A novel mode of Gleevec binding is revealed by the structure of spleen tyrosine kinase

Spleen tyrosine kinase (Syk) is a non-receptor tyrosine kinase required for signaling from immunoreceptors in various hematopoietic cells. Phosphorylation of two tyrosine residues in the activation loop of the Syk kinase catalytic domain is necessary for signaling, a phenomenon typical of tyrosine kinase family members. Syk in vitro enzyme activity, however, does not depend on phosphorylation (activation loop tyrosine --> phenylalanine mutants retain catalytic activity). We have determined the x-ray structure of the unphosphorylated form of the kinase catalytic domain of Syk. The enzyme adopts a conformation of the activation loop typically seen only in activated, phosphorylated tyrosine kinases, explaining why Syk does not require phosphorylation for activation. We also demonstrate that Gleevec (STI-571, Imatinib) inhibits the isolated kinase domains of both unphosphorylated Syk and phosphorylated Abl with comparable potency. Gleevec binds Syk in a novel, compact cis-conformation that differs dramatically from the binding mode observed with unphosphorylated Abl, the more Gleevec-sensitive form of Abl. This finding suggests the existence of two distinct Gleevec binding modes: an extended, trans-conformation characteristic of tight binding to the inactive conformation of a protein kinase and a second compact, cis-conformation characteristic of weaker binding to the active conformation. Finally, the Syk-bound cis-conformation of Gleevec bears a striking resemblance to the rigid structure of the nonspecific, natural product kinase inhibitor staurosporine.     

X-ray structure of translation initiation factor eIF2gamma: implications for tRNA and eIF2alpha binding

The x-ray structure of the gamma-subunit of the heterotrimeric translation initiation factor eIF2 has been determined to 2.4-A resolution. eIF2 is a GTPase that delivers the initiator Met-tRNA to the P site on the small ribosomal subunit during a rate-limiting initiation step in translation. The structure of eIF2gamma closely resembles that of EF1A.GTP, consisting of an N-terminal G domain followed by two beta-barrels arranged in a closed configuration with domain II packed against the G domain in the vicinity of the Switch regions. The G domain of eIF2gamma has an unusual zinc ribbon motif, not previously found in other GTPases. Structure-based site-directed mutagenesis was used to identify two adjacent features on the surface of eIF2gamma that bind the alpha-subunit and Met-tRNA(i)(Met), respectively. These structural, biochemical, and genetic results provide new insights into eIF2 ternary complex assembly.     

Structure of nucleotide-binding domain 1 of the cystic fibrosis transmembrane conductance regulator

Cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-binding cassette (ABC) transporter that functions as a chloride channel. Nucleotide-binding domain 1 (NBD1), one of two ABC domains in CFTR, also contains sites for the predominant CF-causing mutation and, potentially, for regulatory phosphorylation. We have determined crystal structures for mouse NBD1 in unliganded, ADP- and ATP-bound states, with and without phosphorylation. This NBD1 differs from typical ABC domains in having added regulatory segments, a foreshortened subdomain interconnection, and an unusual nucleotide conformation. Moreover, isolated NBD1 has undetectable ATPase activity and its structure is essentially the same independent of ligand state. Phe508, which is commonly deleted in CF, is exposed at a putative NBD1-transmembrane interface. Our results are consistent with a CFTR mechanism, whereby channel gating occurs through ATP binding in an NBD1-NBD2 nucleotide sandwich that forms upon displacement of NBD1 regulatory segments.     

Separation of lipid-free egg yolk proteins by high-pressure liquid chromatography using solvents containing formic acid

A method for obtaining total protein patterns from lipid-containing systems, in particular egg yolk, is described. After dispersion of the yolk in 8 M guanidine hydrochloride solution, lipid is removed by extraction with chloroform-methanol and petrol. The protein solution is applied to a high-pressure liquid chromatograph and eluted with a gradient of formic acid, isopropanol, and acetonitrile. In measurements on a known yolk protein, duck apovitellenin I, the method did not cause irreversible formylation of N-terminal or other residues. The method was used (1) to compare protein patterns of whole yolk from hen and quail eggs; (2) to isolate and partially sequence quail apovitellenin I; and (3) to compare protein patterns of "white yolk" and "yellow yolk."     

AN ISOLATION UNIT FOR SUPPLYING RADIOISOTOPES TO SPECIFIC SEGMENTS OF AN INTACT ROOT

A device useful for supplying radioactive ions to specific segments of intact roots is described. A small volume of aerated, radioactive culture solution may be circulated around an isolated segment of a root, while the remainder of that root and the other roots of the plant are exposed to non-radioactive culture solution. Any root which does not have lateral roots extending beyond the epidermis can be inserted into the device without handling the root itself. No sealants are required to achieve a leak-proof seal above and below the root segment. The use of flexible rubber membranes to isolate root segments causes no detectable damage to root cells where the membranes contact the root epidermis.     

Ecological Load and Balancing Selection in Circumboreal Barnacles

Acorn barnacle adults experience environmental heterogeneity at various spatial scales of their circumboreal habitat, raising the question of how adaptation to high environmental variability is maintained in the face of strong juvenile dispersal and mortality. Here, we show that 4% of genes in the barnacle genome experience balancing selection across the entire range of the species. Many of these genes harbor mutations maintained across 2 My of evolution between the Pacific and Atlantic oceans. These genes are involved in ion regulation, pain reception, and heat tolerance, functions which are essential in highly variable ecosystems. The data also reveal complex population structure within and between basins, driven by the trans-Arctic interchange and the last glaciation. Divergence between Atlantic and Pacific populations is high, foreshadowing the onset of allopatric speciation, and suggesting that balancing selection is strong enough to maintain functional variation for millions of years in the face of complex demography.     

Environmental predictors of forest expansion on open coastal barrens

Rock barrens support rare plant species but may be threatened by forest expansion. We determined the extent of forest expansion onto open coastal barrens and identified environmental correlates of dynamic versus persistent barrens in Nova Scotia, Canada. We used aerial photos to quantify the amount of forest expansion over the last 70 years at five coastal barrens sites and GIS to derive topographic and other environmental predictors to differentiate persistent coastal barrens compared with persistent forests or barrens that succeed to forests. Linear discriminant and classification tree analyses identified the variables associated with each class of habitat. Coastal barrens decreased by an average of 7.9% (from 4.2 to 24.6% depending on the site) in the last 70 years due to forest expansion. The best predictors of persistent barrens were elevation and distance to coast. Environmental factors such as topographical heterogeneity and evidence of fire varied among sites. Climatic and edaphic conditions near the coast and in exposed inland areas may protect coastal barrens vegetation from forest expansion. Evidence of fire was not found at all barrens sites, thus at least some of the persistent open barrens are likely maintained by shallow soils, salt spray, and wind exposure. All three classes of habitat had distinct vegetation, and the only rare species was found in a persistent barren. Management of human activities in such landscapes should take into account the dynamic nature of barrens vegetation, while prioritizing conservation efforts in persistent barrens.