MEK1 and MEK2 are closely related, dual-specificity tyrosine/threonine protein kinases found in the Ras/Raf/MEK/ERK mitogen-activated protein kinase (MAPK) signaling pathway. Approximately 30% of all human cancers have a constitutively activated MAPK pathway, and constitutive activation of MEK1 results in cellular transformation. Here we present the X-ray structures of human MEK1 and MEK2, each determined as a ternary complex with MgATP and an inhibitor to a resolution of 2.4 A and 3.2 A, respectively. The structures reveal that MEK1 and MEK2 each have a unique inhibitor-binding pocket adjacent to the MgATP-binding site. The presence of the potent inhibitor induces several conformational changes in the unphosphorylated MEK1 and MEK2 enzymes that lock them into a closed but catalytically inactive species. Thus, the structures reported here reveal a novel, noncompetitive mechanism for protein kinase inhibition.

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The sequence of the 224 residues of H M G 1 suggests it consists of three domains. We have previously proposed [Cary et al. (1980 Eur. J. Biochern. 131,367-3741 that the A and B domains can fold autonomously and that there is also a small N domain. Several proteases are now found to cut at the end of the B domain (at or close to residue 184). It is shown that the A+B-domain fragment also folds and probably contains all the helix of intact HMG 1. The stability of the B domain is enhanced by the presence of the A domain. The acidic C domain undergoes a coil-+ helix transition on lowering the pH. Several peptides have been prepared by cleavage at tryptophan. Peptide 57-Cterminus contains complete B and C domains but does not fold. In the absence of the A domain the C domain is thus able to destabilise the B domain. It is concluded that the stability of the B domain in HMG 1 is due to interaction with the A domain and the C domain has a separate function from the other domains.The chromosomal proteins high-mobility-group (HMG) 1 and 2 and the equivalent protein HMG T in trout testes, may be involved in the structure of active genes. This idea stems initially from the observation [I, 21 of preferential release on mild digestion of nuclei or chromatin with micrococcal nuclease or DNase I and has been supported by noting a stimulation of transcriptional activity on readdition of HMG 1 and 2 to chromatin previously depleted of these proteins [3]. From the structural point of view HMG1/2 have been designated as DNA unwinding proteins [4] and shown to bind preferentially to single-stranded DNA [4, 51. The present series of structural studies are aimed at elucidating the detailed conformation of the proteins in an attempt to throw light on how they might bind to DNA or chromatin and on what function they perform.The most reasonable proposal to date [l] is that they bind to linker DNA and so displace H1 in a state of chromatin that specifically requires the absence of this histone, i.e. active chromatin. Our earlier study [6] and those of others [7], however, show no conformational resemblance to histone H1. The almost complete sequences of Walker et al.[S] indicated a total of 259 residues in HMG1, including an unbroken sequence of 41 acidic residues close to the C-terminal [9]. The C-terminal region of the H M G 1 sequence has effectively been completed by Dixon and colleagues [lo] from the nucleotide sequence of cloned cDNA. This shows (a) that the total number of residues is probably 224, (b) that the acidic stretch is only 30 residues long and continues right up to the C-terminus of the protein and (c) that the putative cysteine at residue 165 is not present, i.e. there is only one cysteine in the whole molecule. Microheterogeneity at this locus is not excluded, however. For the purposes of this paper, the numbering system of Walker et al. will be used. This puts the C-terminal residue at position 221Abbreviations. HMG, high-mobility group; BPNS-skatole, 2-(2-nitrophenylsulphonyl)-3-methyl-3'-bromoindolenine; SDS, so...

The single cysteine on the cc-subunit of bovine brain S-100a protein has been modified with the thiol specific probe, Acrylodan. When the labelled apoprotein was excited at 380 nm the fluorescence emission maximum was centered at 484 f 2 nm, suggesting that the probe is in a fairly hydrophobic environment. Addition of Ca2+ to the protein caused the emission maximum to undergo a red shift to 504 f 2 nm, implying that the fluorophore is now more exposed to the solvent. Zn2+, when added to the protein, induced only a small perturbation and the emission maximum shifted to 48 1 f 2 nm. Ca2+ was able to perturb the fluorophore in the presence of Zn2+. 2-p-Toluidinylnaphthalene-6-sulfonate (TNS)-labelled cc-subunit when excited at 345 nm exhibited very little fluorescence in the absence of Ca2+. Addition of Ca2+ resulted in an increase in TNS fluorescence accompanied by a blue shift of the emission maximum to 445 f 1 nm indicating that the probe in the presence of Ca2+ moves to a hydrophobic domain. The fact that Ca2+ and Zn2+ can perturb the labelled sulthydryl group in the presence of each other clearly demonstrates that the binding sites for the two metal ions must be different on the m-subunit as well as on the S-IOOa protein.S-100 protein; Ca *+ effect; Znz+ effect; Fluorescence