Pleckstrin homology (PH) domains are small protein modules involved in recruitment of signaling molecules to cellular membranes, in some cases by binding specific phosphoinositides. We describe use of a convenient "dotblot" approach to screen 10 different PH domains for those that recognize particular phosphoinositides. Each PH domain bound phosphoinositides in the assay, but only two (from phospholipase C-␦ 1 and Grp1) showed clear specificity for a single species. Using soluble inositol phosphates, we show that the Grp1 PH domain (originally cloned on the basis of its phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P 3 ) binding) binds specifically to D-myo-inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P 4 ) (the PtdIns(3,4,5)P 3 headgroup) with K D ‫؍‬ 27.3 nM, but binds D-myo-inositol 1,3,4-trisphosphate (Ins(1,3,4)P 3 ) or D-myo-inositol 1,4,5-trisphosphate (Ins-(1,4,5)P 3 ) over 80-fold more weakly. We show that this specificity allows localization of the Grp1 PH domain to the plasma membrane of mammalian cells only when phosphatidylinositol 3-kinase (PI 3-K) is activated. The presence of three adjacent equatorial phosphate groups was critical for inositol phosphate binding by the Grp1 PH domain. By contrast, another PH domain capable of PI 3-K-dependent membrane recruitment (encoded by EST684797) does not distinguish Ins(1,3,4)P 3 from Ins-(1,3,4,5)P 3 (binding both with very high affinity), despite selecting strongly against Ins(1,4,5)P 3 . The remaining PH domains tested appear significantly less specific for particular phosphoinositides. Together with data presented in the literature, our results suggest that many PH domains bind similarly to multiple phosphoinositides (and in some cases phosphatidylserine), and are likely to be regulated in vivo by the most abundant species to which they bind. Thus, using the same simple approach to study several PH domains simultaneously, our studies suggest that highly specific phosphoinositide binding is a characteristic of relatively few cases.

Pleckstrin homology (PH) domains are protein modules of around 120 amino acids found in many proteins involved in cellular signaling. Certain PH domains drive signal-dependent membrane recruitment of their host proteins by binding strongly and specifically to lipid second messengers produced by agonist-stimulated phosphoinositide 3-kinases (PI 3-Ks). We describe X-ray crystal structures of two different PH domains bound to Ins(1,3,4,5)P4, the head group of the major PI 3-K product PtdIns(3,4,5)P3. One of these PH domains (from Grp1) is PtdIns(3,4,5)P3 specific, while the other (from DAPP1/PHISH) binds strongly to both PtdIns(3,4,5)P3 and its 5'-dephosphorylation product, PtdIns(3,4)P2. Comparison of the two structures provides an explanation for the distinct phosphoinositide specificities of the two PH domains and allows us to predict the 3-phosphoinositide selectivity of uncharacterized PH domains.

Standard protein expression systems, such as E. coli, often fail to produce folded, mono-disperse, or functional eukaryotic proteins (see Small-scale Expression of Proteins in E. coli). The expression of these proteins is greatly benefited by using a eukaryotic system, such as mammalian cells, that contains the appropriate folding and posttranslational machinery. Here, we describe methods for both small- and large-scale transient expression in mammalian cells using polyethylenimine (PEI). We find this procedure to be more cost-effective and quicker than the more traditional route of generating stable cell lines. First, optimal transfection conditions are determined on a small-scale, using adherent cells. These conditions are then translated for use in large-scale suspension cultures. For further details on generating stable cell lines please (see Rapid creation of stable mammalian cell lines for regulated expression of proteins using the Gateway® Recombination Cloning Technology and Flp-In T-REx® lines or Generating mammalian stable cell lines by electroporation).

Pyrrolysine (Pyl), the 22nd natural amino acid and genetically encoded by UAG, becomes attached to its cognate tRNA by pyrrolysyl-tRNA synthetase (PylRS). We have determined three crystal structures of the Methanosarcina mazei PylRS complexed with either AMP-PNP, Pyl-AMP plus pyrophosphate, or the Pyl analogue N--[(cylopentyloxy)carbonyl]-L-lysine plus ATP. The structures reveal that PylRS utilizes a deep hydrophobic pocket for recognition of the Pyl side chain. A comparison of these structures with previously determined class II tRNA synthetase complexes illustrates that different substrate specificities derive from changes in a small number of residues that form the substrate side-chainbinding pocket. The knowledge of these structures allowed the placement of PylRS in the aminoacyl-tRNA synthetase (aaRS) tree as the last known synthetase that evolved for genetic code expansion, as well as the finding that Pyl arose before the last universal common ancestral state. The PylRS structure provides an excellent framework for designing new aaRSs with altered amino acid specificity.aminoacyl-tRNA synthetase ͉ evolution ͉ pyrrolysine

The type I insulin-like growth factor receptor (IGF1R) is involved in growth and survival of normal and neoplastic cells. A ligand-dependent conformational change is thought to regulate IGF1R activity, but the nature of this change is unclear. We point out an underappreciated dimer in the crystal structure of the related Insulin Receptor (IR) with Insulin bound that allows direct comparison with unliganded IR and suggests a mechanism by which ligand regulates IR/IGF1R activity. We test this mechanism in a series of biochemical and biophysical assays and find the IGF1R ectodomain maintains an autoinhibited state in which the TMs are held apart. Ligand binding releases this constraint, allowing TM association and unleashing an intrinsic propensity of the intracellular regions to autophosphorylate. Enzymatic studies of full-length and kinase-containing fragments show phosphorylated IGF1R is fully active independent of ligand and the extracellular-TM regions. The key step triggered by ligand binding is thus autophosphorylation.DOI: http://dx.doi.org/10.7554/eLife.03772.001

Receptor-mediated endocytosis via clathrin-coated vesicles has been extensively studied and, while many of the protein players have been identified, much remains unknown about the regulation of coat assembly and the mechanisms that drive vesicle formation [1]. Some components of the endocytic machinery interact with inositol polyphosphates and inositol lipids in vitro, implying a role for phosphatidylinositols in vivo [2] [3]. Specifically, the adaptor protein complex AP2 binds phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2), PtdIns(3)P, PtdIns(3,4,5)P3 and inositol phosphates. Phosphatidylinositol binding regulates AP2 self-assembly and the interactions of AP2 complexes with clathrin and with peptides containing endocytic motifs [4] [5]. The GTPase dynamin contains a pleckstrin homology (PH) domain that binds PtdIns(4,5)P2 and PtdIns(3,4,5)P3 to regulate GTPase activity in vitro [6] [7]. However, no direct evidence for the involvement of phosphatidylinositols in clathrin-mediated endocytosis exists to date. Using well-characterized PH domains as high affinity and high specificity probes in combination with a perforated cell assay that reconstitutes coated vesicle formation, we provide the first direct evidence that PtdIns(4,5)P2 is required for both early and late events in endocytic coated vesicle formation.

Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA Pyl have emerged as ideal translation components for genetic code innovation. Variants of the enzyme facilitate the incorporation >100 noncanonical amino acids (ncAAs) into proteins. PylRS variants were previously selected to acylate N e -acetyl-Lys (AcK) onto tRNA Pyl . Here, we examine an N e -acetyl-lysyl-tRNA synthetase (AcKRS), which is polyspecific (i.e., active with a broad range of ncAAs) and 30-fold more efficient with Phe derivatives than it is with AcK. Structural and biochemical data reveal the molecular basis of polyspecificity in AcKRS and in a PylRS variant [iodo-phenylalanyl-tRNA synthetase (IFRS)] that displays both enhanced activity and substrate promiscuity over a chemical library of 313 ncAAs. IFRS, a product of directed evolution, has distinct binding modes for different ncAAs. These data indicate that in vivo selections do not produce optimally specific tRNA synthetases and suggest that translation fidelity will become an increasingly dominant factor in expanding the genetic code far beyond 20 amino acids.aminoacyl-tRNA synthetase | genetic code | genetic selection | posttranslational modification | synthetic biology T he standard genetic code table relates the 64 nucleotide triplets to three stop signals and 20 canonical amino acids. Some organisms, including humans, naturally evolved expanded genetic codes that accommodate 21 amino acids (1), or possibly 22 amino acids in rare cases (2). Engineering translation system components, including tRNAs (3, 4), aminoacyl-tRNA synthetases (AARSs) (5, 6), elongation factors (7), and the ribosome itself (8), have produced organisms with artificially expanded genetic codes. Products of genetic code engineering include bacterial, yeast, and mammalian cells and animals that are able to synthesize proteins with sitespecifically inserted noncanonical amino acids (ncAAs) (9).Genetic code expansion systems rely on an orthogonal AARS/ tRNA pair (o-AARS, o-tRNA) (5, 6). The o-AARS should be specific in ligating a desired ncAA to a stop codon decoding tRNA, and both the o-tRNA and o-AARS are assumed not to cross-react with endogenous AARSs or tRNAs. Although some AARSs evolved in nature to recognize certain ncAAs (10-12), many genetic code expansion systems require a mutated AARS active site. The active site of the o-AARS is usually redesigned via directed evolution (6), including positive and negative selective rounds, to produce an enzyme that is assumed to be specific for an ncAA and not active with the 20 canonical amino acids. Genetic code expansion technology is rapidly evolving (13), and the ability to incorporate multiple ncAAs into a protein using quadruplet-codon decoding (14) or sense-codon recoding (15-19) is now becoming feasible. Protein synthesis with multiple ncAAs will require o-AARSs that are able to discriminate their ncAA substrate not only from canonical amino acids in the cell but from other ncAAs that are added to the cell.Probing the effects of amino acid analogs on bacterial cell...

The minimal requirements for substrate recognition and turnover by iodotyrosine deiodinase were examined to learn the basis for its catalytic specificity. This enzyme is crucial for iodide homeostasis and the generation of thyroid hormone in chordates. 2-Iodophenol binds only very weakly to the human enzyme and is dehalogenated with a kcat/Km that is more than 4-orders of magnitude lower than that for iodotyrosine. This discrimination likely protects against a futile cycle of iodinating and deiodinating precursors of thyroid hormone biosynthesis. Surprisingly, a very similar catalytic selectivity was expressed by a bacterial homolog from Haliscomenobacter hydrossis. In this example, discrimination was not based on affinity since 4-cyano-2-iodophenol bound to the bacterial deiodinase with a Kd lower than that of iodotyrosine and yet was not detectably deiodinated. Other phenols including 2-iodophenol were deiodinated but only very inefficiently. Crystal structures of the bacterial enzyme with and without bound iodotyrosine are nearly superimposable and quite similar to the corresponding structures of the human enzyme. Likewise, the bacterial enzyme is activated for single electron transfer after binding to the substrate analog fluorotyrosine as previously observed with the human enzyme. A co-crystal structure of bacterial deiodinase and 2-iodophenol indicates that this ligand stacks on the active site FMN in a orientation analogous to that of bound iodotyrosine. However, 2-iodophenol association is not sufficient to activate the FMN chemistry required for catalysis, and thus the bacterial enzyme appears to share a similar specificity for halotyrosines even though their physiological roles are likely very different from those in humans.