Crystal structures of aconitase with isocitrate and nitroisocitrate bound.

The crystal structures of mitochondrial aconitase with isocitrate and nitroisocitrate bound have been solved and refined to R factors of 0.179 and 0.161, respectively, for all observed data in the range 8.0-2.1 A. Porcine heart enzyme was used for determining the structure with isocitrate bound. The presence of isocitrate in the crystals was corroborated by M?ssbauer spectroscopy. Bovine heart enzyme was used for determining the structure with the reaction intermediate analogue nitroisocitrate bound. The inhibitor binds to the enzyme in a manner virtually identical to that of isocitrate. Both compounds bind to the unique Fe atom of the [4Fe-4S] cluster via a hydroxyl oxygen and one carboxyl oxygen. A H2O molecule is also bound, making Fe six-coordinate. The unique Fe is pulled away approximately 0.2 A from the corner of the cubane compared to the position it would occupy in a symmetrically ligated [4Fe-4S] cluster. At least 23 residues from all four domains of aconitase contribute to the active site. These residues participate in substrate recognition (Arg447, Arg452, Arg580, Arg644, Gln72, Ser166, Ser643), cluster ligation and interaction (Cys358, Cys421, Cys424, Asn258, Asn446), and hydrogen bonds supporting active site side chains (Ala74, Asp568, Ser571, Thr567). Residues implicated in catalysis are Ser642 and three histidine-carboxylate pairs (Asp100-His101, Asp165-His147, Glu262-His167). The base necessary for proton abstraction from C beta of isocitrate appears to be Ser642; the O gamma atom is proximal to the calculated hydrogen position, while the environment of O gamma suggests stabilization of an alkoxide (an oxyanion hole formed by the amide and side chain of Arg644). The histidine-carboxylate pairs appear to be required for proton transfer reactions involving two oxygens bound to Fe, one derived from solvent (bound H2O) and one derived from substrate hydroxyl. Each oxygen is in contact with a histidine, and both are in contact with the side chain of Asp165, which bridges the two sites on the six-coordinate Fe.     

Mutational analysis of active site residues in pig heart aconitase.

A cDNA encoding mature porcine heart aconitase was over-expressed in Escherichia coli under the control of a phage T7 promoter. Recombinant aconitase purified from E. coli was identical to the enzyme from pig and beef heart in size, [3Fe-4S] and [4Fe-4S] cluster structure and enzymatic activity. Nine amino acid residues in close proximity to the Fe-S cluster and bound substrate (Lauble, H., Kennedy, M.C., Beinert, H., and Stout, C.D. (1992) Biochemistry, in press) were replaced by site-directed mutagenesis. Fe-S cluster environment as indicated by the EPR spectrum, tight binding of substrate, and enzymatic activity were compared for the mutant and wild type enzymes. Significant perturbations were detected for all of the mutant enzymes. Replacements for Asp100, His101, Asp165, Arg580, and Ser642 result in a 10(3)-10(5)-fold drop in activity, which suggests that these residues are involved in critical aspects of the reaction. Arg580 appears to be a key residue for substrate binding, as shown by a 30-fold increased Km and loss of tight substrate binding. Results of mutagenesis support the interpretation of the x-ray model, namely that Asp100 and His101 form an ion pair for elimination of the substrate hydroxyl and Ser642 may function as a general base for proton abstraction from citrate or isocitrate in the dehydration step and protonation of cis-aconitate in the hydration step. Asp165 appears to play a critical role in the interaction of Fea with substrate.     

The amino acid sequence of human chorionic gonadotropin. The alpha subunit and beta subunit.

The amino acid sequences of both the alpha and beta subunits of human chorionic gonadotropin have been determined. The amino acid sequence of the alpha subunit is: Ala - Asp - Val - Gln - Asp - Cys - Pro - Glu - Cys-10 - Thr - Leu - Gln - Asp - Pro - Phe - Ser - Gln-20 - Pro - Gly - Ala - Pro - Ile - Leu - Gln - Cys - Met - Gly-30 - Cys - Cys - Phe - Ser - Arg - Ala - Tyr - Pro - Thr - Pro-40 - Leu - Arg - Ser - Lys - Lys - Thr - Met - Leu - Val - Gln-50 - Lys - Asn - Val - Thr - Ser - Glu - Ser - Thr - Cys - Cys-60 - Val - Ala - Lys - Ser - Thr - Asn - Arg - Val - Thr - Val-70 - Met - Gly - Gly - Phe - Lys - Val - Glu - Asn - His - Thr-80 - Ala - Cys - His - Cys - Ser - Thr - Cys - Tyr - Tyr - His-90 - Lys - Ser. Oligosaccharide side chains are attached at residues 52 and 78. In the preparations studied approximately 10 and 30% of the chains lack the initial 2 and 3 NH2-terminal residues, respectively. This sequence is almost identical with that of human luteinizing hormone (Sairam, M. R., Papkoff, H., and Li, C. H. (1972) Biochem. Biophys. Res. Commun. 48, 530-537). The amino acid sequence of the beta subunit is: Ser - Lys - Glu - Pro - Leu - Arg - Pro - Arg - Cys - Arg-10 - Pro - Ile - Asn - Ala - Thr - Leu - Ala - Val - Glu - Lys-20 - Glu - Gly - Cys - Pro - Val - Cys - Ile - Thr - Val - Asn-30 - Thr - Thr - Ile - Cys - Ala - Gly - Tyr - Cys - Pro - Thr-40 - Met - Thr - Arg - Val - Leu - Gln - Gly - Val - Leu - Pro-50 - Ala - Leu - Pro - Gin - Val - Val - Cys - Asn - Tyr - Arg-60 - Asp - Val - Arg - Phe - Glu - Ser - Ile - Arg - Leu - Pro-70 - Gly - Cys - Pro - Arg - Gly - Val - Asn - Pro - Val - Val-80 - Ser - Tyr - Ala - Val - Ala - Leu - Ser - Cys - Gln - Cys-90 - Ala - Leu - Cys - Arg - Arg - Ser - Thr - Thr - Asp - Cys-100 - Gly - Gly - Pro - Lys - Asp - His - Pro - Leu - Thr - Cys-110 - Asp - Asp - Pro - Arg - Phe - Gln - Asp - Ser - Ser - Ser - Ser - Lys - Ala - Pro - Pro - Pro - Ser - Leu - Pro - Ser-130 - Pro - Ser - Arg - Leu - Pro - Gly - Pro - Ser - Asp - Thr-140 - Pro - Ile - Leu - Pro - Gln. Oligosaccharide side chains are found at residues 13, 30, 121, 127, 132, and 138. The proteolytic enzyme, thrombin, which appears to cleave a limited number of arginyl bonds, proved helpful in the determination of the beta sequence.     

Identification of structural and catalytic classes of highly conserved amino acid residues in lysine 2,3-aminomutase

Lysine 2,3-aminomutase (LAM) from Clostridium subterminale SB4 catalyzes the interconversion of (S)-lysine and (S)-beta-lysine by a radical mechanism involving coenzymatic actions of S-adenosylmethionine (SAM), a [4Fe-4S] cluster, and pyridoxal 5'-phosphate (PLP). The enzyme contains a number of conserved acidic residues and a cysteine- and arginine-rich motif, which binds iron and sulfide in the [4Fe-4S] cluster. The results of activity and iron, sulfide, and PLP analysis of variants resulting from site-specific mutations of the conserved acidic residues and the arginine residues in the iron-sulfide binding motif indicate two classes of conserved residues of each type. Mutation of the conserved residues Arg134, Asp293, and Asp330 abolishes all enzymatic activity. On the basis of the X-ray crystal structure, these residues bind the epsilon-aminium and alpha-carboxylate groups of (S)-lysine. However, among these residues, only Asp293 appears to be important for stabilizing the [4Fe-4S] cluster. Members of a second group of conserved residues appear to stabilize the structure of LAM. Mutations of arginine 130, 135, and 136 and acidic residues Glu86, Asp165, Glu236, and Asp172 dramatically decrease iron and sulfide contents in the purified variants. Mutation of Asp96 significantly decreases iron and sulfide content. Arg130 or Asp172 variants display no detectable activity, whereas variants mutated at the other positions display low to very low activities. Structural roles are assigned to this latter class of conserved amino acids. In particular, a network of hydrogen bonded interactions of Arg130, Glu86, Arg135, and the main chain carbonyl groups of Cys132 and Leu55 appears to stabilize the [4Fe-4S] cluster.     

Mechanism of the phosphatase component of Clostridium thermocellum polynucleotide kinase-phosphatase

Polynucleotide kinase-phosphatase (Pnkp) from Clostridium thermocellum catalyzes ATP-dependent phosphorylation of 5'-OH termini of DNA or RNA polynucleotides and Ni(2+)/Mn(2+)-dependent dephosphorylation of 2',3' cyclic phosphate, 2'-phosphate, and 3'-phosphate ribonucleotides. CthPnkp is an 870-amino-acid polypeptide composed of three domains: an N-terminal module similar to bacteriophage T4 polynucleotide kinase, a central module that resembles the dinuclear metallo-phosphoesterase superfamily, and a C-terminal ligase-like adenylyltransferase domain. Here we conducted a mutational analysis of CthPnkp that identified 11 residues required for Ni(2+)-dependent phosphatase activity with 2'-AMP and 3'-AMP. Eight of the 11 CthPnkp side chains were also required for Ni(2+)-dependent hydrolysis of p-nitrophenyl phosphate. The ensemble of essential side chains includes the conserved counterparts (Asp187, His189, Asp233, Arg237, Asn263, His264, His323, His376, and Asp392 in CthPnkp) of all of the amino acids that form the dinuclear metal-binding site and the phosphate-binding site of bacteriophage lambda phosphatase. Three residues (Asp236, His264, and Arg237) required for activity with 2'-AMP or 3'-AMP were dispensable for Ni(2+)-dependent hydrolysis of p-nitrophenyl phosphate. Our findings, together with available structural information, provide fresh insights to the metallophosphoesterase mechanism, including the roles of His264 and Asp236 in proton donation to the leaving group. Deletion analysis defined an autonomous phosphatase domain, CthPnkp-(171-424).     

The CRISPR-associated Cas4 protein Pcal_0546 from Pyrobaculum calidifontis contains a [2Fe-2S] cluster: crystal structure and nuclease activity

Cas4 nucleases constitute a core family of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) associated proteins, but little is known about their structure and activity. Here we report the crystal structure of the Cas4 protein Pcal_0546 from Pyrobaculum calidifontis, which revealed a monomeric protein with a RecB-like fold and one [2Fe-2S] cluster coordinated by four conserved Cys residues. Pcal_0546 exhibits metal-dependent 5′ to 3′ exonuclease activity against ssDNA substrates, whereas the Cas4 protein SSO1391 from Sulfolobus solfataricus can cleave ssDNA in both the 5′ to 3′ and 3′ to 5′ directions. The active site of Pcal_0546 contains a bound metal ion coordinated by the side chains of Asp123, Glu136, His146, and the main chain carbonyl of Ile137. Site-directed mutagenesis of Pcal_0546 and SSO1391 revealed that the residues of RecB motifs II, III and QhXXY are critical for nuclease activity, whereas mutations of the conserved Cys residues resulted in a loss of the iron-sulfur cluster, but had no effect on DNA cleavage. Our results revealed the biochemical diversity of Cas4 nucleases, which can have different oligomeric states, contain [4Fe-4S] or [2Fe-2S] clusters, and cleave single stranded DNA in different directions producing single-stranded DNA overhangs, which are potential intermediates for the synthesis of new CRISPR spacers.     

Express analysis of protein amino acid sequences. Primary structure of Penicillium chrysogenum 152A guanyl-specific ribonuclease

The complete amino acid sequence of Penicillium chrysogenum 152A guanyl-specific RNase has been established using automated Edman degradation of two non-fractionated peptide mixtures produced by tryptic and staphylococcal protease digests of the protein. The RNase contains 102 amino acid residues: His2, Arg3, Asp7, Asn8, Thr5, Ser11, Glu4, Gln2, Pro4, Gly11, Ala13, Cys4, Val8, Ile3, Leu3, Tyr9, Phe5 (Mr 10 747).     

NifS-mediated assembly of [4Fe-4S] clusters in the N- and C-terminal domains of the NifU scaffold protein

NifU is a homodimeric modular protein comprising N- and C-terminal domains and a central domain with a redox-active [2Fe-2S](2+,+) cluster. It plays a crucial role as a scaffold protein for the assembly of the Fe-S clusters required for the maturation of nif-specific Fe-S proteins. In this work, the time course and products of in vitro NifS-mediated iron-sulfur cluster assembly on full-length NifU and truncated forms involving only the N-terminal domain or the central and C-terminal domains have been investigated using UV-vis absorption and M?ssbauer spectroscopies, coupled with analytical studies. The results demonstrate sequential assembly of labile [2Fe-2S](2+) and [4Fe-4S](2+) clusters in the U-type N-terminal scaffolding domain and the assembly of [4Fe-4S](2+) clusters in the Nfu-type C-terminal scaffolding domain. Both scaffolding domains of NifU are shown to be competent for in vitro maturation of nitrogenase component proteins, as evidenced by rapid transfer of [4Fe-4S](2+) clusters preassembled on either the N- or C-terminal domains to the apo nitrogenase Fe protein. Mutagenesis studies indicate that a conserved aspartate (Asp37) plays a critical role in mediating cluster transfer. The assembly and transfer of clusters on NifU are compared with results reported for U- and Nfu-type scaffold proteins, and the need for two functional Fe-S cluster scaffolding domains on NifU is discussed.     

Characterization of the iron- and 2-oxoglutarate-binding sites of human prolyl 4-hydroxylase.

Prolyl 4-hydroxylase (EC 1.14.11.2), an alpha2beta2 tetramer, catalyzes the formation of 4-hydroxyproline in collagens. We converted 16 residues in the human alpha subunit individually to other amino acids, and expressed the mutant polypeptides together with the wild-type beta subunit in insect cells. Asp414Ala and Asp414Asn inactivated the enzyme completely, whereas Asp414Glu increased the K(m) for Fe2+ 15-fold and that for 2-oxoglutarate 5-fold. His412Glu, His483Glu and His483Arg inactivated the tetramer completely, as did Lys493Ala and Lys493His, whereas Lys493Arg increased the K(m) for 2-oxoglutarate 15-fold. His501Arg, His501Lys, His501Asn and His501Gln reduced the enzyme activity by 85-95%; all these mutations increased the K(m) for 2-oxoglutarate 2- to 3-fold and enhanced the rate of uncoupled decarboxylation of 2-oxoglutarate as a percentage of the rate of the complete reaction up to 12-fold. These and other data indicate that His412, Asp414 and His483 provide the three ligands required for the binding of Fe2+ to a catalytic site, while Lys493 provides the residue required for binding of the C-5 carboxyl group of 2-oxoglutarate. His501 is an additional critical residue at the catalytic site, probably being involved in both the binding of the C-1 carboxyl group of 2-oxoglutarate and the decarboxylation of this cosubstrate.     

Aspzincin, a family of metalloendopeptidases with a new zinc-binding motif. Identification of new zinc-binding sites (His(128), His(132), and Asp(164)) and three catalytically crucial residues (Glu(129), Asp(143), and Tyr(106)) of deuterolysin from Aspergillus oryzae by site-directed mutagenesis.

Deuterolysin (EC 3.4.24.39; formerly designated as neutral proteinase II) from Aspergillus oryzae, which contains 1 g atom of zinc/mol of enzyme, is a single chain of 177 amino acid residues, includes three disulfide bonds, and has a molecular mass of 19,018 Da. Active-site determination of the recombinant enzyme expressed in Escherichia coli was performed by site-directed mutagenesis. Substitutions of His(128) and His(132) with Arg, of Glu(129) with Gln or Asp, of Asp(143) with Asn or Glu, of Asp(164) with Asn, and of Tyr(106) with Phe resulted in almost complete loss of the activity of the mutant enzymes. It can be concluded that His(128), His(132), and Asp(164) provide the Zn(2+) ligands of the enzyme according to a (65)Zn binding assay. Based on site-directed mutagenesis experiments, it was demonstrated that the three essential amino acid residues Glu(129), Asp(143), and Tyr(106) are catalytically crucial residues in the enzyme. Glu(129) may be implicated in a central role in the catalytic function. We conclude that deuterolysin is a member of a family of Zn(2+) metalloendopeptidases with a new zinc-binding motif, aspzincin, defined by the "HEXXH + D" motif and an aspartic acid as the third zinc ligand.     

Geometry of interaction of the histidine ring with other planar and basic residues

Among the aromatic residues in protein structures, histidine (His) is unique, as it can exist in the neutral or positively charged form at the physiological pH. As such, it can interact with other aromatic residues as well as form hydrogen bonds with polar and charged (both negative and positive) residues. We have analyzed the geometry of interaction of His residues with nine other planar side chains containing aromatic (residues Phe, Tyr, Trp, and His), carboxylate (Asp and Glu), carboxamide (Asn and Gln) and guanidinium (Arg) groups in 432 polypeptide chains. With the exception of the aspartic (Asp) and glutamic (Glu) acid side-chains, all other residues prefer to interact in a face-to-face or offset-face-stacked orientation with the His ring. Such a geometry is different from the edge-to-face relative orientation normally associated with the aromatic-aromatic interaction. His-His pair prefers to interact in a face-to-face orientation; however, when both the residues bind the same metal ion, the interplanar angle is close to 90 degrees. The occurrence of different interactions (including the nonconventional N-H...pi and C-H...pi hydrogen bonds) have been correlated with the relative orientations between the interacting residues. Several structural motifs, mostly involved in binding metal ions, have been identified by considering the cases where His residues are in contact with four other planar moieties. About 10% of His residues used here are also found in sequence patterns in PROSITE database. There are examples of the amino end of the Lys side chain interacting with His residues in such a way that it is located on an arc around a ring nitrogen atom.     

Structural insight into the mechanism of epothilone A bound to beta-tubulin and its mutants at Arg282Gln and Thr274Ile

Epothilone A (EpoA) is under investigation as an antitumor agent. To provide better understanding of the activity of EpoA against cancers, by theoretical studies such as using docking method, molecular dynamics simulation and density functional theory calculations, we identify several key residues located on β-tubulin as the active sites to establish an active pocket responsible for interaction with EpoA. Eight residues (Arg276, Asp224, Asp26, His227, Glu27, Glu22, Thr274, and Met363) are identified as the active sites to form the active pocket on β-tubulin. The interaction energy is predicted to be -121.3?kJ/mol between EpoA and β-tubulin. In the mutant of β-tubulin at Thr274Ile, three residues (Arg359, Glu27, and His227) are identified as the active sites for the binding of EpoA. In the mutant of β-tubulin at Arg282Gln, three residues (Arg276, Lys19, and His227) serve as the active sites. The interaction energy is reduced to -77.2?kJ/mol between EpoA and Arg282Gln mutant and to -50.2?kJ/mol between EpoA and Thr274Ile mutant. The strong interaction with β-tubulin is significant to EpoA's activity against cancer cells. When β-tubulin is mutated either at Arg282Gln or at Thr274Ile, the decreased strength of interaction explains the activity reduced for EpoA. Therefore, this work shows that the structural basis of the active pocket plays an important role in regulating the activity for EpoA with a Taxol-like mechanism of action to be promoted as an antitumor agent.     

The primary structure of the β-subunit of protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa

The complete amino acid sequence of the β-subunit of protocatechuate 3,4-dioxygenase was determined. The β-subunit contained four methionine residues. Thus, five peptides were obtained after cleavage of the carboxymethylated β-subunit with cyanogen bromide, and were isolated on Sephadex G-75 column chromatography. The amino acid sequences of the cyanogen bromide peptides were established by characterization of the peptides obtained after digestion with trypsin, chymotrypsin, thermolysin, or Staphylococcus aureus protease. The major sequencing techniques used were automated and manual Edman degradations. The five cyanogen bromide peptides were aligned by means of the amino acid sequences of the peptides containing methionine purified from the tryptic hydrolysate of the carboxymethylated β-subunit. The amino acid sequence of all the 238 residues was as follows: ProAlaGlnAspAsnSerArgPheValIleArgAsp ArgAsnTrpHis ProLysAlaLeuThrPro-Asp — TyrLysThrSerIleAlaArg SerProArgGlnAla LeuValSerIleProGlnSer — IleSerGluThrThrGly ProAsnPheSerHisLeu GlyPheGlyAlaHisAsp-His — AspLeuLeuLeuAsnPheAsn AsnGlyGlyLeu ProIleGlyGluArgIle-Ile — ValAlaGlyArgValValAsp GlnTyrGlyLysPro ValProAsnThrLeuValGluMet — TrpGlnAlaAsnAla GlyGlyArgTyrArg HisLysAsnAspArgTyrLeuAlaPro — LeuAspProAsn PheGlyGlyValGly ArgCysLeuThrAspSerAspGlyTyrTyr — SerPheArg ThrIleLysProGlyPro TyrProTrpArgAsnGlyProAsnAsp — TrpArgProAla HisIleHisPheGlyIle SerGlyProSerIleAlaThr-Lys — LeuIleThrGlnLeuTyr PheGluGlyAspPro LeuIleProMetCysProIleVal — LysSerIleAlaAsn ProGluAlaValGlnGln LeuIleAlaLysLeuAspMetAsnAsn — AlaAsnProMet AsnCysLeuAlaTyr ArgPheAspIleValLeuArgGlyGlnArgLysThrHis PheGluAsnCys. The sequence published earlier in summary form (Iwaki et al., 1979, J. Biochem.86, 1159–1162) contained a few errors which are pointed out in this paper.     

Steered molecular dynamics simulation of the binding of the β2 and β3 regions in domain-swapped human cystatin C dimer

The crystal structure of the human cystatin C (hCC) dimer revealed that a stable twofold-symmetric dimer was formed via 3D domain swapping. Domain swapping with the need for near-complete unfolding has been proposed as a possible route for amyloid fibril initiation. Thus, the interesting interactions that occur between the two molecules may be important for the further aggregation of the protein. In this work, we performed steered molecular dynamics (SMD) simulations to investigate the dissociation of the β2 and β3 strands in the hCC dimer. The energy changes observed during the SMD simulations showed that electrostatic interactions were the dominant interactions involved in stabilizing the two parts of the dimer during the early stages of SMD simulation, whereas van der Waals (VDW) interactions and electrostatic interactions were equally matched during the latter stages. Furthermore, our data indicated that the two parts of the dimer are stabilized by intermolecular hydrogen bonds among the residues Arg51 (β2), Gln48 (β2), Asp65 (β3), and Glu67 (β3), salt bridges among the residues Arg53 (β2), Arg51 (β2), and Asp65 (β3), and VDW interactions among the residues Gln48 (β2), Arg51 (β2), Glu67 (β3), Asp65 (β3), Phe63 (β3), and Asn61 (β3). The residues Gln48 (β2), Arg51 (β2), Asp65 (β3) and Glu67 (β3) appear to be crucial, as they play important roles in both electrostatic and VDW interactions. Thus, the present study determined the key residues involved in the stabilization of the domain-swapped dimer structure, and also provided molecular-level insights into the dissociation process of the hCC dimer.     

Kinetic interaction analysis of human interleukin 5 receptor alpha mutants reveals a unique binding topology and charge distribution for cytokine recognition

Human interleukin 5 receptor alpha (IL5Ralpha) comprises three fibronectin type III domains (D1, D2, and D3) in the extracellular region. Previous results have indicated that residues in the D1D2 domains are crucial for high affinity interaction with human interleukin 5 (IL5). Yet, it is the D2D3 domains that have sequence homology with the classic cytokine recognition motif that is generally assumed to be the minimum cytokine-recognizing unit. In the present study, we used kinetic interaction analysis of alanine-scanning mutational variants of IL5Ralpha to define the residues involved in IL5 recognition. Soluble forms of IL5Ralpha variants were expressed in S2 cells, selectively captured via their C-terminal V5 tag by anti-V5 tag antibody immobilized onto the sensor chip and examined for IL5 interaction by using a sandwich surface plasmon resonance biosensor method. Marked effects on the interaction kinetics were observed not only in D1 (Asp(55), Asp(56), and Glu(58)) and D2 (Lys(186) and Arg(188)) domains, but also in the D3 (Arg(297)) domain. Modeling of the tertiary structure of IL5Ralpha indicated that these binding residues fell into two clusters. The first cluster consists of D1 domain residues that form a negatively charged patch, whereas the second cluster consists of residues that form a positively charged patch at the interface of D2 and D3 domains. These results suggest that the IL5 x IL5Ralpha system adopts a unique binding topology, in which the cytokine is recognized by a D2D3 tandem domain combined with a D1 domain, to form an extended cytokine recognition interface.     

Constitutive activation of fibroblast growth factor receptor 3 by the transmembrane domain point mutation found in achondroplasia.

Achondroplasia, the most common genetic form of dwarfism, is an autosomal dominant disorder whose underlying mechanism is a defect in the maturation of the cartilage growth plate of long bones. Achondroplasia has recently been shown to result from a Gly to Arg substitution in the transmembrane domain of the fibroblast growth factor receptor 3 (FGFR3), although the molecular consequences of this mutation have not been investigated. By substituting the transmembrane domain of the Neu receptor tyrosine kinase with the transmembrane domains of wild-type and mutant FGFR3, the Arg380 mutation in FGFR3 is shown to activate both the kinase and transforming activities of this chimeric receptor. Residues with side chains capable of participating in hydrogen bond formation, including Glu, Asp, and to a lesser extent, Gln, His and Lys, were able to substitute for the activating Arg380 mutation. The Arg380 point mutation also causes ligand-independent stimulation of the tyrosine kinase activity of FGFR3 itself, and greatly increased constitutive levels of phosphotyrosine on the receptor. These results suggest that the molecular basis of achondroplasia is unregulated signal transduction through FGFR3, which may result in inappropriate cartilage growth plate differentiation and thus abnormal long bone development. Achondroplasia may be one of the number of cogenital disorders where constitutive activation of a member of the FGFR family leads to development abnormalities.     

Identification of essential charged residues in transmembrane segments of the multidrug transporter MexB of Pseudomonas aeruginosa

The MexABM efflux pump exports structurally diverse xenobiotics, utilizing the proton electrochemical gradient to confer drug resistance on Pseudomonas aeruginosa. The MexB subunit traverses the inner membrane 12 times and has two, two, and one charged residues in putative transmembrane segments 2 (TMS-2), TMS-4, and TMS-10, respectively. All five residues were mutated, and MexB function was evaluated by determining the MICs of antibiotics and fluorescent dye efflux. Replacement of Lys342 with Ala, Arg, or Glu and Glu346 with Ala, Gln, or Asp in TMS-2 did not have a discernible effect. Ala, Asn, or Lys substitution for Asp407 in TMS-4, which is well conserved, led to loss of activity. Moreover, a mutant with Glu in place of Asp407 exhibited only marginal function, suggesting that the length of the side chain at this position is important. The only replacements for Asp408 in TMS-4 or Lys939 in TMS-10 that exhibited significant function were Glu and Arg, respectively, suggesting that the native charge at these positions is required. In addition, double neutral mutants or mutants in which the charged residues Asp407 and Lys939 or Asp408 and Lys939 were interchanged completely lost function. An Asp408-->Glu/Lys939-->Arg mutant retained significant activity, while an Asp407-->Glu/Lys939-->Arg mutant exhibited only marginal function. An Asp407-->Glu/Asp408-->Glu double mutant also lost activity, but significant function was restored by replacing Lys939 with Arg (Asp407-->Glu/Asp408-->Glu/Lys939-->Arg). Taken as a whole, the findings indicate that Asp407, Asp408, and Lys939 are functionally important and raise the possibility that Asp407, Asp408, and Lys939 may form a charge network between TMS-4 and TMS-10 that is important for proton translocation and/or energy coupling.     

Molecular structure of human galactokinase: implications for type II galactosemia

Galactokinase functions in the Leloir pathway for galactose metabolism by catalyzing the MgATP-dependent phosphorylation of the C-1 hydroxyl group of alpha-D-galactose. The enzyme is known to belong to the GHMP superfamily of small molecule kinases and has attracted significant research attention for well over 40 years. Approximately 20 mutations have now been identified in human galactokinase, which result in the diseased state referred to as Type II galactosemia. Here we report the three-dimensional architecture of human galactokinase with bound alpha-D-galactose and Mg-AMPPNP. The overall fold of the molecule can be described in terms of two domains with the active site wedged between them. The N-terminal domain is dominated by a six-stranded mixed beta-sheet whereas the C-terminal motif contains six alpha-helices and two layers of anti-parallel beta-sheet. Those residues specifically involved in sugar binding include Arg37, Glu43, His44, Asp46, Gly183, Asp186, and Tyr236. The C-1 hydroxyl group of alpha-D-galactose sits within 3.3 A of the gamma-phosphorus of the nucleotide and 3.4 A of the guanidinium group of Arg37. The carboxylate side chain of Asp186 lies within approximately 3.2 A of the C-2 hydroxyl group of alpha-D-galactose and the guanidinium group of Arg37. Both Arg37 and Asp186 are strictly conserved among both prokaryotic and eukaryotic galactokinases. In addition to providing molecular insight into the active site geometry of the enzyme, the model also provides a structural framework upon which to more fully understand the consequences of the those mutations known to give rise to Type II galactosemia.     

Mechanistic implications from crystalline complexes of wild-type and mutant adenylosuccinate synthetases from Escherichia coli.

Asp13 and His41 are essential residues of adenylosuccinate synthetase, putatively catalyzing the formation of adenylosuccinate from an intermediate of 6-phosphoryl-IMP. Wild-type adenylosuccinate synthetase and three mutant synthetases (Arg143 --> Leu, Lys16 --> Gln, and Arg303 --> Leu) from Eschericha coli have been crystallized in the presence of IMP, hadacidin (an analogue of L-aspartate), Mg2+, and GTP. The active site of each complex contains 6-phosphoryl-IMP, Mg2+, GDP, and hadacidin, except for the Arg303 --> Leu mutant, which does not bind hadacidin. In response to the formation of 6-phosphoryl-IMP, Asp13 enters the inner coordination sphere of the active site Mg2+. His41 hydrogen bonds with 6-phosphoryl-IMP, except in the Arg303 --> Leu complex, where it remains bound to the guanine nucleotide. Hence, recognition of the active site Mg2+ by Asp13 evidently occurs after the formation of 6-phosphoryl-IMP, but recognition of the intermediate by His41 may require the association of L-aspartate with the active site. Structures reported here support a mechanism in which Asp13 and His41 act as the catalytic base and acid, respectively, in the formation of 6-phosphoryl-IMP, and then act together as catalytic acids in the subsequent formation of adenylosuccinate.