| 1. |
- Chi, Celestine N., et al.
(författare)
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Interactions outside the Boundaries of the Canonical Binding Groove of a PDZ Domain Influence Ligand Binding
- 2012
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 51:44, s. 8971-8979
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 2. |
- Dincbas-Renqvist, Vildan, et al.
(författare)
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Thermodynamics of folding, stabilization, and binding in an engineered protein--protein complex.
- 2004
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 126:36, s. 11220-30
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 3. |
- Dogan, Jakob, et al.
(författare)
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Activation Barrier-Limited Folding and Conformational Sampling of a Dynamic Protein Domain
- 2016
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 55:37, s. 5289-5295
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Tidskriftsartikel (refereegranskat)abstract
- 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 (similar to 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.
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| 4. |
- Dogan, Jakob, et al.
(författare)
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Binding Rate Constants Reveal Distinct Features of Disordered Protein Domains
- 2015
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 54:30, s. 4741-4750
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Tidskriftsartikel (refereegranskat)abstract
- 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, k(on), for interactions between NCBD and disordered protein domains is high at low salt concentrations (90-350 x 10(6) M-1 s(-1) at 4 degrees C) but is reduced significantly (10-30-fold) with an increasing ionic strength and reaches a plateau around physiological ionic strength. In contrast, the k(on) for the interaction between NCBD and the folded PNT domain is only 7 x 10(6) M-1 s(-1) (4 degrees 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.
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| 5. |
- Dogan, Jakob, et al.
(författare)
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Fast Association and Slow Transitions in the Interaction between Two Intrinsically Disordered Protein Domains
- 2012
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 287:41, s. 34316-34324
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 6. |
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| 7. |
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| 8. |
- Dogan, Jakob, 1978-
(författare)
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Structural and thermodynamical basis for molecular recognition between engineered binding proteins
- 2006
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The structural determination of interacting proteins, both as individual proteins and in their complex, complemented by thermodynamical studies are vital in order to gain in-depth insights of the phenomena leading to the highly selective protein-protein interactions characteristic of numerous life processes. This thesis describes an investigation of the structural and thermodynamical basis for molecular recognition in two different protein-protein complexes, formed between so-called affibody proteins and their respective targets. Affibody proteins are a class of engineered binding proteins, which can be functionally selected for binding to a given target protein from large collections (libraries) constructed via combinatorial engineering of 13 surface-located positions of the 58-residue three-helix bundle Z domain derived from Staphylococcal protein (SPA). In a first study, an affibody:target protein pair consisting of the ZSPA-1 affibody and the parental Z domain, with a dissociation constant (Kd) of approximately 1 µM, was investigated. ZSPA-1 was in its free state shown to display molten globule-like characteristics. The enthalpy change on binding between Z and ZSPA-1 as measured by isothermal titration calorimetry, was found to be a non-linear function of temperature. This nonlinearity was found to be due to the temperature dependent folded-unfolded equilibrium of ZSPA-1 upon binding to the Z domain and, the energetics of the unfolding equilibrium of the molten globule state of ZSPA-1 could be separated from the binding thermodynamics. Further dissection of the binding entropy revealed that a significant reduction in conformational entropy resulting from the stabilization of the molten globule state of ZSPA-1 upon complex formation could be a major reason for the moderate binding affinity. A second studied affibody:target complex (Kd ~ 0.1 µM) consisted of the ZTaq affibody protein originally selected for binding to Taq DNA polymerase and the anti-ZTaq affibody protein, selected for selective binding to the ZTaq affibody protein, thus constituting an "anti-idiotypic" affinity protein pair. The structure of the ZTaq:anti-ZTaq affibody complex as well as the free state structures of ZTaq and anti-ZTaq were determined using NMR spectroscopy. Both ZTaq and anti-ZTaq are well defined three helix bundles in their free state and do not display the same molten globule-like behaviour of ZSPA-1. The interaction surface was found to involve all of the varied positions in helices 1 and 2 of the anti-ZTaq, the majority of the corresponding side chains in ZTaq, and also several non-mutated residues. The total buried surface area was determined to about 1670 Å2 which is well inside the range of what is typical for many protein-protein complexes, including antibody:antigen complexes. Structural rearrangements, primarily at the side chain level, were observed to take place upon binding. There are similarities between the ZTaq:anti-ZTaq and the Z:ZSPA-1 structure, for instance, the binding interface area in both complexes has a large fraction of non-polar content, the buried surface area is of similar size, and certain residues have the same positioning. However, the relative orientation between the subunits in ZTaq:anti-ZTaq is markedly different from that observed in Z:ZSPA-1. The thermodynamics of ZTaq:anti-ZTaq association were investigated by isothermal titration calorimetry. A dissection of the entropic contributions showed that a large and favourable desolvation entropy of non-polar surface is associated with the binding reaction which is in good agreement with hydrophobic nature of the binding interface, but as in the case for the Z:ZSPA-1 complex a significant loss in conformational entropy opposes complex formation. A comparison with complexes involving affibody proteins or SPA domains suggests that affibody proteins inherit intrinsic binding properties from the original SPA surface. The structural and biophysical data suggest that although extensive mutations are carried out in the Z domain to obtain affibody proteins, this does not necessarily affect the structural integrity or lead to a significant destabilization.
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| 9. |
- Dogan, Jakob, et al.
(författare)
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The binding mechanisms of intrinsically disordered proteins
- 2014
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Ingår i: Physical Chemistry, Chemical Physics - PCCP. - : Royal Society of Chemistry (RSC). - 1463-9076 .- 1463-9084. ; 16:14, s. 6323-6331
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Tidskriftsartikel (refereegranskat)abstract
- 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).
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| 10. |
- Dogan, Jakob, et al.
(författare)
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The transition state structure for coupled binding and folding of disordered protein domains
- 2013
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 3, s. 2076-
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Tidskriftsartikel (refereegranskat)abstract
- 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 (Phi-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.
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