Matrix protein 1 (M1) of the influenza A virus plays multiple roles in virion assembly and infection. Interest in the pH dependence of M1's multiple functions led us to study the effect of subtle pH changes on M1 structure, resulting in the elucidation of a unique low-pH crystal structure of the N1-165-domain of A/WSN/33 (H1N1) M1 that has never been reported. Although the 2.2 Å crystal structure of M1 N-terminus shows a dimer with the two monomers interacting in a face-to-face fashion at low pH as observed earlier, a 44° rotation of the second monomer has led to a significantly different dimer interface that possibly affects dimer stability. More importantly, while one of the monomers is fully defined, the N-terminal half of the second monomer shows considerable disorder that appears inherent in the protein and is potentially physiologically relevant. Such disorder has not been observed in any other previously reported structure at either low or high pH conditions, despite similar crystallization pH conditions. By comparing our novel N1-165-domain structure with other low-pH or neutral-pH M1 structures, it appears that M1 can energetically access different monomer and dimer conformations, as well as oligomeric states, with varying degree of similarities. The study reported here provides further insights into M1 oligomerization that may be essential for viral propagation and infectivity.
The crystallographic analysis of fully liganded Hb ζ2β2
s trapped in a tense conformation is reported.
A variant Hb ζ2β2
s that is formed from sickle hemoglobin (Hb S; α2β2
s) by exchanging adult α-globin with embryonic ζ-globin subunits shows promise as a therapeutic agent for sickle-cell disease (SCD). Hb ζ2β2
s inhibits the polymerization of deoxygenated Hb S in vitro and reverses characteristic features of SCD in vivo in mouse models of the disorder. When compared with either Hb S or with normal human adult Hb A (α2β2), Hb ζ2β2
s exhibits atypical properties that include a high oxygen affinity, reduced cooperativity, a weak Bohr effect and blunted 2,3-diphosphoglycerate allostery. Here, the 1.95 Å resolution crystal structure of human Hb ζ2β2
s that was expressed in complex transgenic knockout mice and purified from their erythrocytes is presented. When fully liganded with carbon monoxide, Hb ζ2β2
s displays a central water cavity, a ζ1–βs2 (or ζ2–βs1) interface, intersubunit salt-bridge/hydrogen-bond interactions, C-terminal βHis146 salt-bridge interactions, and a β-cleft, that are highly unusual for a relaxed hemoglobin structure and are more typical of a tense conformation. These quaternary tense-like features contrast with the tertiary relaxed-like conformations of the ζ1βs1 dimer and the CD and FG corners, as well as the overall structures of the heme cavities. This crystallographic study provides insights into the altered oxygen-transport properties of Hb ζ2β2
s and, moreover, decouples tertiary- and quaternary-structural events that are critical to Hb ligand binding and allosteric function.
hemoglobin; tense state; relaxed state; Bohr effect; 2,3-disphosphoglycerate; cooperativity; allostery
Hb Baden (β18Val→Met) is a rare variant hemoglobin that has never been functionally or clinically characterized. We describe a Hb Baden heterozygote who exhibits normal growth and development, as well as age- and gender-appropriate hematological parameters. Surprisingly, in vitro analyses demonstrate that Hb Baden is relatively unstable and exhibits an abnormally high affinity for O2. These properties are likely to affect the physiologies of individuals who inherit the βBaden mutation in trans to a determinant for either a functionally relevant hemoglobin-opathy or a mild thalassemia. The data also provide insights into the function of the AB-segment/A-helix of the β-globin, supporting a structural model in which this poorly understood region serves as a scaffold that fixes the positions of other helices that directly impact β-globin function.
A new unstable alpha globin variant was detected in a child with hypoxemia and anemia. The child’s mother was found to carry the same mutation. The hemoglobin variant co-eluted with Hb A2 by cation-exchange high performance liquid chromatography (CE-HPLC) and appeared cathodal to Hb A and anodal to Hb F by isoelectric focusing. It represented less than 20% of the total hemoglobin and was unstable by isopropanol testing. Gene sequencing identified a missense mutation in the α2 gene [HBA2:c.140T>C]. Oxygen dissociation and P50 test results were normal.
Hemoglobin (Hb) Lake Tapawingo; α globin variant; unstable hemoglobin (Hb); oxygen saturation
A correction to the paper by Abdulmalik et al. [(2011), Acta Cryst. D67, 920–928].
The affiliation of one of the authors of Abdulmalik et al. (2011) [Acta Cryst. D67, 920–928] is corrected.
hemoglobin; oxygen affinity; sickle-cell disease; polymerization; T state; R2 state; corrigendum
Pyridyl derivatives of vanillin increase the fraction of the more soluble oxygenated sickle hemoglobin and/or directly increase the solubility of deoxygenated sickle hemoglobin. Crystallographic analysis reveals the structural basis of the potent and dual antisickling activity of these derivatives.
Vanillin has previously been studied clinically as an antisickling agent to treat sickle-cell disease. In vitro investigations with pyridyl derivatives of vanillin, including INN-312 and INN-298, showed as much as a 90-fold increase in antisickling activity compared with vanillin. The compounds preferentially bind to and modify sickle hemoglobin (Hb S) to increase the affinity of Hb for oxygen. INN-312 also led to a considerable increase in the solubility of deoxygenated Hb S under completely deoxygenated conditions. Crystallographic studies of normal human Hb with INN-312 and INN-298 showed that the compounds form Schiff-base adducts with the N-terminus of the α-subunits to constrain the liganded (or relaxed-state) Hb conformation relative to the unliganded (or tense-state) Hb conformation. Interestingly, while INN-298 binds and directs its meta-positioned pyridine-methoxy moiety (relative to the aldehyde moiety) further down the central water cavity of the protein, that of INN-312, which is ortho to the aldehyde, extends towards the surface of the protein. These studies suggest that these compounds may act to prevent sickling of SS cells by increasing the fraction of the soluble high-affinity Hb S and/or by stereospecific inhibition of deoxygenated Hb S polymerization.
hemoglobin; oxygen affinity; sickle-cell disease; polymerization; T state; R2 state
The etiology of epilepsy is a very complicated, multifactorial process that is not completely understood. Therefore, the availability of epilepsy animal models induced by different mechanisms is crucial in advancing our knowledge and developing new therapeutic regimens for this disorder. Considering the advantages of zebrafish, we have developed a seizure model in zebrafish larvae using ginkgotoxin, a neurotoxin naturally occurring in Ginkgo biloba and hypothesized to inhibit the formation of the neurotransmitter γ-aminobutyric acid (GABA). We found that a 2-hour exposure to ginkgotoxin induced a seizure-like behavior in zebrafish larvae. This seizure-like swimming pattern was alleviated by the addition of either pyridoxal-5′-phosphate (PLP) or GABA and responded quickly to the anti-convulsing activity of gabapentin and phenytoin, two commonly prescribed anti-epileptic drugs (AEDs). Unexpectedly, the ginkgotoxin-induced PLP depletion in our experimental setting did not affect the homeostasis of folate-mediated one-carbon metabolism, another metabolic pathway playing a crucial role in neural function that also relies on the availability of PLP. This ginkgotoxin-induced seizure behavior was also relieved by primidone, which had been tested on a pentylenetetrazole-induced zebrafish seizure model but failed to rescue the seizure phenotype, highlighting the potential use and complementarity of this ginkgotoxin-induced seizure model for AED development. Structural and morphological characterization showed that a 2-hour ginkgotoxin exposure did not cause appreciable changes in larval morphology and tissues development. In conclusion, our data suggests that this ginkgotoxin-induced seizure in zebrafish larvae could serve as an in vivo model for epileptic seizure research and potential AED screening.
Heme is an important cofactor in a large number of essential proteins and is often involved in small molecule binding and activation. Heme loss from proteins thus negatively affects the function of these proteins, but is also an important component of iron recycling. The characterization of intermediates that form during the loss of heme from proteins has been problematic due, in a large part, to the instability of such intermediates. We have characterized, by X-ray crystallography, three compounds that form during the nitrite-induced degradation of human α2β2 hemoglobin (Hb). The first is an unprecedented complex that exhibits a large β heme displacement of 4.8 Å towards the protein exterior; the heme displacement is stabilized by the binding of the distal His residue to the heme Fe, which in turn allows for the unusual binding of an exogenous ligand at the proximal face of the heme. We have also structurally characterized complexes that display regiospecific nitration of the heme at the 2-vinyl position; we show that heme nitration is not a prerequisite for heme loss. Our results provide structural insight into a possible pathway for nitrite-induced heme loss from human Hb.
iron; heme; binding; heme loss; nitrogen oxides; blood; hemoglobin
Pyridoxal 5′-phosphate (PLP) is a cofactor for dozens of B6 requiring enzymes. PLP reacts with apo-B6 enzymes by forming an aldimine linkage with the ε-amino group of an active site lysine residue, thus yielding the catalytically active holo-B6 enzyme. During protein turnover, the PLP is salvaged by first converting it to pyridoxal by a phosphatase and then back to PLP by pyridoxal kinase. Nonetheless, PLP poses a potential toxicity problem for the cell since its reactive 4′-aldehyde moiety forms covalent adducts with other compounds and non-B6 proteins containing thiol or amino groups. The regulation of PLP homeostasis in the cell is thus an important, yet unresolved issue. In this report, using site-directed mutagenesis, kinetic, spectroscopic and chromatographic studies we show that pyridoxal kinase from E. coli forms a complex with the product PLP to form an inactive enzyme complex. Evidence is presented that, in the inhibited complex, PLP has formed an aldimine bond with an active site lysine residue during catalytic turnover. The rate of dissociation of PLP from the complex is very slow, being only partially released after a 2-hour incubation with PLP phosphatase. Interestingly, the inactive pyridoxal kinase•PLP complex can be partially reactivated by transferring the tightly bound PLP to an apo-B6 enzyme. These results open new perspectives on the mechanism of regulation and role of pyridoxal kinase in the Escherichia coli cell.
Several drugs and natural compounds are known to be highly neurotoxic, triggering epileptic convulsions or seizures, and causing headaches, agitations, as well as other neuronal symptoms. The neurotoxic effects of some of these compounds, including theophylline and ginkgotoxin, have been traced to their inhibitory activity against human pyridoxal kinase (hPL kinase), resulting in deficiency of the active cofactor form of vitamin B6, pyridoxal 5′-phosphate (PLP). Pyridoxal (PL), an inactive form of vitamin B6 is converted to PLP by PL kinase. PLP is the B6 vitamer required as a cofactor for over 160 enzymatic activities essential in primary and secondary metabolism. We have performed structural and kinetic studies on hPL kinase with several potential inhibitors, including ginkgotoxin and theophylline. The structural studies show ginkgotoxin and theophylline bound at the substrate site, and are involved in similar protein interactions as the natural substrate, PL. Interestingly, the phosphorylated product of ginkgotoxin is also observed bound at the active site. This work provides insights into the molecular basis of hPL kinase inhibition and may provide a working hypothesis to quickly screen or identify neurotoxic drugs as potential hPL kinase inhibitors. Such adverse effects may be prevented by administration of an appropriate form of vitamin B6, or provide clues of how to modify these drugs to help reduce their hPL kinase inhibitory effects.
The title molecule, [Fe(C36H44N4)Cl]·1.5CH2Cl2, is a high-spin square-pyramidal iron(III) porphyrinate with an average value for the equatorial Fe—N bond lengths of 2.065 (3) Å and an axial Fe—Cl distance of 2.2430 (13) Å. The iron cation is displaced by 0.518 (1) Å from the 24-atom mean plane of the porphyrin ring. These values are typical for high-spin iron(III) porphyrinates.
The up-and-down binding of dimeric MecI to mecA dyad DNA may account for the cooperative effect of the repressor.
The dimeric repressor MecI regulates the mecA gene that encodes the penicillin-binding protein PBP-2a in methicillin-resistant Staphylococcus aureus (MRSA). MecI is similar to BlaI, the repressor for the blaZ gene of β-lactamase. MecI and BlaI can bind to both operator DNA sequences. The crystal structure of MecI in complex with the 32 base-pair cognate DNA of mec was determined to 3.8 Å resolution. MecI is a homodimer and each monomer consists of a compact N-terminal winged-helix domain, which binds to DNA, and a loosely packed C-terminal helical domain, which intertwines with its counter-monomer. The crystal contains horizontal layers of virtual DNA double helices extending in three directions, which are separated by perpendicular DNA segments. Each DNA segment is bound to two MecI dimers. Similar to the BlaI–mec complex, but unlike the MecI–bla complex, the MecI repressors bind to both sides of the mec DNA dyad that contains four conserved sequences of TACA/TGTA. The results confirm the up-and-down binding to the mec operator, which may account for cooperative effect of the repressor.
The pdxK and pdxY genes have been found to code for pyridoxal kinases, enzymes involved in the pyridoxal phosphate salvage pathway. Two pyridoxal kinase structures have recently been published, including Escherichia coli pyridoxal kinase 2 (ePL kinase 2) and sheep pyridoxal kinase, products of the pdxY and pdxK genes, respectively. We now report the crystal structure of E. coli pyridoxal kinase 1 (ePL kinase 1), encoded by a pdxK gene, and an isoform of ePL kinase 2. The structures were determined in the unliganded and binary complexes with either MgATP or pyridoxal to 2.1-, 2.6-, and 3.2-Å resolutions, respectively. The active site of ePL kinase 1 does not show significant conformational change upon binding of either pyridoxal or MgATP. Like sheep PL kinase, ePL kinase 1 exhibits a sequential random mechanism. Unlike sheep pyridoxal kinase, ePL kinase 1 may not tolerate wide variation in the size and chemical nature of the 4′ substituent on the substrate. This is the result of differences in a key residue at position 59 on a loop (loop II) that partially forms the active site. Residue 59, which is His in ePL kinase 1, interacts with the formyl group at C-4′ of pyridoxal and may also determine if residues from another loop (loop I) can fill the active site in the absence of the substrate. Both loop I and loop II are suggested to play significant roles in the functions of PL kinases.
The 14-kDa BlaI protein represses the transcription of blaZ, the gene encoding β-lactamase. It is homologous to MecI, which regulates the expression of mecA, the gene encoding the penicillin binding protein PBP2a. These genes mediate resistance to β-lactam antibiotics in staphylococci. Both repressors can bind either bla or mec DNA promoter-operator sequences. Regulated resistance genes are activated via receptor-mediated cleavage of the repressors. Cleavage is induced when β-lactam antibiotics bind the extramembrane sensor of the sensor-transducer signaling molecules, BlaR1 or MecR1. The crystal structures of BlaI from Staphylococcus aureus, both in free form and in complex with 32 bp of DNA of the mec operator, have been determined to 2.0- and 2.7-Å resolutions, respectively. The structure of MecI, also in free form and in complex with the bla operator, has been previously reported. Both repressors form homodimers, with each monomer composed of an N-terminal DNA binding domain of winged helix-turn-helix topology and a C-terminal dimerization domain. The structure of BlaI in complex with the mec operator shows a protein-DNA interface that is conserved between both mec and bla targets. The recognition helix α3 interacts specifically with the conserved TACA/TGTA DNA binding motif. BlaI and, probably, MecI dimers bind to opposite faces of the mec DNA double helix in an up-and-down arrangement, whereas MecI and, probably, BlaI dimers bind to the same DNA face of bla promoter-operator DNA. This is due to the different spacing of mec and bla DNA binding sites. Furthermore, the flexibility of the dimeric proteins may make the C-terminal proteolytic cleavage site more accessible when the repressors are bound to DNA than when they are in solution, suggesting that the induction cascade involves bound rather than free repressor.
The crystal structure of Escherichia coli PdxY, the protein product of the pdxY gene, has been determined to a 2.2-Å resolution. PdxY is a member of the ribokinase superfamily of enzymes and has sequence homology with pyridoxal kinases that phosphorylate pyridoxal at the C-5′ hydroxyl. The protein is a homodimer with an active site on each monomer composed of residues that come exclusively from each respective subunit. The active site is filled with a density that fits that of pyridoxal. In monomer A, the ligand appears to be covalently attached to Cys122 as a thiohemiacetal, while in monomer B it is not covalently attached but appears to be partially present as pyridoxal 5′-phosphate. The presence of pyridoxal phosphate and pyridoxal as ligands was confirmed by the activation of aposerine hydroxymethyltransferase after release of the ligand by the denaturation of PdxY. The ligand, which appears to be covalently attached to Cys122, does not dissociate after denaturation of the protein. A detailed comparison (of functional properties, sequence homology, active site and ATP-binding-site residues, and active site flap types) of PdxY with other pyridoxal kinases as well as the ribokinase superfamily in general suggested that PdxY is a member of a new subclass of the ribokinase superfamily. The structure of PdxY also permitted an interpretation of work that was previously published about this enzyme.