- Email: [email protected]
Structural disorder throws new light on moonlighting
Opinion
TRENDS in Biochemical Sciences
Vol.30 No.9 September 2005
Structural disorder throws new light on moonlighting Peter Tompa1, Csilla Sza´sz1 and La´szlo´ Buday2 1
Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, 29 Karolina Street, 1113 Budapest, Hungary Department of Medical Chemistry, Molecular Biology and Pathobiochemistry, Semmelweis University, 9 Puskin Street, 1088 Budapest, Hungary 2
A basic mechanism by which individual proteins can increase network complexity is moonlighting, whereby a given protein fulfils more than one function. Traditionally, this phenomenon is attributed to separate binding surfaces of globular, folded proteins but we suggest that intrinsically unstructured proteins (IUPs) might provide radically different mechanisms. Eleven IUPs have been identified that suggest that the structural malleability of IUPs gives rise to unprecedented cases of moonlighting by eliciting opposing (inhibiting and activating) action on different partners or even the same partner molecule. Unlike classical cases, these proteins use the same region or overlapping interaction surfaces to exert distinct effects and employ non-conventional mechanisms to switch function, enabled by their capacity to adopt different conformations upon binding. Owing to the apparent functional benefits, we expect to see many more examples of this parsimonious use of protein material in complex metabolic networks.
Introduction The recent and near-complete euchromatic sequence of the human genome has revealed that our genetic material encodes only w20–25 000 proteins [1], thus, reviving the issue of the classical C-value paradox, that is, the apparent lack of correlation between the complexity of an organism and the number of its genes [2]. This paradox can be resolved by invoking the capacity of single genes to encode multiple proteins and also by the immense functional potential of non-protein-coding regions of the genome [3]. A further basic means by which increased organismal complexity can be achieved without additional genes stems from the potential of certain proteins to fulfil more then one, apparently unrelated, function. Currently, several dozen such ‘moonlighting’, or multi-tasking, proteins are known [4–6] and, as functionally unbiased genomics programs proceed, many more are expected to be revealed. There are several potential molecular mechanisms that a moonlighting protein could use to switch between functions, for example, changes in cellular localization or ligand binding, expression in different cell types, or variations in oligomerization or complexation state Corresponding author: Tompa, P. ([email protected]). Available online 28 July 2005
(see Box 1). In the few cases for which the 3D structure of the protein is actually known, these distinct phenomena could all be interpreted within the framework of the classical structure–function paradigm by attributing additional function(s) to binding surface(s) of globular proteins that are distinct from the site responsible for their primary function [6]. However, because the classical cases are not even all well-folded proteins [4], we suggest that the flexibility and ensuing structural adaptability of certain proteins offer a radical deviation from this principle. Box 1. The mechanisms of ‘classical’ moonlighting Moonlighting, by definition, is the ability of a protein to fulfil more than one, apparently unrelated, function [4–6]. Moonlighting proteins might serve at distinct points of metabolic networks and might, thus, increase network complexity without increasing the number of underlying proteins. There is a great variety in the combination of functions of such proteins, for example, PGI (phosphoglucose isomerase) has been reported to display both enzymatic activity and a receptor-binding function, CFTR (cystic fibrosis transmembrane conductance regulator) shows channel function plus a regulatory function, cytochrome c has electrontransfer and signaling functions, and crystallins have structural function combined with catalytic activity. Recent X-ray structures have shed light on the possible structural basis of this functional diversity in the case of folded, globular proteins. The structures of I-Anil maturase, PutA proline dehydrogenase and DegP chaperone, for example [6], provided direct and indirect evidence that these proteins use separate surfaces for binding distinct partners and/or catalyzing different reactions. It seems that evolution has recruited their large unused surfaces for alternative purposes. The ensuing capacity to bind distinct partners, however, tells nothing about how or why moonlighting proteins switch function. This switch in function might be initiated by various means [4–6], such as expression in a different cell type (as for neutropilin) or outside rather than inside a cell (PGI). Less dramatic changes that might trigger a switch in function are those such as in cellular localization (as for PutA), oligomeric state (glyceraldehyde-3phosphate dehydrogenase), ligand binding (aconitase) or complexation (ribosomal proteins). The unifying mechanistic theme is a change in the molecular environment. Differential expression reflects changes in the activity of transcription factors, whereas changes in localization, oligomerization or ligand binding are usually caused by changes in the level of a substrate, ligand, cofactor or product. A shift in complexation usually occurs when the availability of partner molecules (i.e. other macromolecules) changes. Although excluded originally, the signal for a switch in function might also be transduced via post-translational modification [5], as in the case of CFTR, the regulatory function of which is controlled by phosphorylation [45] or thymosin-b4, which is Metoxidized and secreted to become an anti-inflammatory agent [49].
www.sciencedirect.com 0968-0004/$ - see front matter Q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibs.2005.07.008
Opinion
TRENDS in Biochemical Sciences
As noted recently [7–11], a marked portion of eukaryotic genomes encodes for intrinsically unstructured proteins (IUPs), which lack a well-defined 3D structure in their native state. These proteins fulfil important functions that are often associated with signal transduction, gene expression and chaperone action [12–14]. For the performance of these functions, structural disorder confers special advantages, such as the capacity of one-tomany signaling or binding promiscuity, whereby an IUP binds distinct partners in a template-induced folding process [7,11,15]. Accordingly, IUPs have the potential to modulate the action of different partner molecules, that is, to moonlight. In fact, as we elaborate here, the underlying structural malleability enables an IUP to elicit both inhibiting and activating effects on different partners or even the same partner molecule. For example, cyclindependent kinase (Cdk) inhibitors p21Cip1 and p27Kip1 [16] were first noted for their capacity for binding promiscuity [15] and then later for their activating effect on Cdk(s) [17]. In a search for such extreme cases of moonlighting, we have identified proteins that can both inhibit and activate their partner(s). Biochemical evidence and bioinformatic predictors demonstrate that these proteins are largely, or completely, disordered and that their structural disorder is crucial for binding with disparate effects. The IUPs identified here throw new light on the mechanism of moonlighting; unlike the classical examples studied in detail thus far [6], they use the same region or interspersed, short interaction motifs to exert different effects. In switching function, they might use three principal, non-exclusive mechanisms. In these, the interaction region might either bind to the same or different partners in different conformations, or undergo structural reorganization in chaperoning or partially releasing the partner. After the individual examples are discussed in some detail, the structural, functional and evolutionary implications of this novel finding are elucidated. Disorder in moonlighting proteins By searching the literature for effectors that can both inhibit and activate their partner(s), we have identified 11 proteins for which the opposing effects can be ascribed to
Vol.30 No.9 September 2005
485
the same binding region; for eight of these proteins, there is direct experimental evidence that the entire protein or its binding region is largely disordered (Table 1). Disorder predictors also confirm that these proteins contain a high level of disorder (Table 1) exceeding that of regulatory and signal-transduction proteins [12,13] and even the most disordered class of proteins, the chaperones [14]. The individual examples discussed here suggest that the structural malleability of the region involved in binding is crucial for their different effects. This region might either bind the same partner in different conformations or bind completely unrelated partners. In the bound state, it might also undergo structural reorganization that results in opposing outcomes. Of course, it is to be emphasized that these mechanisms are not mutually exclusive but could possibly be combined to have a profound effect. Binding to a partner in different conformations A disordered inhibitor/activator protein can bind to the same partner in different conformations and/or at different binding sites, resulting in two opposing effects (Figure 1a). For example, in excitation–contraction coupling in skeletal muscle, the voltage-sensor dihydropyridine receptor (DHPR) has an unstructured loop region (peptide C), which is a multifarious effector of the ryanodine receptor (RyR) [18]. Peptide C can bind RyR with high affinity and activate its opening but, in a different conformation, it can bind to RyR with a lower affinity [18] and inhibit RyR-channel opening. Inhibition and activation are independent processes and it is not known what causes the transition between them. Cystic fibrosis transmembrane-conductance regulator (CFTR) provides another example for a disordered binding region that can bind to the same partner with different outcomes. This chloride channel, which is involved in cystic fibrosis, is blocked and inhibited when its disordered regulatory domain R is dephosphorylated [9]. Upon phosphorylation by protein kinase A, domain R probably undergoes a conformational change that not only relieves internal inhibition to make the pore accessible to chloride ions but also stimulates channel opening [19]. The binding mode determined by the negative charges introduced and/or the
Table 1. Examples of disordered moonlighting proteins Proteina
One (inhibiting) function
Another (activating) function
Calpastatin CFTR (R domain) DHPR (peptide C) EBV-SM
Inhibition of calpain Inhibition of CFTR Inhibition of RyR Down-regulation of introncontaining mRNA Down-regulation of p21Cip1 Inhibition of Cdk Inhibition of activated STAT Inhibition of PP1 Inhibition of MDM2 ubiquitin ligase Inhibition of separase Sequestration of G-actin
Activation of calpain Activation of CFTR Activation of RyR Up-regulation of intron-less mRNA
MDM2 (180–298) p21Cip1 and p27Kip1 PIAS1 (392–541) I-2 Ribosomal L5 Securin Thymosin-b4 (WH2 domain) a
Activation Activation Activation Activation Activation
IUPred (%)b 100 40.8 100 44.1
www.sciencedirect.com
[8d,23] [9d,19] [18d] [33]
Refs
of estrogen receptor a of Cdk of p53 of PP1 (chaperoning) of ribosome
70.0 48.2 41.3 91.7 22.3
70.0 61.0 40.7 77.8 30.8
[34,35] [15d–17] [40,41] [25d–27] [31d,32]
Activation (chaperoning) of separase Activation of actin polymerization, ILK kinase
52.0 100
54.2 64.3
[8d,29,30] [36d,37,39d]
Proteins known to have two opposing functions. If known, the region (domain) to where the functions are localized is indicated. The IUPred algorithm [48] for assessing the percentage of disorder is available at http://iupred.enzim.hu. c The PONDRw version VL-XT algorithm [9] for assessing the percentage of disorder is available at http://www.pondr.com. d Direct evidence for the disorder of the whole protein or its moonlighting segment. b
PONDRw (%)c 80.1 53.7 81.1 44.4
486
Opinion
TRENDS in Biochemical Sciences
(a)
(b)
(c)
Ti BS
Figure 1. Mechanisms by which moonlighting IUPs switch between functions. The scheme depicts the three principal, non-exclusive, molecular mechanisms by which the proteins exert opposing effects. The partner molecule is represented by a yellow square or pentagon before binding, an oval when in an active conformation and a rectangle when in an inactive conformation. Green and red colors indicate activation and inhibition, respectively. (a) A protein can bind to the same partner in two basically different conformations or binding sites, leading to different effects. (b) An inhibitor can function as a chaperone or it can shift the conformational equilibrium of its partner to promote the formation of its active conformation, but blocks its active site. Owing to significant differences in the binding strength of the two sites and/or post-translational modification such as phosphorylation or limited proteolysis, the inhibitory interaction might be partially released so that overall inhibition turns to activation. Release of the partner at a given location might result in targeting. (c) A protein binds to two different partners via two alternative conformations of the same site, or by two different, but overlapping, sites.
conformation of the domain is essential for the inhibition or activation of the channel. Structural reorganization around the partner Another common mode of moonlighting by an IUP is binding to a partner in a manner that shifts its equilibrium towards the active form but blocks its active site. Subsequent release of the block might result in deinhibition, and net activation, of the partner (Figure 1b). For example, fully disordered [15] p21Cip1 binds to both the cyclin and kinase subunits of activated Cdk and inhibits kinase action. However, the assembly of the cyclin–kinase complex, an obligate step in Cdk activation, is also facilitated by p21Cip1 and its homolog, p27Kip1 [17,20]. Because the inhibitor binds to the cyclin markedly www.sciencedirect.com
Vol.30 No.9 September 2005
more strongly than the kinase [21], net activation of the enzyme within the ternary complex [22] might ensue from release of the kinase subunit. The activating effect of calpastatin, the disordered [8] inhibitor of the calciumactivated protease calpain, can be conceived in a similar manner. Calpastatin facilitates binding of calcium to the enzyme but it also inactivates the enzyme via its inhibitory subdomain. When this region is degraded in a manner that leaves the rest of the molecule bound, the enzyme becomes activated [23]; this might ensure its prolonged action in myoblast fusion, for example [24]. A similar partial release is witnessed in the case of inhibitor-2 (I-2) of protein phosphatase 1 (PP1). Complex interactions of this IUP [25] with PP1 elicit fast inhibition, slow inactivation and phosphorylation-induced activation of the enzyme. Principally, the inhibitor binds to the enzyme via two independent sites, one activating and another inhibitory. Its phosphorylation at a third site by glycogen synthase kinase 3 causes conformational changes in both the inhibitor and the enzyme, which initiates the liberation of the active site and further activation via dephosphorylation of the inhibitor [26]. In conjunction, both deletion and overexpression of the yeast homolog of I-2, Glc8p, reduce assayable PP1 activity in yeast, whereas a moderate level stimulates the enzyme [27]. A variation on this theme is when activation of the partner results from chaperoning by the IUP, followed by its partial or full release (Figure 1b). The prime example for this behavior is securin, the disordered [8] inhibitor of separase, the protease that separates sister chromatids at the onset of anaphase. Intriguingly, the genetic ablation of securin does not lead to over-activation of separase and premature chromatid separation, but cripples the enzyme, retards chromosome segregation and causes aneuploidity [28,29], probably due to the lack of the chaperoning effect of securin on separase [30]. Furthermore, securin also activates separase by targeting the action of the enzyme into the nucleus and loading it onto spindles [29,30]. Binding to different partners The malleable structure of IUPs might also enable them to bind to completely different partners with opposing effects (Figure 1c). For example, the partially disordered [31] ribosomal L5 protein facilitates formation of the ribosomal large subunit by co-folding with RNA [31], which is then consolidated by targeting the ribosomal subunit into the nucleolus for proper assembly (Figure 1b). L5, however, can also bind to the oncoprotein mouse double minute 2 (MDM2) and inhibit its E3 ubiquitin-ligase activity [32], which mediates a ribosomal misassembly stress response. Under such conditions, L5 is released and blocks MDM2, which causes cell-cycle arrest by stabilization of the p53 tumor-suppressor protein. Another protein that binds to two different partners is Epstein–Barr virus (EBV)-nuclear protein BS-MLF1 (SM). This protein post-translationally inhibits the expression of intron-containing genes and activates intron-less genes by binding to the distinct premRNA molecules in a different manner [33]. In the case of an intron-containing mRNA, EBV-SM binding inhibits the excision of the terminal intron, causing retention in the nucleus and subsequent degradation of the mRNA. For
Opinion
TRENDS in Biochemical Sciences
inherently unstable, intron-less mRNA molecules, EBV-SM stabilizes the transcript and targets it for cytoplasmic export. This subtle distinction by SM promotes the virus cycle to enter the lytic phase because host genes usually contain introns, whereas lytic viral genes do not. MDM2 also seems to moonlight, as evidenced by the opposing effects of its middle segment. This region down-regulates p21Cip1 by targeting it to the C8 subunit of 20S proteasome for ubiquitin-independent degradation [34], but it can enhance the activity of the nuclear estrogen receptor ERa in a p53-independent manner via direct physical interaction [35]. The WASP (Wiskott–Aldrich syndrome protein) homology domain 2 (WH2) domain of thymosin-b4 also binds to different partners with opposing effects. In early myocardial and epithelial cells, thymosin-b4 promotes cell migration and cell survival. In these cells, the disordered [36] WH2 domain activates integrin-linked kinase (ILK), which subsequently phosphorylates the survival kinase Akt [37]. The classical function of thymosin-b4, however, is to wrap around an ATP-bound G-actin monomer and maintain it in a ‘sequestered’, nonpolymerizable form [38]. Intriguingly, in the homologous actobindin, which contains two WH2 domains, this domain facilitates barbed-end growth of actin polymers, possibly owing to a partial release (Figure 1b) at its C-terminal end [39]. Protein inhibitor of activated STAT (signal transducer and activator of transcription)-1 (PIAS-1) is another protein with multiple activities. STATs are latent cytoplasmic transcription factors that become Tyr-phosphorylated, and dimerize and translocate to the nucleus upon cytokine stimulation. The C-terminal region of PIAS-1 binds to the phosphorylated dimer and inhibits its transcription activity [40]. The same region, however, can also interact with the tetramerization domain of p53 and activate p53-mediated gene expression in a variety of cell types [41]. Furthermore, PIAS-1 is almost identical to RNA Gu-helicase-binding protein, which stimulates degradation of the Gu helicase [42]. Novel mechanisms of moonlighting The complex patterns of effector action presented here illustrate novel mechanistic aspects of protein moonlighting. The traditional view of moonlighting mechanisms derived from the classical structure–function paradigm that equated protein function with a well-defined 3D structure. In accord, most of the moonlighting proteins that have been identified thus far have been globular proteins, with their different functions ascribed to distinct, well-defined binding surfaces [6]. The IUPs described here, however, exemplify a different logic of moonlighting action because they use the structural adaptability of their interaction surfaces to bind to the same or distinct partner(s) with different effects. As discussed in detail, this novel paradigm raises two basic questions. What is the exact mechanism of how these proteins switch function? And what are the functional and evolutionary implications of this moonlighting phenomenon? Structural adaptability As noted, the proteins discussed here are either largely disordered (i.e. I-2, calpastatin, securin, p21Cip1 and www.sciencedirect.com
Vol.30 No.9 September 2005
487
thymosin-b4) or use their disordered domain for the moonlighting function (i.e. PIAS-1, MDM2, DHPR peptide C and CFTR R domain). A basic question is whether the structural adaptability of this region is in fact crucial for their multifarious effect. In general terms, IUPs have the capacity to adapt to different partners in a process called ‘one-to-many signaling’ or ‘binding promiscuity’ [15], that is, they adopt distinct conformations, as demonstrated for HIF-1a [11], DNA-dependent RNA polymerase II (RNAP II) C-terminal domain [43] and SNAP-25 [44], for example. Although direct structural data for the moonlighting IUPs are not available at present, the role of structural adaptability in their function has been suggested for CFTR domain R [19] and DHPR peptide C [18]. The involvement of flexibility is also apparent with these proteins, both of which function via partial binding or release of their partner (e.g. p21Cip1, thymosin-b4 and I-2). Chaperone activity, which has been implicated in the action of securin and ribosomal L5, also involves the ability to bind in alternative conformations (as noted earlier) [14]. Thus, conformational adaptability inherent in structural disorder seems to be essential for the novel moonlighting mechanisms. Another key aspect of moonlighting IUPs is the signal that makes these proteins switch structure and function, a change that they undergo in a manner often similar to the classical moonlighting cases (Box 1). In some cases, such as for CFTR [45] and I-2 [26], an intracellular message that triggers phosphorylation switches the conformation and binding mode of the protein. A signal might also be relayed to the IUP via regulated proteolysis, as in the case of calpastatin [24]. For those capable of binding to distinct partners, it might be the level of the partner molecule itself that controls the switching. For example, improper ribosome assembly causes release of ribosomal L5 so that it can bind to MDM2. Most difficult to rationalize is the control of IUPs that bind to the same partner in distinct conformations and/or at different sites, thus, eliciting disparate responses. It might be, as suggested for DHPR peptide C, that inhibition or activation in these cases is a stochastic process [18]. The intriguing possibility that such a mechanism provides stability to the effector system and contributes to network stability remains to be seen. Function and evolution Structural disorder has already been suggested to confer several functional advantages, such as binding promiscuity, among others [8,9]. We suggest that moonlighting be viewed as a major functional manifestation of binding promiscuity. The examples described here offer functional varieties greater than merely combining activation with inhibition. Chaperoning, transport and targeting, and upor down-regulation of partner molecules is also apparent even in this limited number of cases. Among the partners, we find enzymes, receptors, transcription factors, a structural protein and RNA. Given the great functional versatility of IUPs [8–11] and the range of effects seen in these cases, more functional combinations are conceivable. Furthermore, as protein disorder increases with the increasing complexity of organisms [12,13] and multiple use or connectivity of proteins is common in complex
488
Opinion
TRENDS in Biochemical Sciences
molecular networks [46], many more cases of moonlighting IUPs are expected to be revealed in the future. Another aspect is that, unlike the classical examples (Box 1), the opposing functions of the IUPs discussed in this article are often closely related to each other, either synergistically or antagonistically. This relationship might offer special ways of coordinating cellular activities and also particular stability to protein function, as exemplified by I-2 [26,27] and securin [28–30]. At low levels, I-2 and securin activate their partners, whereas at high levels they inhibit them, ensuring a robust response that is protected from extremities. The opposite is true for domain R of CFTR, which, upon phosphorylation, not only de-inhibits but also stimulates CFTR, possibly to provide a particularly effective bistable switch mechanism for abrupt activation. In terms of evolution, traditional moonlighting proteins are thought to have originally had only one function, and then to have been recruited for another use later by virtue of their large, unused and evolutionarily unconstrained surfaces. IUPs must have followed a different evolutionary path. These proteins use overlapping – or the same – interaction surface(s) for distinct functions. Thus, it is more likely that their functions have co-evolved, neither pre-dating the other. It has already been raised that functional diversity ensuing from conformational diversity might facilitate the evolution of new proteins [47]. This is particularly true for IUPs, which are more likely to have had multiple, less specific, activities than a single function at the outset. These distinct functions might have undergone parallel rounds of refinement in evolution that enabled the use of the same surface for more than one purpose, as seen today. This frugal use of protein material is undoubtedly of substantial benefit to the cell. It is even tempting to speculate that this unique functional capacity has been one of the major reasons for the evolutionary advance of protein disorder [12,13]. Concluding remarks Structural disorder provides unprecedented versatility in partner binding that enables proteins to have distinct functions (i.e. to moonlight). In effect, structural malleability permits various and often opposing functions, such as activation and inhibition, chaperoning, transport and targeting, and up- and down-regulation of partner molecule(s), to reside within the same region. Proteins with such combinations of functions represent unique building blocks for complex metabolic networks. It will be of immense interest to see how, and to what extent, cells actually exploit the great potential inherent in this mechanism. Acknowledgements This work was supported by the Wellcome Trust International Senior Research Fellowship ISRF 067595 and a Bolyai Ja´nos Fellowship (P.T.). We thank Monika Fuxreiter and Veronika Csizmo´k for their critical comments on the manuscript.
References 1 Consortium. (2004) Finishing the euchromatic sequence of the human genome. Nature 431, 931–945 www.sciencedirect.com
Vol.30 No.9 September 2005
2 Petrov, D.A. (2001) Evolution of genome size: new approaches to an old problem. Trends Genet. 17, 23–28 3 Mattick, J.S. (2003) Challenging the dogma: the hidden layer of nonprotein-coding RNAs in complex organisms. Bioessays 25, 930–939 4 Jeffery, C.J. (1999) Moonlighting proteins. Trends Biochem. Sci. 24, 8–11 5 Jeffery, C.J. (2003) Multifunctional proteins: examples of gene sharing. Ann. Med. 35, 28–35 6 Jeffery, C.J. (2004) Molecular mechanisms for multitasking: recent crystal structures of moonlighting proteins. Curr. Opin. Struct. Biol. 14, 663–668 7 Wright, P.E. and Dyson, H.J. (1999) Intrinsically unstructured proteins: re-assessing the protein structure–function paradigm. J. Mol. Biol. 293, 321–331 8 Tompa, P. (2002) Intrinsically unstructured proteins. Trends Biochem. Sci. 27, 527–533 9 Dunker, A.K. et al. (2002) Intrinsic disorder and protein function. Biochemistry 41, 6573–6582 10 Fink, A.L. (2005) Natively unfolded proteins. Curr. Opin. Struct. Biol. 15, 35–41 11 Dyson, H.J. and Wright, P.E. (2005) Intrinsically unstructured proteins and their functions. Nat. Rev. Mol. Cell Biol. 6, 197–208 12 Iakoucheva, L. et al. (2002) Intrinsic disorder in cell-signaling and cancer-associated proteins. J. Mol. Biol. 323, 573–584 13 Ward, J.J. et al. (2004) Prediction and functional analysis of native disorder in proteins from the three kingdoms of life. J. Mol. Biol. 337, 635–645 14 Tompa, P. and Csermely, P. (2004) The role of structural disorder in the function of RNA and protein chaperones. FASEB J. 18, 1169–1175 15 Kriwacki, R.W. et al. (1996) Structural studies of p21Waf1/Cip1/Sdi1 in the free and Cdk2-bound state: conformational disorder mediates binding diversity. Proc. Natl. Acad. Sci. U. S. A. 93, 11504–11509 16 Pavletich, N.P. (1999) Mechanisms of cyclin-dependent kinase regulation: structures of Cdks, their cyclin activators, and Cip and INK4 inhibitors. J. Mol. Biol. 287, 821–828 17 Cheng, M. et al. (1999) The p21(Cip1) and p27(Kip1) CDK ‘inhibitors’ are essential activators of cyclin D-dependent kinases in murine fibroblasts. EMBO J. 18, 1571–1583 18 Haarmann, C.S. et al. (2003) The random-coil ‘C’ fragment of the dihydropyridine receptor II–III loop can activate or inhibit native skeletal ryanodine receptors. Biochem. J. 372, 305–316 19 Ma, J. (2000) Stimulatory and inhibitory functions of the R domain on CFTR chloride channel. News Physiol. Sci. 15, 154–158 20 Olashaw, N. et al. (2004) Cell cycle control: a complex issue. Cell Cycle 3, 263–264 21 Lacy, E.R. et al. (2004) p27 binds cyclin–CDK complexes through a sequential mechanism involving binding-induced protein folding. Nat. Struct. Mol. Biol. 11, 358–364 22 Zhang, H. et al. (1994) p21-containing cyclin kinases exist in both active and inactive states. Genes Dev. 8, 1750–1758 23 Tompa, P. et al. (2002) Calpastatin subdomains A and C are activators of calpain. J. Biol. Chem. 277, 9022–9026 24 Barnoy, S. and Kosower, N.S. (2003) Caspase-1-induced calpastatin degradation in myoblast differentiation and fusion: cross-talk between the caspase and calpain systems. FEBS Lett. 546, 213–217 25 Tompa, P. (2003) The functional benefits of protein disorder. J. Mol. Struct. THEOCHEM 666-667, 361–371 26 Yang, J. et al. (2000) Interaction of inhibitor-2 with the catalytic subunit of type 1 protein phosphatase. Identification of a sequence analogous to the consensus type 1 protein phosphatase-binding motif. J. Biol. Chem. 275, 22635–22644 27 Tung, H.Y. et al. (1995) Regulation of chromosome segregation by Glc8p, a structural homolog of mammalian inhibitor 2 that functions as both an activator and an inhibitor of yeast protein phosphatase 1. Mol. Cell. Biol. 15, 6064–6074 28 Jallepalli, P.V. et al. (2001) Securin is required for chromosomal stability in human cells. Cell 105, 445–457 29 Hornig, N.C. et al. (2002) The dual mechanism of separase regulation by securin. Curr. Biol. 12, 973–982 30 Uhlmann, F. (2003) Separase regulation during mitosis. Biochem. Soc. Symp. 70, 243–251 31 DiNitto, J.P. and Huber, P.W. (2003) Mutual induced fit binding of Xenopus ribosomal protein L5 to 5S rRNA. J. Mol. Biol. 330, 979–992
Opinion
TRENDS in Biochemical Sciences
32 Dai, M.S. and Lu, H. (2004) Inhibition of MDM2-mediated p53 ubiquitination and degradation by ribosomal protein L5. J. Biol. Chem. 279, 44475–44482 33 Ruvolo, V. et al. (1998) The Epstein–Barr virus nuclear protein SM is both a post-transcriptional inhibitor and activator of gene expression. Proc. Natl. Acad. Sci. U. S. A. 95, 8852–8857 34 Zhang, Z. et al. (2004) MDM2 is a negative regulator of p21WAF1/CIP1, independent of p53. J. Biol. Chem. 279, 16000–16006 35 Saji, S. et al. (2001) MDM2 enhances the function of estrogen receptor a in human breast cancer cells. Biochem. Biophys. Res. Commun. 281, 259–265 36 Domanski, M. et al. (2004) Coupling of folding and binding of thymosin b4 upon interaction with monomeric actin monitored by nuclear magnetic resonance. J. Biol. Chem. 279, 23637–23645 37 Bock-Marquette, I. et al. (2004) Thymosin b4 activates integrin-linked kinase and promotes cardiac cell migration, survival and cardiac repair. Nature 432, 466–472 38 Hertzog, M. et al. (2002) Control of actin dynamics by proteins made of b-thymosin repeats: the actobindin family. J. Biol. Chem. 277, 14786–14792 39 Hertzog, M. et al. (2004) The b-thymosin/WH2 domain; structural basis for the switch from inhibition to promotion of actin assembly. Cell 117, 611–623 40 Liao, J. et al. (2000) Distinct roles of the NH2- and COOH-terminal
41 42 43
44 45 46 47
48
49
Vol.30 No.9 September 2005
domains of the protein inhibitor of activated signal transducer and activator of transcription (STAT) 1 (PIAS1) in cytokine-induced PIAS1–Stat1 interaction. Proc. Natl. Acad. Sci. U. S. A. 97, 5267–5272 Megidish, T. et al. (2002) Activation of p53 by protein inhibitor of activated Stat1 (PIAS1). J. Biol. Chem. 277, 8255–8259 Valdez, B.C. et al. (1997) Cloning and characterization of Gu/RH-II binding protein. Biochem. Biophys. Res. Commun. 234, 335–340 Fabrega, C. et al. (2003) Structure of an mRNA capping enzyme bound to the phosphorylated carboxy-terminal domain of RNA polymerase II. Mol. Cell 11, 1549–1561 Breidenbach, M.A. and Brunger, A.T. (2004) Substrate recognition strategy for botulinum neurotoxin serotype A. Nature 432, 925–929 Schwiebert, E.M. et al. (1999) CFTR is a conductance regulator as well as a chloride channel. Physiol. Rev. 79, S145–S166 Gavin, A.C. et al. (2002) Functional organization of the yeast proteome by systematic analysis of protein complexes. Nature 415, 141–147 James, L.C. and Tawfik, D.S. (2003) Conformational diversity and protein evolution – a 60-year-old hypothesis revisited. Trends Biochem. Sci. 28, 361–368 Doszta´nyi, Z. et al. (2005) The pairwise energy content estimated from amino acid composition discriminates between folded and intrinsically unstructured proteins. J. Mol. Biol. 347, 827–839 Young, J.D. et al. (1999) Thymosin b4 sulfoxide is an antiinflammatory agent generated by monocytes in the presence of glucocorticoids. Nat. Med. 5, 1424–1427
Endeavour the quarterly magazine for the history and philosophy of science You can access Endeavour online via ScienceDirect, where you’ll find a collection of beautifully illustrated articles on the history of science, book reviews and editorial comment.
featuring
Selling the silver: country house libraries and the history of science by Roger Gaskell and Patricia Fara Carl Schmidt – a chemical tourist in Victorian Britain by R. Stefan Ross The rise, fall and resurrection of group selection by M.E. Borello Mary Anning: the fossilist as exegete by T.W. Goodhue Caroline Herschel: ‘the unquiet heart’ by M. Hoskin Science in the 19th-century zoo by Oliver Hochadel The melancholy of anatomy by P. Fara and coming soon Etienne Geoffroy St-Hillaire, Napoleon’s Egyptian campaign and a theory of everything by P. Humphries Losing it in New Guinea: The voyage of HMS Rattlesnake by J. Goodman The accidental conservationist by M.A. Andrei Powering the porter brewery by J. Sumner Female scientists in films by B.A. Jones and much, much more . . . Locate Endeavour on ScienceDirect (http://www.sciencedirect.com) www.sciencedirect.com
489
TRENDS in Biochemical Sciences
Vol.30 No.9 September 2005
Structural disorder throws new light on moonlighting Peter Tompa1, Csilla Sza´sz1 and La´szlo´ Buday2 1
Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, 29 Karolina Street, 1113 Budapest, Hungary Department of Medical Chemistry, Molecular Biology and Pathobiochemistry, Semmelweis University, 9 Puskin Street, 1088 Budapest, Hungary 2
A basic mechanism by which individual proteins can increase network complexity is moonlighting, whereby a given protein fulfils more than one function. Traditionally, this phenomenon is attributed to separate binding surfaces of globular, folded proteins but we suggest that intrinsically unstructured proteins (IUPs) might provide radically different mechanisms. Eleven IUPs have been identified that suggest that the structural malleability of IUPs gives rise to unprecedented cases of moonlighting by eliciting opposing (inhibiting and activating) action on different partners or even the same partner molecule. Unlike classical cases, these proteins use the same region or overlapping interaction surfaces to exert distinct effects and employ non-conventional mechanisms to switch function, enabled by their capacity to adopt different conformations upon binding. Owing to the apparent functional benefits, we expect to see many more examples of this parsimonious use of protein material in complex metabolic networks.
Introduction The recent and near-complete euchromatic sequence of the human genome has revealed that our genetic material encodes only w20–25 000 proteins [1], thus, reviving the issue of the classical C-value paradox, that is, the apparent lack of correlation between the complexity of an organism and the number of its genes [2]. This paradox can be resolved by invoking the capacity of single genes to encode multiple proteins and also by the immense functional potential of non-protein-coding regions of the genome [3]. A further basic means by which increased organismal complexity can be achieved without additional genes stems from the potential of certain proteins to fulfil more then one, apparently unrelated, function. Currently, several dozen such ‘moonlighting’, or multi-tasking, proteins are known [4–6] and, as functionally unbiased genomics programs proceed, many more are expected to be revealed. There are several potential molecular mechanisms that a moonlighting protein could use to switch between functions, for example, changes in cellular localization or ligand binding, expression in different cell types, or variations in oligomerization or complexation state Corresponding author: Tompa, P. ([email protected]). Available online 28 July 2005
(see Box 1). In the few cases for which the 3D structure of the protein is actually known, these distinct phenomena could all be interpreted within the framework of the classical structure–function paradigm by attributing additional function(s) to binding surface(s) of globular proteins that are distinct from the site responsible for their primary function [6]. However, because the classical cases are not even all well-folded proteins [4], we suggest that the flexibility and ensuing structural adaptability of certain proteins offer a radical deviation from this principle. Box 1. The mechanisms of ‘classical’ moonlighting Moonlighting, by definition, is the ability of a protein to fulfil more than one, apparently unrelated, function [4–6]. Moonlighting proteins might serve at distinct points of metabolic networks and might, thus, increase network complexity without increasing the number of underlying proteins. There is a great variety in the combination of functions of such proteins, for example, PGI (phosphoglucose isomerase) has been reported to display both enzymatic activity and a receptor-binding function, CFTR (cystic fibrosis transmembrane conductance regulator) shows channel function plus a regulatory function, cytochrome c has electrontransfer and signaling functions, and crystallins have structural function combined with catalytic activity. Recent X-ray structures have shed light on the possible structural basis of this functional diversity in the case of folded, globular proteins. The structures of I-Anil maturase, PutA proline dehydrogenase and DegP chaperone, for example [6], provided direct and indirect evidence that these proteins use separate surfaces for binding distinct partners and/or catalyzing different reactions. It seems that evolution has recruited their large unused surfaces for alternative purposes. The ensuing capacity to bind distinct partners, however, tells nothing about how or why moonlighting proteins switch function. This switch in function might be initiated by various means [4–6], such as expression in a different cell type (as for neutropilin) or outside rather than inside a cell (PGI). Less dramatic changes that might trigger a switch in function are those such as in cellular localization (as for PutA), oligomeric state (glyceraldehyde-3phosphate dehydrogenase), ligand binding (aconitase) or complexation (ribosomal proteins). The unifying mechanistic theme is a change in the molecular environment. Differential expression reflects changes in the activity of transcription factors, whereas changes in localization, oligomerization or ligand binding are usually caused by changes in the level of a substrate, ligand, cofactor or product. A shift in complexation usually occurs when the availability of partner molecules (i.e. other macromolecules) changes. Although excluded originally, the signal for a switch in function might also be transduced via post-translational modification [5], as in the case of CFTR, the regulatory function of which is controlled by phosphorylation [45] or thymosin-b4, which is Metoxidized and secreted to become an anti-inflammatory agent [49].
www.sciencedirect.com 0968-0004/$ - see front matter Q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibs.2005.07.008
Opinion
TRENDS in Biochemical Sciences
As noted recently [7–11], a marked portion of eukaryotic genomes encodes for intrinsically unstructured proteins (IUPs), which lack a well-defined 3D structure in their native state. These proteins fulfil important functions that are often associated with signal transduction, gene expression and chaperone action [12–14]. For the performance of these functions, structural disorder confers special advantages, such as the capacity of one-tomany signaling or binding promiscuity, whereby an IUP binds distinct partners in a template-induced folding process [7,11,15]. Accordingly, IUPs have the potential to modulate the action of different partner molecules, that is, to moonlight. In fact, as we elaborate here, the underlying structural malleability enables an IUP to elicit both inhibiting and activating effects on different partners or even the same partner molecule. For example, cyclindependent kinase (Cdk) inhibitors p21Cip1 and p27Kip1 [16] were first noted for their capacity for binding promiscuity [15] and then later for their activating effect on Cdk(s) [17]. In a search for such extreme cases of moonlighting, we have identified proteins that can both inhibit and activate their partner(s). Biochemical evidence and bioinformatic predictors demonstrate that these proteins are largely, or completely, disordered and that their structural disorder is crucial for binding with disparate effects. The IUPs identified here throw new light on the mechanism of moonlighting; unlike the classical examples studied in detail thus far [6], they use the same region or interspersed, short interaction motifs to exert different effects. In switching function, they might use three principal, non-exclusive mechanisms. In these, the interaction region might either bind to the same or different partners in different conformations, or undergo structural reorganization in chaperoning or partially releasing the partner. After the individual examples are discussed in some detail, the structural, functional and evolutionary implications of this novel finding are elucidated. Disorder in moonlighting proteins By searching the literature for effectors that can both inhibit and activate their partner(s), we have identified 11 proteins for which the opposing effects can be ascribed to
Vol.30 No.9 September 2005
485
the same binding region; for eight of these proteins, there is direct experimental evidence that the entire protein or its binding region is largely disordered (Table 1). Disorder predictors also confirm that these proteins contain a high level of disorder (Table 1) exceeding that of regulatory and signal-transduction proteins [12,13] and even the most disordered class of proteins, the chaperones [14]. The individual examples discussed here suggest that the structural malleability of the region involved in binding is crucial for their different effects. This region might either bind the same partner in different conformations or bind completely unrelated partners. In the bound state, it might also undergo structural reorganization that results in opposing outcomes. Of course, it is to be emphasized that these mechanisms are not mutually exclusive but could possibly be combined to have a profound effect. Binding to a partner in different conformations A disordered inhibitor/activator protein can bind to the same partner in different conformations and/or at different binding sites, resulting in two opposing effects (Figure 1a). For example, in excitation–contraction coupling in skeletal muscle, the voltage-sensor dihydropyridine receptor (DHPR) has an unstructured loop region (peptide C), which is a multifarious effector of the ryanodine receptor (RyR) [18]. Peptide C can bind RyR with high affinity and activate its opening but, in a different conformation, it can bind to RyR with a lower affinity [18] and inhibit RyR-channel opening. Inhibition and activation are independent processes and it is not known what causes the transition between them. Cystic fibrosis transmembrane-conductance regulator (CFTR) provides another example for a disordered binding region that can bind to the same partner with different outcomes. This chloride channel, which is involved in cystic fibrosis, is blocked and inhibited when its disordered regulatory domain R is dephosphorylated [9]. Upon phosphorylation by protein kinase A, domain R probably undergoes a conformational change that not only relieves internal inhibition to make the pore accessible to chloride ions but also stimulates channel opening [19]. The binding mode determined by the negative charges introduced and/or the
Table 1. Examples of disordered moonlighting proteins Proteina
One (inhibiting) function
Another (activating) function
Calpastatin CFTR (R domain) DHPR (peptide C) EBV-SM
Inhibition of calpain Inhibition of CFTR Inhibition of RyR Down-regulation of introncontaining mRNA Down-regulation of p21Cip1 Inhibition of Cdk Inhibition of activated STAT Inhibition of PP1 Inhibition of MDM2 ubiquitin ligase Inhibition of separase Sequestration of G-actin
Activation of calpain Activation of CFTR Activation of RyR Up-regulation of intron-less mRNA
MDM2 (180–298) p21Cip1 and p27Kip1 PIAS1 (392–541) I-2 Ribosomal L5 Securin Thymosin-b4 (WH2 domain) a
Activation Activation Activation Activation Activation
IUPred (%)b 100 40.8 100 44.1
www.sciencedirect.com
[8d,23] [9d,19] [18d] [33]
Refs
of estrogen receptor a of Cdk of p53 of PP1 (chaperoning) of ribosome
70.0 48.2 41.3 91.7 22.3
70.0 61.0 40.7 77.8 30.8
[34,35] [15d–17] [40,41] [25d–27] [31d,32]
Activation (chaperoning) of separase Activation of actin polymerization, ILK kinase
52.0 100
54.2 64.3
[8d,29,30] [36d,37,39d]
Proteins known to have two opposing functions. If known, the region (domain) to where the functions are localized is indicated. The IUPred algorithm [48] for assessing the percentage of disorder is available at http://iupred.enzim.hu. c The PONDRw version VL-XT algorithm [9] for assessing the percentage of disorder is available at http://www.pondr.com. d Direct evidence for the disorder of the whole protein or its moonlighting segment. b
PONDRw (%)c 80.1 53.7 81.1 44.4
486
Opinion
TRENDS in Biochemical Sciences
(a)
(b)
(c)
Ti BS
Figure 1. Mechanisms by which moonlighting IUPs switch between functions. The scheme depicts the three principal, non-exclusive, molecular mechanisms by which the proteins exert opposing effects. The partner molecule is represented by a yellow square or pentagon before binding, an oval when in an active conformation and a rectangle when in an inactive conformation. Green and red colors indicate activation and inhibition, respectively. (a) A protein can bind to the same partner in two basically different conformations or binding sites, leading to different effects. (b) An inhibitor can function as a chaperone or it can shift the conformational equilibrium of its partner to promote the formation of its active conformation, but blocks its active site. Owing to significant differences in the binding strength of the two sites and/or post-translational modification such as phosphorylation or limited proteolysis, the inhibitory interaction might be partially released so that overall inhibition turns to activation. Release of the partner at a given location might result in targeting. (c) A protein binds to two different partners via two alternative conformations of the same site, or by two different, but overlapping, sites.
conformation of the domain is essential for the inhibition or activation of the channel. Structural reorganization around the partner Another common mode of moonlighting by an IUP is binding to a partner in a manner that shifts its equilibrium towards the active form but blocks its active site. Subsequent release of the block might result in deinhibition, and net activation, of the partner (Figure 1b). For example, fully disordered [15] p21Cip1 binds to both the cyclin and kinase subunits of activated Cdk and inhibits kinase action. However, the assembly of the cyclin–kinase complex, an obligate step in Cdk activation, is also facilitated by p21Cip1 and its homolog, p27Kip1 [17,20]. Because the inhibitor binds to the cyclin markedly www.sciencedirect.com
Vol.30 No.9 September 2005
more strongly than the kinase [21], net activation of the enzyme within the ternary complex [22] might ensue from release of the kinase subunit. The activating effect of calpastatin, the disordered [8] inhibitor of the calciumactivated protease calpain, can be conceived in a similar manner. Calpastatin facilitates binding of calcium to the enzyme but it also inactivates the enzyme via its inhibitory subdomain. When this region is degraded in a manner that leaves the rest of the molecule bound, the enzyme becomes activated [23]; this might ensure its prolonged action in myoblast fusion, for example [24]. A similar partial release is witnessed in the case of inhibitor-2 (I-2) of protein phosphatase 1 (PP1). Complex interactions of this IUP [25] with PP1 elicit fast inhibition, slow inactivation and phosphorylation-induced activation of the enzyme. Principally, the inhibitor binds to the enzyme via two independent sites, one activating and another inhibitory. Its phosphorylation at a third site by glycogen synthase kinase 3 causes conformational changes in both the inhibitor and the enzyme, which initiates the liberation of the active site and further activation via dephosphorylation of the inhibitor [26]. In conjunction, both deletion and overexpression of the yeast homolog of I-2, Glc8p, reduce assayable PP1 activity in yeast, whereas a moderate level stimulates the enzyme [27]. A variation on this theme is when activation of the partner results from chaperoning by the IUP, followed by its partial or full release (Figure 1b). The prime example for this behavior is securin, the disordered [8] inhibitor of separase, the protease that separates sister chromatids at the onset of anaphase. Intriguingly, the genetic ablation of securin does not lead to over-activation of separase and premature chromatid separation, but cripples the enzyme, retards chromosome segregation and causes aneuploidity [28,29], probably due to the lack of the chaperoning effect of securin on separase [30]. Furthermore, securin also activates separase by targeting the action of the enzyme into the nucleus and loading it onto spindles [29,30]. Binding to different partners The malleable structure of IUPs might also enable them to bind to completely different partners with opposing effects (Figure 1c). For example, the partially disordered [31] ribosomal L5 protein facilitates formation of the ribosomal large subunit by co-folding with RNA [31], which is then consolidated by targeting the ribosomal subunit into the nucleolus for proper assembly (Figure 1b). L5, however, can also bind to the oncoprotein mouse double minute 2 (MDM2) and inhibit its E3 ubiquitin-ligase activity [32], which mediates a ribosomal misassembly stress response. Under such conditions, L5 is released and blocks MDM2, which causes cell-cycle arrest by stabilization of the p53 tumor-suppressor protein. Another protein that binds to two different partners is Epstein–Barr virus (EBV)-nuclear protein BS-MLF1 (SM). This protein post-translationally inhibits the expression of intron-containing genes and activates intron-less genes by binding to the distinct premRNA molecules in a different manner [33]. In the case of an intron-containing mRNA, EBV-SM binding inhibits the excision of the terminal intron, causing retention in the nucleus and subsequent degradation of the mRNA. For
Opinion
TRENDS in Biochemical Sciences
inherently unstable, intron-less mRNA molecules, EBV-SM stabilizes the transcript and targets it for cytoplasmic export. This subtle distinction by SM promotes the virus cycle to enter the lytic phase because host genes usually contain introns, whereas lytic viral genes do not. MDM2 also seems to moonlight, as evidenced by the opposing effects of its middle segment. This region down-regulates p21Cip1 by targeting it to the C8 subunit of 20S proteasome for ubiquitin-independent degradation [34], but it can enhance the activity of the nuclear estrogen receptor ERa in a p53-independent manner via direct physical interaction [35]. The WASP (Wiskott–Aldrich syndrome protein) homology domain 2 (WH2) domain of thymosin-b4 also binds to different partners with opposing effects. In early myocardial and epithelial cells, thymosin-b4 promotes cell migration and cell survival. In these cells, the disordered [36] WH2 domain activates integrin-linked kinase (ILK), which subsequently phosphorylates the survival kinase Akt [37]. The classical function of thymosin-b4, however, is to wrap around an ATP-bound G-actin monomer and maintain it in a ‘sequestered’, nonpolymerizable form [38]. Intriguingly, in the homologous actobindin, which contains two WH2 domains, this domain facilitates barbed-end growth of actin polymers, possibly owing to a partial release (Figure 1b) at its C-terminal end [39]. Protein inhibitor of activated STAT (signal transducer and activator of transcription)-1 (PIAS-1) is another protein with multiple activities. STATs are latent cytoplasmic transcription factors that become Tyr-phosphorylated, and dimerize and translocate to the nucleus upon cytokine stimulation. The C-terminal region of PIAS-1 binds to the phosphorylated dimer and inhibits its transcription activity [40]. The same region, however, can also interact with the tetramerization domain of p53 and activate p53-mediated gene expression in a variety of cell types [41]. Furthermore, PIAS-1 is almost identical to RNA Gu-helicase-binding protein, which stimulates degradation of the Gu helicase [42]. Novel mechanisms of moonlighting The complex patterns of effector action presented here illustrate novel mechanistic aspects of protein moonlighting. The traditional view of moonlighting mechanisms derived from the classical structure–function paradigm that equated protein function with a well-defined 3D structure. In accord, most of the moonlighting proteins that have been identified thus far have been globular proteins, with their different functions ascribed to distinct, well-defined binding surfaces [6]. The IUPs described here, however, exemplify a different logic of moonlighting action because they use the structural adaptability of their interaction surfaces to bind to the same or distinct partner(s) with different effects. As discussed in detail, this novel paradigm raises two basic questions. What is the exact mechanism of how these proteins switch function? And what are the functional and evolutionary implications of this moonlighting phenomenon? Structural adaptability As noted, the proteins discussed here are either largely disordered (i.e. I-2, calpastatin, securin, p21Cip1 and www.sciencedirect.com
Vol.30 No.9 September 2005
487
thymosin-b4) or use their disordered domain for the moonlighting function (i.e. PIAS-1, MDM2, DHPR peptide C and CFTR R domain). A basic question is whether the structural adaptability of this region is in fact crucial for their multifarious effect. In general terms, IUPs have the capacity to adapt to different partners in a process called ‘one-to-many signaling’ or ‘binding promiscuity’ [15], that is, they adopt distinct conformations, as demonstrated for HIF-1a [11], DNA-dependent RNA polymerase II (RNAP II) C-terminal domain [43] and SNAP-25 [44], for example. Although direct structural data for the moonlighting IUPs are not available at present, the role of structural adaptability in their function has been suggested for CFTR domain R [19] and DHPR peptide C [18]. The involvement of flexibility is also apparent with these proteins, both of which function via partial binding or release of their partner (e.g. p21Cip1, thymosin-b4 and I-2). Chaperone activity, which has been implicated in the action of securin and ribosomal L5, also involves the ability to bind in alternative conformations (as noted earlier) [14]. Thus, conformational adaptability inherent in structural disorder seems to be essential for the novel moonlighting mechanisms. Another key aspect of moonlighting IUPs is the signal that makes these proteins switch structure and function, a change that they undergo in a manner often similar to the classical moonlighting cases (Box 1). In some cases, such as for CFTR [45] and I-2 [26], an intracellular message that triggers phosphorylation switches the conformation and binding mode of the protein. A signal might also be relayed to the IUP via regulated proteolysis, as in the case of calpastatin [24]. For those capable of binding to distinct partners, it might be the level of the partner molecule itself that controls the switching. For example, improper ribosome assembly causes release of ribosomal L5 so that it can bind to MDM2. Most difficult to rationalize is the control of IUPs that bind to the same partner in distinct conformations and/or at different sites, thus, eliciting disparate responses. It might be, as suggested for DHPR peptide C, that inhibition or activation in these cases is a stochastic process [18]. The intriguing possibility that such a mechanism provides stability to the effector system and contributes to network stability remains to be seen. Function and evolution Structural disorder has already been suggested to confer several functional advantages, such as binding promiscuity, among others [8,9]. We suggest that moonlighting be viewed as a major functional manifestation of binding promiscuity. The examples described here offer functional varieties greater than merely combining activation with inhibition. Chaperoning, transport and targeting, and upor down-regulation of partner molecules is also apparent even in this limited number of cases. Among the partners, we find enzymes, receptors, transcription factors, a structural protein and RNA. Given the great functional versatility of IUPs [8–11] and the range of effects seen in these cases, more functional combinations are conceivable. Furthermore, as protein disorder increases with the increasing complexity of organisms [12,13] and multiple use or connectivity of proteins is common in complex
488
Opinion
TRENDS in Biochemical Sciences
molecular networks [46], many more cases of moonlighting IUPs are expected to be revealed in the future. Another aspect is that, unlike the classical examples (Box 1), the opposing functions of the IUPs discussed in this article are often closely related to each other, either synergistically or antagonistically. This relationship might offer special ways of coordinating cellular activities and also particular stability to protein function, as exemplified by I-2 [26,27] and securin [28–30]. At low levels, I-2 and securin activate their partners, whereas at high levels they inhibit them, ensuring a robust response that is protected from extremities. The opposite is true for domain R of CFTR, which, upon phosphorylation, not only de-inhibits but also stimulates CFTR, possibly to provide a particularly effective bistable switch mechanism for abrupt activation. In terms of evolution, traditional moonlighting proteins are thought to have originally had only one function, and then to have been recruited for another use later by virtue of their large, unused and evolutionarily unconstrained surfaces. IUPs must have followed a different evolutionary path. These proteins use overlapping – or the same – interaction surface(s) for distinct functions. Thus, it is more likely that their functions have co-evolved, neither pre-dating the other. It has already been raised that functional diversity ensuing from conformational diversity might facilitate the evolution of new proteins [47]. This is particularly true for IUPs, which are more likely to have had multiple, less specific, activities than a single function at the outset. These distinct functions might have undergone parallel rounds of refinement in evolution that enabled the use of the same surface for more than one purpose, as seen today. This frugal use of protein material is undoubtedly of substantial benefit to the cell. It is even tempting to speculate that this unique functional capacity has been one of the major reasons for the evolutionary advance of protein disorder [12,13]. Concluding remarks Structural disorder provides unprecedented versatility in partner binding that enables proteins to have distinct functions (i.e. to moonlight). In effect, structural malleability permits various and often opposing functions, such as activation and inhibition, chaperoning, transport and targeting, and up- and down-regulation of partner molecule(s), to reside within the same region. Proteins with such combinations of functions represent unique building blocks for complex metabolic networks. It will be of immense interest to see how, and to what extent, cells actually exploit the great potential inherent in this mechanism. Acknowledgements This work was supported by the Wellcome Trust International Senior Research Fellowship ISRF 067595 and a Bolyai Ja´nos Fellowship (P.T.). We thank Monika Fuxreiter and Veronika Csizmo´k for their critical comments on the manuscript.
References 1 Consortium. (2004) Finishing the euchromatic sequence of the human genome. Nature 431, 931–945 www.sciencedirect.com
Vol.30 No.9 September 2005
2 Petrov, D.A. (2001) Evolution of genome size: new approaches to an old problem. Trends Genet. 17, 23–28 3 Mattick, J.S. (2003) Challenging the dogma: the hidden layer of nonprotein-coding RNAs in complex organisms. Bioessays 25, 930–939 4 Jeffery, C.J. (1999) Moonlighting proteins. Trends Biochem. Sci. 24, 8–11 5 Jeffery, C.J. (2003) Multifunctional proteins: examples of gene sharing. Ann. Med. 35, 28–35 6 Jeffery, C.J. (2004) Molecular mechanisms for multitasking: recent crystal structures of moonlighting proteins. Curr. Opin. Struct. Biol. 14, 663–668 7 Wright, P.E. and Dyson, H.J. (1999) Intrinsically unstructured proteins: re-assessing the protein structure–function paradigm. J. Mol. Biol. 293, 321–331 8 Tompa, P. (2002) Intrinsically unstructured proteins. Trends Biochem. Sci. 27, 527–533 9 Dunker, A.K. et al. (2002) Intrinsic disorder and protein function. Biochemistry 41, 6573–6582 10 Fink, A.L. (2005) Natively unfolded proteins. Curr. Opin. Struct. Biol. 15, 35–41 11 Dyson, H.J. and Wright, P.E. (2005) Intrinsically unstructured proteins and their functions. Nat. Rev. Mol. Cell Biol. 6, 197–208 12 Iakoucheva, L. et al. (2002) Intrinsic disorder in cell-signaling and cancer-associated proteins. J. Mol. Biol. 323, 573–584 13 Ward, J.J. et al. (2004) Prediction and functional analysis of native disorder in proteins from the three kingdoms of life. J. Mol. Biol. 337, 635–645 14 Tompa, P. and Csermely, P. (2004) The role of structural disorder in the function of RNA and protein chaperones. FASEB J. 18, 1169–1175 15 Kriwacki, R.W. et al. (1996) Structural studies of p21Waf1/Cip1/Sdi1 in the free and Cdk2-bound state: conformational disorder mediates binding diversity. Proc. Natl. Acad. Sci. U. S. A. 93, 11504–11509 16 Pavletich, N.P. (1999) Mechanisms of cyclin-dependent kinase regulation: structures of Cdks, their cyclin activators, and Cip and INK4 inhibitors. J. Mol. Biol. 287, 821–828 17 Cheng, M. et al. (1999) The p21(Cip1) and p27(Kip1) CDK ‘inhibitors’ are essential activators of cyclin D-dependent kinases in murine fibroblasts. EMBO J. 18, 1571–1583 18 Haarmann, C.S. et al. (2003) The random-coil ‘C’ fragment of the dihydropyridine receptor II–III loop can activate or inhibit native skeletal ryanodine receptors. Biochem. J. 372, 305–316 19 Ma, J. (2000) Stimulatory and inhibitory functions of the R domain on CFTR chloride channel. News Physiol. Sci. 15, 154–158 20 Olashaw, N. et al. (2004) Cell cycle control: a complex issue. Cell Cycle 3, 263–264 21 Lacy, E.R. et al. (2004) p27 binds cyclin–CDK complexes through a sequential mechanism involving binding-induced protein folding. Nat. Struct. Mol. Biol. 11, 358–364 22 Zhang, H. et al. (1994) p21-containing cyclin kinases exist in both active and inactive states. Genes Dev. 8, 1750–1758 23 Tompa, P. et al. (2002) Calpastatin subdomains A and C are activators of calpain. J. Biol. Chem. 277, 9022–9026 24 Barnoy, S. and Kosower, N.S. (2003) Caspase-1-induced calpastatin degradation in myoblast differentiation and fusion: cross-talk between the caspase and calpain systems. FEBS Lett. 546, 213–217 25 Tompa, P. (2003) The functional benefits of protein disorder. J. Mol. Struct. THEOCHEM 666-667, 361–371 26 Yang, J. et al. (2000) Interaction of inhibitor-2 with the catalytic subunit of type 1 protein phosphatase. Identification of a sequence analogous to the consensus type 1 protein phosphatase-binding motif. J. Biol. Chem. 275, 22635–22644 27 Tung, H.Y. et al. (1995) Regulation of chromosome segregation by Glc8p, a structural homolog of mammalian inhibitor 2 that functions as both an activator and an inhibitor of yeast protein phosphatase 1. Mol. Cell. Biol. 15, 6064–6074 28 Jallepalli, P.V. et al. (2001) Securin is required for chromosomal stability in human cells. Cell 105, 445–457 29 Hornig, N.C. et al. (2002) The dual mechanism of separase regulation by securin. Curr. Biol. 12, 973–982 30 Uhlmann, F. (2003) Separase regulation during mitosis. Biochem. Soc. Symp. 70, 243–251 31 DiNitto, J.P. and Huber, P.W. (2003) Mutual induced fit binding of Xenopus ribosomal protein L5 to 5S rRNA. J. Mol. Biol. 330, 979–992
Opinion
TRENDS in Biochemical Sciences
32 Dai, M.S. and Lu, H. (2004) Inhibition of MDM2-mediated p53 ubiquitination and degradation by ribosomal protein L5. J. Biol. Chem. 279, 44475–44482 33 Ruvolo, V. et al. (1998) The Epstein–Barr virus nuclear protein SM is both a post-transcriptional inhibitor and activator of gene expression. Proc. Natl. Acad. Sci. U. S. A. 95, 8852–8857 34 Zhang, Z. et al. (2004) MDM2 is a negative regulator of p21WAF1/CIP1, independent of p53. J. Biol. Chem. 279, 16000–16006 35 Saji, S. et al. (2001) MDM2 enhances the function of estrogen receptor a in human breast cancer cells. Biochem. Biophys. Res. Commun. 281, 259–265 36 Domanski, M. et al. (2004) Coupling of folding and binding of thymosin b4 upon interaction with monomeric actin monitored by nuclear magnetic resonance. J. Biol. Chem. 279, 23637–23645 37 Bock-Marquette, I. et al. (2004) Thymosin b4 activates integrin-linked kinase and promotes cardiac cell migration, survival and cardiac repair. Nature 432, 466–472 38 Hertzog, M. et al. (2002) Control of actin dynamics by proteins made of b-thymosin repeats: the actobindin family. J. Biol. Chem. 277, 14786–14792 39 Hertzog, M. et al. (2004) The b-thymosin/WH2 domain; structural basis for the switch from inhibition to promotion of actin assembly. Cell 117, 611–623 40 Liao, J. et al. (2000) Distinct roles of the NH2- and COOH-terminal
41 42 43
44 45 46 47
48
49
Vol.30 No.9 September 2005
domains of the protein inhibitor of activated signal transducer and activator of transcription (STAT) 1 (PIAS1) in cytokine-induced PIAS1–Stat1 interaction. Proc. Natl. Acad. Sci. U. S. A. 97, 5267–5272 Megidish, T. et al. (2002) Activation of p53 by protein inhibitor of activated Stat1 (PIAS1). J. Biol. Chem. 277, 8255–8259 Valdez, B.C. et al. (1997) Cloning and characterization of Gu/RH-II binding protein. Biochem. Biophys. Res. Commun. 234, 335–340 Fabrega, C. et al. (2003) Structure of an mRNA capping enzyme bound to the phosphorylated carboxy-terminal domain of RNA polymerase II. Mol. Cell 11, 1549–1561 Breidenbach, M.A. and Brunger, A.T. (2004) Substrate recognition strategy for botulinum neurotoxin serotype A. Nature 432, 925–929 Schwiebert, E.M. et al. (1999) CFTR is a conductance regulator as well as a chloride channel. Physiol. Rev. 79, S145–S166 Gavin, A.C. et al. (2002) Functional organization of the yeast proteome by systematic analysis of protein complexes. Nature 415, 141–147 James, L.C. and Tawfik, D.S. (2003) Conformational diversity and protein evolution – a 60-year-old hypothesis revisited. Trends Biochem. Sci. 28, 361–368 Doszta´nyi, Z. et al. (2005) The pairwise energy content estimated from amino acid composition discriminates between folded and intrinsically unstructured proteins. J. Mol. Biol. 347, 827–839 Young, J.D. et al. (1999) Thymosin b4 sulfoxide is an antiinflammatory agent generated by monocytes in the presence of glucocorticoids. Nat. Med. 5, 1424–1427
Endeavour the quarterly magazine for the history and philosophy of science You can access Endeavour online via ScienceDirect, where you’ll find a collection of beautifully illustrated articles on the history of science, book reviews and editorial comment.
featuring
Selling the silver: country house libraries and the history of science by Roger Gaskell and Patricia Fara Carl Schmidt – a chemical tourist in Victorian Britain by R. Stefan Ross The rise, fall and resurrection of group selection by M.E. Borello Mary Anning: the fossilist as exegete by T.W. Goodhue Caroline Herschel: ‘the unquiet heart’ by M. Hoskin Science in the 19th-century zoo by Oliver Hochadel The melancholy of anatomy by P. Fara and coming soon Etienne Geoffroy St-Hillaire, Napoleon’s Egyptian campaign and a theory of everything by P. Humphries Losing it in New Guinea: The voyage of HMS Rattlesnake by J. Goodman The accidental conservationist by M.A. Andrei Powering the porter brewery by J. Sumner Female scientists in films by B.A. Jones and much, much more . . . Locate Endeavour on ScienceDirect (http://www.sciencedirect.com) www.sciencedirect.com
489