Characterization of mevalonate kinase V377I, a mutant implicated in defective isoprenoid biosynthesis and HIDS/periodic fever syndrome

The list of diseases linked to defects in lipid metabolism has recently been augmented by the addition of hyperimmunoglobulinemia D and periodic fever syndrome (HIDS: MIM 260920), which are correlated with depressed levels of mevalonate kinase activity [1,2] and protein [1]. More specifically, a V377I substitution has been proposed to account for this disease. We observed that V377 appears to be far from invariant in eukaryotic mevalonate kinases. Prokaryotic mevalonate kinases are lower in molecular weight and several terminate prior to residue 377 of the eukaryotic proteins. These observations prompted our direct test of the impact of V377 on activity and protein stability by engineering a V377I mutation in a recombinant human mevalonate kinase. The mutant protein has been isolated and kinetically characterized. In comparison with wild-type enzyme, V377I exhibits only modest differences (notably > or = 6-fold inflation of K(m(MVA))) that do not account for the diminished mevalonate kinase activity assayed in HIDS cell extracts. Moreover, thermal inactivation (50 degrees C) of isolated wild-type and V377I enzymes demonstrates little difference in stability between these proteins. We conclude that a single V377I substitution is unlikely to explain the observation of depressed mevalonate kinase stability and catalytic activity in HIDS.     

Cloning,expression, and purification of His-tagged rat mevalonate kinase

Mevalonate kinase catalyzes the phosphorylation of mevalonic acid to form mevalonate 5-phosphate, which plays a key role in regulating cholesterol biosynthesis in animal cells. Deficiency of mevalonate kinase activity in the human body has been linked to mevalonic aciduria and hyperimmunoglobulinemia D/periodic fever syndrome (HIDS). We cloned the gene of rat mevalonate kinase into a bacterial expression vector pLM1 with six continuous histidine codons attached to the 5(') of the gene. The cloned gene was overexpressed in Escherichia coli and the soluble protein was purified with a nickel HiTrap chelating metal affinity column in 90% yield to apparent homogeneity. The purified rat mevalonate kinase had a dimeric structure composed of identical subunits. Based on SDS-PAGE, the subunit was 42 kDa. The specific activity of the purified His-tagged rat mevalonate kinase was 32.7 micromol/min/mg and the optimal pH was found to be 7.0-8.0 in phosphate buffer. The Michaelis constant K(M) was 35 microM for (RS)-mevalonate and 953 microM for ATP, respectively. The V(max) was determined to be 38.7 micromol/min/mg. The overexpression of rat mevalonate kinase in E. coli and one-step purification of the highly active rat mevalonate kinase will facilitate further our investigation of this enzyme through site-directed mutagenesis and enzyme-catalyzed reactions with substrate analogs.     

Molecular mechanism of aminoglycoside antibiotic kinase APH(3')-IIIa: roles of conserved active site residues

The aminoglycoside antibiotic kinases (APHs) constitute a clinically important group of antibiotic resistance enzymes. APHs share structural and functional homology with Ser/Thr and Tyr kinases, yet only five amino acids are invariant between the two groups of enzymes and these residues are all located within the nucleotide binding regions of the proteins. We have performed site-directed mutagenesis on all five conserved residues in the aminoglycoside kinase APH(3')-IIIa: Lys(44) and Glu(60) involved in ATP capture, a putative active site base required for deprotonating the incoming aminoglycoside hydroxyl group Asp(190), and the Mg(2+) ligands Asn(195) and Glu(208), which coordinate two Mg(2+) ions, Mg1 and Mg2. Previous structural and mutagenesis evidence have demonstrated that Lys(44) interacts directly with the phosphate groups of ATP; mutagenesis of invariant Glu(60), which forms a salt bridge with the epsilon-amino group of Lys(44), demonstrated that this residue does not play a critical role in ATP recognition or catalysis. Results of mutagenesis of Asp(190) were consistent with a role in proper positioning of the aminoglycoside hydroxyl during phosphoryl transfer but not as a general base. The Mg1 and Mg2 ligand Asp(208) was found to be absolutely required for enzyme activity and the Mg2 ligand Asn(195) is important for Mg.ATP recognition. The mutagenesis results together with solvent isotope, solvent viscosity, and divalent cation requirements are consistent with a dissociative mechanism of phosphoryl transfer where initial substrate deprotonation is not essential for phosphate transfer and where Mg2 and Asp(208) likely play a critical role in stabilization of a metaphosphate-like transition state. These results lay the foundation for the synthesis of transition state mimics that could reverse aminoglycoside antibiotic resistance in vivo.     

Identification of active site residues in mevalonate diphosphate decarboxylase: implications for a family of phosphotransferases

A combination of sequence homology analyses of mevalonate diphosphate decarboxylase (MDD) proteins and structural information for MDD leads to the hypothesis that Asp 302 and Lys 18 are active site residues in MDD. These residues were mutated to replace acidic/basic side chains and the mutant proteins were isolated and characterized. Binding and competitive displacement studies using trinitrophenyl-ATP, a fluorescent analog of substrate ATP, indicate that these mutant enzymes (D302A, D302N, K18M) retain the ability to stoichiometrically bind nucleotide triphosphates at the active site. These observations suggest the structural integrity of the mutant MDD proteins. The functional importance of mutated residues was evaluated by kinetic analysis. The 10(3) and 10(5)-fold decreases in k(cat) observed for the Asp 302 mutants (D302N and D302A, respectively) support assignment of a crucial catalytic role to Asp 302. A 30-fold decrease in activity and a 16-fold inflation of the K(m) for ATP is documented for the K18M mutant, indicating that Lys 18 influences the active site but is not crucial for reaction chemistry. Demonstration of the influence of conserved aspartate 302 appears to represent the first documentation of the functional importance of a residue in the MDD catalytic site and affords insight into phosphotransferase reactions catalyzed by a variety of enzymes in the galactokinase, homoserine kinase, mevalonate kinase, phosphom-evalonate kinase (GHMP kinase) family.     

Premature ovarian failure

Mevalonic aciduria (MVA) and hyperimmunoglobulinemia D syndrome (HIDS) represent the two ends of a clinical spectrum of disease caused by deficiency of mevalonate kinase (MVK), the first committed enzyme of cholesterol biosynthesis. At least 30 patients with MVA and 180 patients with HIDS have been reported worldwide. MVA is characterized by psychomotor retardation, failure to thrive, progressive cerebellar ataxia, dysmorphic features, progressive visual impairment and recurrent febrile crises. The febrile episodes are commonly accompanied by hepatosplenomegaly, lymphadenopathy, abdominal symptoms, arthralgia and skin rashes. Life expectancy is often compromised. In HIDS, only febrile attacks are present, but a subgroup of patients may also develop neurological abnormalities of varying degree such as mental retardation, ataxia, ocular symptoms and epilepsy. A reduced activity of MVK and pathogenic mutations in the MVK gene have been demonstrated as the common genetic basis in both disorders. In MVA, the diagnosis is established by detection of highly elevated levels of mevalonic acid excreted in urine. Increased levels of immunoglobulin D (IgD) and, in most patients of immunoglobulin A (IgA), in combination with enhanced excretion of mevalonic acid provide strong evidence for HIDS. The diagnosis is confirmed by low activity of mevalonate kinase or by demonstration of disease-causing mutations. Genetic counseling should be offered to families at risk. There is no established successful treatment for MVA. Simvastatin, an inhibitor of HMG-CoA reductase, and anakinra have been shown to have beneficial effect in HIDS.     

Staphylococcus aureus operates protein-tyrosine phosphorylation through a specific mechanism

Protein phosphorylation on tyrosine has been originally characterized in animal systems and has been shown to be involved in several fundamental processes including signal transduction, growth control, and malignancy. It has been later demonstrated to occur also in a number of bacteria, and recent data suggest that it may participate in the control of bacterial pathogenicity. In this work, we provide evidence that the gram-positive human pathogen Staphylococcus aureus harbors a protein-tyrosine kinase activity. This activity is borne by a protein, termed Cap5B2, whose phosphorylating capacity is expressed only in the presence of a stimulatory protein, either Cap5A1 or Cap5A2, that enhances its affinity for the phosphoryl donor ATP. In fact, the last 27/29 amino acids of the C-terminal domain of either polypeptide are sufficient for stimulating Cap5B2 activity. The stimulation of Cap5B2 by Cap5A1 involves essentially three amino acid residues in a helix of Cap5A1 (Asp202, Glu203, and Asp205) and three residues in a helix (helix 7) of Cap5B2 (Glu190, Lys192, and Lys193), thus suggesting helix-helix interaction between these two proteins. This type of helix-helix interaction resembles the interaction required for the activation of MinD ATPase by MinE protein in the process of septum-site determination, MinD sharing sequence similarity with Cap5B2. Such activation mechanism is described here in a gram-positive bacterial tyrosine kinase, and differs from the activation mechanism previously proposed for gram-negative bacteria. Therefore, it appears that S. aureus, and possibly other gram-positive bacteria, utilizes a specific molecular mechanism for triggering protein-tyrosine kinase activity.     

Molecular cloning of human mevalonate kinase and identification of a missense mutation in the genetic disease mevalonic aciduria.

Mevalonic aciduria is the first proposed inherited disorder of the cholesterol/isoprene biosynthetic pathway in humans, and it is presumed to be caused by a mutation in the gene coding for mevalonate kinase. To elucidate the molecular basis of this inherited disorder, a 2.0-kilobase human mevalonate kinase cDNA clone was isolated and sequenced. The 1188-base pair open reading frame coded for a 396-amino acid polypeptide with a deduced M(r) of 42,450. The predicted protein sequence displayed similarity to those of galactokinase and the yeast RAR1 protein, indicating that they may belong to a common gene family. Southern hybridization studies demonstrated that the mevalonate kinase gene is located on human chromosome 12 and is a single copy gene. No major rearrangements were detected in the mevalonic aciduria subject. The relative size (2 kilobases) and amounts of human mevalonate kinase mRNA were not changed in mevalonic aciduria fibroblasts. Approximately half of the mevalonic aciduria cDNA clones encoding mevalonate kinase contained a single base substitution (A to C) in the coding region at nucleotide 902 that changed an asparagine residue to a threonine residue. The presence of this missense mutation was confirmed by polymerase chain reaction amplification and allele-specific hybridization of the genomic DNAs from the proband and the proband's father and brother. Similar analysis failed to detect this mutation in the proband's mother, seven normal subjects, or four additional mevalonic aciduria subjects, indicating that the mutation does not represent a common gene polymorphism. Functional analysis of the defect by transient expression confirmed that the mutation produced an enzyme with diminished activity. Our data suggest that the index case is a compound heterozygote for a mutation in the mevalonate kinase gene.     

Functional consequences of alterations to amino acids located in the hinge domain of the Ca(2+)-ATPase of sarcoplasmic reticulum.

Site-specific mutagenesis of the sarcoplasmic reticulum Ca(2+)-ATPase was used to investigate the functional roles of 18 amino acid residues located at or near the "hinge-domain," a highly conserved region of the cation-transporting ATPases. Mutation of Lys684 to arginine, alanine, histidine, and glutamine resulted in complete loss of calcium transport function and ATPase activity. For the Lys684----Ala, histidine, and glutamine mutants, this coincided with a loss of the ability to form a phosphorylated intermediate from ATP or Pi. The Lys684----Arg mutant retained the ability to phorphorylate from ATP with normal apparent affinity, demonstrating the importance of the positive charge. On the other hand, no phosphorylation was observed with Pi as substrate in this mutant. Examination of the partial reactions after phosphorylation from ATP in the Lys684----Arg mutant demonstrated a reduction of the rate of transformation of the ADP-sensitive phosphoenzyme intermediate (E1P) to the ADP-insensitive phosphoenzyme intermediate (E2P), which could account for the loss of transport function. Once accumulated, the E2P intermediate was able to decompose rapidly in the presence of K+ at neutral pH. These results may be interpreted in terms of a preferential destabilization of protein phosphate interactions in the E2P form of this mutant. The Asp703----Ala and Asn-Asp707----Ala-Ala mutants were completely inactive and unable to form phosphoenzyme intermediates from ATP or Pi. In these mutants as well as in the Lys684----Ala mutant, nucleotides were found to protect with normal affinity against intramolecular cross-linking induced with glutaraldehyde, indicating that the nucleotide binding site was intact. Mutation of Glu646, Glu647, Asp659, Asp660, Glu689, Asp695, Glu696, Glu715, and Glu732 to alanine did not affect the maximum rates of calcium transport and ATP hydrolysis or the apparent affinities for calcium and ATP. Mutation of the 2 highly conserved proline residues, Pro681 and Pro709, as well as Lys728, to alanine resulted in partially inhibited Ca(2+)-ATPase enzymes with retention of the ability to form a phosphoenzyme intermediate from ATP or Pi and with normal apparent affinities for ATP and calcium. The proline mutants retained the biphasic ATP concentration dependence of ATPase activity, characteristic of the wild-type, and therefore the partial inhibition of turnover could not be ascribed to a disruption of the low affinity modulatory ATP site.(ABSTRACT TRUNCATED AT 400 WORDS)     

Stabilities of leucine zipper dimers estimated by an empirical free energy method.

The leucine zipper motif is a characteristic amino acid sequence found in dimeric DNA-binding proteins. Computer-generated models for leucine zippers were constructed as alpha-helical coiled dimers with leucine repeated every seventh residue. An empirical Gibbs free energy, delta G, function which incorporates hydrophobic force, electrostatic interactions, and conformational entropy loss as the major intermolecular interactions was used to estimate the delta G of dimer formation in fos, jun, and GCN4 zipper sequences. The calculations showed that complexes known to form stable homo- or heterodimers have favorable (negative) delta G, while other less stable complexes have unfavorable (positive) delta G. Leucines in position d of the coiled coil contribute large hydrophobic stabilization energies while residues in the a position contribute less to dimer stability. Hydrophobic contributions show little sequence specificity, however, and do not contribute significantly to homo/heterodimer preference. Charged residues in the e and g positions, on the other hand, determine homo/heterodimer specificity. In GCN4 homodimers, residues GLU el, Glu b2, Lys g2, and Lys e4 greatly contribute to dimer stability. The preferential stability of fos-jun heterodimer over the jun-jun and fos-fos homodimers is primarily due to the side chains Asp b1, Glu g1, Asp b2, Glu e2, Glu g2, Glu g3, and Lys a5 of the fos helix, and Arg c1, Lys g1, Lys b2, Lys e2, Arg e4, and Glu g4 of the jun helix.     

Molecular structure of Escherichia coli PurT-encoded glycinamide ribonucleotide transformylase

In Escherichia coli, the PurT-encoded glycinamide ribonucleotide transformylase, or PurT transformylase, catalyzes an alternative formylation of glycinamide ribonucleotide (GAR) in the de novo pathway for purine biosynthesis. On the basis of amino acid sequence analyses, it is known that the PurT transformylase belongs to the ATP-grasp superfamily of proteins. The common theme among members of this superfamily is a catalytic reaction mechanism that requires ATP and proceeds through an acyl phosphate intermediate. All of the enzymes belonging to the ATP-grasp superfamily are composed of three structural motifs, termed the A-, B-, and C-domains, and in each case, the ATP is wedged between the B- and C-domains. Here we describe two high-resolution X-ray crystallographic structures of PurT transformylase from E. coli: one form complexed with the nonhydrolyzable ATP analogue AMPPNP and the second with bound AMPPNP and GAR. The latter structure is of special significance because it represents the first ternary complex to be determined for a member of the ATP-grasp superfamily involved in purine biosynthesis and as such provides new information about the active site region involved in ribonucleotide binding. Specifically in PurT transformylase, the GAR substrate is anchored to the protein via Glu 82, Asp 286, Lys 355, Arg 362, and Arg 363. Key amino acid side chains involved in binding the AMPPNP to the enzyme include Arg 114, Lys 155, Glu 195, Glu 203, and Glu 267. Strikingly, the amino group of GAR that is formylated during the reaction lies at 2.8 A from one of the gamma-phosphoryl oxygens of the AMPPNP.     

Increasing the thermostability of Flavobacterium meningosepticum glycerol kinase by changing Ser329 to Asp in the subunit interface region.

The thermostability enhancement of Flavobacterium meningosepticum glycerol kinase (FGK) by random mutagenesis in the subunit interface region was investigated. A single Escherichia coli transformant, which produced a more thermostable glycerol kinase than the parent enzyme, was obtained. The nucleotide sequence of the gene of the mutant enzyme (FGK2615) was determined, and the four amino acid replacements were identified as Glu327 to Asp, Ser329 to Asp, Thr330 to Ala and Ser334 to Lys. Although the properties of FGK2615 were fundamentally similar to those of the parent enzyme, the thermostability and Km for ATP had changed. The thermostability of FGK2615 was apparently increased; the temperature at which the enzyme activity is inactivated by 50% for a 30-min incubation of FGK2615 was determined to be 72.1 degrees C which was 3.1 degrees C higher than that of the parent FGK. Four additional mutants each having a single amino acid replacement (Glu327 to Asp, Ser329 to Asp, Thr330 to Ala and Ser334 to Lys) were prepared and their thermostability and Km for substrates were evaluated. The effect of the substitution of Ser329 to Asp is discussed.     

Three subunits contribute amino acids to the active site of tetrameric adenylosuccinate lyase: Lys268 and Glu275 are required

Tetrameric adenylosuccinate lyase (ASL) of Bacillus subtilis catalyzes the cleavage of adenylosuccinate to form AMP and fumarate. We previously reported that two distinct subunits contribute residues to each active site, including the His68 and His89 from one and His141 from a second subunit [Brosius, J. L., and Colman, R. F. (2000) Biochemistry 39, 13336-13343]. Glu(275) is 2.8 A from His141 in the ASL crystal structure, and Lys268 is also in the active site region; Glu275 and Lys268 come from a third, distinct subunit. Using site-directed mutagenesis, we have replaced Lys268 by Arg, Gln, Glu, and Ala, with specific activities of the purified mutant enzymes being 0.055, 0.00069, 0.00028, and 0.0, respectively, compared to 1.56 units/mg for wild-type (WT) enzyme. Glu275 was substituted by Gln, Asp, Ala, and Arg; none of these homogeneous mutant enzymes has detectable activity. Circular dichroism and light scattering reveal that neither the secondary structure nor the oligomeric state of the Lys268 mutant enzymes has been perturbed. Native gel electrophoresis and circular dichroism indicate that the Glu275 mutant enzymes are tetramers, but their conformation is altered slightly. For K268R, the K(m)s for all substrates are similar to WT enzyme. Binding studies using [2-3H]-adenylosuccinate reveal that none of the Glu275 mutant enzymes, nor inactive K268A, can bind substrate. We propose that Lys268 participates in binding substrate and that Glu275 is essential for catalysis because of its interaction with His141. Incubation of H89Q with K268Q or E275Q leads to restoration of up to 16% WT activity, while incubation of H141Q with K268Q or E275Q results in 6% WT activity. These complementation studies provide the first functional evidence that a third subunit contributes residues to each intersubunit active site of ASL. Thus, adenylosuccinate lyase has four active sites per enzyme tetramer, each of which is formed from regions of three subunits.     

Snapping of the carboxyl terminal tail of the catalytic subunit of PKA onto its core: characterization of the sites by mutagenesis

A set of 45 mutants of the carboxyl terminal tail of the PKA catalytic subunit was prepared and used to assess the contribution of this tail to the structure and function of the kinase. Ala substitutions of Asp 323, Phe 327, Glu 333, and Phe 350 resulted in a complete loss of enzymatic activity. Other replacements by Ala (Phe 314, Tyr 330, Glu 332, and Phe 347) brought about either a drop in activity to less than 10% of the wild-type enzyme or a reduction of affinity toward ATP (Lys 317, Lys 319, Tyr 330, and Glu 332) or toward Kemptide (Ile 315, Tyr 330, Val 337, Ile 339, Lys 345, and Glu 346). Mutations of Ser 338, a major autophosphorylation site of PKA, by Ala, Glu, Asp, Gln, and Asn showed that the kinetic parameters of these mutants are similar to those of the wild-type. The contribution of each of these tail mutations to the structure and stability of the kinase was assessed by monitoring its effect on the heat stability (when measurable) or by determining the susceptibility of the mutant kinase to cleavage by the Kinase Splitting Membranal Proteinase/Meprin beta. Here we show that the tail of PKA has a key role in creating the active conformation of the kinase. It does so by means of specific amino acid residues, which act as "snapping points" to embrace the two lobes of the kinase and orient them in the correct juxtaposition for substrate docking, biorecognition, and catalysis.     

Protein, lipid and water organization in bacteriorhodopsin crystals: a molecular view of the purple membrane at 1.9 A resolution.

BACKGROUND: Bacteriorhodopsin (bR) from Halobacterium salinarum is a proton pump that converts the energy of light into a proton gradient that drives ATP synthesis. The protein comprises seven transmembrane helices and in vivo is organized into purple patches, in which bR and lipids form a crystalline two-dimensional array. Upon absorption of a photon, retinal, which is covalently bound to Lys216 via a Schiff base, is isomerized to a 13-cis,15-anti configuration. This initiates a sequence of events - the photocycle - during which a proton is transferred from the Schiff base to Asp85, followed by proton release into the extracellular medium and reprotonation from the cytoplasmic side. RESULTS: The structure of bR in the ground state was solved to 1.9 A resolution from non-twinned crystals grown in a lipidic cubic phase. The structure reveals eight well-ordered water molecules in the extracellular half of the putative proton translocation pathway. The water molecules form a continuous hydrogen-bond network from the Schiff-base nitrogen (Lys216) to Glu194 and Glu204 and includes residues Asp85, Asp212 and Arg82. This network is involved both in proton translocation occurring during the photocycle, as well as in stabilizing the structure of the ground state. Nine lipid phytanyl moieties could be modeled into the electron-density maps. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis of single crystals demonstrated the presence of four different charged lipid species. CONCLUSIONS: The structure of protein, lipid and water molecules in the crystals represents the functional entity of bR in the purple membrane of the bacteria at atomic resolution. Proton translocation from the Schiff base to the extracellular medium is mediated by a hydrogen-bond network that involves charged residues and water molecules.     

Localization of subunits D,E, and G in the yeast V-ATPase complex using cysteine-mediated cross-linking to subunit B

Using a combination of cysteine mutagenesis and covalent cross-linking, we have identified subunits in close proximity to specific sites within subunit B of the vacuolar (H(+))-ATPase (V-ATPase) of yeast. Unique cysteine residues were introduced into subunit B by site-directed mutagenesis, and the resultant V-ATPase complexes were reacted with the bifunctional, photoactivatable maleimide reagent 4-(N-maleimido)benzophenone (MBP) followed by irradiation. Cross-linked products were identified by Western blot using subunit-specific antibodies. Introduction of cysteine residues at positions Glu(106) and Asp(199) led to cross-linking of subunits B and E, at positions Asp(341) and Ala(424) to cross-linking of subunits B and D, and at positions Ala(15) and Lys(45) to cross-linking of subunits B and G. Using a molecular model of subunit B constructed on the basis of sequence homology between the V- and F-ATPases, the X-ray coordinates of the F(1)-ATPase, and energy minimization, Glu(106), Asp(199), Ala(15), and Lys(45) are all predicted to be located on the outer surface of the complex, with Ala(15) and Lys(45) located near the top of the complex furthest from the membrane. By contrast, Asp(341) and Ala(424) are predicted to face the interior of the A(3)B(3) hexamer. These results suggest that subunits E and G form part of a peripheral stalk connecting the V(1) and V(0) domains whereas subunit D forms part of a central stalk. Subunit D is thus the most likely homologue to the gamma subunit of F(1), which undergoes rotation during ATP hydrolysis and serves an essential function in rotary catalysis.     

Partial purification and properties of mevalonate kinase from neonatal chick liver

Mevalonate kinase from neonatal chick liver has been partially purified by ammonium sulphate precipitation and Sephadex G100 and DEAE-cellulose fractionation. The kinetic characteristics agreed with the sequential mechanism suggested for the enzyme and provided apparent Km values of 0.01 mM for mevalonic acid and 0.25 mM for ATP. Partially purified mevalonate kinase from neonatal chick liver showed an absolute specificity for ATP. Mn2+ was a better activator than Mg2+ at low concentrations (0.1-1.0 mM). Higher Mn2+ concentrations produced a clear inhibition of mevalonate kinase. Likewise, addition of EDTA, with or without metal ions, clearly inhibited the enzymatic reaction.     

Structural analysis of mevalonate‐3‐kinase provides insight into the mechanisms of isoprenoid pathway decarboxylases

In animals, cholesterol is made from 5‐carbon building blocks produced by the mevalonate pathway. Drugs that inhibit the mevalonate pathway such as atorvastatin (lipitor) have led to successful treatments for high cholesterol in humans. Another potential target for the inhibition of cholesterol synthesis is mevalonate diphosphate decarboxylase (MDD), which catalyzes the phosphorylation of (R)‐mevalonate diphosphate, followed by decarboxylation to yield isopentenyl pyrophosphate. We recently discovered an MDD homolog, mevalonate‐3‐kinase (M3K) from Thermoplasma acidophilum, which catalyzes the identical phosphorylation of (R)‐mevalonate, but without concomitant decarboxylation. Thus, M3K catalyzes half the reaction of the decarboxylase, allowing us to separate features of the active site that are required for decarboxylation from features required for phosphorylation. Here we determine the crystal structure of M3K in the apo form, and with bound substrates, and compare it to MDD structures. Structural and mutagenic analysis reveals modifications that allow M3K to bind mevalonate rather than mevalonate diphosphate. Comparison to homologous MDD structures show that both enzymes employ analogous Arg or Lys residues to catalyze phosphate transfer. However, an invariant active site Asp/Lys pair of MDD previously thought to play a role in phosphorylation is missing in M3K with no functional replacement. Thus, we suggest that the invariant Asp/Lys pair in MDD may be critical for decarboxylation rather than phosphorylation.     

Interleukin 8, neutrophil-activating peptide-2 and GRO-alpha bind to and elicit cell activation via specific and different amino acid residues of CXCR2

The objective of this investigation was to determine the amino acid residues of the human neutrophil CXC chemokine receptor-2 (CXCR2) that are critical for binding the ligands interleukin 8 (IL-8), neutrophil-activating peptide-2 (NAP-2), and growth-related protein alpha (GROalpha) and critical for receptor-mediated signal transduction. Charged residues of the amino terminus and the first extracellular loop of CXCR2 were targeted for point mutagenesis studies. Seven separate CXCR2 mutants (Glu7, Asp9, Glu12, Asp13, Lys108, Asn110, and Lys120, all to Ala) were generated. Based on the Scatchard analysis of radioligand binding studies, the following amino acids were deemed critical for ligand binding: (i) Asp9, Glu12, Lys108, and Lys120 for IL-8 and (ii) Glu7, Asp9, and Glu12 for GROalpha. Point mutations in the amino terminus domain (Asp9 and Glu12) and the first extracellular loop (Lys108, Asn110, and Lys120) of CXCR2 reduced cell activation to all three ligands as measured by changes in intracellular calcium concentration. In conclusion, high-affinity binding of IL-8, NAP-2, and GROalpha to CXCR2 involves interaction with specific and different amino acid residues of CXCR2. Furthermore, we propose that the CXCR2 amino acid residues required for cell activation are not necessarily the same residues required for ligand binding.     

Importance of Glu(282) in transmembrane segment M3 of the Na(+),K(+)-ATPase for control of cation interaction and conformational changes

Glu(282) located in the NH(2)-terminal part of transmembrane helix M3 of the Na(+),K(+)-ATPase was replaced by alanine, glycine, leucine, lysine, aspartate, or glutamine, and the effects of the mutations on the overall and partial reactions of the enzyme were analyzed. The mutations affected at least 3 important functions of the Na(+),K(+)-ATPase: (i) the conformational transitions between E(1) and E(2) forms of dephospho- and phosphoenzyme, (ii) Na(+) binding at the cytoplasmically facing sites of E(1), and (iii) long-range interaction controlling dephosphorylation. In mutants Glu(282) --> Lys and Glu(282) --> Asp, the E(1) form was favored during ATP hydrolysis, whereas the E(2) form was favored in Glu(282) --> Ala and Glu(282) --> Gly. Regardless of the change of conformational equilibrium, all the mutants displayed a reduced apparent affinity for Na(+), at least 3-fold for Glu(282) --> Lys and Glu(282) --> Asp, suggesting a direct effect on the Na(+) binding properties of E(1). Glu(282) --> Ala and Glu(282) --> Gly exhibited an extraordinary high rate of ATP hydrolysis in the mere presence of Na(+) without K(+) ("Na(+)-ATPase activity"), because of an increased rate of dephosphorylation of E(2)P. These results are in accordance with the hypothesis that Glu(282) is involved in the communication between the cation binding pocket and the catalytic site and in control of the cytoplasmic entry pathway for Na(+).