The 20S proteasome is the catalytic portion of the 26S proteasome. Constitutively expressed mammalian 20S proteasomes have three active subunits, beta 1, beta 2, and beta 5, which are replaced in the immunoproteasome by interferon-gamma-inducible subunits beta 1i, beta 2i, and beta 5i, respectively. Here we determined the crystal structure of the bovine 20S proteasome at 2.75 A resolution. The structures of alpha 2, beta 1, beta 5, beta 6, and beta 7 subunits of the bovine enzyme were different from the yeast enzyme but enabled the bovine proteasome to accommodate either the constitutive or the inducible subunits. A novel N-terminal nucleophile hydrolase activity was proposed for the beta 7 subunit. We also determined the site of the nuclear localization signals in the molecule. A model of the immunoproteasome was predicted from this constitutive structure.
Amino acid residues in the ligand binding pocket of human neuroglobin have been identified by site-directed mutagenesis and their properties investigated by resonance Raman and flash photolysis methods. Wildtype neuroglobin has been shown to have six-coordinate heme in both ferric and ferrous states. Substitution of His 96 by alanine leads to complete loss of heme, indicating that His 96 is the proximal ligand. The resonance Raman spectra of M69L and K67T mutants were similar to those of wild-type (WT) neuroglobin in both ferric and ferrous states. By contrast, H64V was six-coordinate high-spin and five-coordinate high-spin in the ferric and ferrous states, respectively, at acidic pH. The spectra were pH-dependent and six-coordinate with the lowspin component dominating at alkaline pH. In a double mutant H64V/K67T, the high-spin component alone was detected in the both ferric and the ferrous states. This implies that His 64 is the endogenous ligand and that Lys 67 is situated nearby in the distal pocket. In the ferrous H64V and H64V/K67T mutants, the (Fe-His) stretching frequency appears at 221 cm ؊1 , which is similar to that of deoxymyoglobin. In the ferrous CO-bound state, the (Fe-CO) stretching frequency was detected at 521 and 494 cm ؊1 in WT, M69L, and K67T, while only the 494 cm ؊1 component was detected in the H64V and H64V/K67T mutants. Thus, the 521 cm ؊1 component is attributed to the presence of polar His 64 . The CO binding kinetics were biphasic for WT, H64V, and K67T and monophasic for H64V/K67T. Thus, His 64 and Lys 67 comprise a unique distal heme pocket in neuroglobin.
CooA is a CO-dependent transcription factor of the bacterium
The binding of the copper(II) complex of water-soluble meso-tetrakis(N-methylpyridinium-4-yl)porphyrin (TMPyP) to double-helical polynucleotides has been studied by optical absorption, circular dichroism (CD), and resonance Raman spectroscopic methods. The target polymers were RNA and RNA.DNA hybrids consisting of rA.rU, rI.rC, rA.dT, and rI.dC base pairs. Relative to the metal-free H(2)TMPyP [Uno, T., Hamasaki, K., Tanigawa, M., and Shimabayashi, S. (1997) Inorg. Chem. 36, 1676-1683], CuTMPyP binds to poly(rA).poly(dT) and poly(rA).poly(rU) with a greatly increased binding constant. The external self-stacking of the porphyrin on the surface of the polymers was evident from the strong conservative-type induced CD signals. The signal intensity correlated almost linearly with the number of stacking sites on the polymer except for poly(rA).poly(dT), which showed extraordinarily strong CD signals. Thus, the bound porphyrin may impose an ordered architecture on the polymer surface, the stacking being facilitated by the more planar nature of the CuTMPyP than the nonmetal counterpart. Resonance Raman spectra of the stacked CuTMPyP were indistinguishable from those of the intercalated one with positive delta(Cbeta-H) and negative delta(Cm-Py) bending shifts, and hence the stacked porphyrins are suggested to adopt a similar structure to that of intercalated ones. Porphyrin flattening by copper insertion opens a new avenue for medical applications of porphyrins, blocking biological events related to RNA and hybrids in malignant cells.
The crystal structure of the 20S proteasome from bovine liver was determined by the molecular replacement method using the structure of the 20S proteasome from the yeast Sacccharomyces cerevisiae. The initial phases were refined by density modification coupled with non-crystallographic symmetry averaging. The structural model was refined with the program CNS. The final R-factor and R(free) were 0.25 and 0.29, respectively. The constitutive proteasome without any contamination by the immunoproteasome was identified in the crystal structure.
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