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This study presents the crystal structures of three functional forms of diacylglycerol kinase, an integral membrane protein that catalyses a crucial step in oligosaccharide and lipopolysaccharide synthesis and assembly; these X-ray structures are markedly different from the only other structure available for this unique kinase that was solved using solution NMR. A small enzyme that packs a punch The integral membrane enzyme diacylglycerol kinase is involved in the synthesis of periplasmic-membrane-derived oligosaccharide and outer-membrane lipopolysaccharide in Gram-negative bacteria. Containing only 121 amino acid residues, this is the smallest known kinase and it has become a model for the study membrane protein behaviour and enzymology. Martin Caffrey and colleagues report here the X-ray crystal structure of native diacylglycerol kinase and of two thermostable, functional mutants at high resolution. Of interest is the fact that the crystal structures are at variance with the only other published structure of diacylglycerol kinase, that solved using solution NMR. In an accompanying News & Views, Jimin Zheng and Zongchao Jia discuss the factors that may have contributed to the discrepancy between the NMR and crystal structures. Diacylglycerol kinase catalyses the ATP-dependent phosphorylation of diacylglycerol to phosphatidic acid for use in shuttling water-soluble components to membrane-derived oligosaccharide and lipopolysaccharide in the cell envelope of Gram-negative bacteria 1 . For half a century, this 121-residue kinase has served as a model for investigating membrane protein enzymology 1 , 2 , 3 , 4 , 5 , 6 , folding 7 , 8 , assembly 9 , 10 , 11 , 12 and stability 1 , 13 . Here we present crystal structures for three functional forms of this unique and paradigmatic kinase, one of which is wild type. These reveal a homo-trimeric enzyme with three transmembrane helices and an amino-terminal amphiphilic helix per monomer. Bound lipid substrate and docked ATP identify the putative active site that is of the composite, shared site type. The crystal structures rationalize extensive biochemical and biophysical data on the enzyme. They are, however, at variance with a published solution NMR model 14 in that domain swapping, a key feature of the solution form, is not observed in the crystal structures.

Background The prevalence of Parkinson’s disease (PD) is increasing in sub-Saharan Africa, but little is known about the genetics of PD in these populations. Due to their unique ancestry and diversity, sub-Saharan African populations have the potential to reveal novel insights into the pathobiology of PD. In this study, we aimed to characterise the genetic variation in known and novel PD genes in a group of Black South African and Nigerian patients. Methods We recruited 33 Black South African and 14 Nigerian PD patients, and screened them for sequence variants in 751 genes using an Ion AmpliSeq™ Neurological Research panel. We used bcftools to filter variants and annovar software for the annotation. Rare variants were prioritised using MetaLR and MetaSVM prediction scores. The effect of a variant on ATP13A2’s protein structure was investigated by molecular modelling. Results We identified 14,655 rare variants with a minor allele frequency ≤ 0.01, which included 2448 missense variants. Notably, no common pathogenic mutations were identified in these patients. Also, none of the known PD-associated mutations were found highlighting the need for more studies in African populations. Altogether, 54 rare variants in 42 genes were considered deleterious and were prioritized, based on MetaLR and MetaSVM scores, for follow-up studies. Protein modelling showed that the S1004R variant in ATP13A2 possibly alters the conformation of the protein. Conclusions We identified several rare variants predicted to be deleterious in sub-Saharan Africa PD patients; however, further studies are required to determine the biological effects of these variants and their possible role in PD. Studies such as these are important to elucidate the genetic aetiology of this disorder in patients of African ancestry.

Background The kinome is made up of a large number of functionally diverse enzymes, with the classification indicating very little about the extent of the conserved kinetic mechanisms associated with phosphoryl transfer. It has been demonstrated that C8-H of ATP plays a critical role in the activity of a range of kinase and synthetase enzymes. Results A number of conserved mechanisms within the prescribed kinase fold families have been identified directly utilizing the C8-H of ATP in the initiation of phosphoryl transfer. These mechanisms are based on structurally conserved amino acid residues that are within hydrogen bonding distance of a co-crystallized nucleotide. On the basis of these conserved mechanisms, the role of the nucleotide C8-H in initiating the formation of a pentavalent intermediate between the γ-phosphate of the ATP and the substrate nucleophile is defined. All reactions can be clustered into two mechanisms by which the C8-H is induced to be labile via the coordination of a backbone carbonyl to C6-NH 2 of the adenyl moiety, namely a \"push\" mechanism, and a \"pull\" mechanism, based on the protonation of N7. Associated with the \"push\" mechanism and \"pull\" mechanisms are a series of proton transfer cascades, initiated from C8-H, via the tri-phosphate backbone, culminating in the formation of the pentavalent transition state between the γ-phosphate of the ATP and the substrate nucleophile. Conclusions The \"push\" mechanism and a \"pull\" mechanism are responsible for inducing the C8-H of adenyl moiety to become more labile. These mechanisms and the associated proton transfer cascades achieve the proton transfer via different family-specific conserved sets of amino acids. Each of these mechanisms would allow for the regulation of the rate of formation of the pentavalent intermediate between the ATP and the substrate nucleophile. Phosphoryl transfer within kinases is therefore a specific event mediated and regulated via the coordination of the adenyl moiety of ATP and the C8-H of the adenyl moiety.

Doc number: 15 Abstract Background: It has been demonstrated that the adenyl moiety of ATP plays a direct role in the regulation of ATP binding and/or phosphoryl transfer within a range of kinase and synthetase enzymes. The role of the C8-H of ATP in the binding and/or phosphoryl transfer on the enzyme activity of a number of kinase and synthetase enzymes has been elucidated. The intrinsic catalysis rate mediated by each kinase enzyme is complex, yielding apparent K M values ranging from less than 0.4 μM to more than 1 mM for ATP in the various kinases. Using a combination of ATP deuterated at the C8 position (C8D-ATP) as a molecular probe with site directed mutagenesis (SDM) of conserved amino acid residues in shikimate kinase and adenylate kinase active sites, we have elucidated a mechanism by which the ATP C8-H is induced to be labile in the broader kinase family. We have demonstrated the direct role of the C8-H in the rate of ATP consumption, and the direct role played by conserved Thr residues interacting with the C8-H. The mechanism by which the vast range in K M might be achieved is also suggested by these findings. Results: We have demonstrated the mechanism by which the enzyme activities of Group 2 kinases, shikimate kinase (SK) and adenylate kinase 1 (AK1), are controlled by the C8-H of ATP. Mutations of the conserved threonine residues associated with the labile C8-H cause the enzymes to lose their saturation kinetics over the concentration range tested. The relationship between the role C8-H of ATP in the reaction mechanism and the ATP concentration as they influence the saturation kinetics of the enzyme activity is also shown. The SDM clearly identified the amino acid residues involved in both the catalysis and regulation of phosphoryl transfer in SK and AK1 as mediated by C8H-ATP. Conclusions: The data outlined serves to demonstrate the \"push\" mechanism associated with the control of the saturation kinetics of Group 2 kinases mediated by ATP C8-H. It is therefore conceivable that kinase enzymes achieve the observed 2,500-fold variation in K M through a combination of the various conserved \"push\" and \"pull\" mechanisms associated with the release of C8-H, the proton transfer cascades unique to the class of kinase in question and the resultant/concomitant creation of a pentavalent species from the γ-phosphate group of ATP. Also demonstrated is the interplay between the role of the C8-H of ATP and the ATP concentration in the observed enzyme activity. The lability of the C8-H mediated by active site residues co-ordinated to the purine ring of ATP therefore plays a significant role in explaining the broad K M range associated with kinase steady state enzyme activities.

This study presents the crystal structures of three functional forms of diacylglycerol kinase, an integral membrane protein that catalyses a crucial step in oligosaccharide and lipopolysaccharide synthesis and assembly; these X-ray structures are markedly different from the only other structure available for this unique kinase that was solved using solution NMR.

This study presents the crystal structures of three functional forms of diacylglycerol kinase, an integral membrane protein that catalyses a crucial step in oligosaccharide and lipopolysaccharide synthesis and assembly; these X-ray structures are markedly different from the only other structure available for this unique kinase that was solved using solution NMR.

This study presents the crystal structures of three functional forms of diacylglycerol kinase, an integral membrane protein that catalyses a crucial step in oligosaccharide and lipopolysaccharide synthesis and assembly; these X-ray structures are markedly different from the only other structure available for this unique kinase that was solved using solution NMR.

This study presents the crystal structures of three functional forms of diacylglycerol kinase, an integral membrane protein that catalyses a crucial step in oligosaccharide and lipopolysaccharide synthesis and assembly; these X-ray structures are markedly different from the only other structure available for this unique kinase that was solved using solution NMR.