The Importance of Intrinsic Order in a Disordered Protein Ligand

Abstract

Over the last decade, it has become increasingly apparent that a significant number of proteins do not form stable tertiary structures, but instead dynamically sample a broad range of largely disordered states (1xIntrinsic protein disorder in complete genomes. Dunker, A.K., Obradovic, Z...., and Brown, C.J. Genome Inform. Ser. Workshop Genome Inform. 2000; 11: 161–171PubMedSee all References1). Generally referred to as intrinsically disordered proteins (IDPs), these species can be associated with a range of functions, and often serve as hub proteins that bind to multiple targets (2xFrom sequence and forces to structure, function, and evolution of intrinsically disordered proteins. Forman-Kay, J.D. and Mittag, T. Structure. 2013; 21: 1492–1499Abstract | Full Text | Full Text PDF | PubMed | Scopus (37)See all References2). Binding events involving IDPs are commonly accompanied by the formation of defined structure. In many cases, these appear to follow an induced-fit model of binding, where the loss of conformational entropy (in going from a largely disordered free state to a structured bound state) is balanced by the extensive number of interactions that can be accessed by the structurally malleable IDP. However, over the last decade it has become clear that the free state does not sample the full range of conformations that would be predicted from random chain diffusion (e.g., the kinase inhibitory domain from Sic (3xCompaction properties of an intrinsically disordered protein: Sic1 and its kinase-inhibitor domain. Brocca, S., Testa, L...., and Grandori, R. Biophys. J. 2011; 100: 2243–2252Abstract | Full Text | Full Text PDF | PubMed | Scopus (30)See all References3) and a polyglutamine binding protein (4xSolution model of the intrinsically disordered polyglutamine tract-binding protein-1. Rees, M., Gorba, C...., and Chen, Y.W. Biophys. J. 2012; 102: 1608–1616Abstract | Full Text | Full Text PDF | PubMed | Scopus (4)See all References4)). Instead, these systems exhibit bias toward more compact, transiently structured states, suggesting that the loss of conformational entropy in the interaction is not as drastic as originally thought.In some cases, the structural propensities of the unbound IDP play a more active role in determining the affinity of the interaction, with significant sampling of conformations resembling the bound state structure (5xp15 (PAF) is an intrinsically disordered protein with nonrandom structural preferences at sites of interaction with other proteins. De Biasio, A., Ibanez de Opakua, A...., and Blanco, F.J. Biophys. J. 2014; 106: 865–874Abstract | Full Text | Full Text PDF | PubMed | Scopus (5)See all References, 6xNMR determines transient structure and dynamics in the disordered C-terminal domain of WASp interacting protein. Haba, N.Y., Gross, R...., and Chill, J.H. Biophys. J. 2013; 105: 481–493Abstract | Full Text | Full Text PDF | PubMed | Scopus (7)See all References). This has renewed interest in the possibility that conformational selection also plays a role in IDP interactions, where binding could be favored by the transient formation of a structured region in the free state that is preferentially bound by the target protein (7xCoupled folding and binding with α-helix-forming molecular recognition elements. Oldfield, C.J., Cheng, Y...., and Dunker, A.K. Biochemistry. 2005; 44: 12454–12470Crossref | PubMed | Scopus (321)See all References7). Evidence that conformational selection may be important for some IDP interactions has been accumulating, in particular for IDPs that form helical structures in the bound state.These studies have shown that perturbations altering the ability of the unbound state to sample a helical conformation correlate with binding affinity (8xMechanism of induced folding: both folding before binding and binding before folding can be realized in staphylococcal nuclease mutants. Onitsuka, M., Kamikubo, H...., and Kataoka, M. Proteins. 2008; 72: 837–847Crossref | PubMed | Scopus (34)See all References, 9xInternal dynamics control activation and activity of the autoinhibited Vav DH domain. Li, P., Martins, I.R.S...., and Rosen, M.K. Nat. Struct. Mol. Biol. 2008; 15: 613–618Crossref | PubMed | Scopus (67)See all References, 10xHelical propensity in an intrinsically disordered protein accelerates ligand binding. Iesmantavicius, V., Dogan, J...., and Kjaergaard, M. Angew. Chem. Int. Ed. Engl. 2014; 53: 1548–1551Crossref | PubMed | Scopus (32)See all References). In this issue of the Biophysical Journal, a similar relationship is demonstrated for an IDP that interacts with its target via an extended structure, providing evidence that conformational selection may also play a role in IDP interactions involving nonhelical binding motifs (11xConformational recognition of an intrinsically disordered protein. Krieger, J.R., Fusco, G...., and De Simone, A. Biophys. J. 2014; 106: 1771–1779Abstract | Full Text | Full Text PDF | PubMed | Scopus (15)See all References11).The work in this issue, by Krieger et al. (11xConformational recognition of an intrinsically disordered protein. Krieger, J.R., Fusco, G...., and De Simone, A. Biophys. J. 2014; 106: 1771–1779Abstract | Full Text | Full Text PDF | PubMed | Scopus (15)See all References11), takes advantage of the information provided by NMR chemical shifts to characterize the unbound state of a peptide from the multisite scaffolding protein Gab2. In its SH3 domain-bound state, an N-terminal polyproline sequence in the Gab2 peptide forms an extended structure that interacts with a hydrophobic groove on the SH3 surface, while its C-terminal RxxK sequence binds via a 310 helix (12xDistinct binding modes of two epitopes in Gab2 that interact with the SH3C domain of Grb2. Harkiolaki, M., Tsirka, T...., and Feller, S.M. Structure. 2009; 17: 809–822Abstract | Full Text | Full Text PDF | PubMed | Scopus (39)See all References12). To characterize the structure of the free state, backbone chemical shifts from the Gab2 peptide were assigned and analyzed for secondary structure content. Two regions of residual structure were identified that contained significant populations of extended β- and polyproline type II structures (highlighted in Fig. 1Fig. 1). In addition, molecular-dynamics simulations were employed using replica-averaged chemical shifts as structural restraints (13xCharacterization of the conformational equilibrium between the two major substates of RNase A using NMR chemical shifts. Camilloni, C., Robustelli, P...., and Vendruscolo, M. J. Am. Chem. Soc. 2012; 134: 3968–3971Crossref | PubMed | Scopus (51)See all References13) in a procedure that has been shown to bias sampling in a way that accurately recapitulates the conformational tendencies of the system (14xAssessment of the use of NMR chemical shifts as replica-averaged structural restraints in molecular dynamics simulations to characterize the dynamics of proteins. Camilloni, C., Cavalli, A., and Vendruscolo, M. J. Phys. Chem. B. 2013; 117: 1838–1843Crossref | PubMed | Scopus (23)See all References14). The application of this chemical shift-restrained molecular-dynamics method, combined with a statistical mechanics analysis of the ensemble, revealed a free energy landscape for Gab2 that favors the formation of a significant population of extended structures resembling the bound state.Figure 1Structure of the Grb2 C-terminal SH3 domain (gray surface; green online) bound to a peptide spanning residues 508–521 from Gab2 (IQPPPVNRNLKPDR). The two regions of the Gab2 peptide that showed extended β- and polyproline II structure in the free state are highlighted (black; red online) for the N-terminal polyproline sequence (dark gray; orange online) for the region C-terminal to the RxxK motif. (PDB:2VWF.) To see this figure in color, go online.View Large Image | View Hi-Res Image | Download PowerPoint SlideFurther raising the significance of these findings were results from a series of mutant peptides that was also subjected to the same measurements and analyses. A reasonable correlation was observed between peptide binding affinity and the propensity of the free state to sample conformations resembling the bound state. The consistency of the trend for free-state conformational tendencies versus binding affinities over the range of mutants tested provides a strong case for the importance of the residual structure in this interaction. These results are in agreement with a model of binding that is facilitated by conformational selection of this bound state structure. Alternatively, the conformational propensities of the free state could influence binding affinity by increasing the rate of binding-induced folding, and/or decreasing the rate of unfolding-coupled dissociation—an interesting possibility that could potentially be resolved by kinetics experiments in the future.These results should be of general interest for groups that study fundamental principles of protein-protein interactions, and particularly those that involve intrinsically structured domains. Although there is a growing body of evidence that bound-state structures can be sampled in IDP ligands, relatively few studies have shown a relationship between binding affinity and the presence of residual bound-state structure, particularly in nonhelical binding motifs. The work presented in this issue helps address this deficiency, and contributes to the evolving view of IDP interactions that utilize a combination of conformational selection and induced fit to increase the affinity and specificity of these interactions (15xMultiscaled exploration of coupled folding and binding of an intrinsically disordered molecular recognition element in measles virus nucleoprotein. Wang, Y., Chu, X...., and Wang, J. Proc. Natl. Acad. Sci. USA. 2013; 110: E3743–E3752Crossref | PubMed | Scopus (36)See all References15).