Kinetics of Folding and Binding of an Intrinsically Disordered Protein: The Inhibitor of Yeast Aspartic Proteinase YPrA

Narayanan, Ranjani; Ganesh, Omjoy; Edison, Arthur S.; Hagen, Stephen J.

doi:10.1021/ja803221c

Cited by 57 publications

(59 citation statements)

References 47 publications

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“…Interestingly, the p53 peptide does not appear to be an unusual case, and evidence has recently accumulated to suggest that induced folding is likely prevalent in IDPprotein interactions 5,22 . Induced folding has been consistently observed in mechanistic studies of IDP interaction from experiments [23][24][25] and simulations [26][27][28][29][30] 38 , the p160 steroid receptor co-activator ACTR 39 , the steroid receptor co-activator 1 (SRC1) 40 , and the interferon regulatory factor 3 (IRF3) 41 , respectively. In these complexes, NCBD adopts two distinct folds, which mainly differ in the tertiary packing of three similar helices.…”

Section: Introductionmentioning

confidence: 79%

Synergistic folding of two intrinsically disordered proteins: searching for conformational selection

2012

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Intrinsically disordered proteins (IDPs) lack stable structures under physiological conditions but often fold into stable structures upon specific binding. These coupled binding and folding processes underlie the organization of cellular regulatory networks, and a mechanistic understanding is thus of fundamental importance. Here, we investigated the synergistic folding of two IDPs, namely, the NCBD domain of transcription coactivator CBP and the p160 steroid receptor coactivator ACTR, using a topology-based model that was carefully calibrated to balance intrinsic folding propensities and intermolecular interactions. As one of the most structured IDPs, NCBD is a plausible candidate that interacts through conformational selectionlike mechanisms, where binding is mainly initiated by pre-existing folded-like conformations.Indeed, the simulations demonstrate that, even though binding and folding of both NCBD and ACTR is highly cooperative on the baseline level, the tertiary folding of NCBD is best described by the "extended conformational selection" model that involves multiple stages of selection and induced folding. The simulations further predict that the NCBD/ACTR recognition is mainly initiated by forming a mini folded core that includes the second and third helices of NCBD and ACTR. These predictions are fully consistent with independent physics-based atomistic simulations as well as a recent experimental mapping of the H/D exchange protection factors.The current work thus adds to the limited number of existing mechanistic studies of coupled binding and folding of IDPs, and provides a first direct demonstration of how conformational selection might contribute to efficient recognition of IDPs. Interestingly, even for highly structured IDPs like NCBD, the recognition is initiated by the more disordered C-terminal segment and with substantial contribution from induced folding. Together with existing studies of IDP interaction mechanisms, this argues that induced folding is likely prevalent in IDP-protein interaction, and emphasizes the importance of understanding how IDPs manage to fold efficiently upon (nonspecific) binding. Success of the current study also further supports the notion that, with careful calibration, topology-based models can be effective tools for mechanistic study of IDP interaction and regulation, especially when combined with physicsbased atomistic simulations and experiments.2

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Section: Introductionmentioning

confidence: 79%

Synergistic folding of two intrinsically disordered proteins: searching for conformational selection

2012

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“…Even when a protein is stably folded in the absence of ligand, binding always enhances stability except in the unlikely event that the binding affinity of the unfolded protein is equal to or higher than that of the folded form. The mechanism by which these coupled folding and binding reactions take place has stimulated much recent interest (29)(30)(31)(32)(33)(34)(35)(36). Kinetically speaking, the key question is: which occurs first, folding or binding?…”

Section: Resultsmentioning

confidence: 99%

“…Several recent studies have sought to determine the order of steps in sequential folding/binding reactions (8,29,32,33,36). The experiments require transient detection of the folding reaction in the presence of various concentrations of ligand (33).…”

Section: Resultsmentioning

confidence: 99%

Conformational selection or induced fit: A flux description of reaction mechanism

Hammes

Chang

Oas

2009

Proc. Natl. Acad. Sci. U.S.A.

521

642

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The mechanism of ligand binding coupled to conformational changes in macromolecules has recently attracted considerable interest. The 2 limiting cases are the ''induced fit'' mechanism (binding first) or ''conformational selection'' (conformational change first). Described here are the criteria by which the sequence of events can be determined quantitatively. The relative importance of the 2 pathways is determined not by comparing rate constants (a common misconception) but instead by comparing the flux through each pathway. The simple rules for calculating flux in multistep mechanisms are described and then applied to 2 examples from the literature, neither of which has previously been analyzed using the concept of flux. The first example is the mechanism of conformational change in the binding of NADPH to dihydrofolate reductase. The second example is the mechanism of flavodoxin folding coupled to binding of its cofactor, flavin mononucleotide. In both cases, the mechanism switches from being dominated by the conformational selection pathway at low ligand concentration to induced fit at high ligand concentration. Over a wide range of conditions, a significant fraction of the flux occurs through both pathways. Such a mixed mechanism likely will be discovered for many cases of coupled conformational change and ligand binding when kinetic data are analyzed by using a fluxbased approach.kinetics ͉ binding ͉ folding ͉ coupled equilibria ͉ mechanism T he binding of ligands by macromolecules is crucial to a multitude of physiological processes. These include the cascade of reactions accompanying hormone binding to receptors (1, 2), enzyme reactions initiated by the binding of substrates to the enzyme (3, 4), gene regulation by the binding of molecules to DNA (5) and RNA (6), and the folding of unstructured proteins to produce biologically active molecules (7,8). In virtually all cases, the binding of ligands and conformational changes go hand in hand. Consequently, considerable effort has been expended in assessing the detailed mechanism by which ligand binding and conformational changes are coupled (3,4,9). Two limiting mechanisms are generally considered: (i) ''conformational selection,'' whereby the ligand selectively binds to a form of the macromolecule that is present only in small amounts, eventually converting the macromolecule to the ligand-bound conformation; and (ii) ''induced fit,'' (9) whereby ligand binds to the predominant free conformation followed by a conformational change in the macromolecule to give the preferred ligandbound conformation.The 2 limiting mechanisms can be written as:Conformational Selection: P weak º P tightInduced Fit: P weak ϩ L º P weak ⅐LIn these equations, P weak and P tight represent the tightly and weakly binding conformations of the protein (or other macromolecule), and L is the ligand. In point of fact, these 2 limiting mechanisms can be distinguished by kinetic measurements of the rate of the conformational change. In the simplest case, which often prevails, the ligand-bind...

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“…IA 3 is a 68 amino acid protein that is unstructured in solution and is a potent inhibitor of yeast aspartic proteinase A (YPRA) [60]. IA 3 undergoes an unstructured to α-helical conformation transition upon binding to YPRA, or in the presence of a high content of TFE in the solution [61]. To obtain an EPR signal, residue E10 at the N-termini of IA 3 was mutated to cysteine and subsequently labeled with an IAP spin label (P1).…”

Section: Structural Transitions In Intrinsically Disordered Proteinsmentioning

confidence: 99%

Toward increased concentration sensitivity for continuous wave EPR investigations of spin-labeled biological macromolecules at high fields

Song

Liu

Kaur

et al. 2016

Journal of Magnetic Resonance

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High-field, high-frequency electron paramagnetic resonance (EPR) spectroscopy at W-(~95 GHz) and D-band (~140 GHz) is important for investigating the conformational dynamics of flexible biological macromolecules because this frequency range has increased spectral sensitivity to nitroxide motion over the 100 ps to 2 ns regime. However, low concentration sensitivity remains a roadblock for studying aqueous samples at high magnetic fields. Here, we examine the sensitivity of a non-resonant thin-layer cylindrical sample holder, coupled to a quasi-optical induction-mode W-band EPR spectrometer (HiPER), for continuous wave (CW) EPR analyses of: (i) the aqueous nitroxide standard, TEMPO; (ii) the unstructured to -helical transition of a model IDP protein; and (iii) the base-stacking transition in a kink-turn motif of a large 232 nt RNA. For sample volumes of ~50 L, concentration sensitivities of 2-20 µM were achieved, representing a ~10-fold enhancement compared to a cylindrical TE 011 resonator on a commercial Bruker W-band spectrometer. These results therefore highlight the sensitivity of the thin-layer sample holders employed in HiPER for spin-labeling studies of biological macromolecules at high fields, where applications can extend to other systems that are facilitated by the modest sample volumes and ease of sample loading and geometry.2

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Kinetics of Folding and Binding of an Intrinsically Disordered Protein: The Inhibitor of Yeast Aspartic Proteinase YPrA

Cited by 57 publications

References 47 publications

Synergistic folding of two intrinsically disordered proteins: searching for conformational selection

Synergistic folding of two intrinsically disordered proteins: searching for conformational selection

Conformational selection or induced fit: A flux description of reaction mechanism

Toward increased concentration sensitivity for continuous wave EPR investigations of spin-labeled biological macromolecules at high fields

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