Behaviour of Intrinsically Disordered Proteins in Protein–Protein Complexes with an Emphasis on Fuzziness

Cell. Mol. Life Sci. (2017) 74:3175–3183 DOI 10.1007/s00018-017-2560-7 Cellular and Molecular Life Sciences MULTI-AUTHOR REVIEW Behaviour of intrinsically disordered proteins in protein–protein complexes with an emphasis on fuzziness 1 1 1 Johan G. Olsen • Kaare Teilum • Birthe B. Kragelund Received: 18 May 2017 / Accepted: 1 June 2017 / Published online: 8 June 2017 Ó The Author(s) 2017. This article is an open access publication Abstract Intrinsically disordered proteins (IDPs) do not, Introduction by themselves, fold into a compact globular structure. They are extremely dynamic and flexible, and are typically Signalling and regulation are essential to all living cells involved in signalling and transduction of information and are based on intermolecular interactions, most of which through binding to other macromolecules. The reason for are mediated by proteins. A substantial fraction of proteins their existence may lie in their malleability, which enables include large regions of disorder without clearly defined them to bind several different partners with high speci- three-dimensional structure. Such intrinsically disordered ficity. In addition, their interactions with other proteins (IDPs) are not only very abundant—30–40% of all macromolecules can be regulated by a variable amount of proteins in the human proteome are disordered or contain chemically diverse post-translational modifications. Four intrinsically disordered regions (IDRs) [1, 2]—they also kinetically and energetically different types of complexes constitute significant parts of membrane proteins [3, 4] and between an IDP and another macromolecule are reviewed: occupy pivotal positions in cellular regulation on all levels (1) simple two-state binding involving a single binding site, [5]. Some even display enzymatic activity [6]. Thus, IDPs (2) avidity, (3) allovalency and (4) fuzzy binding; the last are critically involved in key cellular processes and three involving more than one site. Finally, a qualitative important for understanding life. Although IDP research definition of fuzzy binding is suggested, examples are has grown somewhat independent from traditional biology provided, and its distinction to allovalency and avidity is and biochemistry, it is conceptually important to follow the highlighted and discussed. models, views and nomenclatures used generally for proteins, which have been developed over the past 120 years Keywords IDP Á Allovalency Á Fuzzy complex Á since Fisher proposed the lock-and-key model for ligand Signalling Á Avidity Á Disorder Á Kinetics binding [7]. Thus, throughout this review the IDP is referred to as the ligand (L). The residues involved in binding are expected to be disordered, but that does not exclude the presence of ordered regions in other parts of the peptide chain. In the present discussion, ordered regions are assumed not to be involved in the interaction. The binding partner that may or may not be an IDP is referred to as the receptor (R), although this macromolecule does not need to be a receptor per se. & Birthe B. Kragelund By definition, IDPs have high rotational freedom and [email protected] sample a wide range of conformations [8–10]. Their hyper- dynamical nature renders them malleable and thereby 1 Structural Biology and NMR Laboratory (SBiNLab) and the potentiates their ability to bind multiple structurally diverse Linderstrøm-Lang Centre for Protein Science, Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, 2200 receptors, while retaining specificity. This conjecture Copenhagen, Denmark implies that IDPs are superior to their folded counterparts 123 3176 J. G. Olsen et al. when it comes to binding many different partners. Inter- intrinsically disordered protein PUMA binding to the folded estingly, the thermodynamics of the interaction between an protein MCL-1 [24]. PUMA adopts an a-helix in the bound IDP and a folded partner is essentially similar to the situ- state and the helix forms in a coupled binding and folding ation when two globular proteins interact, only event [24]. For the two-state reaction between a ligand (L) compromised on average by around 2.5 kcal mol-1 due to and its receptor (R) the following equilibrium exists: loss of conformational entropy originating from the struc- kon turing of the disordered chain [11]. However, the L þ R LR: ð1Þ distribution of states and the dynamics of the complexes koff vary. Some IDP binding sites become ordered upon binding to their receptor, a phenomenon called folding upon The binding constant given as the association constant, binding [12]. Several crystal and NMR structures of such Ka is defined by the concentrations of the species in the complexes exist [13, 14] and they highlight details of the solution at equilibrium: interactions [15–18]. In terms of kinetics, these are typical kon ½LR Ka ¼ ¼ : ð2Þ examples of simple two-state reactions, where the energy koff ½L½R landscape of the complex is presented by a very deep well and one single structure can, in essence, represent the complex. At the other extreme, some ligands never ‘rest’ in Avidity complex with a receptor and there is no single conformation for the ‘bound-state’. In this case, the IDP ligand Avidity was originally used to describe the binding retains conformational freedom in the complex. Such between an antibody and an antigen, and is thus not interactions have recently been coined fuzzy complexes exclusively an IDP phenomenon [25]. Avidity arises when [19, 20] and a database has been established, collecting two or more binding sites are present on the ligand, com- examples of the phenomenon [21]. Between these plementing two or more binding sites on the receptor extremes, other binding modes are found. Earlier work has (Fig. 1b). The binding sites on the ligand are connected by provided kinetic interpretations of those modes and their a linker and this linker ensures that once one site is bound mechanisms of binding have been referred to as avidity and to the receptor, other site(s) are spatially close to other allovalency [22, 23]. In the following, we will describe the receptor sites, resulting in cooperative binding, due prin- four different mechanisms in more details. cipally to a lower entropic cost of binding more than one ligand [26]. Avidity requires the receptor and the ligand to have the same number of binding sites, where each site is Simple two-state binding unique and the sites cannot exchange. Once the ligand has bound one site, the probability of establishing an additional The simplest description of the interaction between two binding contact is much higher than for the first binding molecules is that the molecules in their unbound state are event and so forth, introducing cooperativity. separated from the complex state by a single transition state The first binding event is a second order reaction, and that no intermediates are present (Fig. 1a). Such a whereas subsequent binding events are first order (pseudo- scenario is often seen for the interaction between small intramolecular) events. Thus, the entropic loss in subse- molecules that exist mainly in one conformation. The quent binding events is lower. interaction between complex macromolecules can often be If both of the two receptor-sites and the two ligand-sites approximated as two-state binding, even if the binding are identical the order of binding is of no consequence. The involves major conformational changes. The requirement first (second order) reaction is written as a simple two-state for (approximate) two-state binding is that a single site of reaction: the ligand binds a single site on the receptor. In an ordered k protein complex, that can be the end-result of a two-state L þ R 1 LR ; ð3Þ reaction, each back-bone conformation adopts a narrow L0 þ R0 L0 þ R0 k range of angular values in the bound state and all ligand À1 atoms involved in binding are bound to specific receptor and likewise, the second (first order) reaction is written: atoms, as crystal structures of such complexes show. k2 Besides those cooperative events that occur within a single LR LR 0 0 0 0 ; ð4Þ binding site between individual atoms, the binding energy L þ R L R kÀ2 is linearly dependent on the sum of interactions. 0 0 The binding is a second order reaction and there are no where L and R refer to the sites involved in the second subsequent first order reactions. An example is the event. The first binding event can typically be studied 123 Behaviour of intrinsically disordered proteins in protein–protein complexes with an emphasis… 3177 123 3178 J. G. Olsen et al. b Fig. 1 Illustration of four different binding mechanisms involving binding sites on the ligand compete for a single binding site IDPs. Different ligand-receptor interactions involving an IDP ligand on the receptor and only one binding site on the ligand can (wavy black string) and a macromolecular receptor (orange oval) are shown. The binding epitopes on the ligand are highlighted in blue or bind at any given time [22], which is nicely exemplified by (red), whereas a binding site on the receptor is symbolised with an multiple phosphorylations on an IDP binding to and com- indentation. a Simple two-state binding, implying the existence of peting for the same site [30]. Although the affinity for one either the free or the bound state with no intermediates. There is a ligand is low, the presence and competition by multiple linear correlation between the concentration of ligand and the fraction of molecules in the bound state. b Avidity, where two or more tandem sites increase the overall affinity. To explain this epitopes on the ligand and a corresponding number of binding sites on increased affinity a sphere centred at the receptor binding site the receptor will interact. If one interaction is established, a second was defined [22].

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