The c-Kit proto-oncogene is a receptor protein-tyrosine kinase associated with several highly malignant human cancers. Upon binding its ligand, stem cell factor (SCF), c-Kit forms an active dimer that autophosphorylates itself and activates a signaling cascade that induces cell growth. Disease-causing human mutations that activate SCF-independent constitutive expression of c-Kit are found in acute myelogenous leukemia, human mast cell disease, and gastrointestinal stromal tumors. We report on the phosphorylation state and crystal structure of a c-Kit product complex. The c-Kit structure is in a fully active form, with ordered kinase activation and phosphate-binding loops. These results provide key insights into the molecular basis for c-Kit kinase transactivation to assist in the design of new competitive inhibitors targeting activated mutant forms of c-Kit that are resistant to current chemotherapy regimes.Receptor protein-tyrosine kinases (RPTKs) 1 regulate key signal transduction cascades that control cellular growth and proliferation. The stem cell factor (SCF) receptor c-Kit is a type III transmembrane RPTK comprised of five extracellular immunoglobulin domains, a single transmembrane region, an inhibitory cytoplasmic juxtamembrane domain, and a split cytoplasmic kinase domain separated by a kinase insert segment (1, 2). The type III RPTK family includes c-Kit (3), the colonystimulating factor-1 (formerly FMS) (4), the platelet-derived growth factor ␣ and ␤ receptors (1, 5), and the FMS-related receptor FLT-3 (6). Signaling by RPTKs occurs via ligand binding to the extracellular IG domains, inducing the receptors to form dimers, and thereby activating intrinsic tyrosine kinase activity through the transphosphorylation of specific tyrosine residues in the juxtamembrane and kinase domains (7,8). Ligand binding both activates kinase activity and creates tyrosine-phosphorylated receptors that mediate the specific binding of intracellular signaling proteins. Src homology 2 and protein tyrosine binding domains (9), including the proteintyrosine phosphatase SHP-1, act as negative regulators of c-Kit activity (10). These cytoplasmic signaling proteins initiate serine/threonine phosphorylation cascades that activate transcription factors to determine specific cellular responses (Fig. 1).The human c-Kit gene is the cellular homologue of the v-kit oncogene found in the transforming Hardy-Zuckerman 4 feline sarcoma virus (11) and encodes a 976-amino acid residue RPTK. Loss-of-function c-Kit mutations establish its importance for the normal growth of hematopoietic progenitor cells, mast cells, melanocytes, primordial germ cells, and the interstitial cells of Cajal (12-15). Gain-of-function mutations, resulting in SCF-independent, constitutive activation of c-Kit, are found in several highly malignant cancers. Mutations in the c-Kit juxtamembrane region cluster around the two main autophosphorylation sites that mediate protein tyrosine binding, Tyr-568 and Tyr-570, and are associated with human gastrointestinal stromal t...

Fibroblast activation protein ␣ (FAP␣) is highly expressed in epithelial cancers and has been implicated in extracellular matrix remodeling, tumor growth, and metastasis. We present the first high resolution structure for the apoenzyme as well as kinetic data toward small dipeptide substrates.

Microsomal cytochrome P450s participate in xenobiotic detoxification, procarcinogen activation, and steroid hormone synthesis. The first structure of a mammalian microsomal P450 suggests that the association of P450s with the endoplasmic reticulum involves a hydrophobic surface of the protein formed by noncontiguous portions of the polypeptide chain. This interaction places the entrance of the putative substrate access channel in or near the membrane and orients the face of the protein proximal to the heme cofactor perpendicular to the plane of the membrane for interaction with the P450 reductase. This structure offers a template for modeling other mammalian P450s and should aid drug discovery and the prediction of drug-drug interactions.

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We describe the design of Escherichia coli cells that synthesize a structurally perfect, recombinant cytochrome c from the Thermus thermophilus cytochrome c 552 gene. Key features are~1! construction of a plasmid-borne, chimeric cycA gene encoding an Escherichia coli-compatible, N-terminal signal sequence~MetLysIleSerIleTyrAlaThrLeu AlaAlaLeuSerLeuAlaLeuProAlaGlyAla! followed by the amino acid sequence of mature Thermus cytochrome c 552 ; and~2! coexpression of the chimeric cycA gene with plasmid-borne, host-specific cytochrome c maturation genes ccmABCDEFGH !. Approximately 1 mg of purified protein is obtained from 1 L of culture medium. The recombinant protein, cytochrome rsC 552 , and native cytochrome c 552 have identical redox potentials and are equally active as electron transfer substrates toward cytochrome ba 3 , a Thermus heme-copper oxidase. Native and recombinant cytochromes c were compared and found to be identical using circular dichroism, optical absorption, resonance Raman, and 500 MHz 1 H-NMR spectroscopies. The 1.7 Å resolution X-ray crystallographic structure of the recombinant protein was determined and is indistinguishable from that reported for the native protein~Than, ME, Hof P, Huber R, Bourenkov GP, Bartunik HD, Buse G, Soulimane T, 1997, J Mol Biol 271:629-644!. This approach may be generally useful for expression of alien cytochrome c genes in E. coli. Keywords: cytochrome c; Escherichia coli; homologous expression; Thermus thermophilusAlthough studied for many decades as part of the cell's respiratory apparatus~Lemberg & Barrett, 1973;Mathews, 1985;Pettigrew & Moore, 1987!, cytochromes c remain of considerable interest as objects in the study of electron transfer reactions~Ferguson-Miller et al., 1979;Pan et al., 1993;Bjerrum et al., 1995;Geren et al., 1995;Winkler et al., 1995! and of protein folding~Sosnick et al., 1994;Bryngelson et al., 1995;Mines et al., 1996;Bai, 1999!. In addition, recent evidence indicates that cytochrome c released from the mitochondrion is able to initiate apoptosis~Wallace, 1999, and references therein!, suggesting that this protein may have functions other than electron transfer. Today, application of modern experimental approaches to cytochrome c function can be limited by the general unavailability of suitable expression systems for cloned cytochrome c genes. Escherichia coli is certainly the organism of choice, but most attempts to express foreign cytochrome c genes in this bacterium have been unsuccessful; either the yield is imprac-

Transhydrogenase (TH) is a dimeric integral membrane enzyme in mitochondria and prokaryotes that couples proton translocation across a membrane with hydride transfer between NAD(H) and NADP(H) in soluble domains. Crystal structures of the NAD(H) binding R1 subunit (domain I) of Rhodospirillum rubrum TH have been determined at 1.8 Å resolution in the absence of dinucleotide and at 1.9 Å resolution with NADH bound. Each structure contains two domain I dimers in the asymmetric unit (AB and CD); the dimers are intimately associated and related by noncrystallographic 2-fold axes. NADH binds to subunits A and D, consistent with the half-of-the-sites reactivity of the enzyme. The conformation of NADH in subunits A and D is very similar; the nicotinamide is in the anti conformation, the A-face is exposed to solvent, and both N7 and O7 participate in hydrogen bonds. Comparison of subunits A and D to six independent copies of the subunit without bound NADH reveals multiple conformations for residues and loops surrounding the NADH site, indicating flexibility for binding and release of the substrate (product). The NADH-bound structure is also compared to the structures of R. rubrum domain I with NAD bound (PDB code 1F8G) and with NAD bound in complex with domain III of TH (PDB code 1HZZ). The NADH-vs NAD-bound domain I structures reveal conformational differences in conserved residues in the NAD(H) binding site and in dinucleotide conformation that are correlated with the net charge, i.e., oxidation state, of the nicotinamides. The comparisons illustrate how nicotinamide oxidation state can affect the domain I conformation, which is relevant to the hydride transfer step of the overall reaction.The energy-transducing nicotinamide nucleotide transhydrogenases of eukaryotic mitochondria and bacteria are homodimeric integral membrane proteins of monomer molecular mass of about 110 kDa. They catalyze the direct and stereospecific transfer of a hydride ion between the 4A position of NAD(H) 1 and the 4B position of NADP(H) in a reaction that is coupled to transmembrane proton translocation with a H + /H -stoichiometry of n ) 1 (eq 1) (1-4).In bovine submitochondrial particles, the proton motive force (pmf) accelerates the forward reaction 10-12-fold, and shifts the equilibrium toward product formation. In the reverse direction, transhydrogenation from NADPH to NAD results in outward proton translocation and creation of a pmf. Because there is essentially no difference in the reduction potential of the nicotinamide cofactors, the driving force for proton translocation coupled to the reverse reaction is the difference in binding affinities for substrates (NADPH, NAD) and products (NADH, NADP). In mammalian mitochondria, a function of TH is to produce NADPH for reduction of toxic H 2 O 2 by glutathione reductase and glutathione peroxidase.TH monomers are composed of three domains: a 400-430-residue hydrophilic domain I, a 360-400-residue hydrophobic domain II, and a 200-residue hydrophilic domain III. In mammalian TH, the ...

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The [4Fe-4S]2؉/؉ cluster of Azotobacter vinelandii ferredoxin I (FdI) has an unusually low reduction potential (E 0 ) relative to other structurally similar ferredoxins. Previous attempts to raise that E 0 by modification of surface charged residues were unsuccessful. In this study mutants were designed to alter the E 0 by substitution of polar residues for nonpolar residues near the cluster and by modification of backbone amides. Three FdI variants, P21G, I40N, and I40Q, were purified and characterized, and electrochemical E 0 measurements show that all had altered E 0 relative to native FdI. For P21G FdI and I40Q FdI, the E 0 increased by ؉42 and ؉53 mV, respectively validating the importance of dipole orientation in control of E 0 . Protein Dipole Langevin Dipole calculations based on models for those variants accurately predicted the direction of the change in E 0 while overestimating the magnitude. For I40N FdI, initial calculations based on the model predicted a ؉168 mV change in E 0 while a ؊33 mV change was observed. The x-ray structure of that variant, which was determined to 2.8 Å, revealed a number of changes in backbone and side chain dipole orientation and in solvent accessibility, that were not predicted by the model and that were likely to influence E 0 . Subsequent Protein Dipole Langevin Dipole calculations (using the actual I40N x-ray structures) did quite accurately predict the observed change in E 0 .Iron-sulfur ([Fe-S]) proteins contain clusters composed of iron and inorganic sulfide atoms ligated to the protein primarily by cysteine residues. They are ubiquitous, and have diverse functions ranging from electron transfer to regulation of gene expression (for recent reviews, see Refs. 1-7). In order to carry out these different functions, individual proteins can dramatically alter the reactivity of [Fe-S] clusters in a number of ways. For example, by adding or subtracting iron and sulfide atoms to vary the cluster type (1-7), by bridging a cluster between two subunits (8, 9), by introducing non-cysteine ligands (8, 10 -12 2ϩ/ϩ clusters contained within these proteins vary by over 200 mV. Early comparisons of the structures and sequences for these three proteins showed that the peptide folding around the analogous clusters is highly conserved with respect to the location of the four Cys ligands, the Cys dihedral angles, and the eight amide groups H-bonded to sulfur atoms of the cluster (33). These similarities have also been confirmed by the new 1.4-Å structures of AvFdI (34,35) and by the 0.95-Å structure of CaFd (36). Thus, these factors do not appear to be responsible for the observed differences in reduction potential among these proteins that all use the same [4Fe-