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Biomedical subjects

J A Grobler

Publications and source records attributed to J A Grobler.

11 recordsLinked to original sources
PubMed2000

Inhibitors of strand transfer that prevent integration and inhibit HIV-1 replication in cells.

Integrase is essential for human immunodeficiency virus-type 1 (HIV-1) replication; however, potent inhibition of the isolated enzyme in biochemical assays has not readily translated into antiviral activity in a manner consistent with inhibition of integration. In this report, we describe diketo acid inhibitors of HIV-1 integrase that manifest antiviral activity as a consequence of their effect on integration. The antiviral activity of these compounds is due exclusively to inhibition of one of the two catalytic functions of integrase, strand transfer.

Acetoacetates↗
PubMed1999

Stability, activity and flexibility in alpha-lactalbumin.

alpha-Lactalbumins and the type-c lysozymes are homologues with similar folds that differ in function and stability. To determine if the lower stability of alpha-lactalbumin results from specific substitutions required for its adaptation to a new function, the effects of lysozyme-based and other substitutions on thermal stability were determined. Unblocking the upper cleft in alpha-lactalbumin by replacing Tyr103 with Ala, perturbs stability and structure but Pro, which also generates an open cleft, is compatible with normal structure and activity. These effects appear to reflect alternative enthalpic and entropic forms of structural stabilization by Tyr and Pro. Of 23 mutations, only three, which involve substitutions for residues in flexible substructures adjacent to the functional site, increase stability. Two are lysozyme-based substitutions for Leu110, a component of a region with alternative helix and loop conformations, and one is Asn for Lys114, a residue whose microenvironment changes when alpha-lactalbumin interacts with its target enzyme. While all substitutions for Leu110 perturb activity, a Lys114 to Asn mutation increases T(m) by more than 10 degrees C and reduces activity, but two other destabilizing substitutions do not affect activity. It is proposed that increased stability and reduced activity in Lys114Asn result from reduced flexibility in the functional site of alpha-lactalbumin.

Amino Acid Sequence↗
PubMed1998

Catalysis by phospholipase C delta1 requires that Ca2+ bind to the catalytic domain, but not the C2 domain.

The hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) by phosphoinositide-specific phospholipase C (PLC) is absolutely dependent on Ca2+. The PH domain truncated catalytic core of rat phospholipase C delta1 (PLC-delta1) has Ca2+ binding sites in its catalytic and C2 domains, and potential Ca2+ binding sites in two EF-hands. A catalytically inactive PLC-delta1 catalytic core bound with low affinity to PIP2-containing vesicles in the presence of Ca2+. A mutant PLC-delta1 has been engineered which lacks the C2 domain Ca2+ binding site and the surrounding loops known as the jaws. Isothermal calorimetric titration showed four Ca2+ ions bind to the wild-type PLC-delta1 catalytic core in solution but only one binds to the C2 domain jaws deletion mutant. The activity and Ca2+ dependence of wild-type and mutant phospholipase Cs were determined using substrate incorporated in detergent micelles and in large unilamellar vesicles. The activities of wild-type and mutant were identical to each other in both assay systems. Wild-type and the C2 jaws deletion mutant of PLC have Hill coefficients of 1.12-1.16 with respect to [Ca2+]. We conclude that a single Ca2+ bound to the catalytic domain is entirely responsible for the Ca2+ dependence of the basal activity of PLC-delta1.

Amino Acid Sequence↗
PubMed1997

Protein kinase C and phospholipase C: bilayer interactions and regulation.

Protein kinase C and phospholipase C are interfacially active modular enzymes that contain multiple membrane-binding domains. During the past two years, 3D structures and functional data have been reported for the key domains: pleckstrin homology, protein kinase C homology-1 and -2, and the phospholipase C catalytic domain. Roles for membrane bilayer structure and lipid microdomains have become clearly domains has shown how the domains work together to coordinate regulation.

Lipid Bilayers↗
PubMed1996

Functional site in alpha-lactalbumin encompasses a region corresponding to a subsite in lysozyme and parts of two adjacent flexible substructures.

Aromatic cluster 1 of alpha-lactalbumin (LA), a substructure adjacent to the cleft, is important for its interaction with galactosyltransferase (GT) and effects on glucose binding in the lactose synthase complex [Grobler, J. A., Wang, M., Pike, A. K., & Brew, K. (1994) J. Biol. Chem. 269, 5106-5114]. The full extent of the functional region in LA has been probed by mutagenesis of residues that are near aromatic cluster 1 or within the cleft that corresponds to the active site in the homologous type c lysozymes. The conserved residues Val42, Gln54, and Ile59, which correspond to residues of lysozyme that act in substrate binding in subsites C to E, together with residues adjacent to aromatic cluster 1, were found to be not required for activity. In contrast, replacing Leu110, a component of the region corresponding to lysozyme subsite F, with His or Glu greatly reduces the affinity of LA for GT while the introduction of Arg lowers the synergism of LA and glucose binding to GT and also reduces the affinity of LA for GT. Substitutions for Ala106, which is adjacent to Leu110 in the structure, also perturb activity. The region of the cleft corresponding to subsite F is important for function in LA as well as in lysozyme since other components of this subsite, His32 and Phe31, are also crucial for LA activity. The qualitatively different effects of various substitutions for Leu110 may be mediated by their influence on His32 or by changes in the structure of the lactose synthase complex.

Amino Acid Sequence↗
PubMed1996

Second-site suppression of regulatory phosphorylation in Escherichia coli isocitrate dehydrogenase.

Inactivation of Escherichia coli isocitrate dehydrogenase upon phosphorylation at S113 depends upon the direct electrostatic repulsion of the negatively charged gamma-carboxylate of isocitrate by the negatively charged phosphoserine. The effect is mimicked by replacing S113 with aspartate or glutamate, which reduce performance (kcat/K(i).isocitrat/ Km.NADP) by a factor of 10(7). Here, we demonstrate that the inactivating effects of the electrostatic repulsion are completely eliminated by a second-site mutation, and provide the structural basis for this striking example of intragenic suppression. N115 is adjacent to S113 on one face of the D-helix, interacts with isocitrate and NADP+, and has been postulated to serve in both substrate binding and in catalysis. The single N115L substitution reduces affinity for isocitrate by a factor of 50 and performance by a factor of 500. However, the N115L substitution completely suppresses the inactivating electrostatic effects of S113D or S113E: the performance of the double mutants is 10(5) higher than the S113D and S113E single mutants. These mutations have little effect on the kinetics of alternative substrates, which lack the charged gamma-carboxylate of isocitrate. Both glutamate and aspartate at site 113 remain fully ionized in the presence of leucine. In the crystal structure of the N115L mutant, the leucine adopts a different conformer from the wild-type asparagine. Repacking around the leucine forces the amino-terminus of the D-helix away from the rest of the active site. The hydrogen bond between E113 and N115 in the S113E single mutant is broken in the S113E/N115L mutant, allowing the glutamate side chain to move away from the gamma-carboxylate of isocitrate. These movements increase the distance between the carboxylates, diminish the electrostatic repulsion, and lead to the remarkably high activity of the S113E/N115L mutant.

Amino Acids↗
PubMed1996

Expression, characterization, and crystallization of the catalytic core of rat phosphatidylinositide-specific phospholipase C delta 1.

Phosphatidylinositide-specific phospholipase Cs (PI-PLCs) catalyze the calcium-dependent hydrolysis of phosphatidylinositides in response to diverse stimuli in higher eukaryotes. Mammalian PI-PLCs contain divergent regulatory regions, but all share three conserved regions: an N-terminal pleckstrin homology (PH) domain, X, and Y. We report the high-level expression and characterization of a recombinant "catalytic core" of rat PI-PLC delta 1 that contains the catalytically essential X and Y regions, but not the PH domain. The expressed protein, PI-PLC delta delta 1-134, is catalytically active versus phosphatidylinositol 4,5-bisphosphate in deoxycholate micelles with a K(m) of 182 microM and a Vmax of 27 mumol/min/mg. PI-PLC delta delta 1-134 is monomeric and monodisperse as judged by dynamic light scattering. Far-UV CD indicates a structure with approximately 35% alpha-helix. A reversible change in the near-UV CD spectrum is observed on addition of calcium, suggesting that calcium can bind PI-PLC delta delta 1-134 in the absence of phospholipid. Triclinic crystals of PI-PLC delta delta 1-134 have been obtained that diffract beyond 2.4 A resolution under cryogenic conditions. Based on Vm = 2.72 Da/A3 and on the self-rotation function, there are two PI-PLC delta delta 1-134 molecules per asymmetric unit that are related to each other by a noncrystallographic axis of approximate twofold symmetry parallel to a.

Animals↗
PubMed1996

C2 domain conformational changes in phospholipase C-delta 1.

The structure of the PH-domain truncated core of rat phosphoinositide-specific phospholipase C-delta 1 has been determined at 2.4 A resolution and compared to the structure previously determined in a different crystal form. The stereochemical relationship between the EF, catalytic, and C2 domains is essentially identical. The Ca2+ analogue Sm3+ binds at two sites between the jaws of the C2 domain. Sm3+ binding ejects three lysine residues which bridge the gap between the jaws and occupy the Ca2+ site in the apoenzyme, triggering a conformational change in the jaws. The distal sections of the C2 jaws move apart, opening the mouth by 9 A and creating a gap large enough to bind a phospholipid headgroup.

Animals↗
PubMed1994

Study by mutagenesis of the roles of two aromatic clusters of alpha-lactalbumin in aspects of its action in the lactose synthase system.

A new system for the bacterial expression of a variant of bovine alpha-lactalbumin has been developed. Eighteen mutant proteins containing single site substitutions in a cluster of predominantly aromatic residues adjacent to the cleft (aromatic cluster I) and in the hydrophobic box were expressed. The proteins were extracted from inclusion bodies and treated to generate native folding and disulfide bonds to provide appropriately folded protein samples for nine of the mutants. These were characterized with respect to kinetic parameters reflecting aspects of alpha-lactalbumin activity in modulating the specificity of bovine galactosyltransferase. In aromatic cluster I, changes at tryptophan 118 or glutamine 117 were found to specifically reduce affinity for galactosyltransferase, whereas substitutions for phenylalanine 31 or histidine 32 have major effects on the ability to promote glucose binding (13-200-fold) and lesser effects on galactosyltransferase affinity (1.5-70-fold). Substitutions in the hydrophobic box were found to affect folding rather than activity. Thus, the binding of alpha-lactalbumin to galactosyltransferase and its ability to promote glucose binding can be separately perturbed and are associated with distinct but adjacent structures. Aromatic cluster I is directly involved in activity whereas the hydrophobic box appears to have a structural rather than functional role.

Amino Acid Sequence↗
PubMed1994

Sequences of two highly divergent canine type c lysozymes: implications for the evolutionary origins of the lysozyme/alpha-lactalbumin superfamily.

The amino acid sequences of two canine lysozymes, from milk and spleen, have been elucidated by direct sequence analyses of the purified proteins and fragments generated from them. The two enzymes are highly divergent, differing from each other by 45% in sequence, but each is closely similar to lysozymes previously obtained from other mammalian species. The milk lysozyme is similar in sequence to equine and donkey milk lysozymes (83% identity) and, like these enzymes, contains a bound Ca2+ ion while the spleen enzyme is most similar in sequence to the majority of previously studied mammalian and avian lysozymes (80 to 83% identity) and, based on its sequence, does not contain a Ca(2+)-binding site. This demonstrates that Ca(2+)-binding lysozymes are expressed in at least two mammalian orders, the carnivores and perissodactyls, as well as confirming that the genes for the Ca(2+)-binding and conventional lysozymes are paralogous. The latter point was further confirmed by the isolation and partial sequence analysis of a conventional lysozyme from equine spleen. The relationships of these new lysozyme sequences to those of other lysozymes and their homologues, the alpha-lactalbumins, were analyzed using different molecular phylogeny algorithms, producing a new model for the evolutionary origins of the superfamily. The most significant conclusion to be drawn from this model is that Ca(2+)-binding activity was an ancient feature of this protein superfamily which was lost during the evolutionary development of the conventional lysozymes. It also supports a previous suggestion that the alpha-lactalbumins and lysozymes diverged at a time earlier than the divergence of the fishes and tetrapods.

Amino Acid Sequence↗