Structure and Dynamics of a Thermostable Alcohol Dehydrogenase from the Antarctic Psychrophile Moraxella sp. TAE123.

The structure of a recombinant (His-tagged at C-terminus) alcohol dehydrogenase (MoADH) from the cold-adapted bacterium Moraxella sp. TAE123 has been refined with X-ray diffraction data extending to 1.9 Å resolution. The enzyme assumes a homo-tetrameric structure. Each subunit comprises two distinct structural domains: the catalytic domain (residues 1-150 and 288-340/345) and the nucleotide-binding domain (residues 151-287). There are two Zn2+ ions in each protein subunit. Two additional zinc ions have been found in the crystal structure between symmetry-related subunits. The structure has been compared with those of homologous enzymes from Geobacillus stearothermophilus (GsADH), Escherichia coli (EcADH), and Thermus sp. ATN1 (ThADH) that thrive in environments of diverse temperatures. Unexpectedly, MoADH has been found active from 10 to at least 53 °C and unfolds at 89 °C according to circular dichroism spectropolarimetry data. MoADH with substrate ethanol exhibits a small value of activation enthalpy ΔH ‡ of 30 kJ mol-1. Molecular dynamics simulations for single subunits of the closely homologous enzymes MoADH and GsADH performed at 280, 310, and 340 K showed enhanced wide-ranging mobility of MoADH at high temperatures and generally lower but more distinct and localized mobility for GsADH. Principal component analysis of the fluctuations of both ADHs resulted in a prominent open-close transition of the structural domains mainly at 280 K for MoADH and 340 K for GsADH. In conclusion, MoADH is a very thermostable, cold-adapted enzyme and the small value of activation enthalpy allows the enzyme to function adequately at low temperatures.

Structure of dimeric SecA, the Escherichia coli preprotein translocase motor.

SecA is the preprotein translocase ATPase subunit and a superfamily 2 (SF2) RNA helicase. Here we present the 2 A crystal structures of the Escherichia coli SecA homodimer in the apo form and in complex with ATP, ADP and adenosine 5'-[beta,gamma-imido]triphosphate (AMP-PNP). Each monomer contains the SF2 ATPase core (DEAD motor) built of two domains (nucleotide binding domain, NBD and intramolecular regulator of ATPase 2, IRA2), the preprotein binding domain (PBD), which is inserted in NBD and a carboxy-terminal domain (C-domain) linked to IRA2. The structures of the nucleotide complexes of SecA identify an interfacial nucleotide-binding cleft located between the two DEAD motor domains and residues critical for ATP catalysis. The dimer comprises two virtually identical protomers associating in an antiparallel fashion. Dimerization is mediated solely through extensive contacts of the DEAD motor domains leaving the C-domain facing outwards from the dimerization core. This dimerization mode explains the effect of functionally important mutations and is completely different from the dimerization models proposed for other SecA structures. The repercussion of these findings on translocase assembly and catalysis is discussed.

Loopless Rop: structure and dynamics of an engineered homotetrameric variant of the repressor of primer protein.

The repressor of primer (Rop) protein has become a steady source of surprises concerning the relationship between the sequences and the structures of several of its mutants and variants. Here we add another piece to the puzzle of Rop by showing that an engineered deletion mutant of the protein (corresponding to a deletion of residues 30-34 of the wild-type protein and designed to restore the heptad periodicity at the turn region) results in a complete reorganization of the bundle which is converted from a homodimer to a homotetramer. In contrast (and as previously shown), a two-residue insertion, which also restores the heptad periodicity, is essentially identical with wild-type Rop. The new deletion mutant structure is a canonical, left-handed, all-antiparallel bundle with a completely different hydrophobic core and distinct surface properties. The structure agrees and qualitatively explains the results from functional, thermodynamic, and kinetic studies which indicated that this deletion mutant is a biologically inactive hyperstable homotetramer. Additional insight into the stability and dynamics of the mutant structure has been obtained from extensive molecular dynamics simulations in explicit water and with full treatment of electrostatics.

Equilibrium heat-induced denaturation of chitinase 40 from Streptomyces thermoviolaceus.

High-precision differential scanning calorimetry (DSC) and circular dichroism (CD) have been employed to study the thermal unfolding of chitinase 40 (Chi40) from Streptomyces thermoviolaceus. Chi40 belongs to family 18 of glycosyl hydrolase superfamily bearing a catalytic domain with a "TIM barrel"-like fold, which exhibits deviations from the (beta/alpha)8 fold. The thermal unfolding is reversible at pH = 8.0 and 9.0. The denatured state is characterized by extensive structural changes with respect to the native. The process is characterized by slow relaxation kinetics. Even slower refolding rates are recorded upon cooling. It is shown that the denaturation calorimetric data obtained at slow heating rate (0.17 K/min) are in excellent agreement with equilibrium data obtained by extrapolation of the experimental results to zero scanning rate. Analysis of the DSC results reveals that the experimental data can be successfully fitted using either a non-two-state sequential model involving one equilibrium intermediate, or an independent transitions model involving the unfolding of two Chi40 energetic domains to intermediate states. The stability of the native state with respect to the final denatured state is estimated, deltaG = 24.0 kcal/mol at 25 degrees C. The thermal results are in agreement with previous findings from chemical denaturation studies of a wide variety of (beta/alpha)8 barrel proteins, that their unfolding is a non-two-state process, always involving at least one unfolding intermediate.

Thermal denaturation of the BRCT tandem repeat region of human tumour suppressor gene product BRCA1.

Reduced stability of the tandem BRCT domains of human BReast CAncer 1 (BRCA1) due to missense mutations may be critical for loss of function in DNA repair and damage-induced checkpoint control. In the present thermal denaturation study of the BRCA1 BRCT region, high-precision differential scanning calorimetry (DSC) and circular dichroism (CD) spectroscopy provide evidence for the existence of a denatured state that is structurally very similar to the native. Consistency between theoretical structure-based estimates of the enthalpy (DeltaH) and heat capacity change (DeltaCp) and the calorimetric results is obtained when considering partial thermal unfolding contained in the region of the conserved hydrophobic pocket formed at the interface of the two BRCT repeats. The structural integrity of this region has been shown to be crucial for the interaction of BRCA1 with phosphorylated peptides. In addition, cancer-causing missense mutations located at the inter-BRCT-repeat interface have been linked to the destabilization of the tandem BRCT structure.

Crystallization and preliminary X-ray diffraction studies of an alcohol dehydrogenase from the Antarctic psychrophile Moraxella sp. TAE123.

An NAD(+)-dependent psychrophilic alcohol dehydrogenase (ADH) from the Antarctic psychrophile Moraxella sp. TAE123 has been purified to homogeneity. The enzyme consists of four identical subunits, each containing two Zn ions. Protein crystals suitable for X-ray diffraction were obtained under optimized salting-out crystallization conditions using ammonium sulfate as a precipitating agent. The crystals are hexagonal bipyramids and belong to space group P3(1)21 or P3(2)21, with unit-cell parameters a = 136.4, c = 210.7 A. They contain one protein homotetramer in the asymmetric unit. Diffraction data were collected to 2.2 A under cryogenic conditions using synchrotron radiation.

Ionic strength reducers: an efficient approach to protein purification and crystallization. Application to two Rop variants.

Detailed knowledge of the influence of various parameters on macromolecular solubility is essential for crystallization. The concept of so-called 'ionic strength reducers' provides insight into the changes in solubility induced by organic solvents and hydrophilic polymers in aqueous electrolytic solutions. A simple and efficient procedure is presented which exploits the properties of ionic strength reducers in the purification and crystallization of proteins. Using two designed variants of the Rop protein as model systems, superior crystals have been obtained compared with conventional techniques. This procedure is particularly useful in cases where excessive nucleation leads to the growth of a large number of tiny crystals that are useless for crystallographic analysis.

De novo purification scheme and crystallization conditions yield high-resolution structures of chitinase A and its complex with the inhibitor allosamidin.

The purification scheme of chitinase A (ChiA) from S. marcescens has been extensively revised. The pure enzyme crystallizes readily under new crystallization conditions. The ChiA crystal structure has been refined to 1.55 A resolution and the crystal structure of ChiA co-crystallized with the inhibitor allosamidin has been refined to 1.9 A resolution. Allosamidin is located in the deep active-site tunnel of ChiA and interacts with three important residues: Glu315, the proton donor of the catalysis, Asp313, which adopts two conformations in the native structure but is oriented towards Glu315 in the inhibitor complex, and Tyr390, which lies opposite Glu315 in the active-site tunnel.

Application of the effects of ionic strength reducing agents in the purification and crystallization of chitinase A.

The effects of ionic strength reducing agents may find a large number of applications. Based on these effects, we have redesigned the purification scheme of Chitinase A (ChiA) from Serratia marcescen. This scheme led to reproducibly crystallizable enzyme in both salting-in and salting-out conditions, which are presented here. Herein, we demonstrate some experimental applications of the ionic strength reducing agents theory and, in parallel, provide further evidence of the theory's correctness. Finally, we report a new crystal form produced recently in salting-in crystallization experiments. This form may allow the co-crystallization of ChiA mutants with longer substrates.