Ultra-high resolution X-ray structure of orthorhombic bovine pancreatic Ribonuclease A at 100K.

The crystal structure of orthorhombic Bovine Pancreatic Ribonuclease A has been determined to 0.85 Å resolution using low temperature, 100 K, synchrotron X-ray data collected at 16000 keV (λ = 0.77 Å). This is the first ultra-high-resolution structure of a native form of Ribonuclease A to be reported. Refinement carried out with anisotropic displacement parameters, stereochemical restraints, inclusion of H atoms in calculated positions, five [Formula: see text] moieties, eleven ethanol molecules and 293 water molecules, converged with final R values of R1(Free) = 0.129 (4279 reflections) and R1 = 0.112 (85,346 reflections). The refined structure was deposited in the Protein Data Bank as structure 7p4r. Conserved waters, using four high resolution structures, have been investigated. Cluster analysis identified clusters of water molecules that are associated with the active site of Bovine Ribonuclease A. Particular attention has been paid to making detailed comparisons between the present structure and other high quality Bovine Pancreatic Ribonuclease A X-ray crystal structures with special reference to the deposited classic monoclinic structure 3RN3 Howlin et al. (Acta Crystallogr A 45:851-861, 1989). Detailed studies of various aspects of hydrogen bonding and conformation have been carried out with particular reference to active site residues Lys-1, Lys-7, Gln-11, His-12, Lys-41, Asn-44, Thr-45, Lys-66, His-119 and Ser-123. For the two histidine residues in the active site the initial electron density map gives a clear confirmation that the position of His-12 is very similar in the orthorhombic structure to that in 3RN3. In 3RN3 His-119 exhibited poor electron density which was modelled and refined as two distinct sites, A (65%) and B (35%) but with respect to His-119 in the present ultra-high resolution orthorhombic structure there is clear electron density which was modelled and refined as a single conformation distinct from either conformation A or B in 3RN3. Other points of interest include Serine-32 which is disordered at the end of the sidechain in the present orthorhombic form but has been modelled as a single form in 3RN3. Lysine-66: there is density indicating a possible conformation for this residue. However, the density is relatively weak, and the conformation is unclear. Three types of amino acid representation in the ultra-high resolution electron density are examined: (i) sharp with very clearly resolved features, for example Lys-37; (ii) well resolved but clearly divided into two conformations which are well behaved in the refinement, both having high quality geometry, for example Tyr-76; (iii) poor density and difficult or impossible to model, an example is Lys-31 for which density is missing except for Cß. The side chains of Gln-11, His-12, Lys-41, Thr-45 and His-119 are generally recognised as being closely involved in the enzyme activity. It has also been suggested that Lys-7, Asp-44, Lys-66, Phe-120, Asp-121 and Ser-123 may also have possible roles in this mechanism. A molecular dynamics study on both structures has investigated the conformations of His-119 which was modelled as two conformations in 3RN3 but is observed to have a single clearly defined conformation in the present orthorhombic structure. MD has also been used to investigate Lys-31, Lys-41 and Ser32. The form of the Ribonuclease A enzyme used in both the present study and in 3RN3 (Howlin et al. in Acta Crystallogr A 45:851-861, 1989) includes a sulphate anion which occupies approximately the same location as the [Formula: see text] phosphate group in protein nucleotide complexes (Borkakoti et al. in J Mol Biol 169:743-755, 1983). The present structure contains 5 [Formula: see text] groups SO41151-SO41155 two of which, SO41152 and SO41153 are disordered, SO41152 being in the active site, and 11 EtOH molecules, EOH A 201-EOH A 211 all of which have good geometry. H atoms were built into the EtOH molecules geometrically. Illustrations of these features in the present structure are included here. The sulphates are presumably present in the material purchased for use in the present study. 293 water molecules are included in the present structure compared to 134 in 3RN3 (Howlin et al. in Acta Crystallogr A 45:851-861, 1989).

Binding site asymmetry in human transthyretin: insights from a joint neutron and X-ray crystallographic analysis using perdeuterated protein.

Human transthyretin has an intrinsic tendency to form amyloid fibrils and is heavily implicated in senile systemic amyloidosis. Here, detailed neutron structural studies of perdeuterated transthyretin are described. The analyses, which fully exploit the enhanced visibility of isotopically replaced hydrogen atoms, yield new information on the stability of the protein and the possible mechanisms of amyloid formation. Residue Ser117 may play a pivotal role in that a single water molecule is closely associated with the γ-hydrogen atoms in one of the binding pockets, and could be important in determining which of the two sites is available to the substrate. The hydrogen-bond network at the monomer-monomer interface is more extensive than that at the dimer-dimer interface. Additionally, the edge strands of the primary dimer are seen to be favourable for continuation of the ß-sheet and the formation of an extended cross-ß structure through sequential dimer couplings. It is argued that the precursor to fibril formation is the dimeric form of the protein.

The molecular basis of heme oxygenase deficiency in the pcd1 mutant of pea.

The pcd1 mutant of pea lacks heme oxygenase (HO) activity required for the synthesis of the phytochrome chromophore and is consequently severely deficient in all responses mediated by the phytochrome family of plant photoreceptors. Here we describe the isolation of the gene encoding pea heme oxygenase 1 (PsHO1) and confirm the presence of a mutation in this gene in the pcd1 mutant. PsHO1 shows a high degree of sequence homology to other higher plant HOs, in particular with those from other legume species. Expression of PsHO1 increased in response to white light, but did not respond strongly to narrow band light treatments. Analysis of the biochemical activity of PsHO1 expressed in Escherichia coli demonstrated requirements for reduced ferredoxin, a secondary reductant such as ascorbate and an iron chelator for maximum enzyme activity. Using the crystal structure data from homologous animal and bacterial HOs we have modelled the structure of PsHO1 and demonstrated a high degree of structural conservation despite limited primary sequence homology. However, the catalytic site of PsHO1 is larger than that of animal HOs indicating that it may accommodate an ascorbate molecule in close proximity to the heme. This could provide an explanation for why plant HOs show a strong and saturable dependence on this reductant.

The X-ray structure of the plant like 5-aminolaevulinic acid dehydratase from Chlorobium vibrioforme complexed with the inhibitor laevulinic acid at 2.6 A resolution.

5-Aminolaevulinic acid dehydratase (ALAD), an early enzyme of the tetrapyrrole biosynthesis pathway, catalyses the dimerisation of 5-aminolaevulinic acid to form the pyrrole, porphobilinogen. ALAD from Chlorobium vibrioforme is shown to form a homo-octameric structure with 422 symmetry in which each subunit adopts a TIM-barrel fold with a 30 residue N-terminal arm extension. Pairs of monomers associate with their arms wrapped around each other. Four of these dimers interact principally via their arm regions to form octamers in which each active site is located on the surface. The active site contains two invariant lysine residues (200 and 253), one of which (Lys253) forms a Schiff base link with the bound substrate analogue, laevulinic acid. The carboxyl group of the laevulinic acid forms hydrogen bonds with the side-chains of Ser279 and Tyr318. The structure was examined to determine the location of the putative active-site magnesium ion, however, no evidence for the metal ion was found in the electron density map. This is in agreement with previous kinetic studies that have shown that magnesium stimulates but is not required for activity. A different site close to the active site flap, in which a putative magnesium ion is coordinated by a glutamate carboxyl and five solvent molecules may account for the stimulatory properties of magnesium ions on the enzyme.

Atomic resolution analysis of the catalytic site of an aspartic proteinase and an unexpected mode of binding by short peptides.

The X-ray structures of native endothiapepsin and a complex with a hydroxyethylene transition state analog inhibitor (H261) have been determined at atomic resolution. Unrestrained refinement of the carboxyl groups of the enzyme by using the atomic resolution data indicates that both catalytic aspartates in the native enzyme share a single negative charge equally; that is, in the crystal, one half of the active sites have Asp 32 ionized and the other half have Asp 215 ionized. The electron density map of the native enzyme refined at 0.9 A resolution demonstrates that there is a short peptide (probably Ser-Thr) bound noncovalently in the active site cleft. The N-terminal nitrogen of the dipeptide interacts with the aspartate diad of the enzyme by hydrogen bonds involving the carboxyl of Asp 215 and the catalytic water molecule. This is consistent with classical findings that the aspartic proteinases can be inhibited weakly by short peptides and that these enzymes can catalyze transpeptidation reactions. The dipeptide may originate from autolysis of the N-terminal Ser-Thr sequence of the enzyme during crystallization.