Exploring the systematic effect of N-substituted PxxP motifs on peptoid affinity to ARHGEF5/TIM SH3 domain and its relationship with ARHGEF5/TIM activation.

The TIM protein is a short isoform of full-length Rho guanine nucleotide exchange factor 5 (ARHGEF5), which acts as a functional regulator of Rho-dependent signaling pathways by activating the Rho family of GTPases. The activation is auto-inhibited by a putative helix N-terminal to the DH domain of TIM, which is stabilized by the intramolecular interaction of C-terminal SH3 domain with a proline-rich region SSPRQPRKAL (termed as SSP peptide) between the putative helix and the DH domain. Previously, we demonstrate that the auto-inhibitory state of TIM protein can be relieved by targeting its SH3 domain with rationally designed peptide ligands. However, the designed natural peptides have only a moderately increased affinity (~2-fold) as compared to the cognate SH3-SSP interaction and are susceptible to protease degradation. Here, considering that proline is the only endogenous N-substituted amino acid that plays a critical role in SH3-peptide recognition, the two key proline residues Pro49 and Pro52 in the core PxxP motif of SSP peptide are systematically replaced by 19 N-substituted amino acid types to derive a variety of nonnatural peptoid ligands for TIM SH3 domain. Dynamics and energetics analyses reveal that the replacement would impair the active polyproline II (PPII) helical conformation of SSP peptide due to lack of structural constraint introduced by the five-membered ring of native proline side-chains, thus increasing the peptide flexibility that could incur a large entropy penalty upon binding to the domain. However, the impairment is not very significant and the peptide affinity may also be restored and improved if the N-substituted motif of derived peptiod ligands can effectively interact with the PxxP-binding site of TIM SH3 domain. Consequently, a number of potent peptoids are successfully designed by fluorescence spectroscopy confirmation, in which three (ie, SSP[N-Ile49, N-Asn52], SSP[N-Phe49, N-Gln52], and SSP[N-Tyr49, N-Asn52]) exhibit considerably increased affinity (K = 0.09, 0.07, and 0.04 μM, respectively) relative to the native SSP peptide (K = 0.87 μM). In addition, guanine nucleotide exchange assays also substantiate that the designed SH3-targeted peptiods can effectively enhance TIM-catalyzed RhoA exchange activity (EA), which is observed to present an exponential relationship with the measured SH3-peptoid binding affinity (pK ).