Research Reveals the Structure and Self Inhibition Mechanism of Glycosylphosphatidylinositol Transamidase Complex Binding to Ligands

[Source: Shanghai Municipal Government Today Shanghai Industry Information]Recently, NatureCommunications published online the research results of Li exemplary research group of the Center for Excellence and Innovation in Molecular Cell Science of the Chinese Academy of Sciences and Qu Qianhui research group of Fudan UniversityGlycosylphosphatidylinositol TransamidaseIlluminateGPI-APBiogenesis reveals the structural basis of glycosylphosphatidylinositol (GPI) transamidase (GPI-T) complexes for recognizing a wide range of substrates and the self inhibitory mechanism for preventing accidental shear.GPI modification is a common post translational modification in eukaryotes

[Source: Shanghai Municipal Government Today Shanghai Industry Information]

Recently, NatureCommunications published online the research results of Li exemplary research group of the Center for Excellence and Innovation in Molecular Cell Science of the Chinese Academy of Sciences and Qu Qianhui research group of Fudan University
Glycosylphosphatidylinositol TransamidaseIlluminateGPI-APBiogenesis reveals the structural basis of glycosylphosphatidylinositol (GPI) transamidase (GPI-T) complexes for recognizing a wide range of substrates and the self inhibitory mechanism for preventing accidental shear.

GPI modification is a common post translational modification in eukaryotes. Water soluble precursor proteins are anchored to the cell membrane through GPI modification, performing basic biological functions including signal transduction, catalysis, and cell adhesion. GPI-T is a key enzyme in the biosynthesis pathway of GPI-anchored protein (GPI-AP), a five membered transmembrane complex responsible for cleaving the signal peptide at the C-terminus of the precursor protein and connecting the lipid molecule GPI to the newly exposed C-terminus. Unlike most proteolytic enzymes, the peptide sequences recognized by GPI-T do not have sequence uniqueness, but only have fuzzy hydrophilic and hydrophobic arrangement features. Its cleavage site (w site) is usually an amino acid with a smaller side chain, and the cleavage site is connected to the C-terminal hydrophobic segment by a peptide segment of~10 hydrophilic amino acid residues (Figure 1a). How to achieve the "contradictory unity" of substrate broadness and catalytic fidelity is an important biochemical mechanism issue in GPI-AP biosynthesis.

The research team previously analyzed the 2.53 angstrom cryoelectron microscopy three-dimensional structure of human GPI-T and revealed the assembly mechanism of its heteropentamer (Figure 1b), but the mechanism by which GPI-T achieves both substrate broadness (Figure 1a) and catalytic fidelity is still unclear.

To answer the above questions, it is necessary to analyze the complex structure of GPI-T with substrates (precursor proteins, GPI) or products (GPI-AP, signal peptides). This is because GPI molecules cannot currently be synthesized through chemical methods. The team designed cell engineering and protein engineering methods. The study used a combination of gene knockout and point mutation to keep the GPI-T complex in the "substrate binding" and "product binding" states, and analyzed the high-resolution structures of the two (Figure 2a, b). Combined with the activity analysis system, this work reveals the mechanism by which GPI-T simultaneously achieves the "contradictory unity" of substrate broadness and catalytic fidelity.

Research has shown that in terms of fidelity, GPI-T uses a self inhibitory ring to lock in the inactive conformation (Figure 2c). At the same time, the conformational changes in the activation process require breaking down several hydrogen and salt bond interactions and introducing charges and hydrophobic repulsion. This multiple protection mechanism avoids accidental hydrolysis caused by the broad nature of the substrate.

Research has shown that when the substrate binds to GPI-T, the binding energy between the signal peptide and the complex provides binding energy, promoting conformational changes in the GPI-T subunit dominated by rigid movement, initiating the activation process of energy dissipation mentioned above and opening the self inhibitory loop; At the same time, the hydrophilic part of the signal peptide near the catalytic site promotes precise reconstruction of the GPI-T catalytic center by inducing binding (Figure 2d).

Furthermore, the team designed "functional enhanced mutants" and "functional loss mutants" to demonstrate the proposed activation mechanism of self inhibition and substrate binding induction.

In addition, this work also revealed the structural basis and catalytic biochemical mechanism of GPI-T recognition of broad substrates.

The research work was supported by the National Natural Science Foundation of China and the Chinese Academy of Sciences.

Figure 1. Freezing electron microscopy structure and active center composition of GPI-T. a. Schematic diagram of GPI-T catalytic reaction and substrate characteristics; b. Freezing electron microscopy density map of GPI-T.

Figure 2. The structure of GPI-T binding to substrates and products reveals the mechanisms of self inhibition and activation. A-b: Freeze electron microscopy density map of GPI-T with substrate (a) and product (b); c. The self inhibitory ring (red) occupies the substrate (green) combined with the pocket; d. The conformational change of GPI-T from self inhibitory state (blue) to active conformation (magenta); e. The activation process of GPI-T.

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