A Model of a MAPK•Substrate Complex in an Active Conformation: A Computational and Experimental Approach

The mechanisms by which MAP kinases recognize and phosphorylate substrates are not completely understood. Efforts to understand the mechanisms have been compromised by the lack of MAPK-substrate structures. While MAPK-substrate docking is well established as a viable mechanism for bringing MAPKs and substrates into close proximity the molecular details of how such docking promotes phosphorylation is an unresolved issue. In the present study computer modeling approaches, with restraints derived from experimentally known interactions, were used to predict how the N-terminus of Ets-1 associates with ERK2. Interestingly, the N-terminus does not contain a consensus-docking site ((R/K)2-3-X2-6-ΦA-X-ΦB, where Φ is aliphatic hydrophobic) for ERK2. The modeling predicts that the N-terminus of Ets-1 makes important contributions to the stabilization of the complex, but remains largely disordered. The computer-generated model was used to guide mutagenesis experiments, which support the notion that Leu-11 and possibly Ile-13 and Ile-14 of Ets-1 1-138 (Ets) make contributions through binding to the hydrophobic groove of the ERK2 D-recruiting site (DRS). Based on the modeling, a consensus-docking site was introduced through the introduction of an arginine at residue 7, to give the consensus 7RK-X2-ΦA-X-ΦB13. This results in a 2-fold increase in kcat/Km for the phosphorylation of Ets by ERK2. Similarly, the substitution of the N-terminus for two different consensus docking sites derived from Elk-1 and MKK1 also improves kcat/Km by two-fold compared to Ets. Disruption of the N-terminal docking through deletion of residues 1-23 of Ets results in a 14-fold decrease in kcat/Km, with little apparent change in kcat. A peptide that binds to the DRS of ERK2 affects Km, but not kcat. Our kinetic analysis suggests that the unstructured N-terminus provides 10-fold uniform stabilization of the ground state ERK2•Ets•MgATP complex and intermediates of the enzymatic reaction

Cation-Binding Sites of Subtilisin Carlsberg Probed with Eu(III) Luminescence

AbstractTwo Ca2+-binding sites of subtilisin Carlsberg are studied by monitoring static and time-resolved luminescence of selectively substituted Eu3+ at each site, and they are found to be characteristically quite different from each other. Compared with the coordination sphere of free Eu3+, two sites are very similar to each other, so that both have a well-defined binding structure with low coordination symmetry. However, compared with the weak site, the strong site is relatively more polar, more symmetrical, and more easily accessible. Furthermore, despite the absence of water reported in the x-ray crystal structure (Bode et al., 1987, Eur. J. Biochem. 166:673–692), one water molecule is found to exist in the coordination sphere of the strong site in aqueous solution. Thus it is suggested that in solution the Ca2+ bound in the strong site forms an additional coordination bond to a solvent or substrate molecule

Co(II)-binding isotherm of M2 peptide (200 μM in pH 7.4, 20 mM phosphate buffer) was obtained by monitoring changes in absorbance at 360 nm upon stepwise Co(II) addition.

<p>Two molar equivalent Co(II) ion binding to a unimolar M2 peptide was observed. The sigmoidal binding isotherm suggests a concerted binding mode of Co(II) to M2 peptide. Inset, change in absorbance spectra of Co(II)-coordinated M2.</p

Metal-ion dependent oxidation kinetics of M2 peptide.

<p>Ten molar equivalents of Zn(II), Ni(II), and Co(II) ions, respectively, were added to 60 μM M2 peptide in pH 7.4 phosphate buffer solution. The free thiol content was measured for each sample after incubation under accelerated oxidation condition.</p

Zn(II)-ion dependent oxidation kinetics of M2 antibody (HM2) with (solid line) and without (dotted line) Zn(II) ions.

<p>Zn(II)-ion dependent oxidation kinetics of M2 antibody (HM2) with (solid line) and without (dotted line) Zn(II) ions.</p

Eriocaulon nudicuspe Maxim.

原著和名: シラタマホシクサ科名: ホシクサ科 = Eriocaulaceae採集地: 愛知県 新城市 中宇利 (三河 新城市 中宇利)採集日: 1979/9/15採集者: 萩庭丈壽整理番号: JH034806国立科学博物館整理番号: TNS-VS-98480