The cellular response to low tissue oxygen concentrations is mediated by the hypoxia-inducible transcription factor HIF-1. Under hypoxic conditions, HIF-1 activates transcription of critical adaptive genes by recruitment of the general coactivators CBP/p300 through interactions with its α-subunit (Hif-1α). Disruption of the Hif-1α/p300 interaction has been linked to attenuation of tumor growth. To delineate the structural basis for this interaction, we have determined the solution structure of the complex between the carboxy-terminal activation domain (CAD) of Hif-1α and the zinc-binding TAZ1 (CH1) motif of cyclic-AMP response element binding protein (CREB) binding protein (CBP). Despite the overall similarity of the TAZ1 structure to that of the TAZ2 (part of the CH3) domain of CBP, differences occur in the packing of helices that can account for differences in specificity. The unbound CAD is intrinsically disordered and remains relatively extended upon binding, wrapping almost entirely around the TAZ1 domain in a groove through much of its surface. Three short helices are formed upon binding, stabilized by intermolecular interactions. The Asn-803 side chain, which functions as a hypoxic switch, is located on the second of these helices and is buried in the molecular interface. The third helix of the Hif-1α CAD docks in a deep hydrophobic groove in TAZ1, providing extensive intermolecular hydrophobic interactions that contribute to the stability of the complex. The structure of this complex provides new insights into the mechanism through which Hif-1α recruits CBP/p300 in response to hypoxia.

One of the principal peptide components of the amyloid plaque deposits of Alzheimer's disease in humans is the 40‐amino‐acid peptide β‐amyloid A4‐(1–40)‐peptide. The full‐length A4‐(1–40)‐peptide was chemically synthesized and the solution structure determined by two‐dimensional nuclear magnetic resonance spectroscopy and restrained molecular‐dynamics calculations. Synthetic human A4‐(1–40)‐peptide was soluble and non‐aggregating for several days in 40% (by vol.) trifluoroethanol/water. All spin systems could be unambiguously assigned, and a total of 203 sequential and medium‐range cross‐peaks were found in the NOESY (nuclear Overhauser enhancement spectroscopy) spectrum. Long‐range NOE cross‐peaks that would indicate tertiary structure of the peptide were absent. The main secondary‐structure elements found by chemical‐shift analysis, sequential and medium‐range NOESY data, and NOE‐based restrained molecular‐dynamics calculations were two helices, Gln15–Asp23 and IIe31–Met35, whereas the rest of the peptide was in random‐coil conformation. A similar secondary structure is suggested for the aggregation part of prions, the postulated causative agents of the transmissible spongiform encephalopathy. The sequence of the helical part of prion proteins was observed to be remarkably similar to the sequence of the helical part of human A4‐(1–40)‐peptide.

The target of rapamycin (TOR) is a highly conserved Ser/Thr kinase that plays a central role in the control of cellular growth. TOR has a characteristic multidomain structure. Only the kinase domain has catalytic function; the other domains are assumed to mediate interactions with TOR substrates and regulators. Except for the rapamycin-binding domain, there are no high-resolution structural data available for TOR. Here, we present a structural, biophysical, and mutagenesis study of the extremely conserved COOH-terminal FATC domain. The importance of this domain for TOR function has been highlighted in several publications. We show that the FATC domain, in its oxidized form, exhibits a novel structural motif consisting of an α-helix and a COOH-terminal disulfide-bonded loop between two completely conserved cysteine residues. Upon reduction, the flexibility of the loop region increases dramatically. The structural data, the redox potential of the disulfide bridge, and the biochemical data of a cysteine to serine mutant indicate that the intracellular redox potential can affect the cellular amount of the TOR protein via the FATC domain. Because the amount of TOR mRNA is not changed, the redox state of the FATC disulfide bond is probably influencing the degradation of TOR.

Hexim1 is a cellular protein that associates with the positive transcription elongation factor b (P-TEFb) to regulate RNA polymerase II elongation of nascent mRNA transcripts. It directly binds to Cyclin T1 of P-TEFb and inhibits the kinase activity of Cdk9, leading to an arrest of transcription elongation. Here, we report the solution structure of the Cyclin T binding domain (TBD) of Hexim1 that forms a parallel coiled-coil homodimer composed of two segments and a preceding alpha helix that folds back onto the first coiled-coil unit. NMR titration, fluorescence, and immunoprecipitation experiments revealed the binding interface to Cyclin T1, which covers a large surface on the first coiled-coil segment. Electrostatic interactions between an acidic patch on Hexim1 and positively charged residues of Cyclin T1 drive the complex formation that is confirmed by mutagenesis data on Hexim1 mediated transcription regulation in cells. Thus, our studies provide structural insights how Hexim1 recognizes the Cyclin T1 subunit of P-TEFb, which is a key step toward the regulation of transcription elongation.