Abstract
The LIM-only adaptor PINCH (the particularly interesting cysteine- and histidine-rich protein) plays a pivotal role in the assembly of focal adhesions (FAs), supramolecular complexes that transmit mechanical and biochemical information between extracellular matrix and actin cytoskeleton, regulating diverse cell adhesive processes such as cell migration, cell spreading, and survival. A key step for the PINCH function is its localization to FAs, which depends critically on the tight binding of PINCH to integrin-linked kinase (ILK). Here we report the solution NMR structure of the core ILK.PINCH complex (28 kDa, K(D) approximately 68 nm) involving the N-terminal ankyrin repeat domain (ARD) of ILK and the first LIM domain (LIM1) of PINCH. We show that the ILK ARD exhibits five sequentially stacked ankyrin repeat units, which provide a large concave surface to grip the two contiguous zinc fingers of the PINCH LIM1. The highly electrostatic interface is evolutionally conserved but differs drastically from those of known ARD and LIM bound to other types of protein domains. Consistently mutation of a hot spot in LIM1, which is not conserved in other LIM domains, disrupted the PINCH binding to ILK and abolished the PINCH targeting to FAs. These data provide atomic insight into a novel modular recognition and demonstrate how PINCH is specifically recruited by ILK to mediate the FA assembly and cell-extracellular matrix communication.
Highlights
The LIM-only adaptor PINCH plays a pivotal role in the assembly of focal adhesions (FAs), supramolecular complexes that transmit mechanical and biochemical information between extracellular matrix and actin cytoskeleton, regulating diverse cell adhesive processes such as cell migration, cell spreading, and survival
We show that the integrin-linked kinase (ILK) ankyrin repeat domain (ARD) exhibits five sequentially stacked ankyrin repeat units, which provide a large concave surface to grip the two contiguous zinc fingers of the PINCH LIM1
Mutation of a hot spot in LIM1, which is not conserved in other LIM domains, disrupted the PINCH binding to ILK and abolished the PINCH targeting to FAs
Summary
ILK utilizes its N-terminal ARD to recognize the LIM1 domain samples had to be made for the completion of all NMR experiof PINCH, and such binding may promote the targeting of ments as required for the total structure determination. Dipolar couplings for those resonances that are not overlapping and not experiencing broadening due to 1H-1H long range dipolar couplings were included in the structure calculations (86 for the ILK ARD and 23 for the PINCH LIM1). We prepared 100% deuterated and uniformly 15N-labeled ILK ARD in complex with unlabeled LIM1 and collected high sensitivity 15N-edited NOE spectroscopy spectra on a TABLE 1. The final optimized Da values are 11.8 and 8.2 Hz for the ILK ARD and the PINCH LIM1, respectively
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