Structural and Biochemical Characterization of the Interaction between LGN and Frmpd1

Abstract

The tetratricopeptide repeat (TPR) motif-containing protein LGN binds multiple targets and regulates their subcellular localizations and functions during both asymmetric and symmetric cell divisions. Here, we characterized the interaction between LGN-TPR motifs and FERM and PDZ domain containing 1 (Frmpd1) and reported the crystal structure of the complex at 2.4 Å resolution. A highly conserved fragment at the center of Frmpd1 of ~ 20 residues was found to be necessary and sufficient to bind to LGN-TPR. This Frmpd1 fragment forms an extended structure and runs along the concave channel of the TPR superhelix in an antiparallel manner in the complex. Structural comparisons and biochemical studies of LGN/Frmpd1 and other known LGN/target interactions demonstrate that the LGN-TPR motifs are versatile and capable of recognizing multiple targets via diverse binding modes. Nevertheless, a conserved “E/QxEx_4-5E/D/Qx_1-2K/R” motif in LGN/Pins (partner of inscuteable) TPR binding proteins has been identified.

Introduction

A fundamental mechanism for cellular differentiation and self-renewal in invertebrate and vertebrate development is asymmetric cell division, which requires precise orientations of mitotic spindles along intrinsic or extrinsic polarity axes.[1], [2], [3], [4], [5] In Drosophila neuroblasts, a key regulator of cell polarity and spindle orientation is Pins (partner of inscuteable; LGN/AGS3 in mammals), which plays roles in several crucial pathways such as the Par3/Insc (inscuteable; mInsc in mammals)/Pins/Gαi and the Pins/Mud (mushroom body defect; NuMA in mammals)/dynein pathways.[6], [7], [8], [9], [10], [11] Pins/LGN consist of eight tetratricopeptide repeat (TPR) motifs in their N-terminal half[12], [13], [14] and three or four GoLoco motifs (also referred to as G-protein regulatory or GPR motifs) in the C-terminal half. Both the TPR and the GoLoco motifs are implicated in protein–protein interactions.[15], [16] Each GoLoco motif of Pins/LGN is capable of binding to GDP-bound Gαi,[7], [17], [18] and this interaction is responsible for stable cortical localization of Pins/LGN.¹⁹ The TPR motifs of Pins/LGN have multiple binding partners. Pins/LGN localize at the apical cortex with the Par complex by binding to Insc/mInsc via their eight TPR motifs.[6], [12], [20], [21] This apical localized Pins/LGN recruits Mud/NuMA to regulate the mitotic spindle orientation.[8], [9], [10], [22]

The canonical TPR motif is a 34-amino-acid protein–protein interaction module found in multiple copies in a wide range of proteins with diverse functions such as cell cycle regulations, gene transcription and splicing processes, protein trafficking, and protein folding.[16], [23] Each TPR motif adopts a helix–turn–helix fold, and multiple TPR motifs often form a right-handed superhelical structure with a continuous amphipathic groove suitable for the recognition of target proteins. The molecular mechanisms governing the formations of the TPR-mediated LGN/mInsc, Pins/Insc, and LGN/NuMA complexes have been recently elucidated.[12], [13], [14] The TPR motifs of LGN/Pins have distinct structural features compared to those in canonical TPR motifs. The αA and αB helices of LGN/Pins TPRs are longer (40 amino acids per TPR) than the corresponding helices in the canonical TPR motifs, and the packing geometry is also different. Although the extended helix was also observed in other TPR proteins before,[24], [25] it is first reported in LGN/Pins that these longer helices were found to appear regularly in tandem repeats.[12], [13], [14] In the complexes, the LGN/Pins binding fragments of mInsc/Insc and NuMA form elongated structures and bind to the concave channel formed by the TPR motifs. Specifically, the NuMA fragment adopts an extended conformation, whereas the mInsc/Insc fragments form a short α-helix followed by an extended region. It is noted that the LGN/Pins TPR-binding regions of NuMA and mInsc/Insc share very low sequence identity, implying that LGN/Pins TPR motifs are structurally versatile in binding to diverse target proteins.

FERM and PDZ domain containing 1 (Frmpd1) was first reported as a regulatory binding partner of AGS3, and the binding sites between AGS3 and Frmpd1 were mapped to the eight TPR motifs of AGS3 and a short fragment of Frmpd1 (amino acids 901–938).²⁶ Another recent study has demonstrated that Frmpd1 is also a LGN-TPR binder, and a 50-residue fragment (amino acids 901–951) of Frmpd1 was suggested to bind to LGN following a similar mode as mInsc does.¹⁴ As the TPR motifs of AGS3 and LGN are highly similar (77% sequence identity), we assume that they bind to Frmpd1 with a more or less same mechanism. However, the structural basis underlying the interaction between Frmpd1 and LGN/AGS3 is not clear. In this work, we characterized the interaction between LGN-TPR and Frmpd1 in detail. The high-resolution crystal structure of the LGN/Frmpd1 complex solved in this work reveals that LGN binds to Frmpd1 following a mechanism similar to that of NuMA but distinct from that of mInsc. Structural comparison of known LGN/Pins TPR/target complexes reveals the presence of a conserved “E/QxEx_4-5E/D/Qx_1-2K/R” motif in LGN/Pins TPR binding proteins. Further competition experiments demonstrate that NuMA and Frmpd1 compete with each other in binding to LGN, whereas mInsc can efficiently block the associations of both Frmpd1 and NuMA with LGN.