2P051 Differences between solution and crystal structures of a DNA binding protein(29. Protein structure and dynamics (II),Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)

Tsutomu Yamane, Hideyasu Okamura, Mitsunori Ikeguchi, Akinori Kidera, Yoshifumi Nishimura
2006 Seibutsu Butsuri  
TheBiophysical Society of Japan General IncorporatedAssociation 2P049NMR study on the interaction of the transaetiyation ATF-2 with MAP kinase p38 OArjtaka Nagadoi, Ishizu Michiko, Ybshifumi Nishimura Intcrnational Graduate School of Arts and Sciences, Ybkohama city U domain of 11iV. To invcstigate the mechanisrn of transcriptional regutution of aclivating trunscription factar-2 (M'F-2), we have examined lhe direct interaction betwcen thc trunsuctivation domain (TAD) and thc DNA binding domain
more » ... DNA binding domain {DBD) of me-2 or p3S Mitegen-activated pretein kimase (MAPK) hy using NMR lechniques. The MF-2-DgD contains TFIIIA-type Zn finger rnotif in N-termiTuit and exlended structure in C-teTminal, while C-terminul region has phesphorylation metif (TP repeat), MAP kinases are aetivated in response to a divcrse array of extra-cellular stimulus from growth factors and cytokines te enviremiiental stress facter. To study the structure of MF-2-TAD bound to DBD, we performed NMR experiments. Thus. residues 83 to su) of TAD, ttnd even residues flunking this regiun, adopL the ahelical structure when beund to DBD, Thc largcst dcviations arc clustered in the Csubdemain of T)dLD, which are clearly dLEferent frem the locations of maximat chemical shift deviations ebserved upon comp]ex fompation between p3gtz and TAD. The resu]ts sho-・ that the in{eraetion site of the M]F-2 TAD is dtEferent from in inactive or active form. In cunclusion, the ArF-2 DBD intcraczod with thc Csubdemain in inactiyc, whilc p38a interacted with the N-subdomain of AIrF-2 rAD in actiye, Our NMR studies previde thut phosphorylutlen disrupts the TAD and DBD interaction. Consistent with this possibility, phespherylatlon ef MF-2 by p3Sa kinase drarnaticzl ly increases its in vitro DNA-binding activity, Kagaku Institute of Lilb Scienceti (MITILS}, Machida, ]Department ef Biechemical and Chermical Engineering, Gunma University, Kiryu Mastopuran-X (MP-X) isolated from wasp venom is ail ainphiphilic peptidc which activates GTT'-bindmg regulatory protein {G-protein), Tl]c conformation of MP-X bound to G-protein was deterntined by solution NMR. However the interaction sites between MP-X und G-protein are not determined. SoLid-state NMR allowed the detemiinatien ef membrane-beund MP-X structure and the detailed analysis ef the interaction with bilayer membrunes. Thus solid-stale NMR should also be useful ior determinjng thc G-protein-bound MP-X structure and probing the interaction with G-protein. Here we have conducted mugic-angle spinning sel;d-staLe mm [o reveal struet[tre and the interaction sites. Wb havc dctcrmined thc secondary structure of uniformly 'iC, iSN labeled MP-X bound to the deuterated a subunit of G" protein by selid-statc NMR. MP-X was dissolved in an NMR sarnp[e but't'er including G, /a. Then we added to lhe solution 30 times as much Lrehaluse as G/ia in molur rutio as a cryoprotectant. Most NMR spectTa wore recorcled on a solid-sta[e NMR spectrometer at a static field of 11.g T. 2D broadband 'iC-]SC cormlation spectra were obtained with a spin-diffu sion pulse sequence (DARR) at a mixing timc ef ]O ms, This mixing schcme provides inrra-residue eorretations. The chemical shifts of C?. C. und C" suggested that the G"u-buund MP-X a!so ferms an amphiphilic a-hclix from W3 Lo C-teJminal Ll4, Agrcements of these chemical shifts with those for mcmbrane-bound MP-X indicate the similarity between 6protein-andmembrane-boundstructures. 2P051 Differences between solution and crystal binding protein OTsutomu vamane. I{ideyasu Okamur", Mitsunori YbshifumiNishimuru Tnt. Grad. sch. ofArts and Scl., M)kohama City Univ. structures of a DNA Ikeguchi, Akineri Kidera. Protein structures snlvcd by X-ray and NMR are each determined in entirely differcnt environments, However, it is difficult to obtain a ctear-cut explana[ion uf the differences betwcen thc two typcs of stmeturcs, because the lewer quality of NMR strticturcs tends to prevcnt from a rigorous cemparison. Here, by refining the NMR structure using elaborated simulation tcchniqucs. wc tried to analyie the differences between NMR and crysta] structures of a protein-DNA complex. In this study, we supplemented the experimental infermation oi distancc and dthcdral rcst[aints with thc force field used in molecular dynamics simulations, which includes thc fully charged protein and DNA. and explieit wateT melecules, as welt as the proper treatment of electrostarlc intcractions. In addition, we used simu]ated annealing (SA) to search a vast conformational spuce fer most fuverable structurc. The s]'stem ceiisidered here is PhuB DNA binding domain con)plexcd with DNA. Aftcr refining the NMR stmeture, we compared the resultant structure with the crystal struetures of four PheB-DNA complexes contained tn an asymrnetrical unit of thc crystal. In thc analysis. we focused on hydrogen bonding und side-chain conformations in the PhoB-DNA interface, and hydrogen bonding with wutc: As the resu]ts, we fuund thaL the :efined NMR structure actually resembles the crystal structurc morc than observed in the NMR strueture obtained by the conventienal method. 2P052SolutSon Diabetes is defined by a chronic hyperglyeemia, Advanced glycation endpreducts (AGE) resulting from a Maillard reactien increase in diabetie patients and cause hoth macrovascular and microvascu]ar disorders which result in diabetic coTnplicutions, The TeeeptoT (RAGE) has been identified te be a singlc-span transmeinbranc preLcin belunging to thc immunoglobulin superfamily. Due to the clinical siginficances ef the vHsculHr complicntions. RAGE is cttrrently focused as all attraetive turget for drug discoveiy of cundidaLes whieh gnhibit the intracellular signaling by interfering the AGE-RAGE binding. We have determined herc by NMR the three-dimensional strucLure of the v'ariable-typc domain of RAGE (vRAGE), which has been rcvealcd to contain the binding site efAGE. In solution, vRAGE forms a e sandwich structure composed nf seve] 6-strands and three cemplementurity-determinlng region (CDR) -like Loops. The overall stmcrure eihibited a typical V-type immunoglobulin fold with an ttdditional short a-hc]ix, It is churacteristie thaL atmest half of the molecular surfacc is covercd by pesitively chargcd rcsiducs, This positive region should contribute to the binding of negatively cimged AGE, lt was feund that two strands amon.v nine 6-strands in [he Vttype immunoglohulin fold are flcxible. 11iis might be implicated with another feature of RAGE that has binding ability tD various ljgands as a multitigand recep:or related to various diseases, such as inflarnmatlon, Alzheimer's disease und tumors.
doi:10.2142/biophys.46.s308_3 fatcat:mryfuowhzvhvxohmlid25a56pa