Crystal structure of the GTP-binding protein-like domain of AGAP1

Abstract

AGAP1 is often considered to regulate membrane trafficking, protein transport and actin cytoskeleton dynamics. Recent studies have shown that aberrant expression of AGAP1 is associated with many diseases, including neurodevelopmental disorders and acute lymphoblastic leukemia. It has been proposed that the GTP-binding protein-like domain (GLD) is involved in the binding of cofactors and thus regulates the catalytic activity of AGAP1. To obtain a better understanding of the pathogenic mechanism underpinning AGAP1-related diseases, it is essential to obtain structural information. Here, the GLD (residues 70–235) of AGAP1 was overexpressed in Escherichia coli BL21 (DE3) cells. Affinity and gel-filtration chromatography were used to obtain AGAP1GLD with high purity for crystallization. Using the hanging-drop vapor-diffusion method with the protein at a final concentration of 20 mg ml−1, AGAP1GLD protein crystals of suitable size were obtained. The crystals were found to diffract to 3.0 Å resolution and belonged to space group I4, with unit-cell parameters a = 100.39, b = 100.39, c = 48.08 Å. The structure of AGAP1GLD exhibits the highly conserved functional G1–G5 loops and is generally similar to other characterized ADP-ribosylation factor (Arf) GTPase-activating proteins (GAPs), implying an analogous function to Arf GAPs. Additionally, this study indicates that AGAP1 could be classified as a type of NTPase, the activity of which might be regulated by protein partners or by its other domains. Taken together, these results provide insight into the regulatory mechanisms of AGAP1 in cell signaling.

Transcription factor 12‐mediated self‐feedback regulatory mechanism is required in DUX4 fusion leukaemia

“…AGAP1 is an inclusion‐related protein that activates the GTPase of ADP ribosylation factor in a phosphoinositol‐dependent manner, influencing the formation of the actin cytoskeleton. 41 Previous studies have reported that AGAP1 was highly expressed in IGH::DUX4 types of leukaemia. 2 CD371 is a protein associated with cell lectin CLEC12A expression, and studies have shown that CD371 is specifically highly expressed in IGH::DUX4 patients.…”

“…To investigate whether and how TCF12 facilitated IGH::DUX4‐mediated transcription, the abnormal transcriptional activation of IGH::DUX4 targeted genes such as AGAP1 , CLEC12A , ERG alt , CLEC12A alt and C6orf89 alt was measured by qRT‐PCR in leukaemia cell lines REH and NALM‐6. AGAP1 is an inclusion‐related protein that activates the GTPase of ADP ribosylation factor in a phosphoinositol‐dependent manner, influencing the formation of the actin cytoskeleton 41 . Previous studies have reported that AGAP1 was highly expressed in IGH::DUX4 types of leukaemia 2 .…”

Effects of dietary selenium deficiency and supplementation on liver in grazing sheep: insights from transcriptomic and metabolomic analysis

“…Among them, AGAP1 was widely recognized as a gene involved in the regulation of membrane transport and protein transport. Additionally, it has been linked to psychiatric disorders such as autism and schizophrenia ( 32 ). The NFI family is a crucial family of transcription factors involved in adenoviral DNA replication, which consists of 4 genes, including NFIA , NFIB , NFIC , and NFIX , in most vertebrates.…”

Functional Role of AGAP2/PIKE-A in Fcγ Receptor-Mediated Phagocytosis

“…Taken together the data suggest the GAP domain is not sufficient for targeting AGAP2 to the phagocytic cup. Several reports suggest that the GLD domain binds GTP or other nucleotide triphosphates and that intra-molecular interactions with the GAP domain enhance its atypical GTPase activity [6,7,[79][80][81].…”