Biomedical subjects

M Kuti

Publications and source records attributed to M Kuti.

4 recordsLinked to original sources

PubMed2000

Structural basis of SNT PTB domain interactions with distinct neurotrophic receptors.

SNT adaptor proteins transduce activation of fibroblast growth factor receptors (FGFRs) and neurotrophin receptors (TRKs) to common signaling targets. The SNT-1 phosphotyrosine binding (PTB) domain recognizes activated TRKs at a canonical NPXpY motif and, atypically, binds to nonphosphorylated FGFRs in a region lacking tyrosine or asparagine. Here, using NMR and mutational analyses, we show that the PTB domain utilizes distinct sets of amino acid residues to interact with FGFRs or TRKs in a mutually exclusive manner. The FGFR1 peptide wraps around the beta sandwich structure of the PTB domain, and its binding is possibly regulated by conformational change of a unique C-terminal beta strand in the protein. Our results suggest mechanisms by which SNTs serve as molecular switches to mediate the essential interplay between FGFR and TRK signaling during neuronal differentiation.

Adaptor Proteins, Signal Transducing↗

PubMed1999

A refined model for the solution structure of oxidized putidaredoxin.

A refined model for the solution structure of oxidized putidaredoxin (Pdxo), a Cys4Fe2S2 ferredoxin, has been determined. A previous structure (Pochapsky et al. (1994) Biochemistry 33, 6424-6432; PDB entry ) was calculated using the results of homonuclear two-dimensional NMR experiments. New data has made it possible to calculate a refinement of the original Pdxo solution structure. First, essentially complete assignments for diamagnetic 15N and 13C resonances of Pdxo have been made using multidimensional NMR methods, and 15N- and 13C-resolved NOESY experiments have permitted the identification of many new NOE restraints for structural calculations. Stereospecific assignments for leucine and valine CH3 resonances were made using biosynthetically directed fractional 13C labeling, improving the precision of NOE restraints involving these residues. Backbone dihedral angle restraints have been obtained using a combination of two-dimensional J-modulated 15N,1H HSQC and 3D (HN)CO(CO)NH experiments. Second, the solution structure of a diamagnetic form of Pdx, that of the C85S variant of gallium putidaredoxin, in which a nonligand Cys is replaced by Ser, has been determined (Pochapsky et al. (1998) J. Biomol. NMR 12, 407-415), providing information concerning structural features not observable in the native ferredoxin due to paramagnetism. Third, a crystal structure of a closely related ferredoxin, bovine adrenodoxin, has been published (Müller et al. (1998) Structure 6, 269-280). This structure has been used to model the metal binding site structure in Pdx. A family of fourteen structures is presented that exhibits an rmsd of 0.51 A for backbone heavy atoms and 0.83 A for all heavy atoms. Exclusion of the modeled metal binding loop region reduces overall the rmsd to 0.30 A for backbone atoms and 0.71 A for all heavy atoms.

Carbon Isotopes↗

PubMed1999

Solution structure and dynamics of a serpin reactive site loop using interleukin 1beta as a presentation scaffold.

Human interleukin-1beta (IL1beta) was used as a presentation scaffold for the characterization of the reactive site loop (RSL) of the serpin alpha1-antitrypsin (A1AT), the physiological inhibitor of leukocyte elastase. A chimeric protein was generated by replacement of residues 50-53 of IL1beta, corresponding to an exposed reverse turn in IL1beta, with the 10-residue P5-P5' sequence EAIPMSIPPE from A1AT. The chimera (antitrypsin-interleukin, AT-IL) inhibits elastase specifically and also binds the IL1beta receptor. Multinuclear NMR characterization of AT-IL established that, with the exception of the inserted sequence, the structure of the IL1beta scaffold is preserved in the chimera. The structure of the inserted RSL was analyzed relative to that of the isolated 10-residue RSL peptide, which was shown to be essentially disordered in solution. The chimeric RSL was also found to be solvent exposed and conformationally mobile in comparison with the IL1beta scaffold, and there was no evidence of persisting interactions with the scaffold outside of the N- and C-terminal linkages. However, AT-IL exhibits sigificant differences in chemical shift and NOE patterns relative to the isolated RSL that are consistent with local features of non-random structure. The proximity of these features to the P1-P1' residues suggests that they may be responsible for the inhibitory activity of the chimera.

Amino Acid Sequence↗

PubMed1998

The solution structure of a gallium-substituted putidaredoxin mutant: GaPdx C85S.

The Fe2S2 cluster of the ferredoxin putidaredoxin (Pdx) can be replaced by a single gallium ion, giving rise to a colorless, diamagnetic protein in which, apart from the metal binding site, the major structural features of the native ferredoxin are conserved. The solution structure of the C85S variant of gallium putidaredoxin (C85S GaPdx), in which a non-ligand cysteine is replaced by a serine, has been determined via multidimensional NMR methods using uniformly 15N, 13C labeled samples of C85S GaPdx. Stereospecific assignments of leucine and valine methyl resonances were made using 13C, 1H HSQC spectra obtained with fractionally 13C-labeled samples, and backbone dihedral angle restraints were obtained using a combination of two-dimensional J-modulated 15N, 1H HSQC and three-dimensional (HN)CO(CO)NH experiments. A total of 1117 NOE-derived distance restraints were used in the calculations, including 454 short range (i-j < or = 3), 456 long range (i-j > or = 4) interresidue restraints and 207 non-trivial intraresidue restraints. 97 phi and 55 chi 1 angular restraints were also included in the calculation of a family of 20 structures using a combined distance geometry-simulated annealing protocol. Most regions of the protein are well defined in the calculations, with an RMSD of 0.525 A for backbone atoms excluding the metal binding loop (residues 34-48) and the last three C-terminal residues (residues 103-106). Where comparison is possible, these regions show an increase in dynamic behavior over the native protein, as does the loop containing residues 74-76. Structural and dynamic differences between native Pdx and GaPdx are discussed in relation to charge and packing of the metal binding site.

Amino Acid Substitution↗