The interaction between the amino-terminal transactivation domain (TAD) of p53 and TFIIH is directly correlated with the ability of p53 to activate both transcription initiation and elongation. We have identified a region within the p53 TAD that specifically interacts with the pleckstrin homology (PH) domain of the p62 and Tfb1 subunits of human and yeast TFIIH. We have solved the 3D structure of a complex between the p53 TAD and the PH domain of Tfb1 by NMR spectroscopy. Our structure reveals that p53 forms a nine residue amphipathic alpha helix (residues 47-55) upon binding to Tfb1. In addition, we demonstrate that diphosphorylation of p53 at Ser46 and Thr55 leads to a significant enhancement in p53 binding to p62 and Tfb1. These results indicate that a phosphorylation cascade involving Ser46 and Thr55 of p53 could play an important role in the regulation of select p53 target genes.

Inwardly rectifying K+ channels bind intracellular magnesium and polyamines to generate inward rectification. We have examined the architecture of the inner pore of Kir2.1 channels by covalently attaching a constrained number (from one to four) of positively charged moieties of different sizes to the channel. Our results indicate that the inner pore is formed solely by the second transmembrane segment and is unprecedentedly wide. At a position critical for inward rectification (D172), the pore is sufficiently wide to bind three Mg2+ ions or polyamine molecules simultaneously. Single-channel recordings directly demonstrate that partially modified channels exhibit distinct subconductance levels. Such a wide inner pore may greatly facilitate ion permeation and high-affinity binding of multiple pore blockers to generate strong inward rectification.

The functional, spectral, and structural properties of elephant myoglobin and the L29F/H64Q mutant of sperm whale myoglobin have been compared in detail by conventional kinetic techniques, infrared and resonance Raman spectroscopy, 1H NMR, and x-ray crystallography. There is a striking correspondence between the properties of the naturally occurring elephant protein and those of the sperm whale double mutant, both of which are quite distinct from those of native sperm whale myoglobin and the single H64Q mutant. These results and the recent crystal structure determination by Bisig et al. (Bisig, D. A., Di Iorio, E. E., Diederichs, K., Winterhalter, K. H., and Piontek, K. (1995) J. Biol. Chem. 270, 20754-20762) confirm that a Phe residue is present at position 29 (B10) in elephant myoglobin, and not a Leu residue as is reported in the published amino acid sequence. The single Gln64(E7) substitution lowers oxygen affinity approximately 5-fold and increases the rate of autooxidation 3-fold. These unfavorable effects are reversed by the Phe29(B10) replacement in both elephant myoglobin and the sperm whale double mutant. The latter, genetically engineered protein was originally constructed to be a blood substitute prototype with moderately low O2 affinity, large rate constants, and increased resistance to autooxidation. Thus, the same distal pocket combination that we designed rationally on the basis of proposed mechanisms for ligand binding and autooxidation is also found in nature.

Comprehensive 1H NMR assignments of the heme cavity proton resonances of sperm whale metmyoglobin cyanide have provided the dipolar shifts for nonligated residues which, together with the crystal coordinates of carbonyl myoglobin, allow accurate determination of both the anisotropies and orientation of the paramagnetic susceptibility tensor, χ, in the molecular framework. The resulting axial, Δχax = 2.48 × 10-8 m3/mol, and rhombic anisotropy, Δχrh = −0.58 × 10-8 m3/mol, values at 25 °C determined from the most complete set of dipolar shifts are determined to 2% and 6% uncertainty, respectively, and agree well with theoretical estimates (Horrocks, W. D., Jr. and Greenberg, E. S. Mol. Phys. 1974, 27, 993−999). Numerically and spatially restricted input data sets lead to larger uncertainties in Δχax and Δχrh, but do not systematically bias the orientation of the tensor. Determination of the anisotropies and orientation over the temperature range 5−50 °C shows that the susceptibility tensor orientation is minimally influenced, with both anisotropies well-behaved, and with Δχax exhibiting a temperature behavior close to that predicted for the system. The quantitative determination of the magnetic anisotropies over temperature allows the quantitative separation of contact and dipolar shifts for the iron ligands. The heme contact shifts reflect the expected dominant π spin density at pyrrole positions, but the meso-protons exhibit low-field contact shifts indicative of unpaired spin in a σ orbital. Such delocalized σ spin density could arise from either deformation of the heme from planarity or the loss of σ/π separation for the d xz , d yz orbitals when the major magnetic axis is tilted strongly from the heme normal as is experimentally observed. The observed anomalous temperature dependencies of the heme methyl and axial His ring contact shifts, as well as that of the rhombic anisotropy, are all consistent with thermal population of the excited orbital state. The limitations for quanitatively determining the excited orbital state energy separation from the available NMR data are discussed.

FCP1 [transcription factor IIF (TFIIF)-associated carboxyl-terminal domain (CTD) phosphatase] is the only identified phosphatase specific for the phosphorylated CTD of RNA polymerase II (RNAP II).The phosphatase activity of FCP1 is enhanced in the presence of the large subunit of TFIIF (RAP74 in humans). It has been demonstrated that the CTD of RAP74 (cterRAP74; residues 436 -517) directly interacts with the highly acidic CTD of FCP1 (cterFCP; residues 879 -961 in human). In this manuscript, we have determined a high-resolution solution structure of a cterRAP74͞cterFCP complex by NMR spectroscopy. Interestingly, the cterFCP protein is completely disordered in the unbound state, but forms an ␣-helix (H1 ; E945-M961) in the complex. The cterRAP74͞cterFCP binding interface relies extensively on van der Waals contacts between hydrophobic residues from the H2 and H3 helices of cterRAP74 and hydrophobic residues from the H1 helix of cterFCP. The binding interface also contains two critical electrostatic interactions involving aspartic acid residues from H1 of cterFCP and lysine residues from both H2 and H3 of cterRAP74. There are also three additional polar interactions involving highly conserved acidic residues from the H1 helix. The cterRAP74͞cterFCP complex is the first highresolution structure between an acidic residue-rich domain from a holoenzyme-associated regulatory protein and a general transcription factor. The structure defines a clear role for both hydrophobic and acidic residues in protein͞protein complexes involving acidic residue-rich domains in transcription regulatory proteins. R NA polymerase II (RNAP II) is a multisubunit enzyme complex that enters the initiation complex with the carboxylterminal domain (CTD) of its largest subunit in an unphosphorylated form (RNAP IIA). The CTD contains a heptapeptide repeat (YSPTSPS) that becomes extensively phosphorylated (RNAP IIO) primarily at serine-2 and -5 during early stages of transcription (1-3). In the last several years, numerous protein kinases have been implicated in the phosphorylation of the CTD (4-8). This phosphorylation of the CTD enables RNAP II to progress from the initiation phase to a stable elongation complex, and the CTD remains extensively phosphorylated throughout the elongation phase of transcription (4-6). After completion of the transcription cycle, this same RNAP II must be in the unphosphorylated form (RNAP IIA) to be recruited back to the initiation complex (9). Therefore, dephosphorylation of the CTD by a phosphatase(s) is essential to generating a form of the polymerase (RNAP IIA) that is capable of reinitiating transcription.FCP1 [transcription factor IIF (TFIIF)-associating component of the CTD phosphatase], the only known RNAP II CTD-specific phosphatase, was originally partially purified from HeLa cell (10) and yeast (11) extracts. From experiments with this partially purified CTD phosphatase, it was determined that both general transcription factors IIB (TFIIB) and IIF (TFIIF) play important roles in regulating its activity (...

General transcription factor IIH (TFIIH) is recruited to the preinitiation complex (PIC) through direct interactions between its p62 (Tfb1) subunit and the carboxyl-terminal domain of TFIIEalpha. TFIIH has also been shown to interact with a number of transcriptional activator proteins through interactions with the same p62 (Tfb1) subunit. We have determined the NMR solution structure of the amino-terminal domain from the Tfb1 subunit of yeast TFIIH (Tfb1(1-115)). Like the corresponding domain from the human p62 protein, Tfb1(1-115) contains a PH domain fold despite a low level of sequence identity between the two functionally homologous proteins. In addition, we have performed in vitro binding studies that demonstrate that the PH domains of Tfb1 and p62 specifically bind to monophosphorylated inositides [PtdIns(5)P and PtdIns(3)P]. NMR chemical shift mapping demonstrated that the PtdIns(5)P binding site on Tfb1 (p62) is located in the basic pocket formed by beta-strands beta5-beta7 of the PH domain fold. Interestingly, the structural composition of the PtdIns(5)P binding site is different from the composition of the binding sites for phosphoinositides on prototypic PH domains. We have also determined that the PH domains from Tfb1 and p62 are sufficient for binding to the activation domain of VP16. NMR chemical shift mapping demonstrated that the VP16 binding site within the PH domain of Tfb1 (p62) overlaps with the PtdIns(5)P binding site on Tfb1 (p62). These results provide new information about the recognition of phosphoinositides by PH domains, and point to a potential role for phosphoinositides in VP16 regulation.

The bivalve mollusc Lucina pectinata harbors sulfideoxidizing chemoautotrophic bacteria and expresses a monomeric hemoglobin I, HbI, with normal O 2 , but extraordinarily high sulfide affinity. The crystal structure of aquomet Lucina HbI has revealed an active site with three residues not commonly found in vertebrate globins: Phe(B10), Gln(E7), and Phe(E11) (Rizzi, M., Wittenberg, J. B., Coda, A., Fasano, M., Ascenzi, P., and Bolognesi, M. (1994) J. Mol. Biol. 244, 86 -89). Engineering these three residues into sperm whale myoglobin results in a triple mutant with ϳ700-fold higher sulfide affinity than for wild-type. The single crystal x-ray structure of the aquomet derivative of the myoglobin triple mutant and the solution 1 H NMR active site structures of the cyanomet derivatives of both the myoglobin mutant and Lucina HbI have been determined to examine further the structural origin of their unusually high sulfide affinities. The major differences in the distal pocket is that in the aquomet form the carbonyl of Gln 64 (E7) serves as a H-bond acceptor, whereas in the cyanomet form the amido group acts as H-bond donor to the bound ligand. Phe 68 (E11) is rotated ϳ90°about 2 and located ϳ1-2 Å closer to the iron atom in the myoglobin triple mutant relative to its conformation in Lucina HbI. The change in orientation potentially eliminates the stabilizing interaction with sulfide and, together with the decrease in size of the distal pocket, accounts for the 7-fold lower sulfide affinity of the myoglobin mutant compared with that of Lucina HbI.

No abstract