Activation of the ligand binding function of integrin heterodimers requires transmission of an "inside-out" signal from their small intracellular segments to their large extracellular domains. The structure of the cytoplasmic domain of a prototypic integrin alpha(IIb)beta(3) has been solved by NMR and reveals multiple hydrophobic and electrostatic contacts within the membrane-proximal helices of its alpha and the beta cytoplasmic tails. The interface interactions are disrupted by point mutations or the cytoskeletal protein talin that are known to activate the receptor. These results provide a structural mechanism by which a handshake between the alpha and the beta cytoplasmic tails restrains the integrin in a resting state and unclasping of this interaction triggers the inside-out conformational signal that leads to receptor activation.

Cell signaling that culminates in posttranslational modifications directs protein activity. Here we report how multiple Ca 2+ -dependent phosphorylation sites within the transcription activator Ets-1 act additively to produce graded DNA binding affinity. Nuclear magnetic resonance spectroscopic analyses show that phosphorylation shifts Ets-1 from a dynamic conformation poised to bind DNA to a well-folded inhibited state. These phosphates lie in an unstructured flexible region that functions as the allosteric effector of autoinhibition. Variable phosphorylation thus serves as a “rheostat” for cell signaling to fine-tune transcription at the level of DNA binding.

PINCH, integrin-linked kinase (ILK) and calponin homology-containing ILK-binding protein (CH-ILKBP) form a ternary complex that plays crucial roles at cell-extracellular matrix adhesion sites. To understand the mechanism underlying the complex formation and recruitment to cell-adhesion sites we have undertaken a combined structural, mutational and cell biological analysis. Three-dimensional structure-based point mutations identified specific PINCH and ILK sites that mediate the complex formation. Analyses of the binding defective point mutants revealed that the assembly of the PINCH-ILK-CH-ILKBP complex is essential for their localization to cell-extracellular matrix adhesion sites. The formation of the PINCH-ILK-CH-ILKBP complex precedes integrin-mediated cell adhesion and spreading. Furthermore, inhibition of protein kinase C, but not that of actin polymerization, inhibited the PINCH-ILK-CH-ILKBP complex formation, suggesting that the PINCH-ILK-CH-ILKBP complex likely serves as a downstream effector of protein kinase C in the cellular control of focal adhesion assembly. Finally,we provide evidence that the formation of the PINCH-ILK-CH-ILKBP complex,while necessary, is not sufficient for ILK localization to cell-extracellular matrix adhesion sites. These results provide new insights into the molecular mechanism underlying the assembly and regulation of cell-matrix adhesion structures.

ClpB/Hsp100 is an ATP-dependent disaggregase that solubilizes and reactivates protein aggregates in cooperation with the DnaK/ Hsp70 chaperone system. The ClpB-substrate interaction is mediated by conserved tyrosine residues located in flexible loops in nucleotide-binding domain-1 that extend into the ClpB central pore. In addition to the tyrosines, the ClpB N-terminal domain (NTD) was suggested to provide a second substrate-binding site; however, the manner in which the NTD recognizes and binds substrate proteins has remained elusive. Herein, we present an NMR spectroscopy study to structurally characterize the NTD-substrate interaction. We show that the NTD includes a substrate-binding groove that specifically recognizes exposed hydrophobic stretches in unfolded or aggregated client proteins. Using an optimized segmental labeling technique in combination with methyl-transverse relaxation optimized spectroscopy (TROSY) NMR, the interaction of client proteins with both the NTD and the pore-loop tyrosines in the 580-kDa ClpB hexamer has been characterized. Unlike contacts with the tyrosines, the NTD-substrate interaction is independent of the ClpB nucleotide state and protein conformational changes that result from ATP hydrolysis. The NTD interaction destabilizes client proteins, priming them for subsequent unfolding and translocation. Mutations in the NTD substrate-binding groove are shown to have a dramatic effect on protein translocation through the ClpB central pore, suggesting that, before their interaction with substrates, the NTDs block the translocation channel. Together, our findings provide both a detailed characterization of the NTD-substrate complex and insight into the functional regulatory role of the ClpB NTD in protein disaggregation.T he heat shock protein ClpB (Escherichia coli) or Hsp100 (eukaryotes) is the main protein disaggregase in bacteria, yeast, plants, and mitochondria of all eukaryotic cells, and it is essential for cell survival during severe stress (1-4). Recovery of functional proteins from aggregates by ClpB requires the synergistic interaction with a second molecular chaperone, DnaK (1). Through its cochaperone, DnaJ, DnaK initially binds to the aggregates, leading to the exposure of peptide segments that can be recognized by ClpB (5, 6). DnaK then recruits ClpB to the site of aggregation through direct physical interaction (7, 8), transferring the aggregate to ClpB. Using the energy derived from ATP hydrolysis, ClpB unravels the aggregate by threading single polypeptide chains, one at a time, through the central pore of its hexameric ring (9). Once released from the aggregate, the unfolded polypeptides can either refold spontaneously or fold with the help of additional cellular chaperones.Like other Hsp100 proteins, ClpB forms a hexameric ring, with each protomer comprising an N-terminal domain (NTD) and two nucleotide binding domains (NBD1 and NBD2) separated by a unique regulatory coil-coil domain (10) essential for DnaK binding (7, 11) ( Fig. 1 A and B). Both NBDs contain Walk...

Solution NMR spectroscopy is an extremely powerful technology for the study of biomolecular dynamics and site-specific molecular interactions. An important limitation in the past has been molecule size, with molecular weights of targets seldom exceeding 50 kDa. New labeling technology and NMR experiments are changing this paradigm so that applications for investigating supramolecular complexes are starting to become feasible. Here we describe a strategy developed in our laboratory that involves the use of labeled methyl groups of isoleucine, leucine and valine residues in proteins as probes, along with experiments that significantly enhance the lifetimes of the resulting signals. We describe the application of these methods to a number of systems with molecular weights in the hundreds of kilodaltons.

The 306-kDa aspartate transcarbamoylase is a well studied regulatory enzyme, and it has emerged as a paradigm for understanding allostery and cooperative binding processes. Although there is a consensus that the cooperative binding of active site ligands follows the Monod-Wyman-Changeux (MWC) model of allostery, there is some debate about the binding of effectors such as ATP and CTP and how they influence the allosteric equilibrium between R and T states of the enzyme. In this article, the binding of substrates, substrate analogues, and nucleotides is studied, along with their effect on the R-T equilibrium by using highly deuterated, 1 H, 13 C-methyl-labeled protein in concert with methyl-transverse relaxation optimized spectroscopy (TROSY) NMR. Although only the T state of the enzyme can be observed in spectra of wild-type unliganded aspartate transcarbamoylase, binding of active-site substrates shift the equilibrium so that correlations from the R state become visible, allowing the equilibrium constant (L ) between ligand-saturated R and T forms of the enzyme to be measured quantitatively. The equilibrium constant between unliganded R and T forms (L) also is obtained, despite the fact that the R state is ''invisible'' in spectra, by means of an indirect process that makes use of relations that emerge from the fact that ligand binding and the R-T equilibrium are linked. Titrations with MgATP unequivocally establish that its binding directly perturbs the R-T equilibrium, consistent with the Monod-Wyman-Changeux model. This study emphasizes the utility of modern solution NMR spectroscopy in understanding protein function, even for systems with aggregate molecular masses in the hundreds of kilodaltons.allostery ͉ Monod-Wyman-Changeux (MWC) model ͉ NMR spectroscopy ͉ methyl-transverse relaxation optimized spectroscopy ͉ ligand binding Q uantitative, site-specific studies of proteins by NMR spectroscopy for the most part have been restricted to systems with molecular masses on the order of 50 kDa or less. With the development of new labeling schemes along with experiments that optimally preserve NMR signals, it now has become possible to investigate much larger complexes (1). For example, Wüth-rich, Horwich, and coworkers have used 1 H-15 N cross-correlated relaxation-induced polarization transfer (CRIPT) spectroscopy to establish which residues of highly deuterated GroES interact with GroEL in a GroES-GroEL complex that is 900 kDa (2) and to study interactions between substrates and GroEL (3). Sprangers and coworkers have exploited methyl-transverse relaxation optimized spectroscopy (TROSY) of 1 H, 13 C-methyl-labeled probes in the 300-kDa highly deuterated protease ClpP to quantify dynamics and relate it to function (4). In a second study, this methyl-based approach has been used to measure sitespecific dynamics in the 20S proteasome (670 kDa) along with binding to target molecules (5).The methodology that now is available opens the possibility for the study of a wide range of molecular machines in a site-specific...

Weak protein-protein interactions (PPIs) (K(D) > 10(-6) M) are critical determinants of many biological processes. However, in contrast to a large growing number of well-characterized, strong PPIs, the weak PPIs, especially those with K(D) > 10(-4) M, are poorly explored. Genome wide, there exist few 3D structures of weak PPIs with K(D) > 10(-4) M, and none with K(D) > 10(-3) M. Here, we report the NMR structure of an extremely weak focal adhesion complex (K(D) approximately 3 x 10(-3) M) between Nck-2 SH3 domain and PINCH-1 LIM4 domain. The structure exhibits a remarkably small and polar interface with distinct binding modes for both SH3 and LIM domains. Such an interface suggests a transient Nck-2/PINCH-1 association process that may trigger rapid focal adhesion turnover during integrin signaling. Genetic rescue experiments demonstrate that this interface is indeed involved in mediating cell shape change and migration. Together, the data provide a molecular basis for an ultraweak PPI in regulating focal adhesion dynamics during integrin signaling.

Molecular recognition is integral to biological function and frequently involves preferred binding of a molecule to one of several exchanging ligand conformations in solution. In such a process the bound structure can be selected from the ensemble of interconverting ligands a priori (conformational selection, CS) or may form once the ligand is bound (induced fit, IF). Here we focus on the ubiquitous and conserved Hsp70 chaperone which oversees the integrity of the cellular proteome through its ATP-dependent interaction with client proteins. We directly quantify the flux along CS and IF pathways using solution NMR spectroscopy that exploits a methyl TROSY effect and selective isotope-labeling methodologies. Our measurements establish that both bacterial and human Hsp70 chaperones interact with clients by selecting the unfolded state from a pre-existing array of interconverting structures, suggesting a conserved mode of client recognition among Hsp70s and highlighting the importance of molecular dynamics in this recognition event.