Summary
Pin1 is a modular peptidyl-prolyl isomerase specific for phosphorylated Ser/Thr-Pro (pS/T-P) motifs, typically within intrinsically disordered regions (IDRs) of signaling proteins. Pin1 consists of two flexibly linked domains: an N-terminal WW domain for substrate binding and a larger C-terminal peptidyl-prolyl isomerase (PPIase) domain. Previous studies showed that binding of phosphopeptide substrates to Pin1 could alter Pin1 interdomain contact, strengthening or weakening it, depending on the substrate sequence. Thus, substrate-induced changes in interdomain contact may act as a trigger within the Pin1 mechanism. Here, we investigate this possibility via NMR studies of several Pin1 mutants. Our findings provide new mechanistic insights for those substrates that reduce interdomain contact. Specifically, the reduced interdomain contact can allosterically enhance PPIase activity relative to that when the contact is sustained. These findings suggest Pin1 interdomain contact can negatively regulate its activity.
The Pin1 peptidyl-prolyl isomerase (PPIase) catalyzes isomerization of pSer/pThr-Pro motifs in regulating the cell cycle. Peptide substrates, Ac–Phe–Phe–phosphoSer–Pro–Arg–p-nitroaniline, were synthesized in unlabeled form, and with deuterium labeled Ser-d3 and Pro-d7 amino acids. Kinetic data was collected as a function of Pin1 concentration to measure kinetic isotope effects (KIE) on catalytic efficiency (kcat/Km). The normal secondary (2°) KIE value measured for the Ser-d3 substrate (kH/kD = 1.6 ± 0.2) indicates that the serine carbonyl does not rehybridize from sp2 to sp3 in the rate-determining step, ruling out a nucleophilic addition mechanism. The normal 2° KIE can be explained by hyperconjugation between Ser α-C–H/D and C=O, and release of steric strain upon rotation of the amide bond from cis to syn-exo. The inverse 2° KIE value (kH/kD = 0.86 ± 0.08) measured for the Pro-d7 substrate indicates rehybridization of the prolyl nitrogen from sp2 to sp3 during the rate-limiting step of isomerization. No solvent kinetic isotope was measured by NMR exchange spectroscopy (EXSY) (kH2O/kD2O = 0.92 ± 0.12), indicating little or no involvement of exchangeable protons in the mechanism. These results support the formation of a simple twisted-amide transition state as the mechanism for peptidyl prolyl isomerization catalyzed by Pin1. A model of the reaction mechanism is presented using crystal structures of Pin1 with ground state analogues and an inhibitor that resembles a twisted amide transition state.
In this study, we targeted the N-terminal domain (NTD) of transactive response (TAR) DNA binding protein (TDP-43), which is implicated in several neurodegenerative diseases. In silico docking of 50K compounds to the NTD domain of TDP-43 identified a small molecule (nTRD22) that is bound to the N-terminal domain. Interestingly, nTRD22 caused allosteric modulation of the RNA binding domain (RRM) of TDP-43, resulting in decreased binding to RNA in vitro. Moreover, incubation of primary motor neurons with nTRD22 induced a reduction of TDP-43 protein levels, similar to TDP-43 RNA binding-deficient mutants and supporting a disruption of TDP-43 binding to RNA. Finally, nTRD22 mitigated motor impairment in a Drosophila model of amyotrophic lateral sclerosis. Our findings provide an exciting way of allosteric modulation of the RNA-binding region of TDP-43 through the N-terminal domain.
Gram-positive bacteria assemble pili (fimbriae) on their surfaces to adhere to host tissues and to promote polymicrobial interactions. These hair-like structures, although very thin (1 to 5 nm), exhibit impressive tensile strengths because their protein components (pilins) are covalently crosslinked together via lysine–isopeptide bonds by pilus-specific sortase enzymes. While atomic structures of isolated pilins have been determined, how they are joined together by sortases and how these interpilin crosslinks stabilize pilus structure are poorly understood. Using a reconstituted pilus assembly system and hybrid structural biology methods, we elucidated the solution structure and dynamics of the crosslinked interface that is repeated to build the prototypical SpaA pilus from Corynebacterium diphtheriae. We show that sortase-catalyzed introduction of a K190-T494 isopeptide bond between adjacent SpaA pilins causes them to form a rigid interface in which the LPLTG sorting signal is inserted into a large binding groove. Cellular and quantitative kinetic measurements of the crosslinking reaction shed light onto the mechanism of pilus biogenesis. We propose that the pilus-specific sortase in C. diphtheriae uses a latch mechanism to select K190 on SpaA for crosslinking in which the sorting signal is partially transferred from the enzyme to a binding groove in SpaA in order to facilitate catalysis. This process is facilitated by a conserved loop in SpaA, which after crosslinking forms a stabilizing latch that covers the K190-T494 isopeptide bond. General features of the structure and sortase-catalyzed assembly mechanism of the SpaA pilus are likely conserved in Gram-positive bacteria.
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