The structural basis for the oriented assembly of a TBP/TFB/promoter complex.

Recently the definition of the metazoan RNA polymerase II and archaeal core promoters has been expanded to include a region immediately upstream of the TATA box called the B recognition element (BRE), so named because eukaryal transcription factor TFIIB and its archaeal orthologue TFB interact with the element in a sequence-specific manner. Here we present the 2.4-A crystal structure of archaeal TBP and the C-terminal core of TFB (TFB(c)) in a complex with an extended TATA-box-containing promoter that provides a detailed picture of the stereospecific interactions between the BRE and a helix-turn-helix motif in the C-terminal cyclin repeat of TFB(c). This interaction is important in determining the level of basal transcription and explicitly defines the direction of transcription.

Oligomeric integrity--the structural key to thermal stability in bacterial alcohol dehydrogenases.

Principles of protein thermostability have been studied by comparing structures of thermostable proteins with mesophilic counterparts that have a high degree of sequence identity. Two tetrameric NADP(H)-dependent alcohol dehydrogenases, one from Clostridium beijerinckii (CBADH) and the other from Thermoanaerobacter brockii (TBADH), having exceptionally high (75%) sequence identity, differ by 30 degrees in their melting temperatures. The crystal structures of CBADH and TBADH in their holo-enzyme form have been determined at a resolution of 2.05 and 2.5 A, respectively. Comparison of these two very similar structures (RMS difference in Calpha = 0.8 A) revealed several features that can account for the higher thermal stability of TBADH. These include additional ion pairs, "charged-neutral" hydrogen bonds, and prolines as well as improved stability of alpha-helices and tighter molecular packing. However, a deeper structural insight, based on the location of stabilizing elements, suggests that enhanced thermal stability of TBADH is due mainly to the strategic placement of structural determinants at positions that strengthen the interface between its subunits. This is also supported by mutational analysis of structural elements at critical locations. Thus, it is the reinforcement of the quaternary structure that is most likely to be a primary factor in preserving enzymatic activity of this oligomeric bacterial ADH at elevated temperatures.

NADP-dependent bacterial alcohol dehydrogenases: crystal structure, cofactor-binding and cofactor specificity of the ADHs of Clostridium beijerinckii and Thermoanaerobacter brockii.

We have determined the X-ray structures of the NADP(H)-dependent alcohol dehydrogenase of Clostridiim beijerinckii (CBADH) in the apo and holo-enzyme forms at 2.15 A and 2.05 A resolution, respectively, and of the holo-alcohol dehydrogenase of Thermoanaerobacter brockii (TBADH) at 2.5 A. These are the first structures of prokaryotic alcohol dehydrogenase to be determined as well as that of the first NADP(H)-dependent alcohol dehydrogenase. CBADH and TBADH 75% have sequence identity and very similar three-dimensional structures. Both are tetramers of 222 symmetry. The monomers are composed of two domains: a cofactor-binding domain and a catalytic domain. These are separated by a deep cleft at the bottom of which a single zinc atom is bound in the catalytic site. The tetramers are composed of two dimers, each structurally homologous to the dimer of alcohol dehydrogenases of vertebrates. The dimers form tetramers by means of contacts between surfaces opposite the interdomain cleft thus leaving it accessible from the surface of the tetramer. The tetramer encloses a large internal cavity with a positive surface potential. A molecule of NADP(H) binds in the interdomain cleft to the cofactor-binding domain of each monomer. The specificity of the two bacterial alcohol dehydrogenases toward NADP(H) is determined by residues Gly198, Ser199, Arg200 and Tyr218, with the latter three making hydrogen bonds with the 2'-phosphate oxygen atoms of the cofactor. Upon NADP(H) binding to CBADH, Tyr218 undergoes a rotation of approximately 120 degrees about chi1 which facilitates stacking interactions with the adenine moiety and hydrogen bonding with one of the phosphate oxygen atoms. In apo-CBADH the catalytic zinc is tetracoordinated by side-chains of residues Cys37, His59, Asp150 and Glu60; in holo-CBADH, Glu60 is retracted from zinc in three of the four monomers whereas in holo-TBADH, Glu60 does not participate in Zn coordination. In both holo-enzymes, but not in the apo-enzyme, residues Ser39 and Ser113 are in the second coordination sphere of the catalytic zinc. The carboxyl group of Asp150 is oriented with respect to the active carbon of NADP(H) so as to form hydrogen bonds with both pro-S and pro-R hydrogen atoms.

Enhanced thermal stability of Clostridium beijerinckii alcohol dehydrogenase after strategic substitution of amino acid residues with prolines from the homologous thermophilic Thermoanaerobacter brockii alcohol dehydrogenase.

A comparison of the three-dimensional structures of the closely related mesophilic Clostridium beijerinckii alcohol dehydrogenase (CBADH) and the hyperthermophilic Thermoanaerobacter brockii alcohol dehydrogenase (TBADH) suggested that extra proline residues in TBADH located in strategically important positions might contribute to the extreme thermal stability of TBADH. We used site-directed mutagenesis to replace eight complementary residue positions in CBADH, one residue at a time, with proline. All eight single-proline mutants and a double-proline mutant of CBADH were enzymatically active. The critical sites for increasing thermostability parameters in CBADH were Leu-316 and Ser-24, and to a lesser degree, Ala-347. Substituting proline for His-222, Leu-275, and Thr-149, however, reduced thermal stability parameters. Our results show that the thermal stability of the mesophilic CBADH can be moderately enhanced by substituting proline at strategic positions analogous to nonconserved prolines in the homologous thermophilic TBADH. The proline residues that appear to be crucial for the increased thermal stability of CBADH are located at a beta-turn and a terminating external loop in the polypeptide chain. Positioning proline at the N-caps of alpha-helices in CBADH led to adverse effects on thermostability, whereas single-proline mutations in other positions in the polypeptide had varying effects on thermal parameters. The finding presented here support the idea that at least two of the eight extra prolines in TBADH contribute to its thermal stability.

Crystalline alcohol dehydrogenases from the mesophilic bacterium Clostridium beijerinckii and the thermophilic bacterium Thermoanaerobium brockii: preparation, characterization and molecular symmetry.

Two tetrameric NADP(+)-dependent bacterial secondary alcohol dehydrogenases have been crystallized in the apo- and the holo-enzyme forms. Crystals of the holo-enzyme from the mesophilic Clostridium beijerinckii (NCBAD) belong to space group P2(1)2(1)2(1) with unit-cell dimensions a = 90.5, b = 127.9, c = 151.4 A. Crystals of the apo-enzyme (CBAD) belong to the same space group with unit-cell dimensions a = 80.4, b = 102.3, c = 193.5 A. Crystals of the holo-enzyme from the thermophilic Thermoanaerobium brockii (NTBAD) belong to space group P6(1(5)) (a = b = 80.6, c = 400.7 A). Crystals of the apo-form of TBAD (point mutant GI98D) belong to space group P2(1) with cell dimensions a = 123.0, b = 84.8, c = 160.4 A beta = 99.5 degrees. Crystals of CBAD, NCBAD and NTBAD contain one tetramer per asymmetric unit. They diffract to 2.0 A resolution at liquid nitrogen temperature. Crystals of TBAD(GI98D) have two tetramers per asymmetric unit and diffract to 2.7 A at 276 K. Self-rotation analysis shows that both enzymes are tetramers of 222 symmetry.