The adenylate kinases from a mesophilic and three thermophilic methanogenic members of the Archaea.

Adenylate kinase has been isolated from four related methanogenic members of the Archaea. For each, the optimum temperature for enzyme activity was similar to the temperature for optimal microbial growth and was approximately 30 degrees C for Methanococcus voltae, 70 degrees C for Methanococcus thermolithotrophicus, 80 degrees C for Methanococcus igneus, and 80 to 90 degrees C for Methanococcus jannaschii. The enzymes were sensitive to the adenylate kinase inhibitor P1, P5-di(adenosine-5')pentaphosphate, a property that was exploited to purify the enzymes by CIBACRON Blue affinity chromatography. The enzymes had an estimated molecular mass (approximately 23 to 25 kDa) in the range common for adenylate kinases. Each of the enzymes had a region of amino acid sequence close to its N terminus that was similar to the canonical P-loop sequence reported for all adenylate kinases. However, the methanogen sequences lacked a lysine residue that has previously been found to be invariant in adenylate kinases, including an enzyme isolated from the archaeon Sulfolobus acidocaldarius. If verified as a nucleotide-binding domain, the methanogen sequence would represent a novel nucleotide-binding motif. There was no correlation between amino acid abundance and the optimal temperature for enzyme activity.     

Structural basis for thermostability and identification of potential active site residues for adenylate kinases from the archaeal genus Methanococcus

Sequence comparisons of highly related archaeal adenylate kinases (AKs) from the mesophilic Methanococcus voltae, the moderate thermophile Methanococcus thermolithotrophicus, and two extreme thermophiles Methanococcus igneus and Methanococcus jannaschii, allow identification of interactions responsible for the large variation in temperatures for optimal catalytic activity and thermostabilities observed for these proteins. The tertiary structures of the methanococcal AKs have been predicted by using homology modeling to further investigate the potential role of specific interactions on thermal stability and activity. The alignments for the methanococcal AKs have been generated by using an energy-based sequence–structure threading procedure against high-resolution crystal structures of eukaryotic, eubacterial, and mitochondrial adenylate and uridylate (UK) kinases. From these alignments, full atomic model structures have been produced using the program MODELLER. The final structures allow identification of potential active site interactions and place a polyproline region near the active site, both of which are unique to the archaeal AKs. Based on these model structures, the additional polar residues present in the thermophiles could contribute four additional salt bridges and a higher negative surface charge. Since only one of these possible salt bridges is interior, they do not appear significantly to the thermal stability. Instead, our model structures indicate that a larger and more hydrophobic core, due to a specific increase in aliphatic amino acid content and aliphatic side chain volume, in the thermophilic AKs is responsible for increased thermal stability. © 1997 Wiley-Liss Inc.     

Site-directed mutagenesis analysis of amino acids critical for activity of the type I signal peptidase of the archaeon Methanococcus voltae

Site-directed mutagenesis studies of the signal peptidase of the methanogenic archaeon Methanococcus voltae identified three conserved residues (Ser52, His122, and Asp148) critical for activity. The requirement for one conserved aspartic acid residue distinguishes the archaeal enzyme from both the Escherichia coli and yeast Sec11 enzymes.     

Analysis of thermal stabilizing interactions in mesophilic and thermophilic adenylate kinases from the genus Methanococcus.

Adenylate kinases (ADKs) from four closely related methanogenic members of the Archaea (the mesophile Methanococcus voltae (MVO), the thermopile Methanococcus thermolithotrophicus (MTH), and the extreme thermopiles Methanococcus igneus (MIG) and Methanococcus jannaschii (MJA)) were characterized for their resistance to thermal denaturation. Despite possessing between 68 and 81% sequence identity, the methanococcal ADKs significantly differed in their stability against thermal denaturation, with melting points ranging from 69 to 103 degrees C. The high sequence identity between these organisms allowed regions of the MVO and MJA ADKs to be exchanged, producing chimeric ADKs with significantly altered thermal stability. Up to a 20 degrees C increase or decrease in stability was achieved for chimeric ADKs, whereas 88% of the original protein sequence was maintained. Based on our previous structural modeling studies, we conclude that cooperative interactions within the hydrophobic protein core play an integral role in determining the differences in structural stability observed between the methanococcal ADKs. From comparisons of the effects of temperature on protein unfolding and optimal enzymatic activity, we also conclude that thermostability and enzymatic temperature optima are influenced differently by molecular modifications and thus that the protein flexibility required for activity and stability, respectively, is not unconditionally linked within the methanococcal ADKs.     

Novel trimeric adenylate kinase from an extremely thermoacidophilic archaeon,Sulfolobus solfataricus: molecular cloning,nucleotide sequencing,expression in Escherichia coli,and characterization of the recombinant enzyme

A gene coding for adenylate kinase was cloned from an extremely thermoacidophilic archaeon Sulfolobus solfataricus. The open reading frame of the sequenced gene consisted of 585 nucleotides coding for a polypeptide of 195 amino acid residues with a calculated molecular weight of 21,325. Although the S. solfataricus adenylate kinase, which belonged to the small variants of the adenylate kinase family, had low sequence identities with bacterial and eukaryotic enzymes, a functionally important glycine-rich region and also two invariant arginine residues were conserved in the sequence of the S. solfataricus enzyme. The recombinant enzyme, overexpressed in Escherichia coli and purified to homogeneity, had high affinity for AMP and high thermal stability, comparable to the extremely thermostable enzyme from a similar archaeon, S. acidocaldarius. Furthermore, gel filtration and sedimentation analyses showed that the S. solfataricus adenylate kinase was a homotrimer in solution, which is a novel subunit structure for nucleoside monophosphate kinases.     

Characterization of Flagellum Gene Families of Methanogenic Archaea and Localization of Novel Flagellum Accessory Proteins

Archaeal flagella are unique motility structures, and the absence of bacterial structural motility genes in the complete genome sequences of flagellated archaeal species suggests that archaeal flagellar biogenesis is likely mediated by novel components. In this study, a conserved flagellar gene family from each of Methanococcus voltae, Methanococcus maripaludis, Methanococcus thermolithotrophicus, and Methanococcus jannaschii has been characterized. These species possess multiple flagellin genes followed immediately by eight known and supposed flagellar accessory genes, flaCDEFGHIJ. Sequence analyses identified a conserved Walker box A motif in the putative nucleotide binding proteins FlaH and FlaI that may be involved in energy production for flagellin secretion or assembly. Northern blotting studies demonstrated that all the species have abundant polycistronic mRNAs corresponding to some of the structural flagellin genes, and in some cases several flagellar accessory genes were shown to be cotranscribed with the flagellin genes. Cloned flagellar accessory genes of M. voltae were successfully overexpressed as His-tagged proteins in Escherichia coli. These recombinant flagellar accessory proteins were affinity purified and used as antigens to raise polyclonal antibodies for localization studies. Immunoblotting of fractionated M. voltae cells demonstrated that FlaC, FlaD, FlaE, FlaH, and FlaI are all present in the cell as membrane-associated proteins but are not major components of isolated flagellar filaments. Interestingly, flaD was found to encode two proteins, each translated from a separate ribosome binding site. These protein expression data indicate for the first time that the putative flagellar accessory genes of M. voltae, and likely those of other archaeal species, do encode proteins that can be detected in the cell.     

Relationship between local structural entropy and protein thermostability

We developed a technique to compute structural entropy directly from protein sequences. We explored the possibility of using structural entropy to identify residues involved in thermal stabilization of various protein families. Examples include methanococcal adenylate kinase, Ribonuclease HI and holocytochrome c(551). Our results show that the positions of the largest structural entropy differences between wild type and mutant usually coincide with the residues relevant to thermostability. We also observed a good linear relationship between the average structural entropy and the melting temperatures for adenylate kinase and its chimeric constructs. To validate this linear relationship, we compiled a large dataset comprised of 1153 sequences and found that most protein families still display similar linear relationships. Our results suggest that the multitude of interactions involved in thermal stabilization may be generalized into the tendency of proteins to maintain local structural conservation. The linear relationship between structural entropy and protein thermostability should be useful in the study of protein thermal stabilization.     

X-ray structures of the maltose-maltodextrin-binding protein of the thermoacidophilic bacterium Alicyclobacillus acidocaldarius provide insight into acid stability of proteins

Maltose-binding proteins act as primary receptors in bacterial transport and chemotaxis systems. We report here crystal structures of the thermoacidostable maltose-binding protein from Alicyclobacillus acidocaldarius, and explore its modes of binding to maltose and maltotriose. Further, comparison with the structures of related proteins from Escherichia coli (a mesophile), and two hyperthermophiles (Pyrococcus furiosus and Thermococcus litoralis) allows an investigation of the basis of thermo- and acidostability in this family of proteins.The thermoacidophilic protein has fewer charged residues than the other three structures, which is compensated by an increase in the number of polar residues. Although the content of acidic and basic residues is approximately equal, more basic residues are exposed on its surface whereas most acidic residues are buried in the interior. As a consequence, this protein has a highly positive surface charge. Fewer salt bridges are buried than in the other MBP structures, but the number exposed on its surface does not appear to be unusual. These features appear to be correlated with the acidostability of the A. acidocaldarius protein rather than its thermostability.An analysis of cavities within the proteins shows that the extremophile proteins are more closely packed than the mesophilic one. Proline content is slightly higher in the hyperthermophiles and thermoacidophiles than in mesophiles, and this amino acid is more common at the second position of beta-turns, properties that are also probably related to thermostability. Secondary structural content does not vary greatly in the different structures, and so is not a contributing factor.     

Characterization of RNase P holoenzymes from Methanococcus jannaschii and Methanothermobacter thermoautotrophicus

The partial purification and basic biochemical characterization of the RNase P holoenzymes of two species of methanogenic Archaea, Methanothermobacter thermoautotrophicus (previously Methanobacterium thermoautotrophicum strain deltaH) and Methanococcus jannaschii, are described. The properties of these enzymes, particularly buoyant density in Cs2SO4 and recent information about the subunit composition of the archaeal enzymes, suggest that RNase P enzymes in Archaea are much more alike than earlier studies in Sulfolobus acidocaldarius and Haloferax volcanii suggested.     

Structure of the Methanococcus jannaschii mevalonate kinase, a member of the GHMP kinase superfamily.

The mevalonate-dependent pathway is used by many organisms to synthesize isopentenyl pyrophosphate, the building block for the biosynthesis of many biologically important compounds, including farnesyl pyrophosphate, dolichol, and many sterols. Mevalonate kinase (MVK) catalyzes a critical phosphoryl transfer step, producing mevalonate 5'-phosphate. The crystal structure of thermostable MVK from Methanococcus jannaschii has been determined at 2.4 A, revealing an overall fold similar to the homoserine kinase from M. jannaschii. In addition, the enzyme shows structural similarity with mevalonate 5-diphosphate decarboxylase and domain IV of elongation factor G. The active site of MVK is in the cleft between its N- and C-terminal domains. Several structural motifs conserved among species, including a phosphate-binding loop, have been found in this cavity. Asp(155), an invariant residue among MVK sequences, is located close to the putative phosphate-binding site and has been assumed to play the catalytic role. Analysis of the MVK model in the context of the other members of the GHMP kinase family offers the opportunity to understand both the mechanism of these enzymes and the structural details that may lead to the design of novel drugs.     

Deciphering the Arginine-binding preferences at the substrate-binding groove of Ser/Thr kinases by computational surface mapping

Protein kinases are key signaling enzymes that catalyze the transfer of γ-phosphate from an ATP molecule to a phospho-accepting residue in the substrate. Unraveling the molecular features that govern the preference of kinases for particular residues flanking the phosphoacceptor is important for understanding kinase specificities toward their substrates and for designing substrate-like peptidic inhibitors. We applied ANCHORSmap, a new fragment-based computational approach for mapping amino acid side chains on protein surfaces, to predict and characterize the preference of kinases toward Arginine binding. We focus on positions P-2 and P-5, commonly occupied by Arginine (Arg) in substrates of basophilic Ser/Thr kinases. The method accurately identified all the P-2/P-5 Arg binding sites previously determined by X-ray crystallography and produced Arg preferences that corresponded to those experimentally found by peptide arrays. The predicted Arg-binding positions and their associated pockets were analyzed in terms of shape, physicochemical properties, amino acid composition, and in-silico mutagenesis, providing structural rationalization for previously unexplained trends in kinase preferences toward Arg moieties. This methodology sheds light on several kinases that were described in the literature as having non-trivial preferences for Arg, and provides some surprising departures from the prevailing views regarding residues that determine kinase specificity toward Arg. In particular, we found that the preference for a P-5 Arg is not necessarily governed by the 170/230 acidic pair, as was previously assumed, but by several different pairs of acidic residues, selected from positions 133, 169, and 230 (PKA numbering). The acidic residue at position 230 serves as a pivotal element in recognizing Arg from both the P-2 and P-5 positions.     

Structural interpretation of site-directed mutagenesis and specificity of the catalytic subunit of protein kinase CK2 using comparative modelling

The catalytic subunit of protein kinase casein kinase 2 (CK2alpha), which has specificity for both ATP and GTP, shows significant amino acid sequence similarity to the cyclin-dependent kinase 2 (CDK2). We constructed site-directed mutants of CK2alpha and used a three-dimensional model to investigate the basis for the dual specificity. Introduction of Phe and Gly at positions 50 and 51, in order to restore the pattern of the glycine-rich motif, did not seriously affect the specificity for ATP or GTP. We show that the dual specificity probably originates from the loop situated around the position His115 to Asp120 (HVNNTD). The insertion of a residue in this loop in CK2 alpha subunits, compared with CDK2 and other kinases, might orient the backbone to interact with the base A and G; this insertion is conserved in all known CK2alpha. The mutant deltaN118, the design of which was based on the modelling, showed reduced affinity for GTP as predicted from the model. Other mutants were intended to probe the integrity of the catalytic loop, alter the polarity of a buried residue and explore the importance of the carboxy terminus. Introduction of Arg to replace Asn189, which is mapped on the activation loop, results in a mutant with decreased k(cat), possibly as a result of disruption of the interaction between this residue and basic residues in the vicinity. Truncation at position 331 eliminates the last 60 residues of the alpha subunit and this mutant has a reduced catalytic efficiency compared with the wild-type. Catalytic efficiency is restored in the truncation mutant by the replacement of a potentially buried Glu at position 252 by Lys, probably owing to a higher stability resulting from the formation of a salt bridge between Lys252 and Asp208.     

Roles of distal arginine in activity and stability of Coprinus cinereus peroxidase elucidated by kinetic and NMR analysis of the Arg51Gln, -Asn, -Leu, and -Lys mutants

In heme peroxidases, a distal His residue plays an essential role in the initial two electron oxidation of resting state enzyme to compound I by hydrogen peroxide. A distal Arg residue assists in this process. The contributions of the charge, H-bonding capacity, size, and mobility of this Arg residue to Coprinus cinereus peroxidase (CIP) reactivity and stability have been examined by substituting Arg51 with Gln (retains H-bond donor at N epsilon position), Asn (small size, H-bond donor and acceptor), Leu (similar to Asn, but hydrophobic), and Lys (charge and H-bond donor, but at N zeta position). UV-visible spectroscopy was used to monitor pH-linked heme changes, compound I formation and reduction, fluoride binding, and thermostability. (1)H NMR spectroscopy enabled heme pocket differences in both resting and cyanide-ligated states of the enzymes to be evaluated and compared with wild-type CIP. We found that the H-bonding capacity of distal Arg is key to fast compound I formation and ligand binding to heme, whereas charge is important for lowering the pK(a) of distal His and for the binding and stabilisation of anionic ligands at heme iron. The properties of the distal Arg residue in CIP, cytochrome c peroxidase (CCP) and horseradish peroxidase (HRP) differ significantly in their pH induced transitions and dynamics.     

Biochemical and X-ray crystallographic studies on shikimate kinase: the important structural role of the P-loop lysine

Shikimate kinase, despite low sequence identity, has been shown to be structurally a member of the nucleoside monophosphate (NMP) kinase family, which includes adenylate kinase. In this paper we have explored the roles of residues in the P-loop of shikimate kinase, which forms the binding site for nucleotides and is one of the most conserved structural features in proteins. In common with many members of the P-loop family, shikimate kinase contains a cysteine residue 2 amino acids upstream of the essential lysine residue; the side chains of these residues are shown to form an ion pair. The C13S mutant of shikimate kinase was found to be enzymatically active, whereas the K15M mutant was inactive. However, the latter mutant had both increased thermostability and affinity for ATP when compared to the wild-type enzyme. The structure of the K15M mutant protein has been determined at 1.8 A, and shows that the organization of the P-loop and flanking regions is heavily disturbed. This indicates that, besides its role in catalysis, the P-loop lysine also has an important structural role. The structure of the K15M mutant also reveals that the formation of an additional arginine/aspartate ion pair is the most likely reason for its increased thermostability. From studies of ligand binding it appears that, like adenylate kinase, shikimate kinase binds substrates randomly and in a synergistic fashion, indicating that the two enzymes have similar catalytic mechanisms.     

Use of a potential of mean force to analyze free energy contributions in protein folding.

A method for calculation of the free energy of residues as a function of residue burial is proposed. The method is based on the potential of mean force, with a reaction coordinate expressed by residue burial. Residue burials are calculated from high-resolution protein structures. The largest individual contributions to the free energy of a residue are found to be due to the hydrophobic interactions of the nonpolar atoms, interactions of the main chain polar atoms, and interactions of the charged groups of residues Arg and Lys. The contribution to the free energy of folding due to the uncharged side chain polar atoms is small. The contribution to the free energy of folding due to the main chain polar atoms is favorable for partially buried residues and less favorable or unfavorable for fully buried residues. Comparison of the accessible surface areas of proteins and model spheres shows that proteins deviate considerably from a spherical shape and that the deviations increase with the size of a protein. The implications of these results for protein folding are also discussed.     

Phosphotransferase and substrate binding mechanism of the cAMP-dependent protein kinase catalytic subunit from porcine heart as deduced from the 2.0 A structure of the complex with Mn2+ adenylyl imidodiphosphate and inhibitor peptide PKI(5-24).

The crystal structure of the porcine heart catalytic subunit of cAMP-dependent protein kinase in a ternary complex with the MgATP analogue MnAMP-PNP and a pseudosubstrate inhibitor peptide, PKI(5-24), has been solved at 2.0 A resolution from monoclinic crystals of the catalytic subunit isoform CA. The refinement is presently at an R factor of 0.194 and the active site of the molecule is well defined. The glycine-rich phosphate anchor of the nucleotide binding fold motif of the protein kinase is a beta ribbon acting as a flap with conformational flexibility over the triphosphate group. The glycines seem to be conserved to avoid steric clash with ATP. The known synergistic effects of substrate binding can be explained by hydrogen bonds present only in the ternary complex. Implications for the kinetic scheme of binding order are discussed. The structure is assumed to represent a phosphotransfer competent conformation. The invariant conserved residue Asp166 is proposed to be the catalytic base and Lys168 to stabilize the transition state. In some tyrosine kinases Lys168 is functionally replaced by an Arg displaced by two residues in the primary sequence, suggesting invariance in three-dimensional space. The structure supports an in-line transfer with a pentacoordinate transition state at the phosphorus with very few nuclear movements.     

Sulfometuron methyl-sensitive and -resistant acetolactate synthases of the archaebacteria Methanococcus spp.

The herbicide sulfometuron methyl (SM) inhibited growth of some methanococci. Of 28 strains tested, the growth of 7 was completely inhibited by 0.55 mM SM. Growth of an additional 14 strains was partially inhibited, and the growth of 7 strains was unaffected by this concentration of SM. In some cases, the branched-chain amino acids protected growth. Growth inhibition was correlated with the Ki for SM of acetolactate synthase (ALS). For the enzymes from bacteria representative of the sensitive, partially resistant, and resistant methanococci (Methanococcus aeolicus, Methanococcus maripaludis, and Methanococcus voltae, respectively), the Ki for SM was 0.0012, 0.34, and greater than 1.0 mM, respectively. Inhibition was uncompetitive with respect to pyruvate. Based on these observations, ALS appeared to be the major if not the sole site of action of SM in the methanococci. The sensitivity of the ALS from these three methanococci to feedback inhibition by branched-chain amino acids was also quite different. Although all three were sensitive to feedback inhibition by valine, the Ki varied 20-fold, from 0.01 to 0.22 mM. Moreover, only the ALS from M. maripaludis was sensitive to inhibition by leucine, and the Ki was 1.8 mM. The Ki for isoleucine for the ALS from both M. maripaludis and M. voltae was about 0.1 mM. The ALS from M. aeolicus was not inhibited by isoleucine. In other respects, the ALSs from the methanococci were very similar. After dialysis, thiamine pyrophosphate but not FAD and Mg2+ was required for maximal activity, and they were all rapidly inactivated by oxygen. Although the methanococcal ALSs exhibited diverse properties, the range of catalytic and regulatory properties closely resembled those of the eubacterial enzymes.     

Comparative study of the phenylalanyl-tRNA synthetases from Escherichia coli and Thermus thermophlus by the tritium topography method

A comparative study of thermostability and aminoacid composition of the phenylalanyl-tRNA synthetases from E. coli and Thermus thermophilus HB8 has been carried out. Compared with the mesophilic enzyme, a considerable increase of Pro, Leu, Phe, Arg and decrease of Asx, Ile, Ser, Thr and Lys content have been revealed in the thermophilic protein. Using tritium topography, Pro, (Leu + Ile) and Gly were found to be the most accessible on the surfaces of both the enzymes. In the E. coli enzyme, Thr residues were also easy to access while on the surface of the thermophilic enzyme there were more Arg residues. The quantitative assay of the surface compositions revealed the increased exposure of the (Leu + Ile) residues on the thermophilic protein as well as of the charged Asx and Arg residues. A possible correlation of the observed effects with thermostability is discussed.     

Identification of basic residues involved in activation and calmodulin binding of rabbit smooth muscle myosin light chain kinase.

It is postulated that basic residues in the regulatory region of myosin light chain kinase are important for conferring autoinhibition by binding to the catalytic core. To investigate this proposal, 10 basic amino acids within the regulatory region of rabbit smooth muscle myosin light chain kinase (Lys961-Lys979) were replaced either singularly or in combination with acidic or nonpolar residues by site-directed mutagenesis. All active mutant kinases were dependent on Ca2+/calmodulin for catalytic activity. None of the mutants was active in the absence of Ca2+/calmodulin, suggesting that the autoinhibitory region has not been defined completely. Charge reversal mutants at Arg974, Arg975, and Lys976 resulted in loss of high affinity binding of calmodulin and increased the concentration of calmodulin required for half-maximal activation (KCaM). The charge reversal mutant at Lys979 also increased KCaM but to a lesser extent. Charge reversal mutants at Lys965 and Arg967 resulted in an inactive myosin light chain kinase that could not be proteolytically activated. When these residues were mutated to Ala, the expressed kinase was dependent upon Ca2+/calmodulin for activity and exhibited a decrease in KCaM. Charge reversal mutants in Lys961 and Lys962 also had decreased KCaM values. These basic residues amino-terminal of the calmodulin binding domain may play an important role in the activation of the kinase.     

Crystal structure of a trimeric archaeal adenylate kinase from the mesophile Methanococcus maripaludis with an unusually broad functional range and thermal stability

The structure of the trimeric adenylate kinase from the Archaebacteria Methanococcus mariplaludis (AK_MAR) has been solved to 2.5‐Å resolution and the temperature dependent stability and kinetics of the enzyme measured. The K_M and V_max of AK_MAR exhibit only modest temperature dependence from 30°–60°C. Although M. mariplaludis is a mesophile with a maximum growth temperature of 43°C, AK_MAR has a very broad functional range and stability (T_m = 74.0°C) that are more consistent with a thermophilic enzyme with high thermostability and exceptional activity over a wide range of temperatures, suggesting that this microbe may have only recently invaded a mesophilic niche and has yet to fully adapt. A comparison of the Local Structural Entropy (LSE) for AK_MAR to the related adenylate kinases from the mesophile Methanococcus voltae and thermophile Methanococcus thermolithotrophicus show that changes in LSE are able to fully account for the intermediate stability of AK_MAR and highlights a general mechanism for protein adaptation in this class of enzymes. Proteins 2010. © 2009 Wiley‐Liss, Inc.