Kinesin Kar3Cik1 ATPase pathway for microtubule cross-linking

Kar3Cik1 is a Saccharomyces cerevisiae kinesin-14 that functions to shorten cytoplasmic microtubules (MTs) during yeast mating yet maintains mitotic spindle stability by cross-linking anti-parallel interpolar MTs. Kar3 contains both an ATP- and a MT-binding site, yet there is no evidence of a nucleotide-binding site in Cik1. Presteady-state and steady-state kinetic experiments were pursued to define the regulation of Kar3Cik1 interactions with the MT lattice expected during interpolar MT cross-linking. The results reveal that association of Kar3Cik1 with the MT occurs at 4.9 μM(-1) s(-1), followed by a 5-s(-1) structural transition that limits ADP release from the Kar3 head. Mant-ATP binding occurred at 2.1 μM(-1) s(-1), and the pulse-chase experiments revealed an ATP-promoted isomerization at 69 s(-1). ATP hydrolysis was observed as a rapid step at 26 s(-1) and was required for the Kar3Cik1 motor to detach from MT. The conformational change at 5 s(-1) that occurred after Kar3Cik1 MT association and prior to ADP release was hypothesized to be the rate-limiting step for steady-state ATP turnover. We propose a model in which Kar3Cik1 interacts with the MT lattice through an alternating cycle of Cik1 MT collision followed by Kar3 MT binding with head-head communication between Kar3 and Cik1 modulated by the Kar3 nucleotide state and intramolecular strain.     

Structural Insights into the Substrate Specificity of the Rhodopseudomonas palustris Protein Acetyltransferase RpPat: IDENTIFICATION OF A LOOP CRITICAL FOR RECOGNITION BY RpPat*?

Lysine acetylation is a post-translational modification that is important for the regulation of metabolism in both prokaryotes and eukaryotes. In bacteria, the best studied protein acetyltransferase is Pat. In the purple photosynthetic bacterium Rhodopseudomonas palustris, at least 10 AMP-forming acyl-CoA synthetase enzymes are acetylated by the Pat homologue RpPat. All bona fide RpPat substrates contain the conserved motif PX₄GK. Here, we show that the presence of such a motif is necessary but not sufficient for recognition by RpPat. RpPat failed to acetylate the methylmalonyl-CoA synthetase of this bacterium (hereafter RpMatB) in vivo and in vitro, despite the homology of RpMatB to known RpPat substrates. We used RpMatB to identify structural determinants that are recognized by RpPat. To do this, we constructed a series of RpMatB chimeras that became substrates of RpPat. In such chimeras, a short region (11–25 residues) of RpMatB located >20 residues N-terminal to the acetylation site was replaced with the corresponding sequences from other AMP-forming acyl-CoA synthetases that were known RpPat substrates. Strikingly, the enzymatic activity of RpMatB chimeras was regulated by acetylation both in vitro and in vivo. Crystal structures of two of these chimeras showed that the major difference between them and wild-type RpMatB was within a loop region ∼23 Å from the acetylation site. On the basis of these results, we suggest that RpPat likely interacts with a relatively large surface of its substrates, in addition to the PX₄GK motif, and that RpPat probably has relatively narrow substrate specificity.     

X-ray structures of the Dictyostelium discoideum myosin motor domain with six non-nucleotide analogs

The three-dimensional structures of the truncated myosin head from Dictyostelium discoideum myosin II complexed with dinitrophenylaminoethyl-, dinitrophenylaminopropyl-, o-nitrophenylaminoethyl-, m-nitrophenylaminoethyl-, p-nitrophenylaminoethyl-, and o-nitrophenyl-N-methyl-aminoethyl-diphosphate.beryllium fluoride have been determined to better than 2.3-A resolution. The structure of the protein and nucleotide binding pocket in these complexes is very similar to that of S1dC.ADP.BeF(x) (Fisher, A. J., Smith, C. A., Thoden, J., Smith, R., Sutoh, K., Holden, H. M., and Rayment, I. (1995) Biochemistry 34, 8960-8972). The position of the triphosphate-like moiety is essentially identical in all complexes. Furthermore, the alkyl-amino group plays the same role as the ribose by linking the triphosphate to the adenine binding pocket; however, none of the phenyl groups lie in the same position as adenine in S1dC.MgADP.BeF(x), even though several of these nucleotide analogs are functionally equivalent to ATP. Rather the former location of adenine is occupied by water in the nanolog complexes, and the phenyl groups are organized in a manner that attempts to optimize their hydrogen bonding interactions with this constellation of solvent molecules. A comparison of the kinetic and structural properties of the nanologs relative to ATP suggests that the ability of a substrate to sustain tension and to generate movement correlates with a well defined interaction with the active site water structure observed in S1dC.MgADP.BeF(x).     

The three-dimensional structures of nicotinate mononucleotide:5,6- dimethylbenzimidazole phosphoribosyltransferase (CobT) from Salmonella typhimurium complexed with 5,6-dimethybenzimidazole and its reaction products determined to 1.9 A resolution

Nicotinate mononucleotide:5,6-dimethylbenzimidazole phosphoribosyltransferase (CobT) from Salmonella typhimurium plays a central role in the synthesis of alpha-ribazole, which is a key component of the lower ligand of cobalamin. Two X-ray structures of CobT are reported here at 1.9 A resolution. First, a complex of CobT with 5,6-dimethylbenzimidazole, and second, a complex of CobT with its reaction products, nicotinate and alpha-ribazole-5'-phosphate. CobT was cocrystallized with 5,6-dimethylbenzimidazole (DMB) in the space group P2(1)2(1)2 with unit cell dimensions of a = 72.1 A, b = 90.2 A, and c = 47.5 A and one protomer per asymmetric unit. Subsequently, the crystals containing DMB were soaked in nicotinate mononucleotide whereupon the physiological reaction occurred in the crystal lattice to yield nicotinate and alpha-ribazole-5'-phosphate. These studies show that CobT is a dimer where each subunit consists of two domains. The large domain is dominated by a parallel six-stranded beta-sheet with connecting alpha-helices that exhibit the topology of a Rossmann fold. The small domain is made from components of the N- and C-terminal sections of the polypeptide chain and contains a three-helix bundle. The fold of CobT is unrelated to the type I and II phosphoribosylpyrophosphate dependent transferases and does not appear to be related to any other protein whose structure is known. The enzyme active site is located in a large cavity formed by the loops at the C-terminal ends of the beta-strands and the small domain of the neighboring subunit. DMB binds in a hydrophobic pocket created in part by the neighboring small domain. This is consistent with the broad specificity of this enzyme for aromatic substrates [Trzebiatowski, J. R., Escalante-Semerena (1997) J. Biol. Chem. 272, 17662-17667]. The binding site for DMB suggests that Glu317 is the catalytic base required for the reaction. The remainder of the cavity binds the nicotinate and ribose-5'-phosphate moieties, which are nestled within the loops at the ends of the beta-strands. Interestingly, the orientation of the substrate and products are opposite from that expected for a Rossmann fold.     

Frequency and Distribution of Refractive Error in Adult Life: Methodology and Findings of the UK Biobank Study

PurposeTo report the methodology and findings of a large scale investigation of burden and distribution of refractive error, from a contemporary and ethnically diverse study of health and disease in adults, in the UK.MethodsU K Biobank, a unique contemporary resource for the study of health and disease, recruited more than half a million people aged 40–69 years. A subsample of 107,452 subjects undertook an enhanced ophthalmic examination which provided autorefraction data (a measure of refractive error). Refractive error status was categorised using the mean spherical equivalent refraction measure. Information on socio-demographic factors (age, gender, ethnicity, educational qualifications and accommodation tenure) was reported at the time of recruitment by questionnaire and face-to-face interview.ResultsFifty four percent of participants aged 40–69 years had refractive error. Specifically 27% had myopia (4% high myopia), which was more common amongst younger people, those of higher socio-economic status, higher educational attainment, or of White or Chinese ethnicity. The frequency of hypermetropia increased with age (7% at 40–44 years increasing to 46% at 65–69 years), was higher in women and its severity was associated with ethnicity (moderate or high hypermetropia at least 30% less likely in non-White ethnic groups compared to White).ConclusionsRefractive error is a significant public health issue for the UK and this study provides contemporary data on adults for planning services, health economic modelling and monitoring of secular trends. Further investigation of risk factors is necessary to inform strategies for prevention. There is scope to do this through the planned longitudinal extension of the UK Biobank study.     

Chemical force microscopy with active enzymes

The adhesion forces have been measured between an atomic force microscope tip derivatized with an active enzyme, shikimate kinase, and an ATP mimic immobilized on a gold surface. Experiments with competitive binding of other ligands in solution show that the observed adhesion forces arise predominantly from specific interactions between the immobilized enzyme and surface-bound adenine derivative. These experiments represent a step in the development of a screening methodology based upon chemical force microscopy.     

Identification of a gene sequence encoding a putative pyruvate oxidoreductase in Serpulina pilosicoli

Serpulina pilosicoli is a recently described species of intestinal spirochaete which can be identified using a species-specific monoclonal antibody BJL/AC1 reactive with a 29-kDa protein located in the cell envelope. A genomic library of the type strain of S. pilosicoli P43/6/78^T was created in λ zap express™ and screened using BJL/AC1. Single positive clones were isolated and excised into the phagemid vector pBK-CMV. Phagemid DNA was purified and a single clone was selected for sequencing. The size of spirochaetal DNA insert was determined by digestion with restriction endonucleases EcoRI and PstI as being approximately 2.6 kb. The nucleotide sequence of the gene encoding the protein with which the antibody reacted was determined by cycle sequencing. The insert contained an open reading frame of 825 nucleotides. Translation of the nucleotide sequence into amino acid (aa) residues showed a sequence of 275 aa. Comparison of this sequence with databases revealed homology to pyruvate oxidoreductases from various organisms found in the gastrointestinal tract. These included the pyruvate ferredoxin oxidoreductase (POR) α subunit of Helicobacter pylori (38.8% identity in 250 aa), pyruvate-flavodoxin oxidoreductase of Escherichia coli (28.7% identity in 258 aa) and Giardia intestinalis (25.1% identity in 251 aa). A significant level of homology was also observed with hyperthermophilic bacteria such as the POR of Thermatoga maritima (38.6% in 254 aa) and the 2-ketovalerate-ferredoxin oxidoreductase of Pyrococcus furiosus (34% in 262 aa).     

Three-dimensional structure of adenosylcobinamide kinase/adenosylcobinamide phosphate guanylyltransferase (CobU) complexed with GMP: evidence for a substrate-induced transferase active site.

The X-ray crystal structure of adenosylcobinamide kinase/adenosylcobinamide phosphate guanylyltransferase (CobU) from Salmonella typhimurium bound to GMP has been determined by molecular replacement to 2.2 A resolution. CobU is a bifunctional enzyme, which catalyzes the phosphorylation of the 1-amino-O-2-propanol side chain of the adenosylcobinamide ring and subsequently functions as a guanylyltransferase to form adenosylcobinamide.GDP. The transferase activity involves a covalent enzyme-guanylyl intermediate that is most likely a phosphoramidate linkage to His(46). Previous studies have shown that the enzyme is a homotrimer and adopts a pinwheel shape. Each subunit consists of a single domain of six parallel beta-strands and one antiparallel strand flanked on either side by a total of five alpha-helices and one helical turn. Interestingly, His(46) in the apoenzyme is located a considerable distance from the kinase active site or P-loop motif and is solvent-exposed [Thompson, T. B., et al. (1998) Biochemistry 37, 7686-7695]. To examine the structural relationship of the two active sites, CobU was cocrystallized with GTP and pyrophosphate. Crystals belong to space group P2(1)2(1)2(1) with the following unit cell dimensions: a = 58. 4 A, b = 87.8 A, and c = 101.6 A. The structure shows electron density for the hydrolysis product GMP rather than the expected covalent guanylyl intermediate which appears to have been hydrolyzed in the crystal lattice. Even so, CobU exhibits a substantial conformational rearrangement. The helix axis containing His(46), the site of guanylylation, rotates 30 degrees and translates 11 A relative to the apo structure and is accompanied by compensatory unwinding and rewinding at the helix ends to allow the induction of a guanosine binding pocket between beta-strand 2 and alpha-helix 2. This conformational change brings the C(alpha) of His(46) approximately 10 A closer to the P-loop motif such that a phosphate ion located in the P-loop is only 6 A from the alpha-phosphate of GMP. This suggests that the P-loop motif may be used to coordinate the terminal phosphates in both the transferase and kinase reactions and implies that the active sites for both reactions overlap.     

Channel gate! Tension, leak and disclosure.

The crystal structure of a bacterial MscL shows how this homopentameric channel protein is held tightly shut to prevent leakage whilst at rest. By inference, the structure also shows how a stretch force in the lipid bilayer causes the channel to open. We now have a concrete picture as to how a stimulus 'gates' an ion channel.     

Three-dimensional structure of the high-potential iron-sulfur protein isolated from the purple phototrophic bacterium Rhodocyclus tenuis determined and refined at 1.5 A resolution.

The molecular structure of the high-potential iron-sulfur protein (HiPIP) isolated from the phototrophic bacterium, Rhodocyclus tenuis, has been solved and refined to a nominal resolution of 1.5 A with a crystallographic R-factor of 17.3% for all measured X-ray data from 30 A to 1.5 A. It is the smallest of the HiPIP structures studied thus far with 62 amino acid residues. Crystals used in the investigation belonged to the space group P2(1) with unit cell dimensions of a = 36.7 A, b = 52.6 A, c = 27.6 A and beta = 90.8 degrees and contained two molecules per asymmetric unit. The structure was solved by a combination of multiple isomorphous replacement with two heavy-atom derivatives, anomalous scattering from the iron-sulfur cluster, symmetry averaging and solvent flattening. The folding motif for this HiPIP is characterized by one small alpha-helix, six Type I turns, an approximate Type II turn and one Type I' turn. As in other HiPIPs, the iron-sulfur cluster is co-ordinated by four cysteinyl ligands and exhibits a cubane-like motif. These cysteinyl ligands are all located in Type I turns. The hydrogen bonding around the metal cluster in the R. tenuis protein is similar to the patterns observed in the Chromatium vinosum and Ectothiorhodospira halophila HiPIPs. Several of the amino acid residues invariant in the previously determined C. vinosum and E. halophila structures are not retained in the R. tenuis molecule. There are 13 solvent molecules structurally conserved between the two R. tenuis HiPIP molecules in the asymmetric unit, some of which are important for stabilizing surface loops. Interestingly, while it is assumed that this HiPIP functions as a monomer in solution, the two molecules in the asymmetric unit pack as a dimer and are related to each other by an approximate twofold rotation axis.