Biomedical subjects

Wendy F Ochoa

Publications and source records attributed to Wendy F Ochoa.

9 recordsLinked to original sources

PubMed2006

Generation and structural analysis of reactive empty particles derived from an icosahedral virus.

Chemical and genetic modifications on the surface of viral protein cages confer unique properties to the virus particles with potential nano and biotechnological applications. The enclosed space in the interior of the virus particles further increases its versatility as a nanomaterial. In this paper, we report a simple method to generate a high yield of stable cowpea mosaic virus (CPMV) empty capsids from their native nucleoprotein counterparts by removing the encapsidated viral genome without compromising the integrity of the protein coat. Biochemical and structural comparison of artificially generated empty particles did not reveal any distinguishable differences from CPMV particles containing viral RNA. Preliminary results on the use of artificially produced empty CPMV capsids as a carrier capsule are described.

Cryoelectron Microscopy↗

PubMed2005

A virus-based nanoblock with tunable electrostatic properties.

Five different "HIS tag" mutants of cowpea mosaic virus were made by genetically introducing six contiguous histidine residues at various locations on the virus capsid. The mutant particles showed differential affinity for binding nickel, and their electrostatic properties could be controlled as a function of the protonation state of the exposed histidine sequence. The specific addressability of the HIS tag was corroborated by the selective modification of the histidine sequence with nanogold cross-linked to the Ni-NTA moiety.

Amino Acid Sequence↗

PubMed2004

Apo and Holo structures of an NADPH-dependent cinnamyl alcohol dehydrogenase from Saccharomyces cerevisiae.

The crystal structure of Saccharomyces cerevisiae ScAdh6p has been solved using the anomalous signal from the two zinc atoms found per subunit, and it constitutes the first structure determined from a member of the cinnamyl alcohol dehydrogenase family. ScAdh6p subunits exhibit the general fold of the medium-chain dehydrogenases/reductases (MDR) but with distinct specific characteristics. In the three crystal structures solved (two trigonal and one monoclinic), ScAdh6p molecules appear to be structural heterodimers composed of one subunit in the apo and the second subunit in the holo conformation. Between the two conformations, the relative disposition of domains remains unchanged, while two loops, Cys250-Asn260 and Ile277-Lys292, experience large movements. The apo-apo structure is disfavoured because of steric impairment involving the loop Ile277-Lys292, while in the holo-holo conformation some of the hydrogen bonds between subunits would break apart. These suggest that the first NADPH molecule would bind to the enzyme much more tightly than the second. In addition, fluorimetric analysis of NADPH binding demonstrates that only one cofactor molecule binds per dimer. Therefore, ScAdh6p appears to function according to a half-of-the-sites reactivity mechanism, resulting from a pre-existing (prior to cofactor binding) tendency for the structural asymmetry in the dimer. The specificity of ScAdh6p towards NADPH is mainly due to the tripod-like interactions of the terminal phosphate group with Ser210, Arg211 and Lys215. The size and the shape of the substrate-binding pocket correlate well with the substrate specificity of ScAdh6p towards cinnamaldehyde and other aromatic compounds. The structural relationships of ScAdh6p with other MDR structures are analysed.

Alcohol Oxidoreductases↗

PubMed2004

New addresses on an addressable virus nanoblock; uniquely reactive Lys residues on cowpea mosaic virus.

Cowpea mosaic virus (CPMV) is a robust, icosahedrally symmetric platform successfully used for attaching a variety of molecular substrates including proteins, fluorescent labels, and metals. The symmetric distribution and high local concentration of the attached molecules generates novel properties for the 30 nm particles. We report new CPMV reagent particles generated by systematic replacement of surface lysines with arginine residues. The relative reactivity of each lysine on the native particle was determined, and the two most reactive lysine residues were then created as single attachment sites by replacing all other lysines with arginine residues. Structural analysis of gold derivatization not only corroborated the specific reactivity of these unique lysine residues but also demonstrated their dramatically different presentation environment. Combined with site-directed cystine mutations, it is now possible to uniquely double label CPMV, expanding its use as an addressable nanoblock.

Arginine↗

PubMed2003

Complete reversal of coenzyme specificity by concerted mutation of three consecutive residues in alcohol dehydrogenase.

Gastric tissues from amphibian Rana perezi express the only vertebrate alcohol dehydrogenase (ADH8) that is specific for NADP(H) instead of NAD(H). In the crystallographic ADH8-NADP+ complex, a binding pocket for the extra phosphate group of coenzyme is formed by ADH8-specific residues Gly223-Thr224-His225, and the highly conserved Leu200 and Lys228. To investigate the minimal structural determinants for coenzyme specificity, several ADH8 mutants involving residues 223 to 225 were engineered and kinetically characterized. Computer-assisted modeling of the docked coenzymes was also performed with the mutant enzymes and compared with the wild-type crystallographic binary complex. The G223D mutant, having a negative charge in the phosphate-binding site, still preferred NADP(H) over NAD(H), as did the T224I and H225N mutants. Catalytic efficiency with NADP(H) dropped dramatically in the double mutants, G223D/T224I and T224I/H225N, and in the triple mutant, G223D/T224I/H225N (kcat/KmNADPH = 760 mm-1 min-1), as compared with the wild-type enzyme (kcat/KmNADPH = 133330 mm-1 min-1). This was associated with a lower binding affinity for NADP+ and a change in the rate-limiting step. Conversely, in the triple mutant, catalytic efficiency with NAD(H) increased, reaching values (kcat/KmNADH = 155000 mm-1 min-1) similar to those of the wild-type enzyme with NADP(H). The complete reversal of ADH8 coenzyme specificity was therefore attained by the substitution of only three consecutive residues in the phosphate-binding site, an unprecedented achievement within the ADH family.

Alcohol Dehydrogenase↗

PubMed2003

Retinoic acid binds to the C2-domain of protein kinase C(alpha).

Protein kinase C(alpha) (PKC(alpha)) is a key enzyme regulating the physiology of cells and their growth, differentiation, and apoptosis. PKC activity is known to be modulated by all-trans retinoic acid (atRA), although neither the action mechanism nor even the possible binding to PKCs has been established. Crystals of the C2-domain of PKC(alpha), a regulatory module in the protein that binds Ca(2+) and acidic phospholipids, have now been obtained by cocrystallization with atRA. The crystal structure, refined at 2.0 A resolution, shows that RA binds to the C2-domain in two locations coincident with the two binding sites previously reported for acidic phospholipids. The first binding site corresponds to the Ca(2+)-binding pocket, where Ca(2+) ions mediate the interactions of atRA with the protein, as they do with acidic phospholipids. The second binding site corresponds to the conserved lysine-rich cluster localized in beta-strands three and four. These observations are strongly supported by [(3)H]-atRA-binding experiments combined with site-directed mutagenesis. Wild-type C2-domain binds 2 mol of atRA per mol of protein, while the rate reduces to one in the case of C2-domain variants, in which mutations affect either Ca(2+) coordination or the integrity of the lysine-rich cluster site. Competition between atRA and acidic phospholipids to bind to PKC is a possible mechanism for modulating PKC(alpha) activity.

Animals↗

PubMed2003

Crystal structure of the vertebrate NADP(H)-dependent alcohol dehydrogenase (ADH8).

The amphibian enzyme ADH8, previously named class IV-like, is the only known vertebrate alcohol dehydrogenase (ADH) with specificity towards NADP(H). The three-dimensional structures of ADH8 and of the binary complex ADH8-NADP(+) have been now determined and refined to resolutions of 2.2A and 1.8A, respectively. The coenzyme and substrate specificity of ADH8, that has 50-65% sequence identity with vertebrate NAD(H)-dependent ADHs, suggest a role in aldehyde reduction probably as a retinal reductase. The large volume of the substrate-binding pocket can explain both the high catalytic efficiency of ADH8 with retinoids and the high K(m) value for ethanol. Preference of NADP(H) appears to be achieved by the presence in ADH8 of the triad Gly223-Thr224-His225 and the recruitment of conserved Lys228, which define a binding pocket for the terminal phosphate group of the cofactor. NADP(H) binds to ADH8 in an extended conformation that superimposes well with the NAD(H) molecules found in NAD(H)-dependent ADH complexes. No additional reshaping of the dinucleotide-binding site is observed which explains why NAD(H) can also be used as a cofactor by ADH8. The structural features support the classification of ADH8 as an independent ADH class.

Alcohol Dehydrogenase↗

PubMed2003

Crystallization and preliminary X-ray analysis of NADP(H)-dependent alcohol dehydrogenases from Saccharomyces cerevisiae and Rana perezi.

Different crystal forms diffracting to high resolution have been obtained for two NADP(H)-dependent alcohol dehydrogenases, members of the medium-chain dehydrogenase/reductase superfamily: ScADHVI from Saccharomyces cerevisiae and ADH8 from Rana perezi. ScADHVI is a broad-specificity enzyme, with a sequence identity lower than 25% with respect to all other ADHs of known structure. The best crystals of ScADHVI diffracted beyond 2.8 A resolution and belonged to the trigonal space group P3(1)21 (or to its enantiomorph P3(2)21), with unit-cell parameters a = b = 102.2, c = 149.7 A, gamma = 120 degrees. These crystals were produced by the hanging-drop vapour-diffusion method using ammonium sulfate as precipitant. Packing considerations together with the self-rotation function and the native Patterson map seem to indicate the presence of only one subunit per asymmetric unit, with a Volume solvent content of about 80%. ADH8 from R. perezi is the only NADP(H)-dependent ADH from vertebrates characterized to date. Crystals of ADH8 obtained both in the absence and in the presence of NADP(+) using polyethylene glycol and lithium sulfate as precipitants diffracted to 2.2 and 1.8 A, respectively, using synchrotron radiation. These crystals were isomorphous, space group C2, with approximate unit-cell parameters a = 122, b = 79, c = 91 A, beta = 113 degrees and contain one dimer per asymmetric unit, with a Volume solvent content of about 50%.

Alcohol Dehydrogenase↗

PubMed2002

Additional binding sites for anionic phospholipids and calcium ions in the crystal structures of complexes of the C2 domain of protein kinase calpha.

The C2 domain of protein kinase Calpha (PKCalpha) corresponds to the regulatory sequence motif, found in a large variety of membrane trafficking and signal transduction proteins, that mediates the recruitment of proteins by phospholipid membranes. In the PKCalpha isoenzyme, the Ca2+-dependent binding to membranes is highly specific to 1,2-sn-phosphatidyl-l-serine. Intrinsic Ca2+ binding tends to be of low affinity and non-cooperative, while phospholipid membranes enhance the overall affinity of Ca2+ and convert it into cooperative binding. The crystal structure of a ternary complex of the PKCalpha-C2 domain showed the binding of two calcium ions and of one 1,2-dicaproyl-sn-phosphatidyl-l-serine (DCPS) molecule that was coordinated directly to one of the calcium ions. The structures of the C2 domain of PKCalpha crystallised in the presence of Ca2+ with either 1,2-diacetyl-sn-phosphatidyl-l-serine (DAPS) or 1,2-dicaproyl-sn-phosphatidic acid (DCPA) have now been determined and refined at 1.9 A and at 2.0 A, respectively. DAPS, a phospholipid with short hydrocarbon chains, was expected to facilitate the accommodation of the phospholipid ligand inside the Ca2+-binding pocket. DCPA, with a phosphatidic acid (PA) head group, was used to investigate the preference for phospholipids with phosphatidyl-l-serine (PS) head groups. The two structures determined show the presence of an additional binding site for anionic phospholipids in the vicinity of the conserved lysine-rich cluster. Site-directed mutagenesis, on the lysine residues from this cluster that interact directly with the phospholipid, revealed a substantial decrease in C2 domain binding to vesicles when concentrations of either PS or PA were increased in the absence of Ca2+. In the complex of the C2 domain with DAPS a third Ca2+, which binds an extra phosphate group, was identified in the calcium-binding regions (CBRs). The interplay between calcium ions and phosphate groups or phospholipid molecules in the C2 domain of PKCalpha is supported by the specificity and spatial organisation of the binding sites in the domain and by the variable occupancies of ligands found in the different crystal structures. Implications for PKCalpha activity of these structural results, in particular at the level of the binding affinity of the C2 domain to membranes, are discussed.

Animals↗