Focal adhesion kinase (FAK) controls adhesion-dependent cell motility, survival, and proliferation. FAK has kinase-dependent and kinase-independent functions, both of which play major roles in embryogenesis and tumor invasiveness. The precise mechanisms of FAK activation are not known. Using x-ray crystallography, small angle x-ray scattering, and biochemical and functional analyses, we show that the key step for activation of FAK's kinase-dependent functions-autophosphorylation of tyrosine-397-requires site-specific dimerization of FAK. The dimers form via the association of the N-terminal FERM domain of FAK and are stabilized by an interaction between FERM and the C-terminal FAT domain. FAT binds to a basic motif on FERM that regulates co-activation and nuclear localization. FAK dimerization requires local enrichment, which occurs specifically at focal adhesions. Paxillin plays a dual role, by recruiting FAK to focal adhesions and by reinforcing the FAT:FERM interaction. Our results provide a structural and mechanistic framework to explain how FAK combines multiple stimuli into a site-specific function. The dimer interfaces we describe are promising targets for blocking FAK activation.

The canonical action of the p85α regulatory subunit of phosphatidylinositol 3-kinase (PI3K) is to associate with the p110α catalytic subunit to allow stimuli-dependent activation of the PI3K pathway. We elucidate a p110α-independent role of homodimerized p85α in the positive regulation of PTEN stability and activity. p110α-free p85α homodimerizes via two intermolecular interactions (SH3:proline-rich region and BH:BH) to selectively bind unphosphorylated activated PTEN. As a consequence, homodimeric but not monomeric p85α suppresses the PI3K pathway by protecting PTEN from E3 ligase WWP2-mediated proteasomal degradation. Further, the p85α homodimer enhances the lipid phosphatase activity and membrane association of PTEN. Strikingly, we identified cancer patient-derived oncogenic p85α mutations that target the homodimerization or PTEN interaction surface. Collectively, our data suggest the equilibrium of p85α monomer–dimers regulates the PI3K pathway and disrupting this equilibrium could lead to disease development.DOI: http://dx.doi.org/10.7554/eLife.06866.001

In pathogenic Gram-negative bacteria, many virulence factors are secreted via the two-partner secretion pathway, which consists of an exoprotein called TpsA and a cognate outer membrane translocator called TpsB. The HMW1 and HMW2 adhesins are major virulence factors in nontypeable Haemophilus influenzae and are prototype two-partner secretion pathway exoproteins. A key step in the delivery of HMW1 and HMW2 to the bacterial surface involves targeting to the HMW1B and In Gram-negative bacteria, the two-partner secretion (TPS) 2 pathway serves as a common secretion mechanism for large protein virulence factors and is essential for the virulence of many human pathogens, including Haemophilus influenzae, Bordetella pertussis, Serratia marcescens, and Proteus mirabilis, among others. The TPS pathway consists of a secreted exoprotein (referred to as a TpsA protein) and a cognate outer membrane translocator (referred to as a TpsB protein) (1-3). TpsA proteins are synthesized as preproteins 100 -500 kDa in size that are processed in the course of secretion across the bacterial inner and outer membranes, yielding functional TpsA proteins (3, 4). Despite limited overall sequence conservation among the TpsA members, functional studies have established that TpsA proteins contain common features, including an atypical N-terminal signal peptide and an adjacent region of about 250 residues that forms the so-called secretion domain (1-4). Although our knowledge of TpsB proteins remains relatively limited, recent studies have established that TpsB proteins have a modular structure with a C-terminal pore-forming domain (5, 6).The H. influenzae HMW1 and HMW2 proteins are high molecular weight, non-pilus adhesins that were originally identified as major targets of the human serum antibody response during acute otitis media (7). These proteins are present in ϳ80% of nontypeable H. influenzae strains and mediate adherence to a variety of epithelial cell types (4, 8). HMW1 and HMW2 are encoded by separate chromosomal loci, with each locus consisting of three genes, designated hmwA, hmwB, and hmwC (8 -10). The hmwA genes encode the surface-exposed adhesins (HMW1 and HMW2), and the hmwB and hmwC genes encode accessory proteins required for proper processing and secretion of the adhesins (5,(11)(12)(13)(14). In H. influenzae strain 12, the HMW1 and HMW2 proteins are synthesized as preproproteins and undergo two discrete cleavage events during the process of maturation and surface localization, the first releasing the N-terminal signal peptide corresponding to residues 1-68 and the second releasing the pro-piece corresponding to residues 69 -441 (see Fig. 1). In H. influenzae strain 12, mature HMW1 corresponds to residues 442-1536 in the HMW1 preproprotein, and mature HMW2 corresponds to residues 442-1477 in the HMW2 preproprotein. In both HMW1 and HMW2, adhesive activity resides in a ϳ360-amino acid region at the N terminus of the mature species, and anchoring to the bacterial surface is mediated by a 20-amino acid region at the very C te...

In principle, evolutionary outcomes could be largely predicted if all of the relevant physicochemical variants of a particular protein function under selection were known and integrated into an appropriate physiological model. We have tested this principle by generating a family of variants of the tetracycline resistance protein TetX2 and identified the physicochemical properties most correlated with organismal fitness. Surprisingly, small changes in the K m(MCN) , less than twofold, were sufficient to produce highly successful adaptive mutants over clinically relevant drug concentrations. We then built a quantitative model directly relating the in vitro physicochemical properties of the mutant enzymes to the growth rates of bacteria carrying a single chromosomal copy of the tet(X2) variants over a wide range of minocycline (MCN) concentrations. Importantly, this model allows the prediction of enzymatic properties directly from cellular growth rates as well as the physicochemical-fitness landscape of TetX2. Using experimental evolution and deep sequencing to monitor the allelic frequencies of the seven most biochemically efficient TetX2 mutants in 10 independently evolving populations, we showed that the model correctly predicted the success of the two most beneficial variants tet(X2) T280A and tet(X2) N371I . The structure of the most efficient variant, TetX2 T280A , in complex with MCN at 2.7 Å resolution suggests an indirect effect on enzyme kinetics. Taken together, these findings support an important role for readily accessible small steps in protein evolution that can, in turn, greatly increase the fitness of an organism during natural selection. experimental evolution | genome to phenome | oxidoreductase W hat determines success or failure of variants within a population undergoing selection? To answer this challenging question, experimental evolution, genomics, and biochemistry have proved to be powerful approaches to the formulation of specific and testable hypotheses that can be validated in a quantitative manner. Most recently, experimental evolution has provided a wealth of insights into epistasis (1-3), adaptive convergence among populations (4, 5), and the role of mutation supply (6, 7) in asexual populations (8). Dean and Thornton coined the term "functional synthesis" to encompass the increasingly relevant role for molecular biology, biochemistry, and biophysics in elucidating a strong quantitative link between changes in genomes (genotype) to their resultant changes in molecular properties (phenotype) and fitness, the property that selection acts upon (9). Dykhuizen and coworkers were among the first to recognize the importance of the functional synthesis when they successfully modeled the adaptive landscape of the lactose operon to quantitatively predict growth rates (10). Whereas the qualitative link between an enzyme function under selection and fitness is typically straightforward, it has proved very challenging to predict how specific adaptive changes in readily measurable in vitro biophysi...

The focal adhesion kinase (FAK) and the related protein-tyrosine kinase 2-beta (Pyk2) are highly versatile multidomain scaffolds central to cell adhesion, migration, and survival. Due to their key role in cancer metastasis, understanding and inhibiting their functions are important for the development of targeted therapy. Because FAK and Pyk2 are involved in many different cellular functions, designing drugs with partial and function-specific inhibitory effects would be desirable. Here, we summarise recent progress in understanding the structural mechanism of how the tug-of-war between intramolecular and intermolecular interactions allows these protein 'nanomachines' to become activated in a site-specific manner.