Biomedical subjects

S Kazanis

Publications and source records attributed to S Kazanis.

5 recordsLinked to original sources

PubMed1998

Yeast transcript elongation factor (TFIIS), structure and function. I: NMR structural analysis of the minimal transcriptionally active region.

TFIIS is a general transcription elongation factor that helps arrested RNA polymerase II elongation complexes resume transcription. We have previously shown that yeast TFIIS (yTFIIS) comprises three structural domains (I-III). The three-dimensional structures of domain II and part of domain III have been previously reported, but neither domain can autonomously stimulate transcription elongation. Here we report the NMR structural analysis of residues 131-309 of yTFIIS which retains full activity and contains all of domains II and III. We confirm that the structure of domain II in the context of fully active yTFIIS is the same as that determined previously for a shorter construct. We have determined the structure of the C-terminal zinc ribbon domain of active yTFIIS and shown that it is similar to that reported for a shorter construct of human TFIIS. The region linking domain II with the zinc ribbon of domain III appears to be conformationally flexible and does not adopt a single defined tertiary structure. NMR analysis of inactive mutants of yTFIIS support a role for the linker region in interactions with the transcription elongation complex.

Magnetic Resonance Spectroscopy↗

PubMed1998

Yeast transcript elongation factor (TFIIS), structure and function. II: RNA polymerase binding, transcript cleavage, and read-through.

The transcriptionally active fragment of the yeast RNA polymerase II transcription elongation factor, TFIIS, comprises a three-helix bundle and a zinc ribbon motif joined by a linker region. We have probed the function of this fragment of TFIIS using structure-guided mutagenesis. The helix bundle domain binds RNA polymerase II with the same affinity as does the full-length TFIIS, and this interaction is mediated by a basic patch on the outer face of the third helix. TFIIS mutants that were unable to bind RNA polymerase II were inactive for transcription activity, confirming the central role of polymerase binding in the TFIIS mechanism of action. The linker and zinc ribbon regions play roles in promoting cleavage of the nascent transcript and read-through past the block to elongation. Mutation of three aromatic residues in the zinc ribbon domain (Phe269, Phe296, and Phe308) impaired both transcript cleavage and read-through. Mutations introduced in the linker region between residues 240 and 245 and between 250 and 255 also severely impaired both transcript cleavage and read-through activities. Our analysis suggests that the linker region of TFIIS probably adopts a critical structure in the context of the elongation complex.

Models, Molecular↗

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↗

PubMed1997

Structural features of the metal binding site and dynamics of gallium putidaredoxin, a diamagnetic derivative of a Cys4Fe2S2 ferredoxin.

The first reconstitution of an Fe2S2 ferredoxin with a diamagnetic prosthetic group was recently described [Kazanis et al. (1995) J. Am. Chem. Soc., 117, 6625-6626]. The replacement of the iron-sulfur cluster of the bacterial ferredoxin putidaredoxin (Pdx) by gallium (Ga3+) renders the protein diamagnetic and permits the use of high-resolution NMR methods to identify resonances near the metal binding site. We now describe structural features of the metal binding site that are not observable by standard NMR methods in native Pdx due to paramagnetic line broadening. These results provide the first example of high-resolution NMR-derived structural data concerning the metal binding domain of an Fe2S2 ferredoxin, and the first structural information of any sort for the metal binding site of a ferredoxin from this class, which includes adrenodoxin, placental ferredoxin and terpredoxin. Assignments were obtained by applying multidimensional NMR methods to a series of selectively and nonselectively 15N- and 13C/15N-labeled GaPdx samples. For most experiments, a mutant of Pdx was used in which a nonligating Cys85 is replaced by serine. All of the major structural features that were identified in native Pdx are conserved in GaPdx. The overall protein dynamics is considerably faster in GaPdx than in the native protein, as reflected by amide proton exchange rates. The C-terminal residue, Trp106, also exhibits considerable mobility, as indicated by 15N[1H] NOE and 15N T1 values of the C-terminal residue of the protein.

Amino Acid Sequence↗

PubMed1996

A structure-based model for cytochrome P450cam-putidaredoxin interactions.

Putidaredoxin (Pdx) is a Fe2S2 ferredoxin which acts as the physiological reductant of cytochrome P-450cam (CYP101). A model for the solution structure of oxidized Pdx has been determined using NMR methods (Pochapsky et al (1994) Biochemistry 33, 6424-6432). 1H-15N correlations and redox-dependent amide exchange rates have also been described (Lyons et al (1996) Protein Sci 5, 627-639). Data obtained from mutagenesis and kinetic measurements concerning the interactions of Pdx and CYP101 are summarized. A model for the structure of the homologous ferredoxin adrenodoxin (Adx) is also described, and data concerning Adx activity are discussed in relation to this structure. The structures of Pdx and CYP101 were used as starting points for molecular modeling and molecular dynamics simulations. Close approach between the metal centers of the two proteins and interaction between aromatic residues on the surfaces of the proteins are premised. The resulting complex exhibits three intermolecular salt bridges, five intermolecular hydrogen bonds and a 12 A distance between the metal centers. The first direct observations of interaction between Pdx and CYP101 (by two-dimensional NMR of 15N-labeled Pdx in solution with CYP101) are described. The results of the NMR experiments indicate that conformational gating of the electron transfer complex between CYP101 and Pdx may be important.

Adrenodoxin↗