Structure of the ATP binding domain from the Archaeoglobus fulgidus Cu+-ATPase

The P-type ATPases translocate cations across membranes using the energy provided by ATP hydrolysis. CopA from Archaeoglobus fulgidus is a hyperthermophilic ATPase responsible for the cellular export of Cu+ and is a member of the heavy metal P1B-type ATPase subfamily, which includes the related Wilson and Menkes diseases proteins. The Cu+-ATPases are distinct from their P-type counter-parts in ion binding sequences, membrane topology, and the presence of cytoplasmic metal binding domains, suggesting that they employ alternate forms of regulation and novel mechanisms of ion transport. To gain insight into Cu+-ATPase function, the structure of the CopA ATP binding domain (ATPBD) was determined to 2.3 A resolution. Similar to other P-type ATPases, the ATPBD includes nucleotide binding (N-domain) and phosphorylation (P-domain) domains. The ATPBD adopts a closed conformation similar to the nucleotide-bound forms of the Ca2+-ATPase. The CopA ATPBD is much smaller and more compact, however, revealing the minimal elements required for ATP binding, hydrolysis, and enzyme phosphorylation. Structural comparisons to the AMP-PMP-bound form of the Escherichia coli K+-transporting Kdp-ATPase and to the Wilson disease protein N-domain indicate that the five conserved N-domain residues found in P1B-type ATPases, but not in the other families, most likely participate in ATP binding. By contrast, the P-domain includes several residues conserved among all P-type ATPases. Finally, the CopA ATPBD structure provides a basis for understanding the likely structural and functional effects of various mutations that lead to Wilson and Menkes diseases.     

Characterization of a thermophilic P-type Ag+/Cu+-ATPase from the extremophile Archaeoglobus fulgidus.

The thermophilic, sulfur metabolizing Archaeoglobus fulgidus contains two genes, AF0473 and AF0152, encoding for PIB-type heavy metal transport ATPases. In this study, we describe the cloning, heterologous expression, purification, and functional characterization of one of these ATPases, CopA (NCB accession number AAB90763), encoded by AF0473. CopA is active at high temperatures (75 degrees C; E(a) = 103 kJ/mol) and inactive at 37 degrees C. It is activated by Ag+ (ATPase V(max) = 14.82 micromol/mg/h) and to a lesser extent by Cu+ (ATPase V(max) = 3.66 micromol/mg/h). However, Cu+ interacts with the enzyme with higher apparent affinity (ATPase stimulation, Ag+ K(12) = 29.4 microm; Cu+ K(12) = 2.1 microm). This activation by Ag+ or Cu+ is dependent on the presence of millimolar amounts of cysteine. In the presence of ATP, these metals drive the formation of an acid-stable phosphoenzyme with apparent affinities similar to those observed in the ATPase activity determinations (Ag+, K(12) = 23.0 microm; Cu+, K(12) = 3.9 microm). However, comparable levels of phosphoenzyme are reached in the presence of both cations (Ag+, 1.40 nmol/mg; Cu+, 1.08 nmol/mg). The stimulation of phosphorylation by the cations suggests that CopA drives the outward movement of the metal. CopA presents additional functional characteristics similar to other P-type ATPases. ATP interacts with the enzyme with two apparent affinities (ATPase K(m) = 0.25 mm; phosphorylation K(m) = 4.81 microm), and the presence of vanadate leads to enzyme inactivation (IC(50) = 24 microm). This is the first Ag+/Cu+ -ATPase expressed and purified in a functional form. Thus, it provides a model for structure-functional studies of these transporters. Moreover, its characterization will also contribute to an understanding of thermophilic ion transporters.     

Functional roles of metal binding domains of the Archaeoglobus fulgidus Cu(+)-ATPase CopA

CopA, a thermophilic membrane ATPase from Archaeoglobus fulgidus, drives the outward movement of Cu(+) or Ag(+) [Mandal et al. (2002) J. Biol. Chem. 277, 7201-7208]. This, as other P(IB)-ATPases, is characterized by a putative metal binding sequence (C(380)PC(382)) in its sixth transmembrane fragment and cytoplasmic metal binding sequences in its NH(2)- and COOH-terminal ends (C(27)AMC(30) and C(751)HHC(754)). Using isolated CopA, we have studied the functional role of these three putative metal binding domains. Replacement of transmembrane Cys residues by Ala results in nonfunctional enzymes that are unable to hydrolyze ATP. However, the CPC --> APA substituted enzyme binds ATP, indicating its correct folding and suggesting that enzyme turnover is prevented by the lack of metal binding to the transmembrane site. Replacement of C-terminal Cys by Ala (C(751,754)A) has no significant effect on ATPase activity, enzyme phosphorylation, apparent binding affinities of ligands, or E1-E2 equilibrium. In contrast, replacement of Cys in the N-terminal metal binding domain (N-MBD) (C(27,30)A) leads to 40% reduction in enzyme turnover. The C(27,30)A enzyme binds Cu(+), Ag(+), and ATP with the same high apparent affinities as the wild-type CopA. Evidence that N-MBD disruption has no effect on the E1-E2 equilibrium is provided by the normal interaction of ATP acting with low affinity and the unaffected IC(50) for vanadate inhibition observed in the C(27,30)A-substituted enzyme. However, replacement C(27,30)A slowed the dephosphorylation of the E2P(metal) form of the enzyme, suggesting a reduction in the rate of metal release. Other investigators have shown the Cu-dependent interaction of isolated N-MBDs from the Wilson disease Cu-ATPase with the ATP binding cytoplasmic domain [Tsivkovskii et al. (2001) J. Biol. Chem. 276, 2234-2242]. Therefore, the data suggest a regulatory mechanism in which the Cu-dependent N-MBD/ATP binding domain interaction would accelerate cation release, the enzyme rate-limiting step, and consequently Cu(+) transport.     

Structure of the actuator domain from the Archaeoglobus fulgidus Cu(+)-ATPase

Copper homeostasis is maintained in part by membrane-bound P(1B)-type ATPases that are found in all organisms and drive the transport of this essential, yet toxic, metal ion across cellular membranes. CopA from Archaeoglobus fulgidus is a hyperthermophilic member of this ATPase subfamily and is homologous to the human Wilson and Menkes disease ATPases. To gain insight into Cu(+)-ATPase function, the structure of the CopA actuator domain (A-domain) was determined to 1.65 A resolution. The CopA A-domain functions to couple ATP hydrolysis in the ATP binding domain (ATPBD) with structural rearrangements of critical transmembrane segments. Its fold is quite similar to that of the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1) A-domain, with the exception of an external loop region. On the basis of sequence and structural comparisons, specific residues that probably interact with the CopA ATPBD have been identified. Comparisons to the Wilson and Menkes disease A-domains reveal the presence of an additional loop that may be associated with regulatory functions in eukaryotic Cu(+)-ATPases. Finally, several mutations in the Wilson and Menkes disease ATPases occur in the A-domain, and their likely effects on function can be inferred from the CopA A-domain structure.     

Activation of Archaeoglobus fulgidus Cu(+)-ATPase CopA by cysteine

CopA, a thermophilic ATPase from Archaeoglobus fulgidus, drives the outward movement of Cu(+) across the cell membrane. Millimolar concentration of Cys dramatically increases ( congruent with 800%) the activity of CopA and other P(IB)-type ATPases (Escherichia coli ZntA and Arabidopsis thaliana HMA2). The high affinity of CopA for metal ( congruent with 1 microM) together with the low Cu(+)-Cys K(D) (<10(-10)M) suggested a multifaceted interaction of Cys with CopA, perhaps acting as a substitute for the Cu(+) chaperone protein present in vivo. To explain the activation by the amino acid and further understand the mechanism of metal delivery to transport ATPases, Cys effects on the turnover and partial reactions of CopA were studied. 2-20 mM Cys accelerates enzyme turnover with little effect on CopA affinity for Cu(+), suggesting a metal independent activation. Furthermore, Cys activates the p-nitrophenyl phosphatase activity of CopA, even though this activity is metal independent. Cys accelerates enzyme phosphorylation and the forward dephosphorylation rates yielding higher steady state phosphoenzyme levels. The faster dephosphorylation would explain the higher enzyme turnover in the presence of Cys. The amino acid has no significant effect on low affinity ATP K(m) suggesting no changes in the E(1)<-->E(2) equilibrium. Characterization of Cu(+) transport into sealed vesicles indicates that Cys acts on the cytoplasmic side of the enzyme. However, the Cys activation of truncated CopA lacking the N-terminal metal binding domain (N-MBD) indicates that activation by Cys is independent of the regulatory N-MBD. These results suggest that Cys is a non-essential activator of CopA, interacting with the cytoplasmic side of the enzyme while this is in an E1 form. Interestingly, these effects also point out that Cu(+) can reach the cytoplasmic opening of the access path into the transmembrane transport sites either as a free metal or a Cu(+)-Cys complex.     

Structure and interactions of the C‐terminal metal binding domain of Archaeoglobus fulgidus CopA

The Cu⁺‐ATPase CopA from Archaeoglobus fulgidus belongs to the P_1B family of the P‐type ATPases. These integral membrane proteins couple the energy of ATP hydrolysis to heavy metal ion translocation across membranes. A defining feature of P_1B‐1‐type ATPases is the presence of soluble metal binding domains at the N‐terminus (N‐MBDs). The N‐MBDs exhibit a conserved ferredoxin‐like fold, similar to that of soluble copper chaperones, and bind metal ions via a conserved CXXC motif. The N‐MBDs enable Cu⁺ regulation of turnover rates apparently through Cu‐sensitive interactions with catalytic domains. A. fulgidus CopA is unusual in that it contains both an N‐terminal MBD and a C‐terminal MBD (C‐MBD). The functional role of the unique C‐MBD has not been established. Here, we report the crystal structure of the apo, oxidized C‐MBD to 2.0 Å resolution. In the structure, two C‐MBD monomers form a domain‐swapped dimer, which has not been observed previously for similar domains. In addition, the interaction of the C‐MBD with the other cytoplasmic domains of CopA, the ATP binding domain (ATPBD) and actuator domain (A‐domain), has been investigated. Interestingly, the C‐MBD interacts specifically with both of these domains, independent of the presence of Cu⁺ or nucleotides. These data reinforce the uniqueness of the C‐MBD and suggest a distinct structural role for the C‐MBD in CopA transport. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

The Archaeoglobus fulgidus D-lactate dehydrogenase is a Zn(2+) flavoprotein

Archaeoglobus fulgidus, a hyperthermophilic, archaeal sulfate reducer, is one of the few organisms that can utilize D-lactate as a sole source for both carbon and electrons. The A. fulgidus open reading frame, AF0394, which is predicted to encode a D-(-)-lactate dehydrogenase (Dld), was cloned, and its product was expressed in Escherichia coli as a fusion with the maltose binding protein (MBP). The 90-kDa MBP-Dld fusion protein was more efficiently expressed in E. coli when coexpressed with the E. coli dnaY gene, encoding the arginyl tRNA for the codons AGA and AGG. When cleaved from the fusion protein by treatment with factor Xa, the recombinant Dld (rDld) has an apparent molecular mass of 50 kDa, similar to that of the native A. fulgidus Dld enzyme. Both the purified MBP-Dld fusion protein and its rDld cleavage fragment have lactate dehydrogenase activities specific for D-lactate, are stable at 80 degrees C, and retain activity after exposure to oxygen. The flavin cofactor FAD, which binds rDld apoprotein with a 1:1 stoichiometry, is essential for activity.     

A novel regulatory metal binding domain is present in the C terminus of Arabidopsis Zn2+-ATPase HMA2

HMA2 is a Zn2+-ATPase from Arabidopsis thaliana. It contributes to the maintenance of metal homeostasis in cells by driving Zn2+ efflux. Distinct from P1B-type ATPases, plant Zn2+-ATPases have long C-terminal sequences rich in Cys and His. Removal of the 244 amino acid C terminus of HMA2 leads to a 43% reduction in enzyme turnover without significant effect on the Zn2+ K(1/2) for enzyme activation. Characterization of the isolated HMA2 C terminus showed that this fragment binds three Zn2+ with high affinity (Kd = 16 +/- 3 nM). Circular dichroism spectral analysis indicated the presence of 8% alpha-helix, 45% beta-sheet, and 48% random coil in the C-terminal peptide with noticeable structural changes upon metal binding (8% alpha-helix, 39% beta-sheet, and 52% random coil). Zn K-edge XAS of Zn-C-MBD in the presence of one equivalent of Zn2+ shows that the average zinc complex formed is composed of three His and one Cys residues. Upon the addition of two extra Zn2+ ions per C-MBD, these appear coordinated primarily by His residues thus, suggesting that the three Zn2+ binding domains might not be identical. Modification of His residues with diethyl pyrocarbonate completely inhibited Zn2+ binding to the C terminus, pointing out the importance of His residues in Zn2+ coordination. In contrast, alkylation of Cys with iodoacetic acid did not prevent Zn2+ binding to the HMA2 C terminus. Zn K-edge XAS of the Cys-alkylated protein was consistent with (N/O)4 coordination of the zinc site, with three of those ligands fitting for His residues. In summary, plant Zn2+-ATPases contain novel metal binding domains in their cytoplasmic C terminus. Structurally distinct from the well characterized N-terminal metal binding domains present in most P1B-type ATPases, they also appear to regulate enzyme turnover rate.     

Autophosphorylation of Archaeoglobus fulgidus Rio2 and crystal structures of its nucleotide-metal ion complexes

The highly conserved, atypical RIO serine protein kinases are found in all organisms, from archaea to man. In yeast, the kinase activity of Rio2 is necessary for the final processing step of maturing the 18S ribosomal rRNA. We have previously shown that the Rio2 protein from Archaeoglobus fulgidus contains both a small kinase domain and an N-terminal winged helix domain. Previously solved structures using crystals soaked in nucleotides and Mg2+ or Mn2+ showed bound nucleotide but no ordered metal ions, leading us to the conclusion that they did not represent an active conformation of the enzyme. To determine the functional form of Rio2, we crystallized it after incubation with ATP or ADP and Mn2+. Co-crystal structures of Rio2-ATP-Mn and Rio2-ADP-Mn were solved at 1.84 and 1.75 angstroms resolution, respectively. The gamma-phosphate of ATP is firmly positioned in a manner clearly distinct from its location in canonical serine kinases. Comparison of the Rio2-ATP-Mn complex with the Rio2 structure with no added nucleotides and with the ADP complex indicates that a flexible portion of the Rio2 molecule becomes ordered through direct interaction between His126 and the gamma-phosphate oxygen of ATP. Phosphopeptide mapping of the autophosphorylation site of Rio2 identified Ser128, within the flexible loop and directly adjacent to the part that becomes ordered in response to ATP, as the target. These results give us further information about the nature of the active site of Rio2 kinase and suggest a mechanism of regulation of its enzymatic activity.     

Crystal structures of a novel ferric reductase from the hyperthermophilic archaeon Archaeoglobus fulgidus and its complex with NADP+

BACKGROUND: Studies performed within the last decade have indicated that microbial reduction of Fe(III) to Fe(II) is a biologically significant process. The ferric reductase (FeR) from Archaeoglobus fulgidus is the first reported archaeal ferric reductase and it catalyzes the flavin-mediated reduction of ferric iron complexes using NAD(P)H as the electron donor. Based on its catalytic activity, the A. fulgidus FeR resembles the bacterial and eukaryotic assimilatory type of ferric reductases. However, the high cellular abundance of the A. fulgidus FeR (approximately 0.75% of the total soluble protein) suggests a catabolic role for this enzyme as the terminal electron acceptor in a ferric iron-based respiratory pathway [1]. RESULTS: The crystal structure of recombinant A. fulgidus FeR containing a bound FMN has been solved at 1.5 A resolution by multiple isomorphous replacement/ anomalous diffraction (MIRAS) phasing methods, and the NADP+- bound complex of FeR was subsequently determined at 1.65 A resolution. FeR consists of a dimer of two identical subunits, although only one subunit has been observed to bind the redox cofactors. Each subunit is organized around a six-stranded antiparallel beta barrel that is homologous to the FMN binding protein from Desulfovibrio vulgaris. This fold has been shown to be related to a circularly permuted version of the flavin binding domain of the ferredoxin reductase superfamily. The A. fulgidus ferric reductase is further distinguished from the ferredoxin reductase superfamily by the absence of a Rossmann fold domain that is used to bind the NAD(P)H. Instead, FeR uses its single domain to provide both the flavin and the NAD(P)H binding sites. Potential binding sites for ferric iron complexes are identified near the cofactor binding sites. CONCLUSIONS: The work described here details the structures of the enzyme-FMN, enzyme-FMN-NADP+, and possibly the enzyme-FMN-iron intermediates that are present during the reaction mechanism. This structural information helps identify roles for specific residues during the reduction of ferric iron complexes by the A. fulgidus FeR.     

Characterization and structure of a Zn2+ and [2Fe-2S]-containing copper chaperone from Archaeoglobus fulgidus

Bacterial CopZ proteins deliver copper to P1B-type Cu+-ATPases that are homologous to the human Wilson and Menkes disease proteins. The genome of the hyperthermophile Archaeoglobus fulgidus encodes a putative CopZ copper chaperone that contains an unusual cysteine-rich N-terminal domain of 130 amino acids in addition to a C-terminal copper binding domain with a conserved CXXC motif. The N-terminal domain (CopZ-NT) is homologous to proteins found only in extremophiles and is the only such protein that is fused to a copper chaperone. Surprisingly, optical, electron paramagnetic resonance, and x-ray absorption spectroscopic data indicate the presence of a [2Fe-2S] cluster in CopZ-NT. The intact CopZ protein binds two copper ions, one in each domain. The 1.8 A resolution crystal structure of CopZ-NT reveals that the [2Fe-2S] cluster is housed within a novel fold and that the protein also binds a zinc ion at a four-cysteine site. CopZ can deliver Cu+ to the A. fulgidus CopA N-terminal metal binding domain and is capable of reducing Cu2+ to Cu+. This unique fusion of a redox-active domain with a CXXC-containing copper chaperone domain is relevant to the evolution of copper homeostatic mechanisms and suggests new models for copper trafficking.     

Ganglioside GM2 modulates the erythrocyte Ca2+-ATPase through its binding to the calmodulin-binding domain and its 'receptor'

We have previously demonstrated that gangliosides were able to modulate the plasma membrane Ca2+-ATPase (PMCA) from porcine brain synaptosomes and porcine erythrocytes [Y. Zhao, X. Fan, F. Yang, X. Zhang, Arch. Biochem. Biophys. 427 (2004) 204-212 and J. Zhang, Y. Zhao, J. Duan, F. Yang, X. Zhang, Arch. Biochem. Biophys. 444 (2005) 1-6]. The results indicated that the PMCA from porcine erythrocytes responded to gangliosides was different from that from synaptosomes, suggesting that the effects of gangliosides on the PMCA are isoform specific. Most interestingly, GM2 activated the PMCA from porcine erythrocytes at lower concentrations, but inhibited it at higher concentrations. In the present study, we found that GD1b, GM1 and GM3 did not affect the calpain digested PMCA from porcine erythrocytes or the intact enzyme in the presence of calmodulin, while GM2 inhibited it. Moreover, a synthetic peptide of 17 amino acid residues corresponding to the 'receptor' of the calmodulin-binding domain of the enzyme interfered with the inhibition of the enzyme by GM2 in competition assays. Taken together, our results suggested that gangliosides GD1b, GM1, GM2 (lower concentrations) and GM3 stimulated the PMCA by the interaction with calmodulin-binding domain, while the interaction of GM2 with the 'receptor' of the calmodulin-binding domain of the enzyme led to the inhibition of the enzyme.     

Novel Zn2+ coordination by the regulatory N-terminus metal binding domain of Arabidopsis thaliana Zn(2+)-ATPase HMA2

Arabidopsis thaliana HMA2 is a Zn2+ transporting P1B-type ATPase required for maintaining plant metal homeostasis. HMA2 and all eukaryote Zn2+-ATPases have unique conserved N- and C-terminal sequences that differentiate them from other P1B-type ATPases. Homology modeling and structural comparison by circular dichroism indicate that the 75 amino acid long HMA2 N-terminus shares the betaalphabetabetaalpha folding present in most P1B-type ATPase N-terminal metal binding domains (N-MBDs). However, the characteristic metal binding sequence CysXXCys is replaced by Cys17CysXXGlu21, a sequence present in all plant Zn2+-ATPases. The isolated HMA2 N-MBD fragment binds a single Zn2+ (Kd 0.18 microM), Cd2+ (Kd 0.27 microM), or, with less affinity, Cu+ (Kd 13 microM). Mutagenesis studies indicate that Cys17, Cys18, and Glu21 participate in Zn2+ and Cd2+ coordination, while Cys17 and Glu21, but not Cys18, are required for Cu+ binding. Interestingly, the Glu21Cys mutation that generates a CysCysXXCys site is unable to bind Zn2+ or Cd2+ but it binds Cu+ with affinity (Kd 1 microM) higher than wild type N-MBD. Truncated HMA2 lacking the N-MBD showed reduced ATPase activity without significant changes in metal binding to transmembrane metal binding sites. Likewise, ATPase activity of HMA2 carrying mutations Cys17Ala, Cys18Ala, and Glu21Ala/Cys was also reduced but showed a metal dependence similar to the wild type enzyme. These observations suggest that plant Zn2+-ATPase N-MBDs have a folding and function similar to Cu+-ATPase N-MBDs. However, the unique Zn2+ coordination via two thiols and a carboxyl group provides selective binding of the activating metals to these regulatory domains. Metal binding through these side chains, although found in different sequences, appears as a common feature of both bacterial and eukaryotic Zn2+-ATPase N-MBDs.     

Structural and functional analysis of the gpsA gene product of Archaeoglobus fulgidus: a glycerol-3-phosphate dehydrogenase with an unusual NADP+ preference

NAD(+)-dependent glycerol-3-phosphate dehydrogenase (G3PDH) is generally absent in archaea, because archaea, unlike eukaryotes and eubacteria, utilize glycerol-1-phosphate instead of glycerol-3-phosphate for the biosynthesis of membrane lipids. Surprisingly, the genome of the hyperthermophilic archaeon Archaeoglobus fulgidus comprises a G3PDH ortholog, gpsA, most likely due to horizontal gene transfer from a eubacterial organism. Biochemical characterization proved G3PDH-like activity of the recombinant gpsA gene product. However, unlike other G3PDHs, the up to 85 degrees C thermostable A. fulgidus G3PDH exerted a 15-fold preference for NADPH over NADH. The A. fulgidus G3PDH bears the hallmarks of adaptation to halotolerance and thermophilicity, because its 1.7-A crystal structure showed a high surface density for negative charges and 10 additional intramolecular salt bridges compared to a mesophilic G3PDH structure. Whereas all amino acid residues required for dihydroxyacetone phosphate binding and reductive catalysis are highly conserved, the binding site for the adenine moiety of the NAD(P) cosubstrate shows a structural variation that reflects the observed NADPH preference, for example, by a putative salt bridge between R49 and the 2'-phosphate.     

The crystal structure of AF1521 a protein from Archaeoglobus fulgidus with homology to the non-histone domain of macroH2A

MacroH2A is an unusual histone H2A variant that has an extensive C-terminal tail that comprises approximately two thirds of the protein. The C-terminal non-histone domain of macroH2A is also found in a number of other proteins and has been termed the macro domain. Here we report the crystal structure to 1.7A of AF1521, a protein consisting of a stand-alone macro domain from Archaeoglobus fulgidus. The structure has a mixed alpha/beta fold that closely resembles the N-terminal DNA binding domain of the Escherichia coli leucine aminopeptidase PepA. The structure also shows some similarity to members of the P-loop family of nucleotide hydrolases.     

N 5,N 10-Methenyltetrahydromethanopterin cyclohydrolase from the extremely thermophilic sulfate reducing Archaeoglobus fulgidus: comparison of its properties with those of the cyclohydrolase from the extremely thermophilic Methanopyrus kandleri

Archaeoglobus fulgidus and Methanopyrus kandleri are both extremely thermophilic Archaea with a growth temperature optimum at 83°C and 98°C, respectively. Both Archaea contain an active N ⁵,N ¹⁰-methenyltetrahydromethanopterin cyclohydrolase. The enzyme from M. kandleri has recently been characterized. We describe here the purification and properties of the enzyme from A. fulgidus.The cyclohydrolase from A. fulgidus was purified 180-fold to apparent homogeneity and its properties were compared with those recently published for the cyclohydrolase from M. kandleri. The two cytoplasmic enzymes were found to have very similar molecular and catalytic properties. They differed, however, significantly with respect of the effect of K₂HPO₄ and of other salts on the activity and the stability. The cyclohydrolase from A. fulgidus required relatively high concentrations of K₂HPO₄ (1 M) for optimal thermostability at 90°C but did not require salts for activity. Vice versa, the enzyme from M. kandleri was dependent on high K₂HPO₄ concentrations (1.5 M) for optimal activity but not for thermostability. Thus the activity and structural stability of the two thermophilic enzymes depend in a completely different way on the concentration of inorganic salts. The molecular basis for these differences are discussed.Abbreviations H₄MPT tetrahydromethanopterin - MFR methanofuran - CH₃–H₄MPT N ⁵-methyl-H₄MPT - CH₂=H₄MPT N ⁵,N ¹⁰-methylene-H₄MPT - CH₂H₄MPT N ⁵,N ¹⁰-methenyl-H₄MPT - CHO–H₄MPT N ⁵ formyl-H₄MPT - CHO-MFR formyl-MFR - cyclohydrolase N ⁵,N ¹⁰-methenyltetrahydromethanopterin cyclohydrolase - MOPS 3-(N-morpholino) propane sulfonic acid - TRICINE N-tris (hydroxymethyl) methyl glycine - 1 U=1 mol/min     

The C2 domain of PKCdelta is a phosphotyrosine binding domain

In eukaryotic cells, the SH2 and PTB domains mediate protein-protein interactions by recognizing phosphotyrosine residues on target proteins. Here we make the unexpected finding that the C2 domain of PKCdelta directly binds to phosphotyrosine peptides in a sequence-specific manner. We provide evidence that this domain mediates PKCdelta interaction with a Src binding glycoprotein, CDCP1. The crystal structure of the PKCdelta C2 domain in complex with an optimal phosphopeptide reveals a new mode of phosphotyrosine binding in which the phosphotyrosine moiety forms a ring-stacking interaction with a histidine residue of the C2 domain. This is also the first example of a protein Ser/Thr kinase containing a domain that binds phosphotyrosine.     

Substitutions at methionine 295 of Archaeoglobus fulgidus ribulose-1,5-bisphosphate carboxylase/oxygenase affect oxygen binding and CO2/O2 specificity

Archaeoglobus fulgidus RbcL2, a form III ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), exhibits unique properties not found in other well studied form I and II Rubiscos, such as optimal activity from 83 to 93 degrees C and an extremely high kcat value (23 s-1). More interestingly, this protein is unusual in that exposure or assay in the presence of oxygen and high levels of CO2 resulted in substantial loss (85-90%) of activity compared with assays performed under strictly anaerobic conditions. Kinetic studies indicated that A. fulgidus RbcL2 possesses an unusually high affinity for oxygen (Ki=5 microM); O2 is a competitive inhibitor with respect to CO2, yet the high affinity for O2 presumably accounts for the inability of high levels of CO2 to prevent inhibition. Comparative bioinformatic analyses of available archaeal Rubisco sequences were conducted to provide clues as to why the RbcL2 protein might possess such a high affinity for oxygen. These analyses suggested the potential importance of several unique residues, as did additional analyses within the context of available form I-III Rubisco structures. One residue unique to archaeal proteins (Met-295) was of particular interest because of its proximity to known active-site residues. Recombinant M295D A. fulgidus Rubisco was less sensitive to oxygen compared with the wild-type enzyme. This residue, along with other potential changes in conserved residues of form III Rubiscos, may provide an understanding as to how Rubisco may have evolved to function in the presence of air.     

Calcium binding to the transmembrane domain of the sarcoplasmic reticulum Ca2+-ATPase: insights from molecular modeling

Sarcoplasmic reticulum Ca(2+)- ATPase pumps Ca(2+) ions from muscle cells to the sarcoplasmic reticulum. Here we use molecular dynamics and electrostatic modeling to investigate structural and dynamical features of key intermediates in the Ca(2+) binding process of the protein. Structural models of the protein (containing either two, one, or no calcium ions in the transmembrane domain) are constructed based on the X-ray structure by Toyoshima et al. (Nature 2000;405:647-655). The protein is embedded in a water/octane bilayer, which mimics the water/membrane environment. Our calculations provide information on the hydration of the two Ca(2+) ions, not emerging from the X-ray structure. Furthermore, they indicate that uptake of the metal ions causes large structural rearrangements of the metal binding sites. In addition, they suggest that the two ions reach their binding sites via two specific pathways. Finally, they allow identification of residues in the outer mouth of the protein that might interact with the Ca(2+) ions during the binding process.