Recombinant Mutant Proteins Research Articles

Laminin α1 Chain LG4 Module Promotes Cell Attachment through Syndecans and Cell Spreading through Integrin α2β1

The laminin alpha1 chain is a subunit of laminin-1, a heterotrimeric basement membrane protein. The LG4-5 module at the C terminus of laminin alpha1 contains major binding sites for heparin, sulfatide, and alpha-dystroglycan and plays a critical role in early embryonic development. We previously identified active synthetic peptides AG73 and EF-1 from the sequence of laminin alpha1 LG4 for binding to syndecan and integrin alpha2beta1, respectively. However, their activity and functional relationship within the laminin-1 and LG4 as well as the functional relation between these sites and alpha-dystroglycan binding sites in LG4 are not clear. To address these questions, we created mutant recombinant LG4 proteins containing alanine substitutions within the AG73 (M1), EF-1 (M2, M3), and alpha-dystroglycan binding sites (M4, M5) and analyzed their activities. We found that recombinant proteins rec-M1 and rec-M5, containing mutations within M1 and M5, respectively, did not bind heparin or lymphoid cell lines expressing syndecans. These results suggest that LG4 binds to heparin and syndecans through M1 and M5. Rec-M1 and rec-M5 reduced fibroblast attachment, whereas mutant rec-M2 and rec-M3 retained cell attachment activity but did not promote cell spreading. Fibroblast attachment to rec-LG4 was inhibited by heparin but not by integrin antibodies. Spreading of fibroblasts on rec-LG4 was inhibited by anti-integrin alpha2 and beta1 but not by anti-integrin alpha1 and alpha6. These results suggest that the M1 and M5 sites are necessary for cell attachment on LG4 through syndecans and that the EF-1 site is for cell spreading activity through integrin alpha2beta1. In contrast, laminin-1-mediated fibroblast attachment and spreading were not inhibited by heparin or anti-integrin alpha2. Our findings indicate that LG4 has a unique function distinct from laminin-1 and suggest that laminin alpha1 LG4-5 may also be produced by a proteolytic cleavage in certain tissues where it exerts its activity.

A novel missense mutation responsible for factor VII deficiency in research Beagle colonies.

Canine factor VII (cFVII) deficiency, an autosomal recessive trait originally identified in research Beagles, is associated with a mild to moderate bleeding tendency. Our aim was to identify and characterize the mutation causing cFVII deficiency. In order to sequence the coding regions of the cFVII gene, we cloned the cFVII cDNA. Genomic DNA and plasma from FVII-deficient Beagles and obligate carriers were utilized. In all FVII-deficient dogs, we identified a single causative G to A missense mutation in exon 5, encoding the second epidermal growth factor-like domain, resulting in substitution of glycine 96 by glutamic acid, with plasma FVII coagulant activity of <or = 4% in affected Beagles. In vitro expression indicated that the majority (96%) of cFVII-G96E protein was retained intracellularly. In addition, analysis of purified recombinant wild-type and mutant cFVII proteins demonstrated reduced activity of the mutant (< 2%) compared with wild-type. Rotational thromboelastometry revealed a severe impairment of clotting activity in affected Beagles, and heterozygotes also exhibited changes in coagulation-based assays. Using a mutation-specific polymerase chain reaction/restriction digest that allows rapid identification of the G96E mutation, we surveyed a US research Beagle colony and identified a mutant allelic frequency of 31%. We have identified a single causative mutation for cFVII deficiency that may have implications for pharmacotoxicologic research, because reduced FVII coagulant activity may alter hemostatic and/or cardiovascular endpoints in this commonly used animal species.

The Maintenance of High Affinity Plasminogen Binding by Group A Streptococcal Plasminogen-binding M-like Protein Is Mediated by Arginine and Histidine Residues within the a1 and a2 Repeat Domains

Subversion of the plasminogen activation system is implicated in the virulence of group A streptococci (GAS). GAS displays receptors for the human zymogen plasminogen on the cell surface, one of which is the plasminogen-binding group A streptococcal M-like protein (PAM). The plasminogen binding domain of PAM is highly variable, and this variation has been linked to host selective immune pressure. Site-directed mutagenesis of full-length PAM protein from an invasive GAS isolate was undertaken to assess the contribution of residues in the a1 and a2 repeat domains to plasminogen binding function. Mutagenesis to alanine of key plasminogen binding lysine residues in the a1 and a2 repeats (Lys98 and Lys111) did not abrogate plasminogen binding by PAM nor did additional mutagenesis of Arg101 and His102 and Glu104, which have previously been implicated in plasminogen binding. Plasminogen binding was only abolished with the additional mutagenesis of Arg114 and His115 to alanine. Furthermore, mutagenesis of both arginine (Arg101 and Arg114) and histidine (His102 and His115) residues abolished interaction with plasminogen despite the presence of Lys98 and Lys111 in the binding repeats. This study shows for the first time that residues Arg101, Arg114, His102, and His115 in both the a1 and a2 repeat domains of PAM can mediate high affinity plasminogen binding. These data suggest that highly conserved arginine and histidine residues may compensate for variation elsewhere in the a1 and a2 plasminogen binding repeats, and may explain the maintenance of high affinity plasminogen binding by naturally occurring variants of PAM.

Biochemical Activities of Three Pairs of Ehrlichia chaffeensis Two-Component Regulatory System Proteins Involved in Inhibition of Lysosomal Fusion

Ehrlichia chaffeensis, the etiologic agent of human monocytic ehrlichiosis, replicates in early endosomes by avoiding lysosomal fusion in monocytes and macrophages. In E. chaffeensis we predicted three pairs of putative two-component regulatory systems (TCSs) designated PleC-PleD, NtrY-NtrX, and CckA-CtrA based on amino acid sequence homology. In the present study to determine biochemical pairs and specificities of the TCSs, the recombinant proteins of the three putative histidine kinase (HK) kinase domains (rPleCHKD, rNtrYHKD, and MBP-rCckAHKD) and the full-length forms of three putative response regulators (RRs) (rPleD, rNtrX, and rCtrA) as well as the respective mutant recombinant proteins (rPleCHKDH244A, rNtrYHKDH498A, MBP-rCckAHKDH449A, rPleDD53A, rNtrXD59A, and rCtrAD53A) were expressed and purified as soluble proteins. The in vitro HK activity, the specific His residue-dependent autophosphorylation of the kinase domain, was demonstrated in the three HKs. The specific Asp residue-dependent in vitro phosphotransfer from the kinase domain to the putative cognate RR was demonstrated in each of the three RRs. Western blot analysis of E. chaffeensis membrane and soluble fractions using antibodies specific for each recombinant protein detected PleC and CckA in the membrane fraction, whereas it detected NtrY, NtrX, and PleD in the soluble fraction. CtrA was found in the two fractions at similar levels. E. chaffeensis was sensitive to closantel, an HK inhibitor. Closantel treatment induced lysosomal fusion of the E. chaffeensis inclusion in a human monocytic leukemia cell line, THP-1 cells, implying that functional TCSs are essential in preventing lysosomal fusion of the E. chaffeensis inclusion compartment.

Ribosomal S6 kinase-1 modulates interleukin-1β-induced persistent activation of NF-κB through phosphorylation of IκBβ

Activation of NF-kappaB requires the phosphorylation and degradation of its associated inhibitory proteins, IkappaB. Previously, we reported that the extracellular signal-regulated kinase (ERK) is required for IL-1beta to induce persistent activation of NF-kappaB in cultured rat vascular smooth muscle cells (VSMCs). The present study examined the mechanism by which the ERK signaling cascade modulates the duration of NF-kappaB activation. In cultured rat VSMCs, IL-1beta activated ERK and induced degradation of both IkappaBalpha and IkappaBbeta, which was associated with nuclear translocation of both ribosomal S6 kinase (RSK)1 and NF-kappaB p65. RSK1, a downstream kinase of ERK, was associated with an IkappaBbeta/NF-kappaB complex, which was independent of the phosphorylation status of RSK1. Treatment of VSMCs with IL-1beta decreased IkappaBbeta in the RSK1/IkappaBbeta/NF-kappaB complex, an effect that was attenuated by inhibition of ERK activation. Knockdown of RSK1 by small interference RNA attenuated the IL-1beta-induced IkappaBbeta decrease without influencing ether ERK phosphorylation or the earlier IkappaBalpha degradation. By using recombinant wild-type and mutant IkappaBbeta proteins, both active ERK2 and RSK1 were found to directly phosphorylate IkappaBbeta, but only active RSK1 phosphorylated IkappaBbeta on Ser19 and Ser23, two sites known to mediate the subsequent ubiquitination and degradation. In conclusion, in the ERK signaling cascade, RSK1 is a key component that directly phosphorylates IkappaBbeta and contributes to the persistent activation of NF-kappaB by IL-1beta.

Bcl2 Suppresses DNA Repair by Enhancing c-Myc Transcriptional Activity

Bcl2 and c-Myc are two major oncogenic proteins that can functionally promote DNA damage, genetic instability, and tumorigenesis. However, the mechanism(s) remains unclear. Nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is the most potent carcinogen contained in cigarette smoke that induces cellular DNA damage. Here we found that Bcl2 potently suppresses the repair of NNK-induced abasic sites of DNA lesions in association with increased c-Myc transcriptional activity. The Bcl2 BH4 domain (amino acids 6-31) was found to bind directly to c-Myc MBII domain (amino acids 106-143), and this interaction is required for Bcl2 to enhance c-Myc transcriptional activity and inhibit DNA repair. In addition to mitochondria, Bcl2 is also expressed in the nucleus, where it co-localizes with c-Myc. Expression of nuclear-targeted Bcl2 enhances c-Myc transcriptional activity with suppression of DNA repair but fails to prolong cell survival. Depletion of c-Myc expression from cells overexpressing Bcl2 significantly accelerates the repair of NNK-induced DNA damage, indicating that c-Myc may be essential for the Bcl2 effect on DNA repair. It is known that apurinic/apyrimidinic endonuclease (APE1) plays a crucial role in the repair of abasic sites of DNA lesions. That overexpression of Bcl2 results in up-regulation of c-Myc and down-regulation of APE1 suggests APE1 may function as the downstream target of Bcl2/c-Myc in the DNA repair machinery. Thus, Bcl2, in addition to its survival function, may also suppress DNA repair in a novel mechanism involving c-Myc and APE1, which may lead to an accumulation of DNA damage in living cells, genetic instability, and tumorigenesis.

Crystal Structure of Human 3-Hydroxy-3-methylglutaryl-CoA Lyase: INSIGHTS INTO CATALYSIS AND THE MOLECULAR BASIS FOR HYDROXYMETHYLGLUTARIC ACIDURIA

3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase is a key enzyme in the ketogenic pathway that supplies metabolic fuel to extrahepatic tissues. Enzyme deficiency may be due to a variety of human mutations and can be fatal. Diminished activity has been explained based on analyses of recombinant human mutant proteins or, more recently, in the context of structural models for the enzyme. We report the experimental determination of a crystal structure at 2.1 A resolution of the recombinant human mitochondrial HMG-CoA lyase containing a bound activator cation and the dicarboxylic acid 3-hydroxyglutarate. The enzyme adopts a (betaalpha)(8) barrel fold, and the N-terminal barrel end is occluded. The structure of a physiologically relevant dimer suggests that substrate access to the active site involves binding across the cavity located at the C-terminal end of the barrel. An alternative hypothesis that involves substrate insertion through a pore proposed to extend through the barrel is not compatible with the observed structure. The activator cation ligands included Asn(275), Asp(42),His(233), and His(235); the latter three residues had been implicated previously as contributing to metal binding or enzyme activity. Arg(41), previously shown to have a major effect on catalytic efficiency, is also located at the active site. In the observed structure, this residue interacts with a carboxyl group of 3-hydroxyglutarate, the hydrolysis product of the competitive inhibitor 3-hydroxyglutaryl-CoA required for crystallization of human enzyme. The structure provides a rationale for the decrease in enzyme activity due to clinical mutations, including H233R, R41Q, D42H, and D204N, that compromise active site function or enzyme stability.

PYPAF1 nonsense mutation in a patient with an unusual autoinflammatory syndrome: Role of PYPAF1 in inflammation

To gain insight into the pathophysiology of an unusual autoinflammatory syndrome, in a patient of Armenian origin, that mimicked familial Mediterranean fever (FMF) but with episodes triggered by generalized exposure to cold, and to further elucidate the controversial function of the protein encoded by PYPAF1, whose mutations (exclusively missense to date) have been identified in 3 hereditary recurrent fever syndromes. The patient's DNA was screened for mutations in both MEFV, the gene responsible for FMF, and PYPAF1. The ability of different recombinant PYPAF1 isoforms, expressed in HEK 293 cells, to regulate NF-kappaB signaling was subsequently assessed. No disease-causing mutation was found in MEFV. However, a nonsense mutation (p.Arg554X) was identified in PYPAF1; this defect resulted in a truncated protein lacking all leucine-rich repeats. Study of the wild-type and mutant PYPAF1 recombinant proteins revealed that PYPAF1 inhibited NF-kappaB proinflammatory pathways, and that the identified nonsense mutation impaired this property. These molecular and clinical findings, together with the clinical manifestations in the patient, which call into question the current nosology of the hereditary recurrent fever syndromes, are consistent with the hypothesis that PYPAF1 acts as an inhibitor of NF-kappaB signaling. They also provide a clear elucidation of the functional consequences of this nonsense PYPAF1 mutation not previously described in the literature, which result in a partial loss of function and may thereby explain the pathophysiology of the autoinflammatory syndrome observed in this patient.

Saccharomyces cerevisiae Est3p dimerizes in vitro and dimerization contributes to efficient telomere replication in vivo

In Saccharomyces cerevisiae at least five genes, EST1, EST2, EST3, TLC1 and CDC13, are required for telomerase activity in vivo. The telomerase catalytic subunit Est2p and telomerase RNA subunit Tlc1 constitute the telomerase core enzyme. Est1p and Est3p are the other subunits of telomerase holoenzyme. In order to dissect the function of Est3p in telomere replication, we over-expressed and purified recombinant wild-type and mutant Est3 proteins. The wild-type protein, as well as the K71A, E104A and T115A mutants were able to dimerize in vitro, while the Est3p-D49A, -K68A or -D166A mutant showed reduced ability to dimerize. Mutations in Est3p that decreased dimerization also appeared to cause telomere shortening in vivo. Double point mutation of Est3p-D49A-K68A and single point mutation of Est3p-K68A showed similar telomere shortening, suggesting that the K68 residue might be more important for telomerase activity. The ectopic co-expression of K71A or T115A mutant with wild-type Est3p using centromere plasmids caused telomere shortening, while co-expression of the D49A, K68A, D86A or F103A mutants with wild-type Est3p had no effect on telomere length regulation. These data suggested that dimerization is important for Est3p function in vivo.

The interaction site of Flap Endonuclease-1 with WRN helicase suggests a coordination of WRN and PCNA

Werner and Bloom syndromes are genetic RecQ helicase disorders characterized by genomic instability. Biochemical and genetic data indicate that an important protein interaction of WRN and Bloom syndrome (BLM) helicases is with the structure-specific nuclease Flap Endonuclease 1 (FEN-1), an enzyme that is implicated in the processing of DNA intermediates that arise during cellular DNA replication, repair and recombination. To acquire a better understanding of the interaction of WRN and BLM with FEN-1, we have mapped the FEN-1 binding site on the two RecQ helicases. Both WRN and BLM bind to the extreme C-terminal 18 amino acid tail of FEN-1 that is adjacent to the PCNA binding site of FEN-1. The importance of the WRN/BLM physical interaction with the FEN-1 C-terminal tail was confirmed by functional interaction studies with catalytically active purified recombinant FEN-1 deletion mutant proteins that lack either the WRN/BLM binding site or the PCNA interaction site. The distinct binding sites of WRN and PCNA and their combined effect on FEN-1 nuclease activity suggest that they may coordinately act with FEN-1. WRN was shown to facilitate FEN-1 binding to its preferred double-flap substrate through its protein interaction with the FEN-1 C-terminal binding site. WRN retained its ability to physically bind and stimulate acetylated FEN-1 cleavage activity to the same extent as unacetylated FEN-1. These studies provide new insights to the interaction of WRN and BLM helicases with FEN-1, and how these interactions might be regulated with the PCNA–FEN-1 interaction during DNA replication and repair.

A Natural Variant Sequence in the AHSP Gene May Impact Severity of Beta-Thalassemia.

Alpha-hemoglobin stabilizing protein (AHSP) is an erythroid-specific molecular chaperone that binds the alpha chains of hemoglobin, preventing their precipitation and deleterious effects. Loss of AHSP exacerbates alpha globin precipitation and anemia in a murine model for beta thalassemia. In vitro, recombinant AHSP inhibits the production of reactive oxygen species (ROS) by alpha hemoglobin. To further define the role of AHSP as a modifier of beta-thalassemia, we analyzed AHSP sequences for mutations in a large population of beta-thalassemic and control subjects. A single nucleotide change, from A to T at position 12888 (Genebank accession number AC106.730.2) in the third exon of the AHSP gene was identified, converting asparagine 75 to leucine (N75I). We detected a patient who was heterozygous for both beta-thalassemia (b39/bA) and the AHSP-N75I mutation. She presented with an unusually severe anemia that required regular blood transfusion. The father is hematologically normal, without any thalassemia mutation and heterozygous for AHSP-N751. The mother is heterozygous for the beta 39 mutation with classical mild hypochromic microcytic anemia and no mutation in the AHSP gene. Of 282 unrelated control subjects tested, one (0.35%) contained the N75I mutation. Analysis of red blood cells from this subject revealed normal hemoglobin indices but a small number of Heinz bodies, suggesting hemoglobin precipitation. This finding is similar to what we observed previously in mice that are heterozygous for an AHSP null allele. To investigate how the N75I mutation affects AHSP function, we studied the biochemical properties of the recombinant mutant protein. Surface plasmon resonance showed that the binding affinity of AHSP N75I for alpha hemoglobin is normal. Importantly, compared to wild type AHSP, the N75I mutant protein exhibited significantly reduced capacity to inhibit ROS production by alpha hemoglobin. Hence, AHSP N75I may be less effective at conferring protection from oxidative-mediated damage by free alpha hemoglobin in erythrocytes. These effects, when coupled with beta thalassemia, could result in more severe anemia, implicating AHSP N75I as a potential genetic modifier.

Crystallization and preliminary crystallographic analysis of a family 43 beta-D-xylosidase from Geobacillus stearothermophilus T-6.

Beta-D-Xylosidases (EC 3.2.1.37) are hemicellulases that cleave single xylose units from the nonreducing end of xylooligomers. In this study, the crystallization and preliminary X-ray analysis of a beta-D-xylosidase from Geobacillus stearothermophilus T-6 (XynB3), a family 43 glycoside hydrolase, is described. XynB3 is a 535-amino-acid protein with a calculated molecular weight of 61 891 Da. Purified recombinant native and catalytic inactive mutant proteins were crystallized and cocrystallized with xylobiose in two different space groups, P2(1)2(1)2 (unit-cell parameters a = 98.32, b = 99.36, c = 258.64 A) and P4(1)2(1)2 (or the enantiomorphic space group P4(3)2(1)2; unit-cell parameters a = b = 140.15, c = 233.11 A), depending on the detergent. Transferring crystals to cryoconditions required a very careful protocol. Orthorhombic crystals diffract to 2.5 A and tetragonal crystals to 2.2 A.

Structural basis for UTP specificity of RNA editing TUTases from Trypanosoma brucei.

Trypanosomatids are pathogenic protozoa that undergo a unique form of post-transcriptional RNA editing that inserts or deletes uridine nucleotides in many mitochondrial pre-mRNAs. Editing is catalyzed by a large multiprotein complex, the editosome. A key editosome enzyme, RNA editing terminal uridylyl transferase 2 (TUTase 2; RET2) catalyzes the uridylate addition reaction. Here, we report the 1.8 A crystal structure of the Trypanosoma brucei RET2 apoenzyme and its complexes with uridine nucleotides. This structure reveals that the specificity of the TUTase for UTP is determined by a crucial water molecule that is exquisitely positioned by the conserved carboxylates D421 and E424 to sense a hydrogen atom on the N3 position of the uridine base. The three-domain structure also unveils a unique domain arrangement not seen before in the nucleotidyltansferase superfamily, with a large domain insertion between the catalytic aspartates. This insertion is present in all trypanosomatid TUTases. We also show that TbRET2 is essential for survival of the bloodstream form of the parasite and therefore is a potential target for drug therapy.

Apoptotic cell clearance by phagocytes

Macrophages and dendritic cells recognize not only microorganisms foreign to the body but also dying self-cells, which they eliminate by engulfment. This phenomenon prevents dying cells from releasing potentially harmful or immunogenic intracellular materials. We found that milk fat globule-EGF-factor 8 (MFG-E8) linked apoptotic cells and phagocytes, and promoted the phagocytosis of cell corpses. MFG-E8 is expressed in tingible body macrophages (TBM) in the germinal centers of the spleen and lymph nodes. In MFG-E8-deficient mice, a large number of apoptotic lymphocytes were associated with TBM but were not engulfed, indicating that MFG-E8 was involved in the clearance of apoptotic B cells by TBM. The MFG-E8-deficient mice developed splenomegaly and produced autoantibodies such as antinuclear antibodies as well as anti-DNA antibodies in an age-dependent manner. To further examine the consequences of failure in apoptotic cell clearance, we produced a recombinant MFG-E8 mutant protein (D89E), which had inhibitory effects on the phagocytosis of apoptotic cells by a wide variety of phagocytes. When intravenously injected into mice, the D89E protein induced the production of autoantibodies. The autoantibodies production was enhanced by the coinjection of syngeneic apoptotic thymocytes. These results indicated that the impairment of apoptotic cell phagocytosis led to autoantibody production.

Functional Consequences of Hypertrophic and Dilated Cardiomyopathy-causing Mutations in α-Tropomyosin

To study the functional consequences of various cardiomyopathic mutations in human cardiac alpha-tropomyosin (Tm), a method of depletion/reconstitution of native Tm and troponin (Tn) complex (Tm-Tn) in cardiac myofibril preparations has been developed. The endogenous Tm-Tn complex was selectively removed from myofibrils and replaced with recombinant wild-type or mutant proteins. Successful depletion and reconstitution steps were verified by SDS-gel electrophoresis and by the loss and regain of Ca2+-dependent regulation of ATPase activity. Five Tm mutations were chosen for this study: the hypertrophic cardiomyopathy (HCM) mutations E62Q, E180G, and L185R and the dilated cardiomyopathy (DCM) mutations E40K and E54K. Through the use of this new depletion/reconstitution method, the functional consequences of these mutations were determined utilizing myofibrillar ATPase measurements. The results of our studies showed that 1) depletion of >80% of Tm-Tn from myofibrils resulted in a complete loss of the Ca2+-regulated ATPase activity and a significant loss in the maximal ATPase activity, 2) reconstitution of exogenous wild-type Tm-Tn resulted in complete regain in the calcium regulation and in the maximal ATPase activity, and 3) all HCM-associated Tm mutations increased the Ca2+ sensitivity of ATPase activity and all had decreased abilities to inhibit ATPase activity. In contrast, the DCM-associated mutations both decreased the Ca2+ sensitivity of ATPase activity and had no effect on the inhibition of ATPase activity. These findings have demonstrated that the mutations which cause HCM and DCM disrupt discrete mechanisms, which may culminate in the distinct cardiomyopathic phenotypes.

Crystal Structures Representing the Michaelis Complex and the Thiouronium Reaction Intermediate of Pseudomonas aeruginosa Arginine Deiminase

L-arginine deiminase (ADI) catalyzes the irreversible hydrolysis of L-arginine to citrulline and ammonia. In a previous report of the structure of apoADI from Pseudomonas aeruginosa, the four residues that form the catalytic motif were identified as Cys406, His278, Asp280, and Asp166. The function of Cys406 in nucleophilic catalysis has been demonstrated by transient kinetic studies. In this study, the structure of the C406A mutant in complex with L-arginine is reported to provide a snapshot of the enzyme.substrate complex. Through the comparison of the structures of apoenzyme and substrate-bound enzyme, a substrate-induced conformational transition, which might play an important role in activity regulation, was discovered. Furthermore, the position of the guanidinium group of the bound substrate relative to the side chains of His278, Asp280, and Asp166 indicated that these residues mediate multiple proton transfers. His278 and Asp280, which are positioned to activate the water nucleophile in the hydrolysis of the S-alkylthiouronium intermediate, were replaced with alanine to stabilize the intermediate for structure determination. The structures determined for the H278A and D280A mutants co-crystallized with L-arginine provide a snapshot of the S-alkylthiouronium adduct formed by attack of Cys406 on the guanidinium carbon of L-arginine followed by the elimination of ammonia. Asp280 and Asp166 engage in ionic interactions with the guanidinium group in the C406A ADI. L-arginine structure and might orient the reaction center and participate in proton transfer. Structure determination of D166A revealed the apoD166A ADI. The collection of structures is interpreted in the context of recent biochemical data to propose a model for ADI substrate recognition and catalysis.

Leishmania major thialysine Nε-acetyltransferase: Identification of amino acid residues crucial for substrate binding

Thialysine N ε-acetyltransferases and spermidine/spermine N-acetyltransferases (SSAT) are closely related members of the GCN5-related N-acetyltransferase superfamily. Accordingly, a putative orthologue from the human protozoan parasite Leishmania major exhibits an almost equal similarity to human SSAT and thialysine N ε-acetyltransferase. Characterisation of the recombinantly expressed L. major protein indicated that it represents a thialysine N ε-acetyltransferase, preferring thialysine ( S-aminoethyl- l-cysteine) and structurally related amino acids as acceptor molecules. The known thialysine N ε-acetyltransferases contain five conserved amino acid residues that are replaced in SSAT sequences. Kinetic analyses of the respective recombinant mutant proteins suggest that Ser 82 and Thr 83 of L. major thialysine N ε-acetyltransferase are key residues for acceptor binding. In addition, the conserved Leu 130 is tentatively involved in specific interaction with the sulphur-containing side chain of thialysine. The presence of these three amino acid residues is suggested to be a means by which thialysine N ε-acetyltransferases can be distinguished from SSAT sequences.

Dilated Cardiomyopathy Mutations in Three Thin Filament Regulatory Proteins Result in a Common Functional Phenotype

Dilated cardiomyopathy (DCM), characterized by cardiac dilatation and contractile dysfunction, is a major cause of heart failure. Inherited DCM can result from mutations in the genes encoding cardiac troponin T, troponin C, and alpha-tropomyosin; different mutations in the same genes cause hypertrophic cardiomyopathy. To understand how certain mutations lead specifically to DCM, we have investigated their effect on contractile function by comparing wild-type and mutant recombinant proteins. Because initial studies on two troponin T mutations have generated conflicting findings, we analyzed all eight published DCM mutations in troponin T, troponin C, and alpha-tropomyosin in a range of in vitro assays. Thin filaments, reconstituted with a 1:1 ratio of mutant/wild-type proteins (the likely in vivo ratio), all showed reduced Ca(2+) sensitivity of activation in ATPase and motility assays, and except for one alpha-tropomyosin mutant showed lower maximum Ca(2+) activation. Incorporation of either of two troponin T mutants in skinned cardiac trabeculae also decreased Ca(2+) sensitivity of force generation. Structure/function considerations imply that the diverse thin filament DCM mutations affect different aspects of regulatory function yet change contractility in a consistent manner. The DCM mutations depress myofibrillar function, an effect fundamentally opposite to that of hypertrophic cardiomyopathy-causing thin filament mutations, suggesting that decreased contractility may trigger pathways that ultimately lead to the clinical phenotype.

Key Role of Conserved Histidines in Recombinant Mouse β-Carotene 15,15′-Monooxygenase-1 Activity

Alignment of sequences of vertebrate beta-carotene 15,15'-monooxygenase-1 (BCMO1) and related oxygenases revealed four perfectly conserved histidines and five acidic residues (His172, His237, His308, His514, Asp52, Glu140, Glu314, Glu405, and Glu457 in mouse BCMO1). Because BCMO1 activity is iron-dependent, we propose that these residues participate in iron coordination and therefore are essential for catalytic activity. To test this hypothesis, we produced mutant forms of mouse BCMO1 by replacing the conserved histidines and acidic residues as well as four histidines and one glutamate non-conserved in the overall family with alanines by site-directed mutagenesis. Our in vitro and in vivo data showed that mutation of any of the four conserved histidines and Glu405 caused total loss of activity. However, mutations of non-conserved histidines or any of the other conserved acidic residues produced impaired although enzymatically active proteins, with a decrease in activity mostly due to changes in V(max). The iron bound to protein was determined by inductively coupled plasma atomic emission spectrometry. Bound iron was much lower in preparations of inactive mutants than in the wild-type protein. Therefore, the conserved histidines and Glu405 are absolutely required for the catalytic mechanism of BCMO1. Because the mutant proteins are impaired in iron binding, these residues are concluded to coordinate iron required for catalytic activity. These data are discussed in the context of the predicted structure for the related eubacterial apocarotenal oxygenase.

Biochemical Analysis of the DNA Unwinding and Strand Annealing Activities Catalyzed by Human RECQ1*[boxs

RecQ helicases play an important role in preserving genomic integrity, and their cellular roles in DNA repair, recombination, and replication have been of considerable interest. Of the five human RecQ helicases identified, three are associated with genetic disorders characterized by an elevated incidence of cancer or premature aging: Werner syndrome, Bloom syndrome, and Rothmund-Thomson syndrome. Although the biochemical properties and protein interactions of the WRN and BLM helicases defective in Werner syndrome and Bloom syndrome, respectively, have been extensively investigated, less information is available concerning the functions of the other human RecQ helicases. We have focused our attention on human RECQ1, a DNA helicase whose cellular functions remain largely uncharacterized. In this work, we have characterized the DNA substrate specificity and optimal cofactor requirements for efficient RECQ1-catalyzed DNA unwinding and determined that RECQ1 has certain properties that are distinct from those of other RecQ helicases. RECQ1 stably bound to a variety of DNA structures, enabling it to unwind a diverse set of DNA substrates. In addition to its DNA binding and helicase activities, RECQ1 catalyzed efficient strand annealing between complementary single-stranded DNA molecules. The ability of RECQ1 to promote strand annealing was modulated by ATP binding, which induced a conformational change in the protein. The enzymatic properties of the RECQ1 helicase and strand annealing activities are discussed in the context of proposed cellular DNA metabolic pathways that are important in the maintenance of genomic stability.