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Evolution of proteins is constrained by their structure and function. While there is a consensus that the plasticity of intrinsically disordered proteins relaxes the structural constraints on evolution there is a paucity of data on the molecular details of these processes. The Nuclear co-activator binding domain (NCBD) from CREB-binding protein is a protein-protein interaction domain, which contains a hydrophobic core but is not behaving as a typical globular domain, and has been desribed as ‘molten-globule like’. The highly dynamic properties of NCBD makes it an interesting model system for evolutionary structure-function investigation of intrinsically disordered proteins. We have here compared the structure and biophysical properties of an ancient version of NCBD present in a bilaterian animal ancestor living around 600 million years ago with extant human NCBD. Using a combination of NMR spectroscopy, circular dichroism and kinetic methods we show that although NCBD has increased its thermodynamic stability, it has retained its dynamic biophysical properties in the ligand-free state in the evolutionary lineage leading from the last common bilaterian ancestor to humans. Our findings suggest that the dynamic properties of NCBD have been maintained by purifying selection and thus are important for its function, which includes mediating several distinct protein-protein interactions.

Intrinsically disordered proteins (IDPs) are abundant in the eukaryotic proteome. However, little is known about the role of subnanosecond dynamics and the conformational entropy that it represents in protein-protein interactions involving IDPs. Using nuclear magnetic resonance side chain and backbone relaxation, stopped-flow kinetics, isothermal titration calorimetry, and computational studies, we have characterized the interaction between the globular TAZ1 domain of the CREB binding protein and the intrinsically disordered transactivation domain of STAT2 (TAD-STAT2). We show that the TAZ1/TAD-STAT2 complex retains considerable subnanosecond motions, with TAD-STAT2 undergoing only a partial disorder-to-order transition. We report here the first experimental determination of the conformational entropy change for both binding partners in an IDP binding interaction and find that the total change even exceeds in magnitude the binding enthalpy and is comparable to the contribution from the hydrophobic effect, demonstrating its importance in the binding energetics. Furthermore, we show that the conformational entropy change for TAZ1 is also instrumental in maintaining a biologically meaningful binding affinity. Strikingly, a spatial clustering of very high amplitude motions and a cluster of more rigid sites in the complex exist, which through computational studies we found to overlap with regions that experience energetic frustration and are less frustrated, respectively. Thus, the residual dynamics in the bound state could be necessary for faster dissociation, which is important for proteins that interact with multiple binding partners.

A significant fraction of the eukaryotic proteome consists of proteins that are either partially or completely disordered under native-like conditions. Intrinsically disordered proteins (IDPs) are common in protein-protein interactions and are involved in numerous cellular processes. Although many proteins have been identified as disordered, much less is known about the binding mechanisms of the coupled binding and folding reactions involving IDPs. Here we have analyzed the rate-limiting transition state for binding between the TAZ1 domain of CREB binding protein and the intrinsically disordered transactivation domain of STAT2 (TAD-STAT2) by site-directed mutagenesis and kinetic experiments (Φ-value analysis) and found that the native protein-protein binding interface is not formed at the transition state for binding. Instead, native hydrophobic binding interactions form late, after the rate-limiting barrier has been crossed. The association rate constant in the absence of electrostatic enhancement was determined to be rather high. This is consistent with the Φ-value analysis, which showed that there are few or no obligatory native contacts. Also, linear free energy relationships clearly demonstrate that native interactions are cooperatively formed, a scenario that has usually been observed for proteins that fold according to the so-called nucleation-condensation mechanism. Thus, native hydrophobic binding interactions at the rate-limiting transition state for association between TAD-STAT2 and TAZ1 are not a requirement, which is generally in agreement with previous findings on other IDP systems and might be a common mechanism for IDPs.

The interaction between the C-terminal transactivation domain of HIF-1α (CTAD-HIF-1α) and the transcriptional adapter zinc binding 1 (TAZ1) domain of CREB binding protein participate in the initiation of gene transcription during hypoxia. Unbound CTAD-HIF-1α is disordered but undergoes a disorder-to-order transition upon binding to TAZ1. We have here performed NMR side chain and backbone relaxation studies on TAZ1 and side chain relaxation measurements on CTAD-HIF-1α in order to investigate the role of picosecond to nanosecond dynamics. We find that the internal motions are significantly affected upon binding, both on the side chain and the backbone level. The dynamic response corresponds to a conformational entropy change that contributes substantially to the binding thermodynamics for both binding partners. Furthermore, the conformational entropy change for the well-folded TAZ1 varies upon binding to different IDP targets. We further identify a cluster consisting of side chains in bound TAZ1 and CTAD-HIF-1α that experience extensive dynamics and are part of the binding region that involves the N-terminal end of the LPQL motif in CTAD-HIF-1α; a feature that might have an important role in the termination of the hypoxic response.

The globular transcriptional adapter zinc binding 1 (TAZ1) domain of CREB binding protein participates in protein-protein interactions that are involved in transcriptional regulation. TAZ1 binds numerous targets, of which many are intrinsically disordered proteins that undergo a disorder-to-order transition to various degrees. One such target is the disordered transactivation domain of transcription factor RelA (TAD-RelA), which with its interaction with TAZ1 is involved in transcriptional regulation of genes in NF-κB signaling. We have here performed nuclear magnetic resonance backbone and side-chain relaxation studies to investigate the influence of RelA-TA2 (residues 425-490 in TAD-RelA) binding on the subnanosecond internal motions of TAZ1. We find a considerable dynamic response on both the backbone and side-chain levels, which corresponds to a conformational entropy change that contributes significantly to the binding energetics. We further show that the microscopic origins of the dynamic response of TAZ1 vary depending on the target. This study demonstrates that folded protein domains that are able to interact with various targets are not dynamically passive but can have a significant role in the motional response upon target association.

Structural snapshots characterize six hundred million years of evolution of intrinsically disordered proteins., In every established species, protein-protein interactions have evolved such that they are fit for purpose. However, the molecular details of the evolution of new protein-protein interactions are poorly understood. We have used nuclear magnetic resonance spectroscopy to investigate the changes in structure and dynamics during the evolution of a protein-protein interaction involving the intrinsically disordered CREBBP (CREB-binding protein) interaction domain (CID) and nuclear coactivator binding domain (NCBD) from the transcriptional coregulators NCOA (nuclear receptor coactivator) and CREBBP/p300, respectively. The most ancient low-affinity “Cambrian-like” [540 to 600 million years (Ma) ago] CID/NCBD complex contained less secondary structure and was more dynamic than the complexes from an evolutionarily younger “Ordovician-Silurian” fish ancestor (ca. 440 Ma ago) and extant human. The most ancient Cambrian-like CID/NCBD complex lacked one helix and several interdomain interactions, resulting in a larger solvent-accessible surface area. Furthermore, the most ancient complex had a high degree of millisecond-to-microsecond dynamics distributed along the entire sequences of both CID and NCBD. These motions were reduced in the Ordovician-Silurian CID/NCBD complex and further redistributed in the extant human CID/NCBD complex. Isothermal calorimetry experiments show that complex formation is enthalpically favorable and that affinity is modulated by a largely unfavorable entropic contribution to binding. Our data demonstrate how changes in structure and motion conspire to shape affinity during the evolution of a protein-protein complex and provide direct evidence for the role of structural, dynamic, and frustrational plasticity in the evolution of interactions between intrinsically disordered proteins.

Intrinsically disordered proteins (IDPs) are common in eukaryotes. However, relatively few experimental studies have addressed the nature of the rate-limiting transition state for the coupled binding and folding reactions involving IDPs. By using site-directed mutagenesis in combination with kinetics measurements we have here characterized the transition state for binding between the globular TAZ1 domain of CREB binding protein and the intrinsically disordered C-terminal activation domain of Hif-1α (Hif-1α CAD). A total of 17 Hif-1α CAD point-mutations were generated and a Φ-value binding analysis was carried out. We found that native hydrophobic binding interactions are not formed at the transition state. We also investigated the effect the biologically important Hif-1α CAD Asn-803 hydroxylation has on the binding kinetics, and found that the whole destabilization effect due the hydroxylation is within the dissociation rate constant. Thus, the rate-limiting transition state is “disordered-like”, with native hydrophobic binding contacts being formed cooperatively after the rate-limiting barrier, which is clearly shown by linear free energy relationships. The same behavior was observed in a previously characterized TAZ1/IDP interaction, which may suggest common features for the rate-limiting transition state for TAZ1/IDP interactions.

Intrinsically disordered proteins (IDPs) are abundant in the proteome and involved in key cellular functions. However, experimental data about the binding kinetics of IDPs as a function of different environmental conditions are scarce. We have performed an extensive characterization of the ionic strength dependence of the interaction between the molten globular nuclear co-activator binding domain (NCBD) of CREB binding protein and five different protein ligands, including the intrinsically disordered activation domain of p160 transcriptional co-activators (SRC1, TIF2, ACTR), the p53 transactivation domain, and the folded pointed domain (PNT) of transcription factor ETS-2. Direct comparisons of the binding rate constants under identical conditions show that the association rate constant, kon, for interactions between NCBD and disordered protein domains is high at low salt concentrations (90-350 × 10(6) M(-1) s(-1) at 4 °C) but is reduced significantly (10-30-fold) with an increasing ionic strength and reaches a plateau around physiological ionic strength. In contrast, the kon for the interaction between NCBD and the folded PNT domain is only 7 × 10(6) M(-1) s(-1) (4 °C and low salt) and displays weak ionic strength dependence, which could reflect a distinctly different association that relies less on electrostatic interactions. Furthermore, the basal rate constant (in the absence of electrostatic interactions) is high for the NCBD interactions, exceeding those typically observed for folded proteins. One likely interpretation is that disordered proteins have a large number of possible collisions leading to a productive on-pathway encounter complex, while folded proteins are more restricted in terms of orientation. Our results highlight the importance of electrostatic interactions in binding involving IDPs and emphasize the significance of including ionic strength as a factor in studies that compare the binding properties of IDPs to those of ordered proteins.

PSD-95 is a scaffolding protein of the MAGUK protein family, and engages in several vital protein-protein interactions in the brain with its PDZ domains. It has been suggested that PSD-95 is composed of two supramodules, one of which is the PDZ1-2 tandem domain. Here we have developed rigidified high-affinity dimeric ligands that target the PDZ1-2 supramodule, and established the biophysical parameters of the dynamic PDZ1-2/ligand interactions. By employing ITC, protein NMR, and stopped-flow kinetics this study provides a detailed insight into the overall conformational energetics of the interaction between dimeric ligands and tandem PDZ domains. Our findings expand our understanding of the dynamics of PSD-95 with potential relevance to its biological role in interacting with multivalent receptor complexes and development of novel drugs.

Human cell division cycle protein 42 (Cdc42Hs) is a small, Rho-type guanosine triphosphatase involved in multiple cellular processes through its interactions with downstream effectors. The binding domain of one such effector, the actin cytoskeleton-regulating p21-activated kinase 3, is known as PBD46. Nitrogen-15 backbone and carbon-13 methyl NMR relaxation was measured to investigate the dynamical changes in activated GMPPCP·Cdc42Hs upon PBD46 binding. Changes in internal motion of the Cdc42Hs, as revealed by methyl axis order parameters, were observed not only near the Cdc42Hs–PBD46 interface but also in remote sites on the Cdc42Hs molecule. The binding-induced changes in side-chain dynamics propagate along the long axis of Cdc42Hs away from the site of PBD46 binding with sharp distance dependence. Overall, the binding of the PBD46 effector domain on the dynamics of methyl-bearing side chains of Cdc42Hs results in a modest rigidification, which is estimated to correspond to an unfavorable change in conformational entropy of approximately − 10 kcal mol− 1 at 298 K. A cluster of methyl probes closest to the nucleotide-binding pocket of Cdc42Hs becomes more rigid upon binding of PBD46 and is proposed to slow the catalytic hydrolysis of the γ phosphate moiety. An additional cluster of methyl probes surrounding the guanine ring becomes more flexible on binding of PBD46, presumably facilitating nucleotide exchange mediated by a guanosine exchange factor. In addition, the Rho insert helix, which is located at a site remote from the PBD46 binding interface, shows a significant dynamic response to PBD46 binding.

Intrinsically disordered proteins are very common in the eukaryotic proteome, and many of them are associated with diseases. Disordered proteins usually undergo a coupled binding and folding reaction and often interact with many different binding partners. Using double mutant cycles, we mapped the energy landscape of the binding interface for two interacting disordered domains and found it to be largely suboptimal in terms of interaction free energies, despite relatively high affinity. These data depict a frustrated energy landscape for interactions involving intrinsically disordered proteins, which is likely a result of their functional promiscuity.

Protein-protein interactions involving intrinsically disordered proteins are important for cellular function and common in all organisms. However, it is not clear how such interactions emerge and evolve on a molecular level. We performed phylogenetic reconstruction, resurrection and biophysical characterization of two interacting disordered protein domains, CID and NCBD. CID appeared after the divergence of protostomes and deuterostomes 450–600 million years ago, while NCBD was present in the protostome/deuterostome ancestor. The most ancient CID/NCBD formed a relatively weak complex (Kd∼5 µM). At the time of the first vertebrate-specific whole genome duplication, the affinity had increased (Kd∼200 nM) and was maintained in further speciation. Experiments together with molecular modeling using NMR chemical shifts suggest that new interactions involving intrinsically disordered proteins may evolve via a low-affinity complex which is optimized by modulating direct interactions as well as dynamics, while tolerating several potentially disruptive mutations., eLife, 6, ISSN:2050-084X

Partially or fully disordered proteins are instrumental for signal-transduction pathways; however, many mechanistic aspects of these proteins are not well-understood. For example, the number and nature of intermediate states along the binding pathway is still a topic of intense debate. To shed light on the conformational heterogeneity of disordered protein domains and their complexes, we performed single-molecule experiments by translocating disordered proteins through a nanopore embedded within a thin dielectric membrane. This platform allows for single-molecule statistics to be generated without the need of fluorescent labels or other modification groups. These studies were performed on two different intrinsically disordered protein domains, a binding domain from activator of thyroid hormone and retinoid receptors (ACTR) and the nuclear coactivator binding domain of CREB-binding protein (NCBD), along with their bimolecular complex. Our results demonstrate that both ACTR and NCBD populate distinct conformations upon translocation through the nanopore. The folded complex of the two disordered domains, on the other hand, translocated as one conformation. Somewhat surprisingly, we found that NCBD undergoes a charge reversal under high salt concentrations. This was verified by both translocation statistics as well as by measuring the ζ-potential. Electrostatic interactions have been previously suggested to play a key role in the association of intrinsically disordered proteins, and the observed behavior adds further complexity to their binding reactions.

Folding reaction mechanisms of globular protein domains have been extensively studied by both experiment and simulation and found to be highly concerted chemical reactions in which numerous noncovalent bonds form in an apparent two-state fashion. However, less is known regarding intrinsically disordered proteins because their folding can usually be studied only in conjunction with binding to a ligand. We have investigated by kinetics the folding mechanism of such a disordered protein domain, the nuclear coactivator-binding domain (NCBD) from CREB-binding protein. While a previous computational study suggested that NCBD folds without an activation free energy barrier, our experimental data demonstrate that NCBD, despite its highly dynamic structure, displays relatively slow folding (∼10 ms at 277 K) consistent with a barrier-limited process. Furthermore, the folding kinetics corroborate previous nuclear magnetic resonance data showing that NCBD exists in two folded conformations and one more denatured conformation at equilibrium and, thus, that the folding mechanism is a three-state mechanism. The refolding kinetics is limited by unfolding of the less populated folded conformation, suggesting that the major route for interconversion between the two folded states is via the denatured state. Because the two folded conformations have been suggested to bind distinct ligands, our results have mechanistic implications for conformational sampling in protein-protein interactions.

We describe the development of a novel serum albumin binding protein showing an extremely high affinity (K(D)) for HSA in the femtomolar range. Using a naturally occurring 46-residue three-helix bundle albumin binding domain (ABD) of nanomolar affinity for HSA as template, 15 residues were targeted for a combinatorial protein engineering strategy to identify variants showing improved HSA affinities. Sequencing of 55 unique phage display-selected clones showed a strong bias for wild-type residues at nine positions, whereas various changes were observed at other positions, including charge shifts. Additionally, a few non-designed substitutions appeared. On the basis of the sequences of 12 variants showing high overall binding affinities and slow dissociation rate kinetics, a set of seven 'second generation' variants were constructed. One variant denoted ABD035 displaying wild-type-like secondary structure content and excellent thermal denaturation/renaturation properties showed an apparent affinity for HSA in the range of 50-500 fM, corresponding to several orders of magnitude improvement compared with the wild-type domain. The ABD035 variant also showed an improved affinity toward serum albumin from a number of other species, and a capture experiment involving human serum indicated that the selectivity for serum albumin had not been compromised from the affinity engineering.

Protein or protein regions that are not forming well-defined structures in their free states under native-like conditions are called intrinsically disordered proteins. Such proteins are very common in protein-protein interactions, where their disorder apparently gives several advantages including optimal binding properties. To fully appreciate why protein disorder is advantageous for protein-protein interactions we need to understand the mechanism(s) of interaction. However, elucidating mechanisms in protein-protein interactions is usually very challenging. Here we discuss how kinetics in combination with protein engineering and structural information can be used to depict details of protein-protein interactions involving intrinsically disordered proteins.

Affibody binding proteins are selected from phage-displayed libraries of variants of the 58 residue Z domain. Z(Taq) is an affibody originally selected as a binder to Taq DNA polymerase. The anti-Z(Taq) affibody was selected as a binder to Z(Taq) and the Z(Taq):anti-Z(Taq) complex is formed with a dissociation constant K(d)=0.1 microM. We have determined the structure of the Z(Taq):anti-Z(Taq) complex as well as the free state structures of Z(Taq) and anti-Z(Taq) using NMR. Here we complement the structural data with thermodynamic studies of Z(Taq) and anti-Z(Taq) folding and complex formation. Both affibody proteins show cooperative two-state thermal denaturation at melting temperatures T(M) approximately 56 degrees C. Z(Taq):anti-Z(Taq) complex formation at 25 degrees C in 50 mM NaCl and 20 mM phosphate buffer (pH 6.4) is enthalpy driven with DeltaH degrees (bind) = -9.0 (+/-0.1) kcal mol(-1)(.) The heat capacity change DeltaC(P) degrees (,bind)=-0.43 (+/-0.01) kcal mol(-1) K(-1) is in accordance with the predominantly non-polar character of the binding surface, as judged from calculations based on changes in accessible surface areas. A further dissection of the small binding entropy at 25 degrees C (-TDeltaS degrees (bind) = -0.6 (+/-0.1) kcal mol(-1)) suggests that a favourable desolvation of non-polar surface is almost completely balanced by unfavourable conformational entropy changes and loss of rotational and translational entropy. Such effects can therefore be limiting for strong binding also when interacting protein components are stable and homogeneously folded. The combined structure and thermodynamics data suggest that protein properties are not likely to be a serious limitation for the development of engineered binding proteins based on the Z domain.

Affibody molecules constitute a class of engineered binding proteins based on the 58-residue three-helix bundle Z domain derived from staphylococcal protein A (SPA). Affibody proteins are selected as binders to target proteins by phage display of combinatorial libraries in which typically 13 side-chains on the surface of helices 1 and 2 in the Z domain have been randomized. The Z(Taq):anti-Z(Taq) affibody-affibody complex, consisting of Z(Taq), originally selected as a binder to Taq DNA polymerase, and anti-Z(Taq), selected as binder to Z(Taq), is formed with a dissociation constant K(d) approximately 100 nM. We have determined high-precision solution structures of free Z(Taq) and anti-Z(Taq), and the Z(Taq):anti-Z(Taq) complex under identical experimental conditions (25 degrees C in 50 mM NaCl with 20 mM potassium phosphate buffer at pH 6.4). The complex is formed with helices 1 and 2 of anti-Z(Taq) in perpendicular contact with helices 1 and 2 of Z(Taq). The interaction surface is large ( approximately 1670 A(2)) and unusually non-polar (70 %) compared to other protein-protein complexes. It involves all varied residues on anti-Z(Taq), most corresponding (Taq DNA polymerase binding) side-chains on Z(Taq), and several additional side-chain and backbone contacts. Other notable features include a substantial rearrangement (induced fit) of aromatic side-chains in Z(Taq) upon binding, a close contact between glycine residues in the two subunits that might involve aliphatic glycine Halpha to backbone carbonyl hydrogen bonds, and four hydrogen bonds made by the two guanidinium N(eta)H(2) groups of an arginine side-chain. Comparisons of the present structure with other data for affibody proteins and the Z domain suggest that intrinsic binding properties of the originating SPA surface might be inherited by the affibody binders. A thermodynamic characterization of Z(Taq) and anti-Z(Taq) is presented in an accompanying paper.

We analyzed the thermodynamics of a complex protein-protein binding interaction using the (engineered) Z(SPA)(-)(1) affibody and it's Z domain binding partner as a model. Free Z(SPA)(-)(1) exists in an equilibrium between a molten-globule-like (MG) state and a completely unfolded state, wheras a well-ordered structure is observed in the Z:Z(SPA)(-)(1) complex. The thermodynamics of the MG state unfolding equilibrium can be separated from the thermodynamics of binding and stabilization by combined analysis of isothermal titration calorimetry data and a separate van't Hoff analysis of thermal unfolding. We find that (i) the unfolding equilibrium of free Z(SPA)(-)(1) has only a small influence on effective binding affinity, that (ii) the Z:Z(SPA)(-)(1) interface is inconspicuous and structure-based energetics calculations suggest that it should be capable of supporting strong binding, but that (iii) the conformational stabilization of the MG state to a well-ordered structure in the Z:Z(SPA)(-)(1) complex is associated with a large change in conformational entropy that opposes binding.

The Φ value analysis is a method to analyze the structure of metastable states in reaction pathways. Such a methodology is based on the quantitative analysis of the effect of point mutations on the kinetics and thermodynamics of the probed reaction. The Φ value analysis is routinely used in protein folding studies and is potentially an extremely powerful tool to analyze the mechanism of binding induced folding of intrinsically disordered proteins. In this review we recapitulate the key equations and experimental advices to perform the Φ value analysis in the perspective of the possible caveats arising in intrinsically disordered systems. Finally, we briefly discuss some few examples already available in the literature.

Many intrinsically disordered proteins fold upon binding to other macromolecules. The secondary structure present in the well-ordered complex is often formed transiently in the unbound state. The consequence of such transient structure for the binding process is, however, not clear. The activation domain of the activator for thyroid hormone and retinoid receptors (ACTR) is intrinsically disordered and folds upon binding to the nuclear coactivator binding domain (NCBD) of the CREB binding protein. A number of mutants was designed that selectively perturbs the amount of secondary structure in unbound ACTR without interfering with the intermolecular interactions between ACTR and NCBD. Using NMR spectroscopy and fluorescence-monitored stopped-flow kinetic measurements we show that the secondary structure content in helix1 of ACTR indeed influences the binding kinetics. The results thus support the notion of preformed secondary structure as an important determinant for molecular recognition in intrinsically disordered proteins. De två första författarna delar första författarskapet.

Intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) of proteins are very common and instrumental for cellular signaling. Recently, a number of studies have investigated the kinetic binding mechanisms of IDPs and IDRs. These results allow us to draw conclusions about the energy landscape for the coupled binding and folding of disordered proteins. The association rate constants of IDPs cover a wide range (10(5)-10(9) M-1 s(-1)) and are largely governed by long-range charge-charge interactions, similarly to interactions between well-folded proteins. Off-rate constants also differ significantly among IDPs (with half-lives of up to several minutes) but are usually around 0.1-1000 s(-1), allowing for rapid dissociation of complexes. Likewise, affinities span from pM to mu M suggesting that the low-affinity high-specificity concept for IDPs is not straightforward. Overall, it appears that binding precedes global folding although secondary structure elements such as helices may form before the protein-protein interaction. Short IDPs bind in apparent two-state reactions whereas larger IDPs often display complex multi-step binding reactions. While the two extreme cases of two-step binding (conformational selection and induced fit) or their combination into a square mechanism is an attractive model in theory, it is too simplistic in practice. Experiment and simulation suggest a more complex energy landscape in which IDPs bind targets through a combination of conformational selection before binding (e. g., secondary structure formation) and induced fit after binding (global folding and formation of short-range intermolecular interactions).

International audience; The interactions between proteins and ligands often involve a conformational change in the protein. This conformational change can occur before (conformational selection) or after (induced fit) the association with ligand. It is often very difficult to distinguish induced fit from conformational selection when hyperbolic binding kinetics are observed. In light of a recent paper in this journal (Vogt et al., Biophys. Chem., 186, 2014, 13-21) and the current interest in binding mechanisms emerging from observed sampling of distinct conformations in protein domains, as well as from the field of intrinsically disordered proteins, we here describe a kinetic method that, at least in some cases, unequivocally distinguishes induced fit from conformational selection. The method relies on measuring the observed rate constant λ for binding and varying both the protein and the ligand in separate experiments. Whereas induced fit always yields a hyperbolic dependence of increasing λ values, the conformational selection mechanism gives rise to distinct kinetics when the ligand and protein (displaying the conformational change) concentration is varied in separate experiments. We provide examples from the literature and discuss the limitations of the approach.

Intrinsically disordered proteins are abundant in the eukaryotic proteome and they are implicated in a range of different diseases. However, there is a paucity of experimental data on molecular details of the coupled binding and folding of such proteins. Two interacting and relatively well studied disordered protein domains are the activation domain from the p160 transcriptional co-activator ACTR and the nuclear co-activator binding domain (NCBD) of CREB binding protein. We have analyzed the transition state for their coupled binding and folding by protein engineering and kinetic experiments (Φ-value analysis) and found that it involves weak native interactions between the N-terminal helices of ACTR and NCBD, but is otherwise "disordered-like". Most native hydrophobic interactions in the interface between the two domains form later, after the rate-limiting barrier for association. Linear free energy relationships suggest a cooperative formation of native interactions, reminiscent of the nucleation-condensation mechanism in protein folding.

The postsynaptic density protein-95/discs large/zonula occludens-1 (PDZ) domain is a protein-protein interaction module with a shallow binding groove where protein ligands bind. However, interactions that are not part of this canonical binding groove are likely to modulate peptide binding. We have investigated such interactions beyond the binding groove for PDZ3 from PSD-95 and a peptide derived from the C-terminus of the natural ligand CRIPT. We found via nuclear magnetic resonance experiments that up to eight residues of the peptide ligand interact with the PDZ domain, showing that the interaction surface extends far outside of the binding groove as defined by the crystal structure. PDZ3 contains an extra structural element, a C-terminal helix (α3), which is known to affect affinity. Deletion of this helix resulted in the loss of several intermolecular nuclear Overhauser enhancements from peptide residues outside of the binding pocket, suggesting that α3 forms part of the extra binding surface in wild-type PDZ3. Site-directed mutagenesis, isothermal titration calorimetry, and fluorescence intensity experiments confirmed the importance of both α3 and the N-terminal part of the peptide for the affinity. Our data suggest a general mechanism in which different binding surfaces outside of the PDZ binding groove could provide sites for specific interactions.

Proteins that contain long disordered regions are prevalent in the proteome and frequently associated with diseases. However, the mechanisms by which such intrinsically disordered proteins (IDPs) recognize their targets are not well understood. Here, we report the first experimental investigation of the interaction kinetics of the nuclear co-activator binding domain of CREB-binding protein and the activation domain from the p160 transcriptional co-activator for thyroid hormone and retinoid receptors. Both protein domains are intrinsically disordered in the free state and synergistically fold upon binding each other. Using the stopped-flow technique, we found that the binding reaction is fast, with an association rate constant of 3 x 10(7) M-1 s(-1) at 277 K. Mutation of a conserved buried intermolecular salt bridge showed that electrostatics govern the rapid association. Furthermore, upon mutation of the salt bridge or at high salt concentration, an additional kinetic phase was detected (similar to 20 and similar to 40 s(-1), respectively, at 277 K), suggesting that the salt bridge may steer formation of the productive bimolecular complex in an intramolecular step. Finally, we directly measured slow kinetics for the IDP domains (similar to 1 s(-1) at 277 K) related to conformational transitions upon binding. Together, the experiments demonstrate that the interaction involves several steps and accumulation of intermediate states. Our data are consistent with an induced fit mechanism, in agreement with previous simulations. We propose that the slow transitions may be a consequence of the multipartner interactions of IDPs.

Intrinsically disordered proteins are very common and mediate numerous protein-protein and protein-DNA interactions. While it is clear that these interactions are instrumental for the life of the mammalian cell, there is a paucity of data regarding their molecular binding mechanisms. Here we have used short peptides as a model system for intrinsically disordered proteins. Linear free energy relationships based on rate and equilibrium constants for the binding of these peptides to ordered target proteins, PDZ domains, demonstrate that native side-chain interactions form mainly after the rate-limiting barrier for binding and in a cooperative fashion. This finding suggests that these disordered peptides first form a weak encounter complex with non-native interactions. The data do not support the recent notion that the affinities of intrinsically disordered proteins toward their targets are generally governed by their association rate constants. Instead, we observed the opposite for peptide-PDZ interactions, namely, that changes in K(d) correlate with changes in k(off).

Comprehensive application of solution NMR spectroscopy to studies of macromolecules remains fundamentally limited by the molecular rotational correlation time. For proteins, molecules larger than 30 kDa require complex experimental methods, such as TROSY in conjunction with isotopic labeling schemes that are often expensive and generally reduce the potential information available. We have developed the reverse micelle encapsulation strategy as an alternative approach. Encapsulation of proteins within the protective nano-scale water pool of a reverse micelle dissolved in ultra-low viscosity nonpolar solvents overcomes the slow tumbling problem presented by large proteins. Here, we characterize the contributions from the various components of the protein-containing reverse micelle system to the rotational correlation time of the encapsulated protein. Importantly, we demonstrate that the protein encapsulated in the reverse micelle maintains a hydration shell comparable in size to that seen in bulk solution. Using moderate pressures, encapsulation in ultra-low viscosity propane or ethane can be used to magnify this advantage. We show that encapsulation in liquid ethane can be used to reduce the tumbling time of the 43 kDa maltose binding protein from ~23 ns to ~10 ns. These conditions enable, for example, acquisition of TOCSY-type data resolved on the adjacent amide NH for the 42 kDa encapsulated maltose binding protein dissolved in liquid ethane, which is typically impossible for proteins of such size without use of extensive deuteration or the TROSY effect.

We are writing to make an important and general comment that relates to the conclusions of a paper recently published in the Biophysical Journal by Krieger et al. (1xConformational recognition of an intrinsically disordered protein. Krieger, J.M., Fusco, G...., and De Simone, A. Biophys. J. 2014; 106: 1771–1779Abstract | Full Text | Full Text PDF | PubMed | Scopus (14)See all References1). Intrinsically disordered proteins (IDPs) and their binding reactions are currently being studied intensively by protein chemists and biologists (2xMultiparametric analysis of intrinsically disordered proteins: looking at intrinsic disorder through compound eyes. Uversky, V.N. and Dunker, A.K. Anal. Chem. 2012; 84: 2096–2104Crossref | PubMed | Scopus (30)See all References2). It was suggested early on that these disordered regions may transiently populate folded structures, which may act as recognition elements (3xPreformed structural elements feature in partner recognition by intrinsically unstructured proteins. Fuxreiter, M., Simon, I...., and Tompa, P. J. Mol. Biol. 2004; 338: 1015–1026Crossref | PubMed | Scopus (308)See all References3). The paper by Krieger et al. (1xConformational recognition of an intrinsically disordered protein. Krieger, J.M., Fusco, G...., and De Simone, A. Biophys. J. 2014; 106: 1771–1779Abstract | Full Text | Full Text PDF | PubMed | Scopus (14)See all References1) addresses this pertinent question by assessing the sampling of a preformed structure in an IDP (Gab2503-524), and in particular the amount of conformation that is similar to that in the bound state, and its role in ligand binding. But which species is involved in the initial binding—the structured one or the disordered one?The authors employ an experimental strategy of modulating the propensity to form the secondary structure by mutation, similar to what was described in another recent paper by Iesmantavicius et al. (4xHelical 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 (29)See all References4), which we co-authored. In both studies, the amount of preformed, bound-like secondary structure in the free state was measured by NMR methods to probe the effect of mutation. In addition, both studies reported a positive correlation between the preformed bound-like structure in the free IDP and the binding affinity, although in the work by Krieger et al., the mutations modulated not only the amount of free-state residual structure but also direct interactions with its partner. Nevertheless, the correlation is there, but the question is, what does it mean? In the Discussion, Krieger et al. (1xConformational recognition of an intrinsically disordered protein. Krieger, J.M., Fusco, G...., and De Simone, A. Biophys. J. 2014; 106: 1771–1779Abstract | Full Text | Full Text PDF | PubMed | Scopus (14)See all References1) write, “Therefore, the results reported here suggest that the binding of Gab2503-524 [the IDP] requires a selection of conformations that appear to be intrinsically encoded in the energy landscape of this disordered state.” We wish to point out that this is not a requirement. It is formally equally possible that the IDP binds in a disordered conformation and that the increased propensity of the IDP to adopt a bound-like conformation lowers the energetic barrier for a subsequent rate-limiting step (affecting the rate constants of binding) as well as the energy of the bound complex (affecting the overall affinity) (4xHelical 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 (29)See all References4).Thus, it is important to emphasize that neither the results from Krieger et al. (1xConformational recognition of an intrinsically disordered protein. Krieger, J.M., Fusco, G...., and De Simone, A. Biophys. J. 2014; 106: 1771–1779Abstract | Full Text | Full Text PDF | PubMed | Scopus (14)See all References1) nor the previous data (4xHelical 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 (29)See all References4) prove that it is the folded conformation of the IDP that binds the target. It could be the disordered one, it could be the ordered one, or it could even be a whole range of conformations (5xLigand concentration regulates the pathways of coupled protein folding and binding. Daniels, K.G., Tonthat, N.K...., and Oas, T.G. J. Am. Chem. Soc. 2014; 136: 822–825Crossref | PubMed | Scopus (16)See all References, 6xThe binding mechanisms of intrinsically disordered proteins. Dogan, J., Gianni, S., and Jemth, P. Phys. Chem. Chem. Phys. 2013; 16: 6323–6331Crossref | PubMed | Scopus (18)See all References), with ligand-concentration-dependent flux through different pathways (7xConformational selection or induced fit: a flux description of reaction mechanism. Hammes, G.G., Chang, Y.-C., and Oas, T.G. Proc. Natl. Acad. Sci. USA. 2009; 106: 13737–13741Crossref | PubMed | Scopus (210)See all References7). To prove a mechanism, it is necessary, but perhaps not sufficient, to perform kinetic studies. Several studies on IDPs (8xMapping backbone and side-chain interactions in the transition state of a coupled protein folding and binding reaction. Bachmann, A., Wildemann, D...., and Kiefhaber, T. Proc. Natl. Acad. Sci. USA. 2011; 108: 3952–3957Crossref | PubMed | Scopus (53)See all References, 9xThe transition state structure for coupled binding and folding of disordered protein domains. Dogan, J., Mu, X...., and Jemth, P. Sci. Rep. 2013; 3: 2076Crossref | PubMed | Scopus (19)See all References, 10xStructure of the transition state for the binding of c-Myb and KIX highlights an unexpected order for a disordered system. Giri, R., Morrone, A...., and Gianni, S. Proc. Natl. Acad. Sci. USA. 2013; 110: 14942–14947Crossref | PubMed | Scopus (17)See all References, 11xCoupled folding and binding of the disordered protein PUMA does not require particular residual structure. Rogers, J.M., Wong, C.T., and Clarke, J. J. Am. Chem. Soc. 2014; 136: 5197–5200Crossref | PubMed | Scopus (22)See all References), including the one by Iesmantavicius et al. (4xHelical 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 (29)See all References4), have employed mutagenesis in combination with kinetics to understand the binding reaction. However, these studies addressed the nature of the rate-limiting transition state of the binding reaction, and the role of the preformed structure could not be decisively defined. To prove or disprove a mechanism such as conformational selection (i.e., to determine the simplest mechanism that is consistent with all available data), one must measure the observed rate constants for formation of the preformed structure in question, in the binding reaction, and subject them to careful analysis (12xDistinguishing induced fit from conformational selection. Gianni, S., Dogan, J., and Jemth, P. Biophys. Chem. 2014; 189: 33–39Crossref | PubMed | Scopus (15)See all References12). In brief, such an analysis involves measuring the observed rate constant for binding over as wide a range of concentrations as possible for both interacting species. If a hyperbolic behavior of the rate constant is observed, then conformational selection may be distinguished from induced fit as detailed in Gianni et al. (12xDistinguishing induced fit from conformational selection. Gianni, S., Dogan, J., and Jemth, P. Biophys. Chem. 2014; 189: 33–39Crossref | PubMed | Scopus (15)See all References12). However, since helix-coil transitions and similar equilibria involving secondary structure elements occur on a very fast timescale (13xSubmillisecond kinetics of protein folding. Eaton, W.A., Munoz, V...., and Hofrichter, J. Curr. Opin. Struct. Biol. 1997; 7: 10–14Crossref | PubMed | Scopus (171)See all References, 14xLocal conformational dynamics in α-helices measured by fast triplet transfer. Fierz, B., Reiner, A., and Kiefhaber, T. Proc. Natl. Acad. Sci. USA. 2009; 106: 1057–1062Crossref | PubMed | Scopus (36)See all References), this is not a trivial pursuit, but something we must tackle experimentally if we want to settle this issue.In conclusion, although it is tempting to suggest a conformational selection mechanism based on observations of bound-like conformations in the free state, we must all exercise caution in our interpretations in the absence of direct evidence.

The physical basis for high affinity interactions involving proteins is complex and potentially involves a range of energetic contributions. Among these are changes in protein conformational entropy, which cannot yet be reliably computed from molecular structures. We have recently employed changes in conformational dynamics as a proxy for changes in conformational entropy of calmodulin upon association with domains from regulated proteins. The apparent change in conformational entropy was linearly related to the overall binding entropy. This view warrants a more quantitative foundation. Here we calibrate an “entropy meter” employing an experimental dynamical proxy based on NMR relaxation and show that changes in the conformational entropy of calmodulin are a significant component of the energetics of binding. Furthermore, the distribution of motion at the interface between the target domain and calmodulin are surprisingly non-complementary. These observations promote modification of our understanding of the energetics of protein-ligand interactions.

Affibodies are a novel class of binding proteins selected from phagemid libraries of the Z domain from staphylococcal protein A. The Z(SPA-1) affibody was selected as a binder to protein A, and it binds the parental Z domain with micromolar affinity. In earlier work we determined the structure of the Z:Z(SPA-1) complex and noted that Z(SPA-1) in the free state exhibits several properties characteristic of a molten globule. Here we present a more detailed biophysical investigation of Z(SPA-1) and four Z(SPA-1) mutants with the objective to understand these properties. The characterization includes thermal and chemical denaturation profiles, ANS binding assays, size exclusion chromatography, isothermal titration calorimetry, and an investigation of structure and dynamics by NMR. The NMR characterization of Z(SPA-1) was facilitated by the finding that trimethylamine N-oxide (TMAO) stabilizes the molten globule conformation in favor of the fully unfolded state. All data taken together lead us to conclude the following: (1) The topology of the molten globule conformation of free Z(SPA-1) is similar to that of the fully folded structure in the Z-bound state; (2) the extensive mutations in helices 1 and 2 destabilize these without affecting the intrinsic stability of helix 3; (3) stabilization and reduced aggregation can be achieved by replacing mutated residues in Z(SPA-1) with the corresponding wild-type Z residues. This stabilization is better correlated to changes in helix propensity than to an expected increase in polar versus nonpolar surface area of the fully folded state.

NMR assignments of the free and bound-state protein components of an anti-idiotypic affibody complex