Structural modeling of osteoarthritis ADAMTS4 complex with its cognate inhibitory protein TIMP3 and rational derivation of cyclic peptide inhibitors from the complex interface to target ADAMTS4.

The ADAMTS4 (a disintegrin and metalloproteinase with thrombospondin motifs 4) enzyme is a matrix-associated zinc metalloendopeptidase that plays an essential role in the degradation of cartilage aggrecan in arthritic diseases and has been recognized as one of the most primary targets for therapeutic intervention in osteoarthritis (OA). Here, we reported computational modeling of the atomic-level complex structure of ADAMTS4 with its cognate inhibitory protein TIMP3 based on high-resolution crystal template. By systematically examining the modeled complex structure we successfully identified a short inhibitory loop (62EASESLC68) in TIMP3 N-terminal inhibitory domain (NID) that directly participates in blocking the enzyme's active site, which, and its extended versions, were then broken from the full-length protein to serve as the peptide inhibitor candidates of ADAMTS4. Atomistic molecular dynamics simulation, binding energetic analysis, and fluorescence-based assay revealed that the TIMP3-derived linear peptides can only bind weakly to the enzyme (Kd = 74 ±â€¯8 µM), which would incur a considerable entropy penalty due to the high conformational flexibility and intrinsic disorder of these linear peptides. In this respect, we proposed a cyclization strategy to improve enzyme-peptide binding affinity by, instead of traditionally maximizing enthalpy contribution, minimizing entropy cost of the binding, where a disulfide bond was added across the two terminal residues of linear peptides, resulting in a number of TIMP3-derived cyclic peptides. Our studies confirmed that the cyclization, as might be expected, can promote peptide binding capability against ADAMTS4 substantially, with affinity increase by 3-fold, 9-fold and 7-fold for cyclic peptides , and , respectively.

Computational studies of the principle of dynamic-change-driven protein interactions.

Dynamic allostery emphasizes a role of entropy change manifested as a sole change in protein fluctuations without structural changes. This kind of entropy-driven effect remains largely understudied. The most significant examples involve protein-ligand interactions, leaving protein-protein interactions, which are critical in signaling and other cellular events, largely unexplored. Here we study an example of how protein-protein interaction (binding of Ras to the Ras binding domain [RBD] of the effector protein Raf) affects a subsequent protein association process (Ras dimerization) by quenching Ras internal motions through dynamic allostery. We also investigate the influence of point mutations or ambient temperature, respectively, on the protein dynamics and interaction of two other systems: in adenylate kinase (ADK) and in the EphA2 SAM:Ship2 SAM complex. Based on these examples, we postulate that there are different ways in which dynamic-change-driven protein interactions are manifested and that it is likely a general biological phenomenon.

Rational cyclization-based minimization of entropy penalty upon the binding of Nrf2-derived linear peptides to Keap1: A new strategy to improve therapeutic peptide activity against sepsis.

Nrf2 is a critical regulator of innate immune response and survival during sepsis, which is constitutively degraded through binding to the Keap1 adapter protein of E3 ubiquitin ligase. Two linear peptides DLG and ETG derived from, respectively, the low-affinity and high-affinity motifs of Nrf2 binding site exhibit self-binding affinity to Keap1 central hole (active pocket); they can be exploited as therapeutic self-inhibitory peptides to disrupt the Nrf2-Keap1 interaction. Molecular dynamics simulation and binding energetics decomposition reveal that the two peptides possess large flexibility and intrinsic disorder in unbound free state, and thus would incur a considerable entropy penalty upon binding to Keap1. In order to improve Keap1-peptide binding affinity (or free energy ΔG), instead of traditionally increasing favorable enthalpy contribution (ΔH) we herein describe a rational peptide cyclization strategy to minimize unfavorable entropy penalty (ΔS) upon the binding of Nrf2-derived linear peptides to Keap1. Crystal structure analysis impart that the native active conformations of DLG and ETG peptides bound with Keap1 are folded into U-shape and hairpin configurations, respectively, and adopt their turning head to insert into the central hole of Keap1. Here, cyclization is designed by adding a disulfide bond across the two arms of DLG U-shape or ETG hairpin, which would not influence the direct intermolecular interaction between Keap1 and peptide as well as desolvation effect involved in the interaction, but can effectively constrain the conformational flexibility and disorder of the two peptides in free state, thus largely minimizing entropy penalty upon the binding. Both free energy calculation and binding affinity assay substantiate that the cyclization, as might be expected, can moderately or considerably enhance peptide binding potency to Keap1, with affinity (dissociation constant Kd) increase by 1.4-7.5-fold for designed cyclic peptides relative to their linear counterparts.