No abstract

Iterative dual-space direct methods based on phase refinement in reciprocal space and peak picking in real space are able to locate relatively large numbers of anomalous scatterers efficiently from MAD or SAD data. Truncation of the data at a particular resolution, typically in the range 3.0-3.5 A, can be critical to success. The efficiency can be improved by roughly an order of magnitude by Patterson-based seeding instead of starting from random phases or sites; Patterson superposition methods also provide useful validation. The program SHELXD implementing this approach is available as part of the SHELX package.

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The activity of the c-Kit receptor protein-tyrosine kinase is tightly regulated in normal cells, whereas deregulated c-Kit kinase activity is implicated in the pathogenesis of human cancers. The c-Kit juxtamembrane region is known to have an autoinhibitory function; however the precise mechanism by which c-Kit is maintained in an autoinhibited state is not known. We report the 1.9-Å resolution crystal structure of native c-Kit kinase in an autoinhibited conformation and compare it with active c-Kit kinase. Autoinhibited c-Kit is stabilized by the juxtamembrane domain, which inserts into the kinase-active site and disrupts formation of the activated structure. A 1.6-Å crystal structure of c-Kit in complex with STI-571 (Imatinib® or Gleevec®) demonstrates that inhibitor binding disrupts this natural mechanism for maintaining c-Kit in an autoinhibited state. Together, these results provide a structural basis for understanding c-Kit kinase autoinhibition and will facilitate the structure-guided design of specific inhibitors that target the activated and autoinhibited conformations of c-Kit kinase.The stem cell factor receptor c-Kit is a receptor proteintyrosine kinase (RPTK) 1 that initiates cell growth and proliferation signal transduction cascades in response to stem cell factor binding (1). c-Kit, named after its viral homolog v-Kit (2), is a member of the Type III transmembrane RPTK subfamily, which includes the colony-stimulating factor-1 receptor (3), also known as the FMS receptor, the related Flt-3 receptor (4), and the platelet-derived growth factor ␣-and ␤-receptors (5, 6), as well as c-Kit (7). The Type III RPTK family is characterized by five extracellular immunoglobulin (Ig) domains, a single transmembrane helix, an autoinhibitory juxtamembrane domain, and a cytoplasmic kinase domain that is split by a kinase insertion domain (KID) (see Fig. 1A) (6,8).The binding of a stem cell factor dimer to the extracellular Ig domains of c-Kit causes two c-Kit RPTKs to dimerize and permits the kinase domains to act in trans as a substrate and enzyme for one another. The result of stem cell factor binding is the phosphorylation of specific tyrosine residues located in c-Kit juxtamembrane regions (9 -12). Tyrosine residue 568 is the primary site of in vivo autophosphorylation (see Fig. 1B). Phosphorylation of the tyrosine initiates a cytoplasmic serine/ threonine phosphorylation cascade that promotes cell growth and proliferation (12). Mutations that cause constitutive activation of c-Kit kinase activity in the absence of stem cell factor binding are implicated in highly malignant human cancers, including gastrointestinal stromal tumors (13, 14), germ cell tumors (15), mast cell and myeloid leukemias (16), and in mastocytosis (17). Moreover, activating c-Kit mutations that occur in the kinase domain are resistant to many kinase inhibitors currently in use as chemotherapy treatments (18 -21).The kinase activity of c-Kit is tightly regulated throughout its signaling cycle. Binding of the protein-tyrosine phosphatase S...