Post COVID-19 ischemic stroke in a 15-Year-old patient.

Coronavirus disease 2019 (COVID-19) has been reported in the literature to be associated with a higher risk of stroke in young individuals with no previous risk factors. We present here one such case of a 15-year-old girl with posterior circulation ischemic stroke resulting in dense right hemiplegia and cerebellar incoordination. The patient tested positive for COVID-19 infection without displaying any symptoms of active COVID-19 infection at the time of the stroke. An MRI brain scan showed acute infarcts in the pons and left cerebellar hemisphere, and a CT angiogram of the cerebrovascular system showed occluded left vertebral and basilar arteries.The most salient feature of this case is COVID-19 infection manifesting clinically as cerebrovascular thrombosis in an otherwise healthy young girl with no pre-existing comorbidities and no laboratory findings of coagulopathy except for mildly elevated D-dimer.

Crystallization and preliminary X-ray diffraction analysis of the Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase I86A mutant.

The Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase I86A mutant is stereospecific for (R)-alcohols instead of (S)-alcohols. Pyramidal crystals grown in the presence of (R)-phenylethanol via the hanging-drop vapour-diffusion method diffracted to 3.2 A resolution at the Canadian Light Source. The crystal belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 80.23, b = 124.90, c = 164.80 A. The structure was solved by molecular replacement using the structure of T. brockii SADH (PDB entry 1ykf).

Crystallization and preliminary X-ray studies of the N-domain of the Wilson disease associated protein.

Wilson disease associated protein (ATP7B) is essential for copper transport in human cells. Mutations that affect ATP7B function result in Wilson's disease, a chronic copper toxicosis. Disease-causing mutations within the N-domain of ATP7B (WND) are known to disrupt ATP binding, but a high-resolution X-ray structure of the ATP-binding site has not been reported. The N-domain was modified to delete the disordered loop comprising residues His1115-Asp1138 (WNDDelta(1115-1138)). Unlike the wild-type N-domain, WNDDelta(1115-1138) formed good-quality crystals. Synchrotron diffraction data have been collected from WNDDelta(1115-1138) at the Canadian Light Source. A native WNDDelta(1115-1138) crystal diffracted to 1.7 A resolution and belonged to space group P4(2)2(1)2, with unit-cell parameters a = 39.2, b = 39.2, c = 168.9 A. MAD data were collected to 2.7 A resolution from a SeMet-derivative crystal with unit-cell parameters a = 38.4, b = 38.4, c = 166.7 A. The WNDDelta(1115-1138) structure is likely to be solved by phasing from multiwavelength anomalous diffraction (MAD) experiments.

Structural investigation of a phosphorylation-catalyzed, isoaspartate-free, protein succinimide: crystallographic structure of post-succinimide His15Asp histidine-containing protein.

Aspartates and asparagines can spontaneously cyclize with neighboring main-chain amides to form succinimides. These succinimides hydrolyze to a mixture of isoaspartate and aspartate products. Phosphorylation of aspartates is a common mechanism of protein regulation and increases the propensity for succinimide formation. Although typically regarded as a form of protein damage, we hypothesize succinimides could represent an effective mechanism of phosphoaspartate autophosphatase activity, provided hydrolysis is limited to aspartate products. We previously reported the serendipitous creation of a protein, His15Asp histidine-containing protein (HPr), which undergoes phosphorylation-catalyzed formation of a succinimide whose hydrolysis is seemingly exclusive for aspartate formation. Here, through the high-resolution structure of postsuccinimide His15Asp HPr, we confirm the absence of isoaspartate residues and propose mechanisms for phosphorylation-catalyzed succinimide formation and its directed hydrolysis to aspartate. His15Asp HPr represents the first characterized protein example of an isoaspartate-free succinimide and lends credence to the hypothesis that intramolecular cyclization could represent a physiological mechanism of autophosphatase activity. Furthermore, this indicates that current strategies for succinimide evaluation, based on isoaspartate detection, underestimate the frequencies of these reactions. This is considerably significant for evaluation of protein stability and integrity.

Structure of a GTP-dependent bacterial PEP-carboxykinase from Corynebacterium glutamicum.

GTP-dependent phosphoenolpyruvate carboxykinase (PCK) is the key enzyme that controls the blood glucose level during fasting in higher animals. Here we report the first substrate-free structure of a GTP-dependent phosphoenolpyruvate (PEP) carboxykinase from a bacterium, Corynebacterium glutamicum (CgPCK). The protein crystallizes in space group P2(1) with four molecules per asymmetric unit. The 2.3A resolution structure was solved by molecular replacement using the human cytosolic PCK (hcPCK) structure (PDB ID: 1KHF) as the starting model. The four molecules in the asymmetric unit pack as two dimers, and is an artifact of crystal packing. However, the P-loop and the guanine binding loop of the substrate-free CgPCK structure have different conformations from the other published GTP-specific PCK structures, which all have bound substrates and/or metal ions. It appears that a change in the P-loop and guanine binding loop conformation is necessary for substrate binding in GTP-specific PCKs, as opposed to overall domain movement in ATP-specific PCKs.

Substrate-free structure of a monomeric NADP isocitrate dehydrogenase: an open conformation phylogenetic relationship of isocitrate dehydrogenase.

Both monomeric and dimeric NADP+-dependent isocitrate dehydrogenase (IDH) belong to the metal-dependent beta-decarboxylating dehydrogenase family and catalyze the oxidative decarboxylation from 2R,3S-isocitrate to yield 2-oxoglutarate, CO2, and NADPH. It is important to solve the structures of IDHs from various species to correlate with its function and evolutionary significance. So far, only two crystal structures of substrate/cofactor-bound (isocitrate/NADP) NADP+-dependent monomeric IDH from Azotobacter vinelandii (AvIDH) have been solved. Herein, we report for the first time the substrate/cofactor-free structure of a monomeric NADP+-dependent IDH from Corynebacterium glutamicum (CgIDH) in the presence of Mg2+. The 1.75 A structure of CgIDH-Mg2+ showed a distinct open conformation in contrast to the closed conformation of AvIDH-isocitrate/NADP+ complexes. Fluorescence studies on CgIDH in the presence of isocitrate/or NADP+ suggest the presence of low energy barrier conformers. In CgIDH, the amino acid residues corresponding to the Escherichia coli IDH phosphorylation-loop are alpha-helical compared with the more flexible random-coil region in the E. coli protein where IDH activation is controlled by phosphorylation. This more structured region supports the idea that activation of CgIDH is not controlled by phosphorylation. Monomeric NADP+-specific IDHs have been identified from about 50 different bacterial species, such as proteobacteria, actinobacteria, and planctomycetes, whereas, dimeric NADP+-dependent IDHs are diversified in both prokaryotes and eukaryotes. We have constructed a phylogenetic tree based on amino acid sequences of all bacterial monomeric NADP+-dependent IDHs and also another one with specifically chosen species which either contains both monomeric and dimeric NADP+-dependent IDHs or have monomeric NADP+-dependent, as well as NAD+-dependent IDHs. This is done to examine evolutionary relationships.

Structure/function studies of phosphoryl transfer by phosphoenolpyruvate carboxykinase.

Phosphoenolpyruvate carboxykinase (PCK) catalyzes the conversion of oxaloacetate (OAA) to PEP and carbon dioxide with the subsequent conversion of nucleoside triphosphate to nucleoside diphosphate (NDP). The 1.9 A resolution structure of Escherichia coli PCK consisted of a 275-residue N-terminal domain and a 265-residue C-terminal domain with the active site located in a cleft between these domains. Each domain has an alpha/beta topology and the overall structure represents a new protein fold. Furthermore, PCK has a unique mononucleotide-binding fold. The 1.8 A resolution structure of the complex of ATP/Mg(2+)/oxalate with PCK revealed a 20 degrees hinge-like rotation of the N- and C-terminal domains, which closed the active site cleft. The ATP was found in the unusual syn conformation as a result of binding to the enzyme. Along with the side chain of Lys254, Mg(2+) neutralizes charges on the P beta and P gamma oxygen atoms of ATP and stabilizes an extended, eclipsed conformation of the P beta and P gamma phosphoryl groups. The sterically strained high-energy conformation likely lowers the free energy of activation for phosphoryl transfer. Additionally, the gamma-phosphoryl group becomes oriented in-line with the appropriate enolate oxygen atom, which strongly supports a direct S(N)2-type displacement of this gamma-phosphoryl group by the enolate anion. In the 2.0 A resolution structure of the complex of PCK/ADP/Mg(2+)/AlF(3), the AlF(3) moiety represents the phosphoryl group being transferred during catalysis. There are three positively charged groups that interact with the fluorine atoms, which are complementary to the three negative charges that would occur for an associative transition state.

Crystal structure of Anaerobiospirillum succiniciproducens PEP carboxykinase reveals an important active site loop.

The 2.2 Angstroms resolution crystal structure of the enzyme phosphoenolpyruvate carboxykinase (PCK) from the bacterium Anaerobiospirillum succiniciproducens complexed with ATP, Mg(2+), Mn(2+) and the transition state analogue oxalate has been solved. The 2.4 Angstroms resolution native structure of A. succiniciproducens PCK has also been determined. It has been found that upon binding of substrate, PCK undergoes a conformational change. Two domains of the molecule fold towards each other, with the substrates and metal ions held in a cleft formed between the two domains. This domain movement is believed to accelerate the reaction PCK catalyzes by forcing bulk solvent molecules out of the active site. Although the crystal structure of A. succiniciproducens PCK with bound substrate and metal ions is related to the structures of PCK from Escherichia coli and Trypanosoma cruzi, it is the first crystal structure from this class of enzymes that clearly shows an important surface loop (residues 383-397) from the C-terminal domain, hydrogen bonding with the peptide backbone of the active site residue Arg60. The interaction between the surface loop and the active site backbone, which is a parallel beta-sheet, seems to be a feature unique of A. succiniciproducens PCK. The association between the loop and the active site is the third type of interaction found in PCK that is thought to play a part in the domain closure. This loop also appears to help accelerate catalysis by functioning as a 'lid' that shields water molecules from the active site.

Activation of the SspA serine protease zymogen of Staphylococcus aureus proceeds through unique variations of a trypsinogen-like mechanism and is dependent on both autocatalytic and metalloprotease-specific processing.

The serine and cysteine proteases SspA and SspB of Staphylococcus aureus are secreted as inactive zymogens, zSspA and zSspB. Mature SspA is a trypsin-like glutamyl endopeptidase and is required to activate zSspB. Although a metalloprotease Aureolysin (Aur) is in turn thought to contribute to activation of zSspA, a specific role has not been demonstrated. We found that pre-zSspA is processed by signal peptidase at ANA(29) downward arrow, releasing a Leu(30) isoform that is first processed exclusively through autocatalytic intramolecular cleavage within a glutamine-rich propeptide segment, (40)QQTQSSKQQTPKIQ(53). The preferred site is Gln(43) with secondary processing at Gln(47) and Gln(53). This initial processing is necessary for optimal and subsequent Aur-dependent processing at Leu(58) and then Val(69) to release mature SspA. Although processing by Aur is rate-limiting in zSspA activation, the first active molecules of Val(69)SspA promote rapid intermolecular processing of remaining zSspA at Glu(65), producing an N-terminal (66)HANVILP isoform that is inactive until removal of the HAN tripeptide by Aur. Modeling indicated that His(66) of this penultimate isoform blocks the active site by hydrogen bonding to Ser(237) and occlusion of substrate. Binding of glutamate within the active site of zSspA is energetically unfavorable, but glutamine fits into the primary specificity pocket and is predicted to hydrogen bond to Thr(232) proximal to Ser(237), permitting autocatalytic cleavage of the glutamine-rich propeptide segment. These and other observations suggest that zSspA is activated through a trypsinogen-like mechanism where supplementary features of the propeptide must be sequentially processed in the correct order to allow efficient activation.

Structure of PEP carboxykinase from the succinate-producing Actinobacillus succinogenes: a new conserved active-site motif.

Actinobacillus succinogenes can produce, via fermentation, high concentrations of succinate, an important industrial commodity. A key enzyme in this pathway is phosphoenolpyruvate carboxykinase (PCK), which catalyzes the production of oxaloacetate from phosphoenolpyruvate and carbon dioxide, with the concomitant conversion of adenosine 5'-diphosphate to adenosine 5'-triphosphate. 1.85 and 1.70 A resolution structures of the native and a pyruvate/Mn(2+)/phosphate complex have been solved, respectively. The structure of the complex contains sulfhydryl reducing agents covalently bound to three cysteine residues via disulfide bonds. One of these cysteine residues (Cys285) is located in the active-site cleft and may be analogous to the putative reactive cysteine of PCK from Trypanosoma cruzi. Cys285 is also part of a previously unreported conserved motif comprising residues 280-287 and containing the pattern NXEXGXY(/F)A(/G); this new motif appears to have a structural role in stabilizing and positioning side chains that bind substrates and metal ions. The first few residues of this motif connect the two domains of the enzyme and a fulcrum point appears to be located near Asn280. In addition, an active-site Asp residue forms two coordinate bonds with the Mn(2+) ion present in the structure of the complex in a symmetrical bidentate manner, unlike in other PCK structures that contain a manganese ion.

The structure of a universally employed enzyme: V8 protease from Staphylococcus aureus.

V8 protease, an extracellular protease of Staphylococcus aureus, is related to the pancreatic serine proteases. The enzyme cleaves peptide bonds exclusively on the carbonyl side of aspartate and glutamate residues. Unlike the pancreatic serine proteases, V8 protease possesses no disulfide bridges. This is a major evolutionary difference, as all pancreatic proteases have at least two disulfide bridges. The structure of V8 protease shows structural similarity with several other serine proteases, specifically the epidermolytic toxins A and B from S. aureus and trypsin, in which the conformation of the active site is almost identical. V8 protease is also unique in that the positively charged N-terminus is involved in determining the substrate-specificity of the enzyme.

Mechanisms of activation of phosphoenolpyruvate carboxykinase from Escherichia coli by Ca2+ and of desensitization by trypsin.

The 1.8-A resolution structure of the ATP-Mg(2+)-Ca(2+)-pyruvate quinary complex of Escherichia coli phosphoenolpyruvate carboxykinase (PCK) is isomorphous to the published complex ATP-Mg(2+)-Mn(2+)-pyruvate-PCK, except for the Ca(2+) and Mn(2+) binding sites. Ca(2+) was formerly implicated as a possible allosteric regulator of PCK, binding at the active site and at a surface activating site (Glu508 and Glu511). This report found that Ca(2+) bound only at the active site, indicating that there is likely no surface allosteric site. (45)Ca(2+) bound to PCK with a K(d) of 85 micro M and n of 0.92. Glu508Gln Glu511Gln mutant PCK had normal activation by Ca(2+). Separate roles of Mg(2+), which binds the nucleotide, and Ca(2+), which bridges the nucleotide and the anionic substrate, are implied, and the catalytic mechanism of PCK is better explained by studies of the Ca(2+)-bound structure. Partial trypsin digestion abolishes Ca(2+) activation (desensitizes PCK). N-terminal sequencing identified sensitive sites, i.e., Arg2 and Arg396. Arg2Ser, Arg396Ser, and Arg2Ser Arg396Ser (double mutant) PCKs altered the kinetics of desensitization. C-terminal residues 397 to 540 were removed by trypsin when wild-type PCK was completely desensitized. Phe409 and Phe413 interact with residues in the Ca(2+) binding site, probably stabilizing the C terminus. Phe409Ala, DeltaPhe409, Phe413Ala, Delta397-521 (deletion of residues 397 to 521), Arg396(TAA) (stop codon), and Asp269Glu (Ca(2+) site) mutations failed to desensitize PCK and, with the exception of Phe409Ala, appeared to have defects in the synthesis or assembly of PCK, suggesting that the structure of the C-terminal domain is important in these processes.