AntBio GST Antibody Supports Human KICSTOR Complex Research

Introduction

On November 6, 2025, the research group of Su Mingyuan from the Department of Biochemistry, School of Medicine, Southern University of Science and Technology, together with collaborators, published an article entitled "Architecture of the human KICSTOR and GATOR1–KICSTOR complexes" in 《Nature Structural & Molecular Biology》. The article mainly uses cryo-electron microscopy technology combined with biochemical and cell biology methods to resolve, for the first time, the high-resolution three-dimensional structure of the human KICSTOR complex and the supercomplex composed of it and GATOR1, and systematically clarifies the molecular mechanism by which KICSTOR, as a scaffold protein, anchors GATOR1 to the lysosomal membrane, thereby regulating the mTORC1 signaling pathway.

Research Background

KICSTOR is a heterotetrameric complex composed of KPTN, ITFG2, C12orf66 and the scaffolding protein SZT2. Its function is to anchor GATOR1, an inhibitor of mTORC1, to the lysosomal surface, playing a crucial role in nutrient sensing and the regulation of mTORC1 signaling. Mutations in KICSTOR subunits are associated with severe neurodevelopmental disorders and epilepsy, but the mechanisms of its structural assembly and interaction with GATOR1 remain unclear. Figure 1 Cryo-EM structure of the human KICSTOR complex a. Schematic diagram of the structure of human KICSTOR subunits. Dashed lines indicate unresolved regions. b. Purified KICSTOR protein assay. c. Superose 6 size-exclusion chromatography profile of the human KICSTOR complex.

The asterisk marks the chromatographic peak corresponding to KICSTOR. d. The four components: KPTN, ITFG2, C12orf66, and the scaffold protein SZT2. e. Cryo-EM density map (left) and model (right) of human KICSTOR.

Article discovery

Structure Analysis of KICSTOR

Four protein components of KICSTOR were expressed the authors. After purification, multiple conformational states of the full-length KICSTOR and its C-terminal core complex (CCC) were analyzed by cryo-electron microscopy with resolutions between 2.90 and 3.19.

⭐ SZT2 exhibits a crescent-shaped long-shank structure, consisting of 12 SZ units in series. Its C-terminal is bound to C12orf66 through SZ7 units, and interacts with KPTN-ITFG2 heterodimers through SZ4 and SZ5 units, respectively.

⭐ KPTN and ITFG2 form a WD40β-propeller structure and stabilize each other through the C-terminal helix.

⭐ C12orf66 has structural features similar to the Roadblock domain and Brox family, its N-terminal domain is helically bound to the C-terminal of SZT2.

Interaction between KICSTOR and GATOR1

⭐ By resolving the structure of the KICSTOR–GATOR1 supercomplex (with a resolution of 2.97 Å), it was found that GATOR1 binds to the N-terminal domain of SZT2 through its subunit NPRL3.

⭐ Key interface residues include E115, D119, E194, and D195 of SZT2, as well as R147, R148, and H155 of NPRL3. Mutations in these residues significantly weaken the binding between the two.

⭐ After reintroducing wild-type NPRL3 into NPRL3 knockout cells, the sensitivity of mTORC1 to amino acids was restored. In contrast, mutants with SZT2 binding defects (including NPRL3Δ216−231 and single or combined mutants of NPRL3 such as R147G, R148G, and H155A) failed to restore sensitivity to amino acid deprivation. This indicates that the KICSTOR complex is necessary for GATOR1 to fully inhibit mTORC1 activity.

⭐ Cross-linking mass spectrometry analysis further verified the specific interaction between SZT2 and NPRL3, and no cross-linking between SZT2 and other subunits of GATOR1 was detected.

KICSTOR does not alter the GAP activity of GATOR1

In vitro GTPase assays showed that the presence of KICSTOR does not affect the GAP activity of GATOR1 towards RagA/B, indicating that its main function is to act as a scaffold to localize GATOR1 to lysosomes rather than to regulate its catalytic efficiency.

KICSTOR binds to the lysosomal membrane through electrostatic interactions

⭐ Liposome binding experiments showed that KICSTOR preferentially binds to negatively charged lipids, such as phosphatidylserine and various phosphoinositides.

⭐ SZT2 and C12orf66 are key membrane-binding subunits, and both have positively charged patches on their surfaces, which may interact with the negatively charged lipid head groups.

⭐ The KPTN–ITFG2 heterodimer itself has no membrane-binding ability, and its surface potential is mainly negative.

The lysosomal localization of KICSTOR depends on SZT2 and C12orf66

⭐ Cell imaging experiments showed that KICSTOR can be localized to lysosomes only when SZT2 and C12orf66 are co-expressed.

⭐ Disruption of the SZT2–C12orf66 interaction (such as the C12orf66 L216D/F269D mutation) leads to the mislocalization of KICSTOR in the cytoplasm.

6. Functional verification: The SZT2–GATOR1 interaction is crucial for mTORC1 regulation

⭐ In NPRL3 or SZT2 knockout cells, mutants that disrupt the SZT2–NPRL3 interaction cannot restore the sensitivity of mTORC1 to amino acid starvation.

⭐ The TFE3–GFP nucleocytoplasmic shuttling experiment further confirmed that SZT2 mutation leads to the continuous activation of mTORC1 and the retention of TFE3 in the cytoplasm.

Conclusion

This study is the first to structurally reveal that KICSTOR, acting as a long-handled crescent-shaped scaffold, assembles KPTN–ITFG2 and C12orf6 through its C-terminal, and binds to NPRL3 of GATOR1 via its N-terminal, thereby anchoring GATOR1 to the lysosomal membrane. KICSTOR achieves its membrane localization through the interaction of SZT2 and C12orf66 with negatively charged lysosomal membrane lipids. This work clarifies the scaffolding function of KICSTOR in the nutrient-sensing pathway and provides a structural basis for understanding the mechanism by which its mutations cause neurodevelopmental diseases.