A structurally heterogeneous transition state underlies coupled binding and folding of disordered proteins.

Many intrinsically disordered proteins (IDPs) attain a well-defined structure in a coupled folding and binding reaction with another protein. Such reactions may involve early to late formation of different native structural regions along the reaction pathway. To obtain insights into the transition state for a coupled binding and folding reaction, we performed restrained molecular dynamics simulations using previously determined experimental binding Φb values of the interaction between two IDP domains: the activation domain from the p160 transcriptional co-activator for thyroid hormone and retinoid receptors (ACTR) and the nuclear co-activator binding domain (NCBD) of CREB-binding protein, each forming three well-defined α-helices upon binding. These simulations revealed that both proteins are largely disordered in the transition state for complex formation, except for two helices, one from each domain, that display a native-like structure. The overall transition state structure was extended and largely dynamic with many weakly populated contacts. To test the transition state model, we combined site-directed mutagenesis with kinetic experiments, yielding results consistent with overall diffuse interactions and formation of native intramolecular interactions in the third NCBD helix during the binding reaction. Our findings support the view that the transition state and, by inference, any encounter complex in coupled binding and folding reactions are structurally heterogeneous and largely independent of specific interactions. Furthermore, experimental Φb values and Brønsted plots suggested that the transition state is globally robust with respect to most mutations but can display more native-like features for some highly destabilizing mutations, possibly because of Hammond behavior or ground-state effects.

Native Hydrophobic Binding Interactions at the Transition State for Association between the TAZ1 Domain of CBP and the Disordered TAD-STAT2 Are Not a Requirement.

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.

Activation Barrier-Limited Folding and Conformational Sampling of a Dynamic Protein Domain.

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.

Binding Rate Constants Reveal Distinct Features of Disordered Protein Domains.

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.

A frustrated binding interface for intrinsically disordered proteins.

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.

Rigidified clicked dimeric ligands for studying the dynamics of the PDZ1-2 supramodule of PSD-95.

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.

The binding mechanisms of intrinsically disordered proteins.

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 µM 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).

Helical propensity in an intrinsically disordered protein accelerates ligand binding.

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 helix 1 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.

Fast association and slow transitions in the interaction between two intrinsically disordered protein domains.

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 × 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 (∼20 and ∼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 (∼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.

Single-molecule studies of intrinsically disordered proteins using solid-state nanopores.

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.

Interactions outside the boundaries of the canonical binding groove of a PDZ domain influence ligand binding.

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.

Side-chain interactions form late and cooperatively in the binding reaction between disordered peptides and PDZ domains.

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).

Characterization of the endopeptidase activity of tripeptidyl-peptidase II.

Tripeptidyl-peptidase II (TPP II) is a giant cytosolic peptidase with a proposed role in cellular protein degradation and protection against apoptosis. Beside its well-characterised exopeptidase activity, TPP II also has an endopeptidase activity. Little is known about this activity, and since it could be important for the physiological role of TPP II, we have investigated it in more detail. Two peptides, Nef(69-87) and LL37, were incubated with wild-type murine TPP II and variants thereof as well as TPP II from human and Drosophila melanogaster. Two intrinsically disordered proteins were also included in the study. We conclude that the endopeptidase activity is more promiscuous than previously reported. It is also clear that TPP II can attack longer disordered peptides up to 75 amino acid residues. Using a novel FRET substrate, the catalytic efficiency of the endopeptidase activity could be determined to be 5 orders of magnitude lower than for the exopeptidase activity.

The role of conformational entropy in molecular recognition by calmodulin.

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 used 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' using 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 is surprisingly noncomplementary. These observations promote modification of our understanding of the energetics of protein-ligand interactions.

The dynamic properties of a nuclear coactivator binding domain are evolutionarily conserved.

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 Coactivator Binding Domain (NCBD) from CREB-binding protein is a protein interaction domain, which contains a hydrophobic core but is not behaving as a typical globular domain, and has been described 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 kinetics 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.

Optimization of NMR spectroscopy of encapsulated proteins dissolved in low viscosity fluids.

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 to ~10 ns. These conditions enable, for example, acquisition of TOCSY-type data resolved on the adjacent amide NH for the 43 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.

Characterization of the dynamics and the conformational entropy in the binding between TAZ1 and CTAD-HIF-1α.

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.

Role of Conformational Entropy in Molecular Recognition by TAZ1 of CBP.

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.

Engineering of a femtomolar affinity binding protein to human serum albumin.

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.