No abstract

The effects of N-terminal acetylation and C-terminal tail structure on the orientation of binding of imidazole/pyrrole polyamide DNA ligands has been investigated. We find that N-terminal acetylation leads to an intramolecular steric clash for hairpin ligands bound in the minor groove, promoting a rotation of the spatially close C-terminal pyrrole ring. This in turn leads to loss of contacts between the tail and the groove, removing the preference for 5‘-to-3‘ orientational binding typical of this class of ligand. Similarly, introduction of a glycine linker into the tail leads to a direct steric clash with the groove, again promoting rotation of the attached ligand ring. The effects of acetylation and a glycine in the tail are additive. The implications for the design of sequence-specific ligands are discussed.

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 ILK⅐PINCH complex (28 kDa, K D ϳ 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 ␤ 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...