Molecular architecture of the PTG/PP1 holoenzyme: a promising target for Lafora disease

Glycogen metabolism is coordinated by regulatory enzymes that modulate the activity of glycogen synthase (GYS) and glycogen phosphorylase (PYG), the two key proteins responsible for glycogen synthesis and degradation. Phosphorylation of both enzymes gives a signal for glycogenolysis, while dephosphorylation, carried out by type 1 protein phosphatase (PP1), promotes the glycogenesis. PP1 is a pleiotropic serine/threonine phosphatase involved in a variety of cellular processes, whose specificity relies on its scaffolding proteins. PTG (protein targeting to glycogen) is one of those regulatory subunits which brings PP1 to GYS and PYG, leading to their dephosphorylation and, thus, initiation of glycogen synthesis. In Lafora Disease (LD), a severe form of pediatric progressive myoclonus epilepsy, genetic alternations induce PTG accumulation causing formation of neurotoxic insoluble polyglucosans called Lafora bodies (LB). In LD mice models, knocking out PTG resulted in a nearly complete disappearance of LB, and resolution of the devastating symptoms, suggesting that interfering with the PTG/PP1 interaction could be a promising therapeutic strategy for LD cure. In this work, we present the first structural characterization of PTG and PTG/PP1 holoenzyme bound to a carbohydrate, both in cristallo and in solution, reveling their mechanism of interaction and giving the bases for developing small molecules targeting PTG.

In the Protein Facility at the Structural Biology Lab of Elettra, we produced a number of protein constructs of PTG and PP1 that could be co-crystallized in different forms depicting the structural organization of the PTG bound to a sugar, and to its functional partner PP1. From data collected at Elettra’s XRD2 beamline, we first determined the crystal structure of the human PTG carbohydrate binding domain (CBM21) in complex with β-cyclodextrin, revealing a typical immunoglobulin-like fold containing two carbohydrate binding sites. Site II has been identified as the main driving force of the interaction with sugars, and this motif is preserved in other CBM21 proteins. Site I could play a role as an additional support for binding long-chain polysaccharides, which could further facilitate interaction with glycogen (see Fig. 1a). Moreover, we analyzed the crystal structure of PP1 bound to N-terminal PTG peptide containing RVXF signature motif highly conserved within PP1 regulatory subunits, which reveals primarily hydrophobic and stacking interaction with two principal PP1 grooves (Fig. 1b and 1c). In collaboration with IC-CNR group (Trieste Outstation), Isothermal Titration Calorimetry (ITC) and Grating-Coupled Interferometry (GCI) were applied to measure the binding stoichiometry and the affinity. All data collected confirm that the RVXF motif is the main driving force for PTG interaction with PP1 with the additional contribution of SALK sequence, while no affinity was detected from CBM21.

Figure 1, top-story G. Petrone et al., C 1s and TPD spectra

Figure 1: Figure 1: Structural organization of the ternary PP1/PTG/cyclodextrin complex. a) Crystallographic structure of the PTG CBM21 (yellow) in complex with (β-cyclodextrin (green) showing two biding sites; b) Crystal structure of PP1 (green) in complex with the PTG peptide 81-107 (yellow). The PTG peptide is arranged in two β-strands and an intervening 310 helix and accommodates itself on PP1 β sheet; c) The peptide (yellow) perfectly adapts to the PP1 surface (green) filling the two grooves with hydrophobic side chains. d) The PTG (yellow) region (aa. 111-128) crosses the PP1 surface (green) to connect its N-terminal stretch to the CBM21 on the opposite PP1 side. e) The butterfly-shaped pseudo-knotted PP1/PTG dimer.

Finally, we were able to obtain the x-ray crystal structure of the PTG/PP1 complex resulting in being the first PP1 holoenzyme reported that includes a large portion of PTG regulatory subunit. Our data confirms that binding to PP1 occurs mainly due to the N-terminal RVXF conserved motif which is further supported by a downstream SALK sequence, while poor contribution is observed from CBM21 (Fig 1d). Additionally, the complex assembles itself as a peculiar pseudo-knotted dimer of dimers via a hydrophobic core formed by residues in all four chains (Fig. 1e). We also managed to compare the behavior in cristallo with in solution of the complex.  In collaboration with Gabriele Giachin from the University of Padua we performed a SAXS data analysis (data collected at ESRF) that suggest that PTG CBM21 can oscillate in solution, assuming multiple orientations with respect to PP1, while its N-terminal region stays bound. We hypothesize that for CBM21 domain to be able to change its position, SALK sequence detaches from PP1 leaving the only interaction via RVXF motif. This could facilitate substrate recognition and recruitment and explains the particular tetrameric crystal organization observed in the crystal structure.

In conclusion, here we performed a comprehensive structural analysis of the PTG and its complex with PP1 and sugars, shedding light on the mechanisms that tightly regulate the glycogen metabolism in brain. Given the link of PTG accumulation and Lafora bodies, this work also gives the basis for the development of novel drugs for Lafora therapy.

This research was conducted by the following research team:

Marta Stefania Semrau1,2, Gabriele Giachin3, Sonia Covaceuszach4, Alberto Cassetta4, Paola Storici2 and Graziano Lolli1

1 Department of Cellular, Computational and Integrative Biology - CIBIO, University of Trento, Trento, Italy
2 Elettra-Sincrotrone Trieste S.C.p.A., Trieste, Italy
3 Department of Chemical Sciences (DiSC), University of Padua, Padova, Italy
4 Institute of Crystallography - C.N.R. - Trieste Outstation, Trieste, Italy

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M.S. Semrau, G. Giachin, S. Covaceuszach, A. Cassetta, N. Demitri, P. Storici,  G. Lolli, "Molecular architecture of the glycogen- committed PP1/PTG holoenzyme", Nat. Commun. 13, 6199 (2022); DOI: 10.1038/s41467-022-33693-z

Last Updated on Friday, 27 January 2023 15:03