented here will be certainly useful, both for the interpretation of the experimental results, and for the design of new experiments e.g.
those based on domain truncations or domain swaps.
according to our analysis, in some proteins the pci domain is restricted to a c-terminal wh-like part.
as these proteins are also components of pci-complexes, a role of the wh domain in pci:pci domain interaction is very likely.
on the other hand, tpr-repeats in general form versatile protein-interaction surfaces  <cit>  and we expect the same to be true for the tpr-like repeats found in the pci proteins.
tsuge et al.
analysed truncated forms of human csn <dig> for its interaction with other pci subunits of the csn complex  <cit> .
a construct containing residues 197– <dig>  was able to bind to csn <dig>  csn <dig> and csn <dig>  another construct starting at position  <dig>  and thus lacking the helical-repeat region, no longer bound to csn <dig> and csn <dig> but maintained binding to csn <dig>  by contrast, a construct 197– <dig> that lacks the wh-like region was only able to bind to csn <dig>  these experiments suggest that both the wh portion and the helical-repeat part of the pci proteins have a role in pci:pci interactions, although they seem to interact with different subunits of the complex.
the importance of both subdomains is confirmed by a recent study of isono et al., who analyse multiple point mutations in the lid subunit rpn <dig>  <cit> .
in their hands, both mutations in the n-terminal helical repeat part of rpn <dig> and mutations within the wh-like region are able to abrogate binding to rpn <dig>  another pci subunit of the proteasome lid.
so far, no information is available on the pci regions involved in binding to the mpn subunits.
CONCLUSIONS
in summary, we believe the pci domain could play a role as a universal binding domain supporting intra-complex interactions as well as recruitment of additional ligands.
the model presented here is a first step to the understanding of the supramolecular architecture of three important complexes and certainly will facilitate the interpretation of further experimental results.
nevertheless, a full understanding of the interaction mode between pci- and mpn-domain proteins will certainly require experimentally determined high-resolution structures of the components – or ideally, that of an intact complex.

 in eukaryotic organisms, there exist at least three distinct multi-protein assemblies that are jointly referred to as'pci complexes' and have a similar subunit architecture despite their fundamentally different function: i) the proteasome lid, a subcomplex of the 19s proteasome regulator and the 26s proteasome, ii) the cop signalosome or csn complex, and iii) the eukaryotic translation initiation factor eif as a common feature, these complexes are composed of multiple subunits harbouring the pci domain, named after the three participating complexes, sometimes also referred to as the pint domain. other subunits of these complexes are characterized by a second shared homology domain called mpn. among these complexes, the proteasome lid and the csn share a particular degree of analogy. both complexes consist of eight core subunits, six of them of the pci class and two of the mpn class. as described previously and summarized in table there is a clear 1: correspondence between the paralogous sets of pci and mpn subunits. in addition, a similar ring-shaped structure was observed for the two complexes and there is evidence that in those rings paralogous subunits occupy equivalent positions. by contrast, the eif complex has a smaller number of pci subunits and its two mpn subunits are absent in several unicellular eukaryotes. unlike the proteasome lid and the csn, the eif complex contains a number of non-pci/non-mpn subunits, which are required for its function in translation. despite the common homology domains and a similar structure, the functions of the three pci complexes are very different. the proteasome lid, in combination with the'base' complex containing a hexameric ring of aaa-atpases, forms the 19s regulatory particle, which in turn constitutes an essential subcomplex of the 26s proteasome. the lid complex contains an intrinsic deubiquitinating activity, which is encoded by the mpn subunit rpn that has the hallmarks of a metalloprotease. no specific function has been described for the pci subunits of the lid. the csn complex has been first described as a regulator of photomorphogenesis in plants, but seems to regulate diverse cellular processes like signal transduction, regulation of transcription or cell proliferation. csn an mpn-bearing subunit of the signalosome, which is analogous to rpn also encodes a metalloprotease that is essential for the removal of the ubiquitin-like protein nedd from cullins the third pci complex, the translation initiation factor eif promotes the formation of preinitiation complexes and works as a scaffold by binding to other initiation factors, to ribosomes and to mrna. both mpn subunits of eif lack the residues necessary for metal binding and are most likely catalytically inactive. so far, the metal-containing mpn subunits and the non-pci/non-mpn portion of the complex constitute the only known carriers of functionality. the pci proteins themselves seem to be the main building blocks of the complexes, a fact already suggested by their high abundance. there are several hints that the pci subunits are crucial for proper complex assembly. the mpn subunits of the three complexes are rather well conserved and the detection of mpn domains and their boundaries is relatively straightforward. by contrast, the degree of conservation between pci subunits is highly variable. sequence similarity between the corresponding subunits of proteasome lid and csn is generally easy to spot, while the detection of similarity between other paralogous pci subunits typically requires sophisticated sequence comparison approaches, such as the generalized profile method. a particular challenge is the detection of the highly divergent pci domains in the budding yeast csn-like complex and those of the eif complex, where only three pci subunits could be detected in the initial survey. due to this difficulties, it is to be expected that there are still a number of highly divergent pci domain proteins in eukaryotic genomes, which have eluded detection so far. a second issue in the bioinformatical definition of the pci domain concerns the position of its n-terminal boundary. in general, homology domains are thought to correspond to structural domains in the sense of autonomous folding units; they are typically characterized by a pronounced loss of sequence similarity at the domain boundaries. while this is true for the pci domain c-terminus, the n-terminal domain boundary is blurred through a gradual decay in sequence similarity instead of a sharp drop. as a consequence, different pci domain boundaries have been used in the literature and in various domain databases like prosite, pfam and smart. the corresponding accession numbers are ps pf and sm respectively. during an exhaustive bioinformatical analysis of proteasome subunits and other components of the ubiquitin/proteasome system, we obtained two independent results jointly suggesting that a structure-based redefinition of the pci domain is appropriate: on one hand, we detected multiple instances of tpr-like repeats in the n-terminus of many pci proteins, which suggests that the homology between the proteasome and csn components is not restricted to the pci domain itself. on the other hand, we detected a previously overlooked pci domain in the novel eif subunit eif3k. most interestingly, an x-ray structure of eif3k has been published recently. based on this structure and on our alignment data, we suggest a bipartite consensus model for the canonical pci proteins, consisting of a c-terminal'winged helix' domain preceded by an extended helical repeat region. we use this model to re-evaluate some bioinformatical and experimental findings that have been enigmatic so far.  tpr-like helical repeats in pci proteins in most pci proteins, the canonical pci domain occupies a region of approximately residues close to the carboxy-terminus of the sequence. the n-terminal non-pci portion of the proteins is moderately conserved between species and only poorly conserved between different pci subunits – even between the analogous subunits of the lid and the csn. upon submitting those pci proteins to profile- or hmm-based domain detection services, no significant matches were obtained for the n-termini of the proteins. however, the prosite profile for the tetratrico-peptide repeat yielded a number of closely sub-significant matches in multiple pci proteins, e.g. rpn from s. bayanus and csn from e. histolytica. the tpr repeat family is very heterogeneous, and tpr motif descriptors such as the prosite profile are known to miss several instances of bona fide tpr repeats. upon closer inspection, most pci proteins exhibit multiple regions of similarity to profiles derived from established tpr repeats, although the similarity scores for each of the single regions do not reach statistical significance. no relevant similarity scores were obtained for other helical repeat motifs, such as heat or armadillo repeats. to further investigate if a tpr-like structure should be assumed for the n-terminal portions of all pci proteins, we performed a secondary structure prediction for each of the protein families individually. to that aim, we constructed multiple alignments for representative members of each subunit family and submitted the alignment to phd and jpred prediction servers. as a result, all pci subunits of lid and csn are predicted to adopt an all-helical secondary structure upstream of the pci domain. interestingly, these helical regions merge seamlessly into the pci domain, at least if the longer pci versions of prosite and pfam are used. this finding is in agreement with the observation of several regions with weak tpr-similarity within the n-terminal part of the pci domain itself. further support for a tpr-like structure comes from a sequence-based fold recognition for lid and csn subunits using the superfamily-service. several subunits like rpn from budding yeast and human csn were found to have significant scores for the tpr fold upstream of the pci domain. the predicted all-helical secondary structure of the non-pci portion of lid and csn subunits consists of several short helices that appear to occur in pairs. to test whether those bi-helical segments correspond to the structural elements of a tpr-like repeat, we selected several examples starting immediately upstream of the predicted pci domains. when multiple alignments of those bi-helical segments were used for profile construction and in subsequent database searches several bona fide tpr proteins were found to match within the tpr region, with the bi-helices being in the correct tpr register, these segments were also classified as tpr-like. no matches to established heat- or armadillo-repeat proteins were found, demonstrating that the scores are not just caused by an arbitrary helical repeat arrangement. it should be pointed out that none of the singular observations described above is able to prove a statistically significant sequence relationship between the n-terminal portions of pci proteins and true tpr-repeats. taken as a whole, the results strongly suggest that there is a general tendency of pci domains to be preceded by an α-helical repeat structure that has at least some specific relationship to the tetratrico-peptide repeat. a previously unrecognized pci domain in eif3k in the first surveys of recognizable pci domains, only three pci subunits of the eif complex had been detected. more recently, a number of novel eif components have been identified: eif3j, eif3k and eif3l. among these novel subunits, only eif3l has been reported to harbour a pci domain, interestingly also preceded by a tpr-region. in order to find further indications of divergent pci domains, we performed a thorough profile analysis of all uncharacterized eif subunits. a generalized profile was constructed from the conserved portion of representative eif3k orthologs from vertebrates, invertebrates, plants and fungi. after a scaling step, the resulting profile was run against a nonredundant protein database. apart from the eif3k proteins already used for profile construction, the only other sequences matching with significance were selected pci subunits of the proteasome and the csn, among them rice csn and the drosophila rpn homologue. all of the twenty top-scoring sequences could be identified as either csn8- or rpn12-homologs. as shown in table csn and rpn are the corresponding pci subunits in the csn and the lid, respectively. csn and rpn are the most divergent pci subunits of the proteasome and the signalosome, respectively, and their pci domains appear to be shorter than that of the more typical family members. our observations provide good bioinformatical evidence that eif3k is the fifth pci-containing subunit of the eif complex and most likely a direct analogue of csn and rpn. a structural model for the canonical pci domain the discovery of a pci domain in eif3k is of particular importance, as a three-dimensional structure of eif has been solved recently. so far, no structural information on the pci domain has been available, and a structural model for the canonical pci domain based on the alignment shown in figure should allow interesting insights into the architecture of the pci complexes. a detailed analysis of the eif3k structure reveals a bipartite structure of two subdomains that are in close contact through a large inter-domain surface patch. the c-terminal half-domain is a globular α/β structure with an "αβααββ" arrangement. the three β-strands are very short and form an antiparallel sheet. the whole c-terminal part can be classified as a "winged helix" fold and thus is referred to as "wh-domain". by contrast, the n-terminal half-domain is entirely helical with a core of six regularly-spaced helices that form three antiparallel helical hairpin elements. the resulting superhelix is reminiscent of the solenoids found in helical repeats such as heat, armadillo and tpr. somewhat unusual are the short 3– helices that connect the consecutive α-hairpins. according to wei et al. the n-terminal half-domain resembles structurally mainly heat and armadillo repeats, and thus the name "ham-domain" was proposed. the bipartite structure of eif3k is in good overall agreement with the secondary structure predictions for the single pci domain families and also with our result of tpr-like helical repeats partially overlapping the pci domain. it was therefore of special interest to make a detailed comparison of the eif3k structure and the profile-guided alignment of the canonical pci superfamily shown in figure within the n-terminal subdomain, the sequence conservation between the different pci domain families is relatively poor and some aspects of the alignment shown in figure are not very reliable. nevertheless, there is a good correspondence between the helices that build the α-hairpins of eif3k and the uninterrupted sequence blocks in the pci alignment. the gap-regions in the pci alignment are typically caused by insertion events in selected pci subfamilies. in no case, a deletion of one or more of the hairpin helices is observed. this finding suggests that the helical hairpin structure is conserved in most or all pci domains. our alignment and the derived secondary structure predictions suggest that the short helices that connect the helical hairpins in eif3k are absent in most other pci proteins. as mentioned in the previous paragraphs, there are several instances of subsignificant sequence similarity to tpr repeats found also within the n-terminal subdomain of the pci domain. by contrast, no similarity to heat or armadillo-repeats has been observed. thus, we prefer to interpret the helical hairpin structure of the n-terminal subdomain as atypical tpr-like repeats rather than as the heat/armadillo repeats suggested by wei et al.. the globular c-terminal subdomain is generally better conserved than the helical n-terminal domain and as a consequence, the part of the alignment covering this structural subdomain shown in figure is more reliable. the "αβααββ" arrangement of α- and β-regions is distributed over two large sequence blocks with a single major gap region between "αβα" and "αββ". as can be seen in figure no important secondary structure element is interrupted by a gap found in the pci alignment. like in the n-terminal subdomain, the wh portion shows a good concordance between the secondary structure predicted from the canonical pci families and the structural elements of the eif3k structure, apart from minor problems in predicting one of the very short β-strands. taken together, the comparison of the pci alignment with the eif3k structure shows that the two structures are clearly compatible and suggests that the canonical pci domains will have an analogous bipartite fold similar to that shown in figure the prediction of tpr-like helical repeats n-terminal of the proper pci domain suggests that they form an extension of the helical repeat region of the first pci subdomain. the implications of this model for the overall pci structure will be discussed below. discussion revision of the eif complex stoichiometry while the proteasome lid and the csn complex have an analogous architecture of six pci-subunits and two mpn-subunits, the more distantly related eif complex has so far only three readily detectable pci proteins: eif3a, eif3c and eif3e. recent work by morris-desbois et al. has grouped eif3l with the pci components of eif and our work described above adds eif3k to the ranks of pci proteins. besides the pci subunits, vertebrate eif complexes also contain two mpn proteins: eif3f and eif3h. unlike the situation in the lid and csn complexes, both mpn subunits of eif have lost their metal-coordinating residues and are most likely catalytically inactive. in addition, several unicellular eukaryotes, including budding yeasts, do not seem to have any eif3-associated mpn proteins. comparing the stoichiometry of eif with the two better-conserved pci complexes, only one pci subunit seems to be missing. our sequence analysis efforts have also included other known eif subunits, but no indications for further pci domains could be obtained. given the high degree of pci sequence divergence, it cannot be fully excluded that one of the non-pci/non-mpn subunits harbours a cryptic pci domain that has eluded our detection. on the other hand, it is well conceivable that eif has a deviating subunit composition. in yeast and several other organisms, not only the mpn proteins are missing but also the number of pci components is reduced, as eif3e and eif3l are absent. at present, it is not clear whether the corresponding positions in the complex are left empty or are filled by additional copies of the remaining pci components. in evolutionary terms, it appears likely that the eif complex is a'degraded' copy of an ancient lid-like complex, which has lost its mpn+/jamm mediated catalytic activity and potentially some of its pci subunits. in turn, by acquiring a group of novel non-pci/non-mpn subunits, the eif complex has gained a functionality that is different to the proteasome lid and its cousin, the csn complex. the bipartite structure of the pci domain in our original discovery note, we had defined the pci domain as a homology domain, i.e. as a region of localized similarity found within multiple proteins that are otherwise unrelated. the results presented here suggest that this view should be revised. the sequence regions detected as pci domains by bioinformatical methods seems to consist of two structurally distinct domains. the c-terminal portion, which in eif3k is referred to as the wh-domain, is much better conserved in sequence than the n-terminal portion, and the c-terminal boundary of the pci homology domain is relatively well defined by a notable loss of sequence conservation. by contrast, the n-terminal boundary of the homology domain has always been ill-defined, as the overall sequence conservation in this region is low and different families of pci proteins appear to lose their similarity at different positions. as a consequence, different domain databases and their associated web-servers detect pci-domains of varying length in the order prosite > pfam > smart. using the eif3k-derived structural model, most of these observations can be readily explained. the c-terminal pci/pint boundary, which is agreed on by all domain databases, corresponds to the c-terminal boundary of the structural wh-like domain. the n-terminal boundary of the pint domain, as described in the smart database, essentially corresponds to the n-terminus of the wh-like domain. the pci domain of the pfam database corresponds to the wh-portion plus a single α-helical hairpin repeat. finally, the pci domain as described in the prosite database covers the wh-portion and all three helical hairpin repeats found in the eif3k structure. of the three representations, the pint domain of the smart database is structurally most correct, as it describes a true autonomously folding domain. the observation that some pci families lose their sequence conservation at different n-terminal positions can be explained by assuming a variable number of helical repeat motifs for those proteins. as an extreme example, only the wh-like region could be detected in eif3e by our profile searches, and the secondary structure prediction for the eif3e family suggests a β-structure instead of the usual helical-hairpin repeats upstream of the wh region. this finding can be taken as a further hint for structural and functional independence of the n- and c-terminal sub-regions of the pci homology domain. the nature of the n-terminal helical repeat extension our finding of tpr-like repeats preceding many pci domains, combined with the helical repeat structure of the n-terminal portion of the pci domain itself, leads to the interesting question if these repeats are of the same type and may form a continuous solenoid structure. the authors of the eif3k crystal structure propose a structural relationship between the eif3k n-terminus and the heat motif based on superposition calculations with dali. by contrast, our sequence-based analysis methods rather point to an evolutionary relationship to the tpr motif, both for the region preceding the pci domain and for the first helical hairpin of the pci domain itself. a related finding was reported for rpn and csn elsewhere, where a homology domain termed "pam" with tpr-like properties has been proposed. our findings suggest that the pam-domain is a special case of a more widespread preference of the wh-portion of the pci domain to be preceded by tpr-like repeats. in addition, both our results and those of ciccarelli et al. argue in favour of a continuity between the n-terminal repeats and those found within the pci domain. due to the borderline sequence similarity between the classical tpr motif and the distinct helical hairpins of the pci proteins, a completely novel type of bi-helical repeats distinct from tpr and heat/armadillo or some kind of intermediary form cannot be ruled out. structurally, heat and tpr repeats are relatively similar and both tend to form superhelical solenoid structures. without assuming a particular repeat family, we have attempted a rough estimation of what a typical pci component of the lid or the csn complex might look like. figure 3b shows schematically a pci protein with a wh-like domain at the c-terminus, preceded by three helical-repeats assumed to lie within the pci boundaries according to prosite, which are in turn preceded by three additional helical repeats that represent the tpr-related n-terminal extension. as we do not assume a particular repeat family with a well-known radius of solenoid curvature, we use the values derived from the first two helical hairpins of the eif structure instead. it should be stressed that the model of figure 3b with its'boomerang'-shaped architecture can only give a very coarse approximation of the real situation. both the solenoid curvature and the exact number of n-terminal repeat extensions are rough estimations. nevertheless, the model appears to be roughly compatible with the electron density maps of the lid and csn complexes. a structural scaffold for three multi-protein complexes pci proteins constitute the main components of the proteasome lid and the csn complex and also form the structural core of the translation initiation factor eif so far, no catalytic activity has been described for pci proteins. given the lack of invariant polar residues, such a role appears unlikely. the role of the pci domains is most likely that of a scaffold for the other complex subunits and other binding partners. there are at least three distinct structural roles that pci proteins have to fulfil: i) maintaining the integrity of the complex by binding to other pci proteins, ii) attaching the mpn-subunits to the complex, and iii) binding to other partners such as the base-complex in the case of the proteasome lid or the rna-binding subunits of the eif complex. the assignment of these functionalities to the different regions of the pci proteins, and equally important, the source for the subunit interaction specificity or promiscuity have been subject to several experimental studies, some of them published while others have been presented at a recent meeting on pci complexes. the pci model presented here will be certainly useful, both for the interpretation of the experimental results, and for the design of new experiments e.g. those based on domain truncations or domain swaps. according to our analysis, in some proteins the pci domain is restricted to a c-terminal wh-like part. as these proteins are also components of pci-complexes, a role of the wh domain in pci:pci domain interaction is very likely. on the other hand, tpr-repeats in general form versatile protein-interaction surfaces and we expect the same to be true for the tpr-like repeats found in the pci proteins. tsuge et al. analysed truncated forms of human csn for its interaction with other pci subunits of the csn complex. a construct containing residues 197– was able to bind to csn csn and csn another construct starting at position and thus lacking the helical-repeat region, no longer bound to csn and csn but maintained binding to csn by contrast, a construct 197– that lacks the wh-like region was only able to bind to csn these experiments suggest that both the wh portion and the helical-repeat part of the pci proteins have a role in pci:pci interactions, although they seem to interact with different subunits of the complex. the importance of both subdomains is confirmed by a recent study of isono et al., who analyse multiple point mutations in the lid subunit rpn. in their hands, both mutations in the n-terminal helical repeat part of rpn and mutations within the wh-like region are able to abrogate binding to rpn another pci subunit of the proteasome lid. so far, no information is available on the pci regions involved in binding to the mpn subunits.  in summary, we believe the pci domain could play a role as a universal binding domain supporting intra-complex interactions as well as recruitment of additional ligands. the model presented here is a first step to the understanding of the supramolecular architecture of three important complexes and certainly will facilitate the interpretation of further experimental results. nevertheless, a full understanding of the interaction mode between pci- and mpn-domain proteins will certainly require experimentally determined high-resolution structures of the components – or ideally, that of an intact complex. 
during an exhaustive bioinformatical analysis of proteasome components, we detected multiple instances of tetratrico-peptide repeats in the n-terminal region of most pci proteins, suggesting that their homology is not restricted to the pci domain. the tpr-like repeats detected in the n-terminal region of pci proteins most likely form an uninterrupted extension of the repeats found within the pci domain boundaries. the translation initiation complex eif also contains pci- and mpn-domain proteins, but seems to deviate from the 6+ stoichiometry. this model allows an interpretation of several puzzling experimental results. our model predicts that the homology domain pci is not a true domain in the structural sense but rather consists of two subdomains: a c-terminal'winged helix' domain with a key role in pci:pci interaction, preceded by a helical repeat region. the'lid' subcomplex of the 26s proteasome and the cop signalosome share a common architecture consisting of six subunits harbouring a so-called pci domain at their c-terminus, plus two subunits containing mpn domains. initially, the pci domain was defined as the region of detectable sequence similarity between the components mentioned above. we also detected a previously unrecognized pci domain in the eif component eif3k, a protein whose 3d-structure has been determined recently. by using profile-guided alignment techniques, we show that the structural elements found in eif3k are most likely conserved in all pci proteins, resulting in a structural model for the canonical pci domain.
 the'lid' subcomplex of the 26s proteasome and the cop signalosome share a common architecture consisting of six subunits harbouring a so-called pci domain at their c-terminus, plus two subunits containing mpn domains. the translation initiation complex eif also contains pci- and mpn-domain proteins, but seems to deviate from the 6+ stoichiometry. initially, the pci domain was defined as the region of detectable sequence similarity between the components mentioned above.  during an exhaustive bioinformatical analysis of proteasome components, we detected multiple instances of tetratrico-peptide repeats in the n-terminal region of most pci proteins, suggesting that their homology is not restricted to the pci domain. we also detected a previously unrecognized pci domain in the eif component eif3k, a protein whose 3d-structure has been determined recently. by using profile-guided alignment techniques, we show that the structural elements found in eif3k are most likely conserved in all pci proteins, resulting in a structural model for the canonical pci domain.  our model predicts that the homology domain pci is not a true domain in the structural sense but rather consists of two subdomains: a c-terminal'winged helix' domain with a key role in pci:pci interaction, preceded by a helical repeat region. the tpr-like repeats detected in the n-terminal region of pci proteins most likely form an uninterrupted extension of the repeats found within the pci domain boundaries. this model allows an interpretation of several puzzling experimental results. 