Global Conformational Selection and Local Induced Fit for the Recognition between Intrinsic Disordered p53 and CBP

<div><p>The transactivation domain (TAD) of tumor suppressor p53 can bind with the nuclear coactivator binding domain (NCBD) of cyclic-AMP response element binding protein (CBP) and activate transcription. NMR experiments demonstrate that both apo-NCBD and TAD are intrinsic disordered and bound NCBD/TAD undergoes a transition to well folded. The recognition mechanism between intrinsic disordered proteins is still hotly debated. Molecular dynamics (MD) simulations in explicit solvent are used to study the recognition mechanism between intrinsic disordered TAD and NCBD. The average RMSD values between bound and corresponding apo states and Kolmogorov-Smirnov <i>P</i> test analysis indicate that TAD and NCBD may follow an induced fit mechanism. Quantitative analysis indicates there is also a global conformational selection. In summary, the recognition of TAD and NCBD might obey a local induced fit and global conformational selection. These conclusions are further supported by high-temperature unbinding kinetics and room temperature landscape analysis. These methods can be used to study the recognition mechanism of other intrinsic disordered proteins.</p> </div

Ribbon representation of the NMR structure for TAD-NCBD complex (pdb code: 2L14).

<p>Helices α1, α2 and α3 of NCBD are colored with blue, cyan and green, respectively. Helices α4 and α5 of TAD are colored with yellow and red, respectively. N and C-terminal domains are labeled.large number of proteins (between 25% and 41%) are intrinsically disordered, however, these proteins also play important function in cell signaling and cancer upon binding with multiple interaction partners. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059627#pone.0059627-Liu1" target="_blank">[11]</a> In this study, NMR experiments indicate that apo-TAD is intrinsic disordered protein and apo-NCBD is not entirely unstructured with a helical molten globule <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059627#pone.0059627-Demarest1" target="_blank">[3] </a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059627#pone.0059627-Wells1" target="_blank">[12]</a>. Upon binding each other, both NCBD and TAD undergo a transition from disordered to well folded. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059627#pone.0059627-Lee1" target="_blank">[10]</a> This suggests that both NCBD and TAD have significant conformational adjustment in complex. These experimental observations raise an interesting question if these intrinsic disordered NCBD and TAD obey an induced fit upon binding. To reveal this question, we utilize all atom molecular dynamics (MD) simulations in explicit solvent to analyze the coupling between binding and folding in the NCBD-TAD complex. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059627#pone.0059627-Henkels1" target="_blank">[13]</a>.</p

Variations and landscapes of distance difference for apo-NCBD, apo-TAD and complex.

<p>A: Cα atomic fluctuation for NCBD and TAD. B: Φ/Ψ variation for NCBD and TAD. C: landscapes of distance difference for NCBD. D: landscapes of distance difference for TAD.</p

Local conformational RMSD differences between bound and apo conformations as a function of distance from the centroid of binding partner and statistical significance of conformational selection in NCBD and TAD binding.

<p>Average local RMSD for 10 pairs of bound conformations and the most similar apo conformation and for 90 pairs of bound NCBD and the other apo conformations, as a function of distance from the centroid of binding partner. A: NCBD. B: TAD. C: NCBD. D: TAD.</p

Identification of transition state.

<p>A: Cα RMSD of complex at 498K, the first 1400 ps in 498K trajectory are inset, the time point of transition state is labeled. B: two-dimension projection with root mean square deviation (RMSD), the time span of each cluster is labeled. C: average structure of transition state for bound and apo states of NCBD and TAD.</p

Predicted Ф-values for apo and bound states for NCBD and TAD.

<p>Predicted Ф-values for apo and bound states for NCBD and TAD.</p

Free energy landscapes with respect to Rg and RMSD for apo and bound states of NCBD and TAD.

<p>A: apo-NCBD. B: apo-TAD. C: bound NCBD. D: bound TAD.</p

Interactions between NCBD and TAD.

<p>A: Hydrophobic contact. 1 for Ala42/Leu73; 2 for Ile44/Met87; 3 for Ala42/Pro74; 4 for Leu17/Phe101; 5 for Met40/Met87; 6 for Leu17/Ile97; 7 for Leu14/Phe101; and 8 for Phe43/Trp100. B: Electrostatic interaction. 1 for Arg47/Asp96; 2 for Lys45/Glu75; 3 for Lys50/Asp96; 4 for Arg3/Glu75; and 5 for Lys18/Asp104. C: Hydrogen bond. 1 for OD1(Asp96)/NH2(Arg47); and 2 for OD2(Asp96)/NH2(Arg47).</p

Test and Evaluation of <i>ff99IDPs</i> Force Field for Intrinsically Disordered Proteins

Over 40% of eukaryotic proteomic sequences have been predicted to be intrinsically disordered proteins (IDPs) or intrinsically disordered regions (IDRs) and confirmed to be associated with many diseases. However, widely used force fields cannot well reproduce the conformers of IDPs. Previously the <i>ff99IDPs</i> force field was released to simulate IDPs with CMAP energy corrections for the eight disorder-promoting residues. In order to further confirm the performance of <i>ff99IDPs</i>, three representative IDP systems (arginine-rich HIV-1 Rev, aspartic proteinase inhibitor IA₃, and α-synuclein) were used to test and evaluate the simulation results. The results show that for free disordered proteins, the chemical shifts from the <i>ff99IDPs</i> simulations are in quantitative agreement with those from reported NMR measurements and better than those from <i>ff99SBildn</i>. Thus, <i>ff99IDPs</i> can sample more clusters of disordered conformers than <i>ff99SBildn</i>. For structural proteins, both <i>ff99IDPs</i> and <i>ff99SBildn</i> can well reproduce the conformations. In general, <i>ff99IDPs</i> can successfully be used to simulate the conformations of IDPs and IDRs in both bound and free states. However, relative errors could still be found at the boundaries of ordered residues scattered in long disorder-promoting sequences. Therefore, polarizable force fields might be one of the possible ways to further improve the performance on IDPs

Allosteric Autoinhibition Pathway in Transcription Factor ERG: Dynamics Network and Mutant Experimental Evaluations

Allosteric autoinhibition exists in many transcription factors. The ERG proteins exhibit autoinhibition on DNA binding by the C-terminal and N-terminal inhibitory domains (CID and NID). However, the autoinhibition mechanism and allosteric pathway of ERG are unknown. In this study we intend to elucidate the residue-level allosteric mechanism and pathway via a combined approach of computational and experimental analyses. Specifically computational residue-level fluctuation correlation data was analyzed to reveal detailed dynamics signatures in the allosteric autoinhibition process. A hypothesis of “NID/CID binding induced allostery” is proposed to link similar structures and different protein functions, which is subsequently validated by perturbation and mutation analyses in both computation and experiment. Two possible allosteric autoinhibition pathways of L286-L382-A379-G377-I360-Y355-R353 and L286-L382-A379-G377-I360-Y355- A351-K347-R350 were identified computationally and were confirmed by the computational and experimental mutations. Specifically we identified two mutation sites on the allosteric inhibition pathways, L286P/Q383P (NID/CID binding site) and I360G (pathway junction), which completely restore the wild type DNA binding affinity. These results suggest that the putative protein structure–function relationship may be augmented with a general relationship of protein “structure/fluctuation–correlation/function” for more thorough analyses of protein functions