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[Preprint]. 2025 Jun 25:2025.06.24.661328.
doi: 10.1101/2025.06.24.661328.

Rac1 Selectively Binds a Specific Lamellipodin Isoform via a Noncanonical Helical Interface

Tong Gao  1 , Pingfeng Zhang  1 , Alison M Kurimchak  1 , James S Duncan  1 , Jinhua Wu  1
Affiliations

Rac1 Selectively Binds a Specific Lamellipodin Isoform via a Noncanonical Helical Interface

Tong Gao et al. bioRxiv. .

Update in

Abstract

Lamellipodin (Lpd) is a multifunctional adapter protein that regulates cell migration and adhesion by recruiting Ena/VASP proteins to the leading edge and modulating actin polymerization. The interaction of Lpd and Rho family or Ras family GTPases is crucial for regulating actin dynamics. Contrary to previous assumptions that the main Lpd isoform interacts with Rac1, here we show that strong and specific binding to Rac1 is instead mediated by the short isoform Lpds. This interaction is dependent on Rac1's GTPase activity and a short insertion (cs2) within the coiled-coil (CC) region unique to the Lpds isoform. Structural modeling and mutagenesis analyses further reveal that Lpds engages Rac1 through a noncanonical, single-helix binding mode distinct from the classical helical pair configuration. Our results reveal a novel isoform-dependent GTPase:effector binding mode and suggest a critical regulatory pathway that may represent a promising therapeutic target in Rac1-driven cancer progression.

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Conflict of interest statement

Declaration of Interests The authors declare no competing financial interests.

Figures

Figure 1.
Figure 1.. The Lpds isoform of Lpd has strong binding affinity with activated Rac1 specifically.
(A). Schematic representation of Lpd isoforms. TBS: talin binding site; IN: autoinhibitory segment; CC: coiled-coil region; CS: insertion of cc; RA, Ras-association domain; PH, pleckstrin homology domain. A talin-binding site (green) is located at the N-terminal end of Lpd and is conserved across all isoforms. The CS is depicted in orange, the CC is in red, the RA (Ras-associating) domain is in yellow, and the PH (pleckstrin homology) domain is in cyan. (B). The Co-IP assay of GFP-tagged full-length Lpd and Lpds and HA-tagged constitutively active Rac1 form (Rac1 Q61L). Cell lysate and IP samples are detected by Western blot using anti-HA antibody and anti-GFP antibody.
Figure 2.
Figure 2.. Rac1:Lpds interaction requires the GTPase activity of Rac1 and the CS insertion of Lpds.
(A). Co-IP assay of GFP-tagged Lpds1-PH with HA-tagged Rac1 Q61L, Rap1 G12V, M-Ras Q71L, M-Ras WT, H-Ras G12V and R-Ras G12V. (B). Co-IP assay of HA-tagged wild-type Rac1 and Rac1 Q61L with GFP-tagged Lpds FL, 1-PH, csRAPH, cs-cc and Lpd ccRAPH. The schematic diagrams of the constructs are shown above, with the same domain color codes as in Figure 1A. (C). Co-IP assay of HA-tagged wild-type Rac1, constitutively actived Rac1 form (Rac1 Q61L) and inactivated Rac1 form (Rac1 Q61L/T17N) with GFP-tagged Lpds csRAPH. (D). Pulldown assay of GST-tagged wild-type Rac1, Rac1 Q61L and Rac1 Q61L/G12V with His-tagged Lpds csRAPH.
Figure 3.
Figure 3.. Lpds binds Rac1 through a non–β-sheet interface distinct from the RIAM:Rap1 interaction.
(A). Structural alignment of the AlphaFold-predicted Lpds model with the crystal structure of the RIAM:Rap1 complex. Lpds domains are colored as follows: CS insertion (orange), CC (red), RA domain (yellow), and PH domain (cyan). The RIAM:Rap1 complex is shown in gray. The zoomed-in panel (bottom right) highlights key salt bridges in the RIAM:Rap1 interface (Asp33:Lys213 and Tyr40:Lys193). The corresponding residues in Lpds (Lys335 and Lys355), selected for mutagenesis, are labeled. (B). Sequence alignment of the Switch I region from Rac1, Rap1, H-Ras, and M-Ras. Conserved residues are highlighted in green. Rac1 residues Ile33 and Tyr40, corresponding to salt bridge–forming residues in Rap1, are marked in red. (C) Sequence alignment of Lpd, Lpds and RIAM, with lysine residues involved in Rap1:RIAM interaction highlighted in the boxes. Lpds numbering is based on the sequence includes the CS insertion. (D). Co-IP assay of HA-tagged Rac1 Q61L with GFP-tagged Lpds csRAPH, csRAPH K335A, and Lpd ccRAPH. (E). Co-IP assay of HA-tagged Rac1 Q61L with various truncations of GFP-tagged Lpds csRAPH and csRAPH K355A.
Figure 4.
Figure 4.. Structure prediction and co-IP assay suggest that the cs2 insertion stabilize the CC region via hydrophobic interaction.
(A). Amino acid sequence of the two Lpd splicing insertions, cs1 and cs2, are shown. The cs2 insertion residues are numbered 1’ to 25’. Two hydrophobic leucine clusters, LLL (3L) and LL (2L), in the cs2 insertion are highlighted in cyan. Leu4’ and Ile15’ of cs2 are colored in pink. (B). Predicted interface between the cs2 insertion (in pink) and CC region (in red). Hydrophobic residues contributing to the cs2:CC interaction are shown in surface representation. Residue numbers of CC are based on the Lpd main isoform to avoid confusion. (C). Co-IP assay of HA-tagged Rac1 Q61L and GFP-tagged Lpds csRAPH, Lpds csRAPH 3L3A, Lpds csRAPH 2L2A and Lpds csRAPH 3L3A/2L2A.
Figure 5.
Figure 5.. AlphaFold3–predicted Lpds:Rac1 structure reveals a noncanonical single-helix binding mode.
(A). Structure of Rac1 (in grey) in complex with the helical-pair for Prk1 (in pink). (B). Structure Lpds:Rac1 complex predicted by AlphaFold3. Upper: Lpds is colored as indicated in Fig. 1A. Rac1 is colored in Gray. The interface between Lpds-cs2-CC and Rac1 is highlighted in a zoomed-in view below. Lower: Switch I and Switch II regions of Rac1 are colored in dark grey. Residues forming the hydrophobic interaction interface are shown in surface representation. These includes Ile256, Leu260, and Ile263 (in red) from the Lpds CC region; residues 3 (Tyr3’) and 4 (Leu4’) from the cs2 insertion (in orange); Val36 and Phe37 from Switch I of Rac1 (in grey); and Leu67 and Leu70 from Switch II of Rac1 (in dark grey); and Trp56 in Rac1.
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