| 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, KD ∼ 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.Cell-extracellular matrix (ECM)3 adhesion, migration, and survival are essential for the development and maintenance of tissues and organs in living organisms. They are mediated by integrin transmembrane receptors, which function by adhering to ECM proteins via their large extracellular domains while connecting to the actin cytoskeleton via their small cytoplasmic tails (20-70 residues) (1). The integrin-actin connection supports strong cell-ECM adhesion, and its alteration leads to dynamic cell shape change, migration, and survival (2). The molecular details of such connection, however, are highly complex, involving a large protein complex network called focal adhesions (FAs) (3, 4).Integrin-linked kinase (ILK) is a 50-kDa FA protein that contains an N-terminal ankyrin repeat domain (ARD), a middle pleckstrin homology domain, and a C-terminal kinase domain. Originally discovered as an integrin β cytoplasmic tail-binding protein (5), ILK has been established as a major regulator that controls the complex FA assembly and transmits many cell adhesive signals between integrins and actin (6-8). Soon after the discovery of ILK, Tu et al. (9) identified an ILK binding partner called PINCH that contains five LIM domains. Extensive studies have shown that the PINCH binding to ILK is essential for triggering the FA assembly and for relaying diverse mechanical and biochemical signals between ECM and the actin cytoskeleton (9-11). Consistent with the importance of the ILK/PINCH association in almost all cellular behavior and fate, ablation of either ILK (12) or PINCH in mice is embryonically lethal (13, 14). PINCH also has a highly homologous isoform called PINCH-2. However, although complementary to PINCH in many cellular behaviors (for reviews, see Refs. 8 and 15), PINCH-2 appears to be involved at the later stage of development (16), and thus its ablation in mice is not embryonically lethal (17). At the clinical level, dysregulation of the ILK/PINCH interaction has been implicated in the development of numerous human disorders such as cancer (6, 18) and heart diseases (19, 20). A Phase I clinical trial is ongoing on a drug called thymosin β-4 (RegeneRx) that appears to specifically target ILK/PINCH for treating myocardial infarction, a major heart failure disorder (19).Despite the cellular, physiological, and pathological importance of the ILK/PINCH interaction, the structural basis for how exactly PINCH binds to ILK has not been well understood. Previous biochemical/structural analyses have indicated that ILK utilizes its N-terminal ARD to recognize the LIM1 domain of PINCH, and such binding may promote the targeting of PINCH to FAs (9, 21). However, the precise atomic basis for such targeting process is elusive. No structure of any ARD·LIM complex has been reported. Using a combination of NMR-based techniques, we have solved the solution structure of the ILK ARD·PINCH LIM1 complex that revealed an interface that is distinct from other ARD and LIM bound to non-ARD/LIM domains. Structure-based mutation of a hot spot in PINCH LIM1, which is not conserved in other LIM domains, abolished the PINCH binding to ILK and its localization to FAs. These results not only reveal a unique LIM/ARD recognition mode but also provide a definitive functional basis for how PINCH is recruited by ILK to focal adhesion site, a major step toward the dynamic cell adhesion and migration processes. |
