Localization of N-terminal sequences in human AMP deaminase isoforms that influence contractile protein binding.

The reversible association of AMP deaminase (AMPD, EC 3.5.4.6) with elements of the contractile apparatus is an identified mechanism of enzyme regulation in mammalian skeletal muscle. All three members of the human AMPD multigene family contain coding information for polypeptides with divergent N-terminal and conserved C-terminal domains. In this study, serial N-terminal deletion mutants of up to 111 (AMPD1), 214 (AMPD2), and 126 (AMPD3) residues have been constructed without significant alteration of catalytic function or protein solubility. The entire sets of active enzymes are used to extend our understanding of the contractile protein binding of AMPD. Analysis of the most truncated active enzymes demonstrates that all three isoforms can associate with skeletal muscle actomyosin and suggests that a primary binding domain is located within the C-terminal 635-640 residues of each polypeptide. However, discrete stretches of N-terminal sequence alter this behavior. Residues 54-83 in the AMPD1 polypeptide contribute to a high actomyosin binding capacity of both isoform M spliceoforms, although the exon 2- enzyme exhibits significantly greater association compared to its exon 2+ counterpart. Conversely, residues 129-183 in the AMPD2 polypeptide reduce actomyosin binding of isoform L. In addition, residues 1-48 in the AMPD3 polypeptide dramatically suppress contractile protein binding of isoform E, thus allowing this enzyme to participate in other intracellular interactions.     

Molecular cloning of AMP deaminase isoform L. Sequence and bacterial expression of human AMPD2 cDNA.

Human AMPD2 cDNA clones have been isolated from T-lymphoblast and placental lambda gt11 libraries utilizing a previously cloned rat partial AMPD2 cDNA as the probe. Alignment analysis of all cDNA clones indicates the presence of intervening sequences in several placental isolates. This has been confirmed by sequencing human AMPD2 genomic clones. Intervening sequences can be removed from the cDNA clones by restriction with endonucleases at unique sites within the proposed open reading frame. This results in a 3292-base pair cDNA proposed to contain the entire AMPD2 open reading frame, which would encode a 760-amino acid polypeptide with a predicted subunit molecular mass of 88.1 kDa. Nucleotide and predicted amino acid comparisons with the 264 base pairs of proposed coding sequences in the rat AMPD2 cDNA demonstrate 91% similarity and identity, respectively. A comparison of the predicted human AMPD1 and AMPD2 polypeptides demonstrates homology in their C-terminal domains. Included in this region is the conserved motif, SLSTDDP, proposed to be part of the catalytic site of all AMP deaminases. In contrast, the predicted N-terminal domains of the human AMPD1 and AMPD2 polypeptides are unique. When placed in a prokaryotic expression vector, the human AMPD2 cDNA expresses AMP deaminase activity which can be precipitated with polyclonal antisera specific for isoform L.     

N-terminal sequence and distal histidine residues are responsible for pH-regulated cytoplasmic membrane binding of human AMP deaminase isoform E

Mammalian AMP deaminase 3 (AMPD3) enzymes reportedly bind to intracellular membranes, plasma lipid vesicles, and artificial lipid bilayers with associated alterations in enzyme conformation and function. However, proteolytic sensitivity of AMPD polypeptides makes it likely that prior studies were performed with N-truncated enzymes. This study uses erythrocyte ghosts to characterize the reversible cytoplasmic membrane association of human full-sized recombinant isoform E (AMPD3). Membrane-bound isoform E exhibits diminished catalytic activity whereas low micromolar concentrations of the cationic antibiotic, neomycin, disrupt this protein-lipid interaction and relieve catalytic inhibition. The cytoplasmic membrane association of isoform E also displays an inverse correlation with pH in the physiological range. Diethyl pyrocarbonate (DEPC) modification of isoform E nearly abolishes its cytoplasmic membrane binding capacity, and this effect can be reversed by hydroxylamine. Difference spectra reveal that 18 of 29 histidine residues in each isoform E subunit are N-carbethoxylated by DEPC. These combined data demonstrate that protonated imidazole rings of histidine residues mediate a pH-responsive association of isoform E with anionic charges on the surface of the cytoplasmic membrane, possibly phosphatidylinositol 4,5-bisphosphate, a pure noncompetitive inhibitor of the enzyme. Finally, AMPD1 and a series of N-truncated AMPD3 enzymes are used to show that these behaviors are specific to isoform E and require up to 48 N-terminal amino acids, even though this stretch of sequence contains no histidine residues. The pH-responsive cytosol-membrane partitioning of isoform E may be an important mechanism for branch point regulation of adenylate catabolism.     

Component X of mammalian pyruvate dehydrogenase complex: structural and functional relationship to the lipoate acetyltransferase (E2) component

The lipoate acetyltransferase (E2, Mr 70,000) and protein X (Mr 51,000) subunits of the bovine pyruvate dehydrogenase multienzyme complex (PDC) core assembly are antigenically distinct polypeptides. However comparison of the N-terminal amino acid sequence of the E2 and X polypeptides reveals significant homology between the two components. Selective tryptic release of the 14C-labelled acetylated lipoyl domains of E2 and protein X from native PDC generates stable, radiolabelled 34 and 15 kDa fragments, respectively. Thus, in contrast to E2 which contains two tandemly-arranged lipoyl domains, protein X appears to contain only a single lipoyl domain located at its N-terminus.     

The closely related homomeric and heterodimeric mannose-binding lectins from garlic are encoded by one-domain and two-domain lectin genes, respectively.

Lectin cDNA clones for two different lectins from garlic (Allium sativum L.) bulbs, ASAI and ASAII (ASA, Allium sativum agglutinin), were isolated and characterized. The first lectin, ASAI, is a heterodimer composed of two different subunits of 11.5 kDa and 12.5 kDa. It is translated from an mRNA of 1400 nucleotides encoding a polypeptide of 306 amino acids with two very similar domains. N-terminal sequencing of the two polypeptides of the mature lectin confirmed that both subunits are derived from the same precursor and that each corresponds to one of the two domains in the sequence. In contrast to ASAI, the second garlic lectin, ASAII, is a homodimer of two identical 12-kDa subunits. It is translated from an mRNA of approximately 800 nucleotides encoding a polypeptide of 154 amino acids. Interestingly, the coding region of the ASAII cDNA clones is almost identical to that of the second domain of the ASAI cDNA clones.     

Structure of the genes encoding the rod-core linker polypeptides of Mastigocladus laminosus phycobilisomes and functional aspects of the phycobiliprotein/linker-polypeptide interactions.

The 3' portion of the cpc operon in Mastigocladus laminosus encloses the genes 5'-cpcF-cpcG1-cpcG2-cpcG3 3'. The three cpcG genes encode different phycocyanin-associated rod-core linker polypeptides of the phycobilisomes with predicted 279, 247 and 254 amino acids in length. The gene products CpcG show a high similarity at their N-terminal domains (190 amino acids) and an overall identity of 47-53% to one another. Each of the three CpcG polypeptides is highly related to one of the four CpcG gene products of Anabaena sp. PCC 7120 (66-81% identity). It is suggested that these pairs of rod-core linker polypeptides mediate the same specific type of phycocyanin----allophycocyanin interaction in the similar phycobilisomes of M. laminosus and Anabaena sp. PCC 7120. The similarity of the CpcG1, CpcG2 and CpcG3 polypeptides to the single CpcG rod-core linker polypeptide of Synechococcus sp. PCC 7002 (36-41% identity) is lower. The rod-core linker polypeptides are more distantly related to the rod linker polypeptides associated with phycocyanin or phycoerythrin. However, six conserved domains were identified within the N-terminal 190 amino acids of these linker proteins, which bear similar amino acid sequences, including highly conserved basic amino acids. A similar amino acid sequence but with conserved acidic amino acids can be found in the beta subunits of phycocyanin, phycoerythrin and phycoerythrocyanin, which is protruding into the central cavity of the phycobiliprotein hexamers. It is suggested that these domains are sites of phycobiliprotein-hexamer/rod and rod-core linker interactions.     

Structural basis for the activation of phenylalanine in the non-ribosomal biosynthesis of gramicidin S.

The non-ribosomal synthesis of the cyclic peptide antibiotic gramicidin S is accomplished by two large multifunctional enzymes, the peptide synthetases 1 and 2. The enzyme complex contains five conserved subunits of approximately 60 kDa which carry out ATP-dependent activation of specific amino acids and share extensive regions of sequence similarity with adenylating enzymes such as firefly luciferases and acyl-CoA ligases. We have determined the crystal structure of the N-terminal adenylation subunit in a complex with AMP and L-phenylalanine to 1.9 A resolution. The 556 amino acid residue fragment is folded into two domains with the active site situated at their interface. Each domain of the enzyme has a similar topology to the corresponding domain of unliganded firefly luciferase, but a remarkable relative domain rotation of 94 degrees occurs. This conformation places the absolutely conserved Lys517 in a position to form electrostatic interactions with both ligands. The AMP is bound with the phosphate moiety interacting with Lys517 and the hydroxyl groups of the ribose forming hydrogen bonds with Asp413. The phenylalanine substrate binds in a hydrophobic pocket with the carboxylate group interacting with Lys517 and the alpha-amino group with Asp235. The structure reveals the role of the invariant residues within the superfamily of adenylate-forming enzymes and indicates a conserved mechanism of nucleotide binding and substrate activation.     

N-terminal intramolecularly conserved histidines of three domains in Gonyaulax luciferase are responsible for loss of activity in the alkaline region

Gonyaulax luciferase is a single-chain ( approximately 137 kDa) polypeptide comprising 111 N-terminal amino acids followed by three contiguous homologous domains (377 amino acids each). Each domain has luciferase activity, accounting for the earlier observation that proteolytic fragments ( approximately 35 kDa) of luciferase are active. The activity of the full-length native enzyme is maximal at pH 6.3, dropping to near zero at pH 8; the activity of fragments also peaks at pH 6.3 but remains high at 8. While the activity loss at higher pH might be thought to be associated with the conformation of the full-length protein, we show here that this is a property of individual domains. The three intramolecularly homologous domains, separately cloned and expressed in Escherichia coli as fusion proteins, exhibit pH-activity curves similar to that of the full-length enzyme. For each domain the removal of approximately 50 N-terminal amino acids resulted in an increase in the ratio of luciferase activity at pH 8 relative to that at pH 6.3, such that their pH-activity profiles mimicked that of the proteolytic fragments reported earlier. Replacement of N-terminal histidines by alanine by site-directed mutagenesis identified four that are involved in the loss of activity at high pH. This system illustrates an unusual, possibly unique mechanism for pH regulation of enzyme activity, which has been postulated to be responsible for the control of the characteristic flashes of bioluminescence.     

Construction and analyses of tetrameric forms of yeast NAD+-specific isocitrate dehydrogenase

Yeast NAD(+)-specific isocitrate dehydrogenase (IDH) is an octameric enzyme composed of four heterodimers of regulatory IDH1 and catalytic IDH2 subunits. The crystal structure suggested that the interactions between tetramers in the octamer are restricted to defined regions in IDH1 subunits from each tetramer. Using truncation and mutagenesis, we constructed three tetrameric forms of IDH. Truncation of five residues from the amino terminus of IDH1 did not alter the octameric form of the enzyme, but this truncation with an IDH1 G15D or IDH1 D168K residue substitution produced tetrameric enzymes as assessed by sedimentation velocity ultracentrifugation. The IDH1 G15D substitution in the absence of any truncation of IDH1 was subsequently found to be sufficient for production of a tetrameric enzyme. The tetrameric forms of IDH exhibited ～50% reductions in V(max) and in cooperativity with respect to isocitrate relative to those of the wild-type enzyme, but they retained the property of allosteric activation by AMP. The truncated (-5)IDH1/IDH2 and tetrameric enzymes were much more sensitive than the wild-type enzyme to inhibition by the oxidant diamide and concomitant formation of a disulfide bond between IDH2 Cys-150 residues. Binding of ligands reduced the sensitivity of the wild-type enzyme to diamide but had no effect on inhibition of the truncated or tetrameric enzymes. These results suggest that the octameric structure of IDH has in part evolved for regulation of disulfide bond formation and activity by ensuring the proximity of the amino terminus of an IDH1 subunit of one tetramer to the IDH2 Cys-150 residues in the other tetramer.     

Low-molecular-weight polypeptides of vicilin from Vicia faba L. are products of proteolytic breakdown

Vicilin, the main 7-S globulin of Vicia faba L., undergoes cleavage during prolonged treatment at room temperature, which can be inhibited by protease inhibitors such as 1 microM leupeptin. The cleavage products show identical electrophoretic mobilities with the polypeptides normally visible after sodium dodecylsulphate gel electrophoresis of vicilin prepared from mature seeds. N-terminal amino acid analysis of electrophoretically prepared polypeptides reveals serine as common N terminus of the two largest polypeptides of Mr approximately equal to 50000 and 35000. According to serological experiments and peptide mapping the low-molecular-weight polypeptides (Mr approximately equal to 35000; 31000; 19000 and below) have antigenic determinants and amino acid sequences, respectively, that are similar to each other and are all contained within the structure of the large polypeptide of Mr approximately equal to 50000. This leads of the conclusion that polypeptides of Mr approximately equal to 35000 and below are derived by proteolysis from one (or a few closely related) polypeptide(s) of Mr approximately equal to 50000 and that proteolysis starts soon after biosynthesis as a post-translational process within the developing seed. Some experiments indicate the existence of 'nicking' points within the vicilin polypeptides, which are the major cleavage sites during preparation. These observations strongly support the view that 'native' vicilin is a trimeric or tetrameric globulin with polypeptides of Mr greater than or equal to 50000.     

Carcinoembryonic antigen family: characterization of cDNAs coding for NCA and CEA and suggestion of nonrandom sequence variation in their conserved loop-domains

We have isolated cDNAs for carcinoembryonic antigen (CEA) and for a normal cross-reacting antigen (NCA) and report here their nucleotide and derived amino acid sequences. Our data show that both the CEA and NCA polypeptides are organized into extracellular domains, some with cysteine-linked loops, that share extensive sequence homology (approximately 78% overall) with each other and appear similar to immunoglobulin superfamily members. A major difference between the two apoproteins is the presence of a single loop-domain in NCA compared to three tandemly repeated loop-domains in CEA. Sequence comparisons between the extracellular domains of CEA and NCA show that the N-terminal and adjacent loop domains of each apoprotein have high homology (85-90%) to each other, while comparison of loop-domain regions reveals a possible nonrandom distribution of base changes and altered amino acids near certain cysteine residues that are inferred to be involved in forming disulfide loops. Both apoproteins show high identity in their hydrophobic C-termini that are reminiscent of the type of transmembrane tails seen in proteins that potentiate signal transduction. These findings, coupled with distinct expression profiles of CEA and NCA mRNAs, suggest that these apoproteins may function as unique cell-surface molecules mediating cell-specific interactions in normal and neoplastic cells.     

Membrane association, mechanism of action, and structure of Arabidopsis embryonic factor 1 (FAC1)

Embryonic factor 1 (FAC1) is one of the earliest expressed plant genes and encodes an AMP deaminase (AMPD), which is also an identified herbicide target. This report identifies an N-terminal transmembrane domain in Arabidopsis FAC1, explores subcellular fractionation, and presents a 3.3-A globular catalytic domain x-ray crystal structure with a bound herbicide-based transition state inhibitor that provides the first glimpse of a complete AMPD active site. FAC1 contains an (alpha/beta)(8)-barrel characterized by loops in place of strands 5 and 6 that places it in a small subset of the amidohydrolase superfamily with imperfect folds. Unlike tetrameric animal orthologs, FAC1 is a dimer and each subunit contains an exposed Walker A motif that may be involved in the dramatic combined K(m) (25-80-fold lower) and V(max) (5-6-fold higher) activation by ATP. Normal mode analysis predicts a hinge motion that flattens basic surfaces on each monomer that flank the dimer interface, which suggests a reversible association between the FAC1 globular catalytic domain and intracellular membranes, with N-terminal transmembrane and disordered linker regions serving as the anchor and attachment to the globular catalytic domain, respectively.     

Structure of histone acetyltransferases

Histone acetyltranferase (HAT) enzymes are the catalytic subunits of multisubunit protein complexes that acetylate specific lysine residues on the N-terminal regions of the histone components of chromatin to promote gene activation. These enzymes, which now include more than 20 members, fall into distinct families that generally have high sequence similarity and related substrate specificity within families, but have divergent sequence and substrate specificity between families. Significant insights into the mode of catalysis and histone substrate binding have been provided by the structure determination of the divergent HAT enzymes Hat1, Gcn5/PCAF and Esa1. A comparison of these structures reveals a structurally conserved central core domain that mediates extensive interactions with the acetyl-coenzyme A cofactor, and structurally divergent N and C-terminal domains. A correlation of these structures with other studies reveals that the core domain plays a particularly important role in histone substrate catalysis and that the N and C-terminal domains play important roles in histone substrate binding. These correlations imply a related mode of catalysis and histone substrate binding by a diverse group of HAT enzymes.     

Characterization of the metallocenter of rabbit skeletal muscle AMP deaminase. Evidence for a dinuclear zinc site

XAS of Zn-peptide binary and ternary complexes prepared using peptides mimicking the potential metal binding sites of rabbit skeletal muscle AMP deaminase (AMPD) strongly suggest that the region 48-61 of the enzyme contains a zinc binding site, whilst the region 360-372 of the enzyme is not able to form 1:1 complexes with zinc, in contrast with what has been suggested for the corresponding region of yeast AMPD. XAS performed on fresh preparations of rabbit skeletal muscle AMPD provides evidence for a dinuclear zinc site in the enzyme compatible with a (mu-aqua)(mu-carboxylato)dizinc(II) core with an average of two histidine residues at each metal site and a Zn-Zn distance of about 3.3 Angstrom. The data indicate that zinc is not required for HPRG/AMPD interaction, both zinc ions being bound to the catalytic subunit of the enzyme, one to the three conserved amino acid residues among those four assumed to be in contact with zinc in yeast AMPD, and the other at the N-terminal region, probably to His-52, Glu-53 and His-57. Tryptic digests of different enzyme preparations demonstrate the existence of two different protein conformations and of a zinc ion connecting the N-terminal and C-terminal regions of AMPD.     

A structural comparison of the A and B subunits of Griffonia simplicifolia I isolectins

A structural comparison between the A and B subunits of the five tetrameric Griffonia simplicifolia I isolectins (A4, A3B, A2B2, AB3, B4) was undertaken to determine the extent of homology between the subunits. The first 25 N-terminal amino acids of both A and B subunits were determined following the enzymatic removal of N-terminal pyroglutamate blocking groups with pyroglutamate aminopeptidase. Although 21 amino acids were common to both subunits, there were four unique amino acids in the N-terminal sequence of A and B. Residues 8, 9, 17, and 19 were asparagine, leucine, lysine, and asparagine in subunit A and threonine, phenylalanine, glutamic acid, and serine in subunit B. The last six C-terminal amino acids, released by digestion with carboxypeptidase Y, were the same for both subunits: Arg-(Phe, Val)-Leu-Thr-Ser-COOH. Subunit B, which contains one methionyl residue, was cleaved by cyanogen bromide into two fragments, a large (Mr = 31,000) and a small (Mr = 2700) polypeptide. Failure of the small fragment to undergo manual Edman degradation indicated an N-terminal blocking group, presumably pyroglutamate. Both subunits were digested with trypsin and the tryptic peptides were analyzed using reverse-phase HPLC. Tryptic glycopeptides were identified by labeling the carbohydrate moiety of the A and B subunit using sodium [3H] borohydride. Cysteine-containing tryptic peptides were similarly identified by using [1-14C]iodoacetamide. Approximately 30% of the tryptic peptides were common to both subunits. Thus, although the N- and C-terminal regions of A and B are similar, the subunits each possess unique sequences.     

Comparison of the NH2-terminal amino acid sequences of the four non-identical subunits of the NAD-linked hydrogenases from Nocardia opaca 1b and Alcaligenes eutrophus H16

The cytoplasmic, NAD-linked hydrogenase of the Gram-positive hydrogen-oxidizing bacterium Nocardia opaca 1b was compared with the analogous enzyme isolated from the Gram-negative bacterium Alcaligenes eutrophus H16. The hydrogenase of N. opaca 1b was purified by a new procedure applying chromatography on phenyl-Sepharose and DEAE-Sephacel with two columns in series. A homogeneous enzyme preparation with a specific activity of 74 mumol H2 oxidized.min-1.mg protein-1 and a yield of 32% was isolated. The A. eutrophus enzyme was purified as previously published. Both enzymes are tetrameric proteins composed of four non-identical subunits (alpha, beta, gamma, delta). The four subunits of both of these enzymes were separated and isolated as single polypeptides by preparative polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Immunological comparison of the four subunits of the Nocardia hydrogenase with those of the Alcaligenes enzyme showed that the alpha, beta, gamma, and delta subunits of one organism were serologically related to the analogous subunits of the other organism. Among themselves, the four subunits do not have any serological relationship. The eight individual polypeptides were also compared with respect to the NH2-terminal amino acid sequences determined by automated Edman degradation and to the amino acid compositions. Strong sequence similarities exist between the analogous subunits isolated from the two bacteria. Within the established N-terminal sequences the similarities between both alpha, beta, gamma and delta subunits amount to 63%, 79%, 80% and 65%, respectively. No similarities exist between the different, non-analogous subunits alpha, beta, gamma and delta.     

Comparison of the aspartate transcarbamoylases from Serratia marcescens and Escherichia coli

The aspartate transcarbamoylases (ATCase, EC 2.1.3.2) of Escherichia coli and Serratia marcescens have similar dodecameric enzyme structures (2(c3):3(r2] but differ in both regulatory and catalytic characteristics. The catalytic cistrons (pyrB) of the ATCases from E. coli and S. marcescens encode polypeptides of 311 and 306 amino acids, respectively; there is a 76% identity between the DNA sequences and an overall amino acid homology of 88% (38 differences). The regulatory cistrons (pyrI) of these ATCases encode polypeptides of 153 and 154 amino acids, respectively, and there is a 75% identity between the DNA sequences and an overall amino acid homology of 77% (36 differences). In both species, the two genes are arranged as a bicistronic operon, with pyrB promoter proximal. A comparison of the deduced amino acid sequences reveals that the active site and the allosteric binding sites, as well as most of the intrasubunit interactions and intersubunit associations, are conserved in the E. coli and the S. marcescens enzymes; however, there are specific differences which undoubtedly contribute to the catalytic and regulatory differences between the enzymes of the two species. These differences include residues that have been implicated in the T-R transition, c1:r1 interface interactions, and the CTP binding site. A hybrid ATCase assembled in vivo with catalytic subunits from E. coli and regulatory subunits from S. marcescens has a 6 mM requirement for aspartate at half-maximal saturation, similar to the 5.5 mM aspartate requirement of the native E. coli holoenzyme at half-maximal saturation. However, the heterotropic response of this hybrid enzyme is characteristic of the heterotropic response of the native S. marcescens holoenzyme: ATP activation and CTP activation. Activation by both allosteric effectors indicates that the heterotropic response of this hybrid holoenzyme (Cec:Rsm) is determined by the associated S. marcescens regulatory subunits.     

Topology of subunits of the mammalian cytochrome c oxidase: relationship to the assembly of the enzyme complex.

The arrangement of three subunits of beef heart cytochrome c oxidase, subunits Va, VIa, and VIII, has been explored by chemical labeling and protease digestion studies. Subunit Va is an extrinsic protein located on the C side of the mitochondrial inner membrane. This subunit was found to label with N-(4-azido-2-nitrophenyl)-2-aminoethane[35S]sulfonate and sodium methyl 4-[3H]formylphenyl phosphate in reconstituted vesicles in which 90% of cytochrome c oxidase complexes were oriented with the C domain outermost. Subunit VIa was cleaved by trypsin both in these reconstituted vesicles and in submitochondrial particles, indicating a transmembrane orientation. The epitope for a monoclonal antibody (mAb) to subunit VIa was lost or destroyed when cleavage occurred in reconstituted vesicles. This epitope was localized to the C-terminal part of the subunit by antibody binding to a fusion protein consisting of glutathione S-transferase (G-ST) and the C-terminal amino acids 55-85 of subunit VIa. No antibody binding was obtained with a fusion protein containing G-ST and the N-terminal amino acids 1-55. The mAb reaction orients subunit VIa with its C-terminus in the C domain. Subunit VIII was cleaved by trypsin in submitochondrial particles but not in reconstituted vesicles. N-Terminal sequencing of the subunit VIII cleavage product from submitochondrial particles gave the same sequence as the untreated subunit, i.e., ITA, indicating that it is the C-terminus which is cleaved from the M side. Subunits Va and VIII each contain N-terminal extensions or leader sequences in the precursor polypeptides; subunit VIa is made without an N-terminal extension.