Abstract
Ras suppressor-1 (Rsu-1) is a leucine-rich repeat (LRR)-containing protein that is crucial for regulating cell adhesion and is involved in such physiological and pathological processes as focal adhesion assembly and tumor metastasis. Rsu-1 interacts with zinc-finger type multi-LIM domain-containing adaptor protein PINCH-1, known to be involved in the integrin-mediated consensus adhesome, but not with its highly homologous family member PINCH-2. However, the structural basis for and regulatory mechanisms of this specific interaction remain unclear. Here, we determined the crystal structures of Rsu-1 and its complex with the PINCH-1 LIM4-5 domains. Rsu-1 displays an arc-shaped solenoid architecture, with eight LRRs shielded by N- and C-terminal capping modules. We showed that the conserved concave surface of the Rsu-1 LRR domain binds and stabilizes the PINCH-1 LIM5 domain via salt bridge and hydrophobic interactions, while the C-terminal non-LIM region of PINCH-2 sterically disfavors Rsu-1 binding. We also showed that Rsu-1 can be assembled, via PINCH-1-binding, into a heteropentamer complex comprising Rsu-1, PINCH-1, ILK, Parvin, and Kindlin-2, which constitute a major consensus integrin adhesome crucial for focal adhesion assembly. Our mutagenesis and cell biological data emphasize the significance of the Rsu-1/PINCH-1 interaction in focal adhesion assembly and cell spreading, providing crucial molecular insights into Rsu-1-mediated cell adhesion with implications for disease development.
Highlights
The ninth β-strand in the following leucine-rich repeat (LRR) motif of Ras suppressor protein-1 (Rsu-1) is aligned to a parallel β-sheet in the concave surface but a preceded four-residue insertion and a following helix-turn-helix module build up a distinct structural organization from the LRR motif, resulting in the structural part of the C-terminal cap that stabilizes the internal domain of LRR, as seen in other canonical LRR-containing proteins [11]
Apart from LRRcontaining genes from human pathogen Leptospira interrogans such as LIC11098 that exhibits a high structural similarity with Z-scores of 26.6 [24], it is noteworthy that the structure of Rsu-1 resembles that of a human protein phosphatase 1 regulatory subunit 7
Rsu1 comprises two distinct variable sequence lengths (23- and 25-residues) in the LRRs that resemble in part those in the plant-specific (PS) subfamily in the LRR superfamily [10], our structural analysis strongly suggests that Rsu-1 falls into the SDS22-like subfamily rather than the PS subfamily [22]
Summary
QuantStudio 5 RealTime PCR System from Thermo Fisher Scientific. Proteins were buffer-exchanged in 20 mM Tris, pH 7.5, 150 mM NaCl and prepared in a MicroAmp Optical 96-well plate from Thermo Fisher Scientific at a final concentration of 2 μM in 20 μl reaction volume that contained a Protein Thermal Shift Dye (Thermo Fisher Scientific) as a fluorescence probe at a 1× concentration. The thermal melting measurements were performed for three independent experiments in four replicates of each reaction. No protein control (NPC) was prepared in the reaction mixture consisting buffer and dye without protein. The melting temperature (Tm) was determined by fitting the melt curve to the Boltzmann equation using the Protein Thermal Shift Software version 1.0 from Thermo Fisher Scientific
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