Abstract: Cys-59 and Cys-62, forming a disulfide bond in the four-residue loop of Shewanella violacea cytochrome c (5) (SV cytc (5)), contribute to protein stability but not to redox function. These Cys residues were substituted with Ala in SV cytc (5), and the structural and functional properties of the resulting C59A/C62A variant were determined and compared with those of the wild-type. The variant had similar features to those of the wild-type in absorption, circular dichroic, and paramagnetic (1)H NMR spectra. In addition, the redox potentials of the wild-type and variant were essentially the same, indicating that removal of the disulfide bond from SV cytc (5) does not affect the redox function generated in the vicinity of heme. However, calorimetric analysis of the wild-type and variant showed that the mutations caused a drastic decrease in the protein stability through enthalpy, but not entropy. Four residues are encompassed by the SV cytc (5) disulfide bond, which is the shortest one that has been proved to affect protein stability. The protein stability of SV cytc (5) can be controlled without changing the redox function, providing a new strategy for regulating the stability and function of cytochrome c.
Abstract: In the NAD biosynthetic pathway, nicotinamide phosphoribosyltransferase (NMPRTase; EC 2.4.2.12) plays an important role in catalyzing the synthesis of nicotinamide mononucleotide from nicotinamide and 5'-phosphoribosyl-1'-pyrophosphate. Because the diffraction pattern of the initially obtained crystals was not suitable for structure analysis, the crystal quality was improved by successive use of the microseeding technique. The resultant crystals diffracted to 2.0 A resolution. These crystals belonged to space group P21, with unit-cell parameters a = 60.56, b = 106.40, c = 82.78 A. Here, the crystallization of human NMPRTase is reported in the free form; the crystals should be useful for inhibitor-soaking experiments on the enzyme.
Abstract: The NAD(P)(+)-dependent enzyme 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) catalyzes the reversible interconversion of hydroxyl and oxo groups at position 3 of the steroid nucleus. The complex of NADH and 3alpha-HSD from Pseudomonas sp. B-0831 was crystallized by the hanging-drop vapour-diffusion method. Refinement of crystallization conditions with microseeding improved the quality of the X-ray diffraction data to a resolution of 1.8 A. The crystals belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 62.46, b = 82.25, c = 86.57 A, and contained two molecules, reflecting dimer formation of 3alpha-HSD, in the asymmetric unit.
Abstract: Bacterial 3alpha-hydroxysteroid dehydrogenase, which belongs to a short-chain dehydrogenase/reductase family and forms a dimer composed of two 26-kDa subunits, catalyzes the oxidoreduction of hydroxysteroids in a coenzyme-dependent manner. This enzyme also catalyzes the oxidoreduction of nonsteroid compounds that play an important role in xenobiotic metabolism of bacteria. We performed an x-ray analysis on the crystal of Ps3alphaHSD, the enzyme from Pseudomonas sp. B-0831 complexed with NADH. The resulting crystal structure at 1.8A resolution showed that Ps3alphaHSD exists as a structural heterodimer composed of apo- and holo-subunits. A distinct structural difference between them was found in the 185-207-amino acid region, where the structure in the apo-subunit is disordered whereas that in the holo-subunit consists of two alpha-helices. This fact proved that the NADH binding allows the helical structures to form the substrate binding pocket even in the absence of the substrate, although the region corresponds to the so-called "substrate-binding loop." The induction of alpha-helices in solution by the coenzyme binding was also confirmed by the CD experiment. In addition, the CD experiment revealed that the helix-inducing ability of NADH is stronger than that of NAD. We discuss the negative cooperativity for the coenzyme binding, which is caused by the effect of the structural change transferred between the subunits of the heterodimer.
Abstract: We have studied the structure-thermostability relationship using cytochromes c from mesophilic and thermophilic bacteria; Pseudomonas aeruginosa (PAc(551)) growing at 37 degrees C and Hydrogenobacter thermophilus (HTc(552)) at 72 degrees C and showed that only five residues primarily differentiate their stabilities. For a more comprehensive study, we found Hydrogenophilus thermoluteolus (Pseudomonas hydrogenothermophila) growing at 52 degrees C and showed the moderate stability of the cytochrome c from this bacterium (PHc(552)). To explore the stabilization mechanisms, the crystal structure of PHc(552) was determined by X-ray analysis. The solution structure of HTc(552) elucidated previously by NMR was refined using distributed computational implementation. Furthermore, the recently reported crystal structure of HTc(552) has become available [Travaglini-Allocatelli, C. et al. (2005) J. Biol. Chem. 280, 25729-25734]. When the structures of these three cytochromes c were combined, this revealed that the five residues, corresponding to those mentioned above, determine the difference of stabilities among them as well. These facts suggested the stabilization mechanisms as follows: (1) improved van der Waals interactions by packing optimization at the N-terminal helix, (2) attractive electrostatic interactions with the heme propionate group, and (3) favorable van der Waals interaction with the heme. This comparative study, by supplementing the structural information of PHc(552) with its complementary feature, demonstrates that just a small number of amino acid residues determine the overall molecular stability by means of additivity of the effects of their substitutions. It is interesting that, in naturally occurring proteins, these adaptation strategies are accommodated by these bacteria to survive in the wide range of thermal conditions.
Abstract: Five amino acid residues responsible for extreme stability have been identified in cytochrome c(552) (HT c(552)) from a thermophilic bacterium, Hydrogenobacter thermophilus. The five residues, which are spatially distributed in three regions of HT c(552), were replaced with the corresponding residues in the homologous but less stable cytochrome c(551) (PA c(551)) from Pseudomonas aeruginosa. The quintuple HT c(552) variant (A7F/M13V/Y34F/Y43E/I78V) showed the same stability against guanidine hydrochloride denaturation as that of PA c(551), suggesting that the five residues in HT c(552) necessarily and sufficiently contribute to the overall stability. In the three HT c(552) variants carrying mutations in each of the three regions, the Y34F/Y43E mutations resulted in the greatest destabilization, by -13.3 kJ mol(-1), followed by A7F/M13V (-3.3 kJ mol(-1)) and then I78V (-1.5 kJ mol(-1)). The order of destabilization in HT c(552) was the same as that of stabilization in PA c(551) with reverse mutations such as F34Y/E43Y, F7A/V13M, and V78I (13.4, 10.3, and 0.3 kJ mol(-1), respectively). The results of guanidine hydrochloride denaturation were consistent with those of thermal denaturation for the same variants. The present study established a method for reciprocal mutation analysis. The effects of side-chain contacts were experimentally evaluated by swapping the residues between the two homologous proteins that differ in stability. A comparative study of the two proteins was a useful tool for assessing the amino acid contribution to the overall stability.
Abstract: X-ray analysis has been carried out on a crystal of the collagen model peptide (Hyp(R)-Hyp(R)-Gly)10 [where Hyp(R) is 4(R)-hydroxyproline] with 1.5 A resolution. The triple-helical structure of (Hyp(R)-Hyp(R)-Gly)10 has the same helical parameters and Rich and Crick II hydrogen bond patterns as those of other collagen model peptides. However, our full-length crystal structure revealed that almost all consecutive Hyp(R) residues take the up-up pucker in contrast to putative down-up puckering propensities of other collagen model peptides. The unique feature of thermodynamic parameters associated with the conformational transition of this peptide from triple helix to single coil is that both enthalpy and entropy changes of the transition are much smaller than those of other model peptides such as (Pro-Pro-Gly)10 and (Pro-Hyp(R)-Gly)10. To corroborate the precise structural information including main- and side-chain dihedral angles and intra- and interwater bridge networks, we estimated the degrees of hydration by comparing molecular volumes observed experimentally in solution to those calculated ones from the crystal structure. The results showed that the degree of hydration of (Hyp(R)-Hyp(R)-Gly)10 is comparable to that of (Pro-Hyp(R)-Gly)10 in the triple-helical state, but the former was more highly hydrated than (Pro-Hyp(R)-Gly)10 in the single-coil state. Because hydration reduces the enthalpy due to the formation of a hydrogen bond with a water molecule and diminishes the entropy due to the restriction of water molecules surrounding a peptide molecule, we concluded that the high thermal stability of (Hyp(R)-Hyp(R)-Gly)10 is able to be described by its high hydration in the single-coil state.
Abstract: The amino-acid sequence of cytochrome c552 (PH c552) from a moderately thermophilic bacterium, Hydrogenophilus thermoluteolus, was more than 50% identical to that of cytochrome c from an extreme thermophile, Hydrogenobacter thermophilus (HT c552), and from a mesophile, Pseudomonas aeruginosa (PA c551). The PH c552 gene was overexpressed as a correctly processed holoprotein in the Escherichia coli periplasm. The overexpressed PH c552 has been crystallized by vapour diffusion from polyethylene glycol 4000 pH 6.5. The crystals belong to space group C222(1), with unit-cell parameters a = 48.98, b = 57.99, c = 56.20 A. The crystals diffract X-rays to around 2.1 A resolution.
Abstract: A plant- and prokaryote-conserved domain (PPC) has previously been found in AT-hook motif nuclear localized protein 1 (AHL1) localized in the nuclear matrix of Arabidopsis thaliana (AtAHL1). AtAHL1 has a DNA-binding function. Mutation analyses of AtAHL1 has previously revealed that the hydrophobic region of the PPC domain is essential for its nuclear localization. In this study, the PPC of the hyperthermophilic archaebacterium Pyrococcus horikoshii (PhPPC) was crystallized using the hanging-drop vapour-diffusion method. The crystals belonged to the hexagonal space group P6(3)22, with unit-cell parameters a = b = 53.69, c = 159.2 A. Data were obtained at 100 K, with diffraction being observed to a resolution of 1.7 A. A complete data set from crystals of the SeMet-substituted protein was also obtained.
Abstract: The complete thermal-unfolding profiles of both oxidized and reduced forms of cytochrome c551 (PA) from mesophilic Pseudomonas aeruginosa and cytochrome c552 (HT) from thermophilic Hydrogenobacter thermophilus were obtained by the newly developed pressure-proof cell compartment installed in a circular dichroic spectrometer, which facilitates protein thermal-unfolding experiments up to 180 degrees C. The thermodynamic cycle, which relates protein stability and redox function, indicated that the redox potentials of PA and HT in the native state are regulated by the stability of the oxidized proteins rather than by that of the reduced ones.
