Flexible C-terminal Region Research Articles

The Tail of KdsC: CONFORMATIONAL CHANGES CONTROL THE ACTIVITY OF A HALOACID DEHALOGENASE SUPERFAMILY PHOSPHATASE

The phosphatase KdsC cleaves 3-deoxy-D-manno-octulosonate 8-phosphate to generate a molecule of inorganic phosphate and Kdo. Kdo is an essential component of the lipopolysaccharide envelope in Gram-negative bacteria. Because lipopolysaccharide is an important determinant of bacterial resistance and toxicity, KdsC is a potential target for novel antibacterial agents. KdsC belongs to the broad haloacid dehalogenase superfamily. In haloacid dehalogenase superfamily enzymes, substrate specificity and catalytic efficiency are generally dictated by a fold feature called the cap domain. It is therefore not clear why KdsC, which lacks a cap domain, is catalytically efficient and highly specific to 3-deoxy-D-manno-octulosonate 8-phosphate. Here, we present a set of seven structures of tetrameric Escherichia coli KdsC (ranging from 1.4 to 3.06 A in resolution) that model different intermediate states in its catalytic mechanism. A crystal structure of product-bound E. coli KdsC shows how the interface between adjacent monomers defines the active site pocket. Kdo is engaged in a network of polar and nonpolar interactions with residues at this interface, which explains substrate specificity. Furthermore, this structural and kinetic analysis strongly suggests that the binding of the flexible C-terminal region (tail) to the active site makes KdsC catalytically efficient by facilitating product release.

Structure–function correlation of human programmed cell death 5 protein

Human programmed cell death 5 (PDCD5) is a translocatory protein playing an important role in the apoptotic process of cells. Although there are accumulated data about PDCD5 function, the correlation of the structure with the function of PDCD5 has not been investigated. Here, we report the studies of structure–function relationship of PDCD5 by multidimensional NMR methods and by FACScan flow cytometer and fluorescence microscope. The 3D structure of intact PDCD5 and the internal motions of PDCD5 have been determined. PDCD5 has a compact core structure of low flexibility with two mobile α-helices at N-terminal region and a flexible unstructured C-terminal region. The flow cytometry and internalization measurements of different PDCD5 fragments indicate that the charged residues are crucial for the ability of apoptosis-promoting and cell translocation of the protein. Combined analyses reveal a fact that the regions that seem to be most involved in the function also are more flexible in PDCD5.

Exceptional Amyloid β Peptide Hydrolyzing Activity of Nonphysiological Immunoglobulin Variable Domain Scaffolds

Nucleophilic sites in the paired variable domains of the light and heavy chains (VL and VH domains) of Ig can catalyze peptide bond hydrolysis. Amyloid beta (Abeta)-binding Igs are under consideration for immunotherapy of Alzheimer disease. We searched for Abeta-hydrolyzing human IgV domains (IgVs) in a library containing a majority of single chain Fv clones mimicking physiological VL-VH-combining sites and minority IgV populations with nonphysiological structures generated by cloning errors. Random screening and covalent selection of phage-displayed IgVs with an electrophilic Abeta analog identified rare IgVs that hydrolyzed Abeta mainly at His14-Gln15. Inhibition of IgV catalysis and irreversible binding by an electrophilic hapten suggested a nucleophilic catalytic mechanism. Structural analysis indicated that the catalytic IgVs are nonphysiological structures, a two domain heterodimeric VL (IgVL2-t) and single domain VL clones with aberrant polypeptide tags (IgVL-t'). The IgVs hydrolyzed Abeta at rates superior to naturally occurring Igs by 3-4 orders of magnitude. Forced pairing of the single domain VL with VH or VL domains resulted in reduced Abeta hydrolysis, suggesting catalysis by the unpaired VL domain.Angstrom level amino acid displacements evident in molecular models of the two domain and unpaired VL domain clones explain alterations of catalytic activity. In view of their superior catalytic activity, the VL domain IgVs may help attain clearance of medically important antigens more efficiently than natural Igs.

Importance of C-terminal flexible region of 4E-binding protein in binding with eukaryotic initiation factor 4E

Although the α-helical Y(X)4Lϕ containing region of eIF4E-binding protein (4EBP) is the major binding region with eukaryotic initiation factor 4E (eIF4E), the roles of its N- and C-terminal regions in the binding are hardly known. To clarify the roles of these flexible regions in the interaction, the binding features of the sequentially N-, C-, or both-terminal-residue-deleted 4EBP2 mutants were investigated by surface plasmon resonance (SPR) analysis. It was shown that the C-terminal His74-Glu89 sequence has an auxiliary, but indispensable, function in stabilizing the binding to eIF4E. The possible interaction with eIF4E was estimated by molecular dynamics simulation. This is the first report on the importance of the C-terminal flexible region in the eIF4E-binding regulation of 4EBP.

Identification of the Functionally Critical Amino Acid Segment and its Role in the Flexible C-Terminal Region of the Chaperonin GroEL

final 23 residues ( 526 KNDAADLGAAGGMGGMGGMGGMM 548 ) in the C-terminal region of the GroEL are invisible in crystallographic analyses due to high flexibility. In order to clarify the functional role of these residues in the chaperonin mechanism, we generated and characterized C-terminal truncated, double ring and single ring mutants of GroEL. The ability to assist the refolding of substrate proteins rhodanese and malate dehydrogenase decreased suddenly when 23 amino acids were truncated, indicating that a sudden change in the environment within the central cavity had occurred. From further experiments and analyses of the hydropathy of the C-terminal region, we focused on the hydrophilicity of the sequence region 526 KNDAAD 531 and generated two GroEL mutants where these residues were changed to a neutral hydropathy sequence ( 526 GGGAAG 531 )and a hydrophobic sequence ( 526 IGIAAI 531 ), respectively. Very interestingly, the two mutants were found to be defective in function both in vitro and in vivo. Deterioration of function was not observed in mutants where this region was replaced by a scrambled ( 526 NKADDA 531 ) or homologous ( 526 RQEGGE 531 ) sequence, indicating that the hydrophilicity of this sequence was important. These results highlight the importance of the hydrophilic nature of 526 KNDAAD 531 residues in the flexible C-terminal region for proper protein folding within the central cavity of GroEL. doi:10.4172/jpb.s1000191

Hydrophilic Residues 526KNDAAD531 in the Flexible C-terminal Region of the Chaperonin GroEL Are Critical for Substrate Protein Folding within the Central Cavity

The final 23 residues in the C-terminal region of Escherichia coli GroEL are invisible in crystallographic analyses due to high flexibility. To probe the functional role of these residues in the chaperonin mechanism, we generated and characterized C-terminal truncated, double ring, and single ring mutants of GroEL. The ability to assist the refolding of substrate proteins rhodanese and malate dehydrogenase decreased suddenly when 23 amino acids were truncated, indicating that a sudden change in the environment within the central cavity had occurred. From further experiments and analyses of the hydropathy of the C-terminal region, we focused on the hydrophilicity of the sequence region (26 KNDAAD 531 and generated two GroEL mutants where these residues were changed to a neutral hydropathy sequence (526 GGGAAG 531) and a hydrophobic sequence (526 IGIAAI 531), respectively. Very interestingly, the two mutants were found to be defective in function both in vitro and in vivo. Deterioration of function was not observed in mutants where this region was replaced by a scrambled (526 NKADDA 531) or homologous (526 RQEGGE 531) sequence, indicating that the hydrophilicity of this sequence was important. These results highlight the importance of the hydrophilic nature of 526 KNDAAD 531 residues in the flexible C-terminal region for proper protein folding within the central cavity of GroEL.

NELF-E RRM Undergoes Major Structural Changes in Flexible Protein Regions on Target RNA Binding

The E subunit of the human heterotetrameric negative transcription elongation factor (NELF-E) contains a canonical betaalphabetabetaalphabeta RNA recognition motif (RRM) that binds to a wide variety of RNA sequences. These induce very similar conformational changes in the RRM as determined by nuclear magnetic resonance spectroscopy. Although the RNA binding interface of a canonical RRM is mainly located at its beta-sheet surface, for NELF-E RRM large chemical shift perturbations are observed for residues in the flexible C-terminal region and the loop between beta 3 and alpha 2, and both regions are distant from the interface. We determined the solution structure of single-stranded transactivator responsive element (TAR) RNA-bound NELF-E RRM. This structure clearly shows that RNA binding to NELF-E RRM induces formation of a helix in the C-terminus. The RNA-bound form of NELF-E RRM is very similar to the RNA-bound form of U1A RRM, although the C-terminus of the NELF-E RRM is unstructured in the free protein, whereas it is helical in the U1A protein. Thus, RNA binding to NELF-E RRM induces a conformational change toward the U1A structure, resulting in highly similar RNA binding conformations for both proteins.

The solution structure of the periplasmic domain of the TonB system ExbD protein reveals an unexpected structural homology with siderophore‐binding proteins

The transport of iron complexes through outer membrane transporters from Gram-negative bacteria is highly dependent on the TonB system. Together, the three components of the system, TonB, ExbB and ExbD, energize the transport of iron complexes through the outer membrane by utilizing the proton motive force across the cytoplasmic membrane. The three-dimensional (3D) structure of the periplasmic domain of TonB has previously been determined. However, no detailed structural information for the other two components of the TonB system is currently available and their role in the iron-uptake process is not yet clearly understood. ExbD from Escherichia coli contains 141 residues distributed in three domains: a small N-terminal cytoplasmic region, a single transmembrane helix and a C-terminal periplasmic domain. Here we describe the first well-defined solution structure of the periplasmic domain of ExbD (residues 44-141) solved by multidimensional nuclear magnetic resonance (NMR) spectroscopy. The monomeric structure presents three clearly distinct regions: an N-terminal flexible tail (residues 44-63), a well-defined folded region (residues 64-133) followed by a small C-terminal flexible region (residues 134-141). The folded region is formed by two alpha-helices that are located on one side of a single beta-sheet. The central beta-sheet is composed of five beta-strands, with a mixed parallel and antiparallel arrangement. Unexpectedly, this fold closely resembles that found in the C-terminal lobe of the siderophore-binding proteins FhuD and CeuE. The ExbD periplasmic domain has a strong tendency to aggregate in vitro and 3D-TROSY (transverse relaxation optimized spectroscopy) NMR experiments of the deuterated protein indicate that the multimeric protein has nearly identical secondary structure to that of the monomeric form. Chemical shift perturbation studies suggest that the Glu-Pro region (residues 70-83) of TonB can bind weakly to the surface and the flexible C-terminal region of ExbD. At the same time the Lys-Pro region (residues 84-102) and the folded C-terminal domain (residues 150-239) of TonB do not show significant binding to ExbD, suggesting that the main interactions forming the TonB complex occur in the cytoplasmic membrane.

3D structure of Thermus aquaticus single-stranded DNA-binding protein gives insight into the functioning of SSB proteins.

In contrast to the majority of tetrameric SSB proteins, the recently discovered SSB proteins from the Thermus/Deinoccus group form dimers. We solved the crystal structures of the SSB protein from Thermus aquaticus (TaqSSB) and a deletion mutant of the protein and show the structure of their ssDNA binding domains to be similar to the structure of tetrameric SSBs. Two conformations accompanied by proline cis–trans isomerization are observed in the flexible C-terminal region. For the first time, we were able to trace 6 out of 10 amino acids at the C-terminus of an SSB protein. This highly conserved region is essential for interaction with other proteins and we show it to adopt an extended conformation devoid of secondary structure. A model for binding this region to the χ subunit of DNA polymerase III is proposed. It explains at a molecular level the reason for the ssb113 phenotype observed in Escherichia coli.

Structural Basis for Nucleic Acid and Toxin Recognition of the Bacterial Antitoxin CcdA

Toxin–antitoxin systems are highly abundant in plasmids and bacterial chromosomes. They ensure plasmid maintenance by killing bacteria that have lost the plasmid. Their expression is autoregulated at the level of transcription. Here, we present the solution structure of CcdA, the antitoxin of the ccd system, as a free protein (16.7 kDa) and in complex with its cognate DNA (25.3 kDa). CcdA is composed of two distinct and independent domains: the N-terminal domain, responsible for DNA binding, which establishes a new family of the ribbon-helix-helix fold and the C-terminal region, which is responsible for the interaction with the toxin CcdB. The C-terminal domain is intrinsically unstructured and forms a tight complex with the toxin. We show that CcdA specifically recognizes a 6 bp palindromic DNA sequence within the operator–promoter (OP) region of the ccd operon and binds to DNA by insertion of the positively charged N-terminal β-sheet into the major groove. The binding of up to three CcdA dimers to a 33mer DNA of its operator–promoter region was studied by NMR spectroscopy, isothermal titration calorimetry and single point mutation. The highly flexible C-terminal region of free CcdA explains its susceptibility to proteolysis by the Lon ATP-dependent protease.

1P072 Flexible C-terminal region of GroEL contributes to maintaining the environment of central cavity for proper protein folding(2. Protein function (I),Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)

1P072 Flexible C-terminal region of GroEL contributes to maintaining the environment of central cavity for proper protein folding(2. Protein function (I),Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)

Nitrotyrosination of plant α-tubulin: potential mechanisms of influence on cellular processes

3-Nitrotyrosine, the product of interaction between the free tyrosine with nitric oxide, is characterized by its posttranslational incorporation into carboxyl terminus of α-tubulin molecule via tubulin-tyrosine ligase, similar to unmodified tyrosine residue. However, α-tubulin C-terminal nitrotyrosination is irreversible unlike an enzymatically regulated tyrosination/detyrosination cycle. The nitrotyrosination of α-tubulin results in an appearance of some changes in animal cell morphology and their microtubule organization, a loss of some cellular functions, and in intracellular redistribution of cytoplasmic dynein. Since, nitrotyrosination in plant cell has not been investigated, the aim of our study was to reconstruct the spatial structure of nitrotyrosinated plant α-tubulin with following modeling of possible mechanisms of this posttranslational modification influence on cellular processes. The last 10–12 amino acid residues of α-tubulin C-terminal region, unlike the main part of the molecule, form a free oscillating tail that can be considered as individual structural domain. The results of reconstructing the spatial structure of detyrosinated, tyrosinated and nitrotyrosinated goosegrass (Eleusine indica (L.) Gaerth.) α-tubulin forms and studies of their behavior testify that these modifications change substantially the level of mobility of flexible α-tubulin C-terminal domain. We found that tyrosination increases substantially the mobility of C-terminus (see Figure ​Figure11). Figure 1 Modelling of different allowed conformation of C-terminal tails of α-tubulin from goosegrass: A. Detyrosinated molecule; B. Tyrosinated molecule; C. Nitrotyrosinated molecule The root mean square deviation (RMSD) in coordinates of amino acid residues composing this region grows from 0.48 A for detyrosinated form, to 0.55 A for tyrosinated α-tubulin as was calculated on the basis of molecular fluctuations trajectory in 500 ps interval. These results correlate with data about predomination of detyrosinated α-tubulin in long-time living microtubules and vice versa. The breakdown of tyrosination/detyrosination cycle due to incorporation the nitrotyrosine into C-terminus should resulted in predomination of short-time living microtubules over long-time living microtubules. It is necessary to note that mobility of nitrotyrosinated terminus is lower than the tyrosinated one, but it is higher than the detyrosinated terminus. In the last case RMSD is 0.52 A. It can lead to the appearance of microtubules with a certain intermediate lifetime and, as a consequence, modify the microtubules organization in cells. It is known that the tubulin C-terminal region is responsible for interactions with structural and motor MAPs. In combination with the above mentioned data about the redistribution of cytoplasmic dynein after nitrotyrosination of α-tubulin we can speculate that nitrotyrosination can influence processes of tubulin-MAP recognition and interaction. This assumption is confirmed by our results on modeling the interaction of α-tubulin with the MT-binding domain of dynein heavy chains. The high content of dicarboxylic amino acid residues, especially glutamate residues, is the hallmark of the flexible C-terminal region of both tubulin subunits. It has to be correlated with the necessity of presence, a surface enriched with diamidate amino acid residues in the dynein MT-binding domain structure. The surface of dynein MT-binding domain formed by residues Lys3148, Lys3151, Lys3154, Lys3155, Arg3162, Lys3287, Lys3300, Arg3327, Lys3328 is correspondent to this factor. The surface is flanked by residues Asp3144, Asp3146 and Asp3320, Asp3321 on both sides. Included in nitrotyrosine nitro-group possesses an additional negative charge, that is not typical for dynein-tubulin interaction, and locates close to flanking residues. This location results in a strong decrease in the affinity between the dynein contact surface and α-tubulin C-terminal region.

Characterization of the Structure and the Amyloidogenic Properties of the Josephin Domain of the Polyglutamine-containing Protein Ataxin-3

Expansion of the polyglutamine (polyQ) region in the protein ataxin-3 is associated with spinocerebellar ataxia type 3, an inherited neurodegenerative disorder that belongs to the family of polyQ diseases. Increasing evidence indicates that protein aggregation and fibre formation play an important role in these pathologies. In a previous study, we determined the domain architecture of ataxin-3, suggesting that it comprises a globular domain, named Josephin, and a more flexible C-terminal region, that includes the polyQ tract. Here, we have characterised for the first time the biophysical properties of the isolated Josephin motif, showing that it is an autonomously folded unit and that it has no significant interactions with the C-terminal region. Study of its thermodynamic stability indicates that Josephin has an intrinsic tendency to aggregate and forms temperature-induced fibrils similar to those described for expanded ataxin-3. We show that, under destabilising conditions, the behaviours of the isolated Josephin domain and ataxin-3 are extremely similar. Our data therefore strongly suggest that the stability and aggregation properties of non-expanded ataxin-3 are determined by those of the Josephin domain, which is sufficient to reproduce the behaviour of the full-length protein. Our data support a mechanism in which the thermodynamic stability of ataxin-3 is governed by the properties of the Josephin domain, but the presence of an expanded polyQ tract increases dramatically the protein's tendency to aggregate.

The effect of an ionic detergent on the natively unfolded beta-dystroglycan ectodomain and on its interaction with alpha-dystroglycan.

Dystroglycan (DG) is an adhesion complex, expressed in a wide variety of tissues, formed by an extracellular and a transmembrane subunit, alpha-DG and beta-DG, respectively, interacting noncovalently. Recently, we have shown that the recombinant ectodomain of beta-DG, beta-DG(654-750), behaves as a natively unfolded protein, as it is able to bind the C-terminal domain of alpha-DG, while not displaying a defined structural organization. We monitored the effect of a commonly used denaturing agent, the anionic detergent sodium dodecylsulphate (SDS), on beta-DG(654-750) using a number of biophysical techniques. Very low concentrations of SDS (< or =2 mM) affect both tryptophan fluorescence and circular dichroism of beta-DG, and significantly perturb the interaction with the alpha-DG subunit as shown by solid-phase binding assays and fluorescence titrations in solution. This result confirms, as recently proposed for natively unfolded proteins, that beta-DG(654-750) exists in a native state, which is crucial to fulfill its biological function. Two-dimensional NMR analysis shows that SDS does not induce any evident conformational rearrangement within the ectodomain of beta-DG. Its first 70 amino acids, which show a lower degree of mobility, interact with the detergent, but this does not change the amount of secondary structure, whereas the highly flexible and mobile C-terminal region of beta-DG(654-750) remains largely unaffected, even at a very high SDS concentration (up to 50 mM). Our data indicate that SDS can be used as a useful tool for investigating natively unfolded proteins, and confirm that the beta-DG ectodomain is an interesting model system.

Structural Features of Human Initiation Factor 4E, Studied by X-ray Crystal Analyses and Molecular Dynamics Simulations

The structural features of human eIF4E were investigated by X-ray crystal analyses of its cap analog (m 7GTP and m 7GpppA) complexes and molecular dynamics (MD) simulations of cap-free and cap-bound eIF4Es, as well as the cap-bound Ser209-phosphorylated eIF4E. Crystal structure analyses at 2.0 Å resolution revealed that the molecule forms a temple-bell-shaped surface of eight antiparallel β-structures, three α-helices and ten loop structures, where the N-terminal region corresponds to the handle of the bell. This concave backbone provides a scaffold for the mRNA cap-recognition pocket consisting of three receiving parts for the 5′-terminal m 7G base, the triphosphate, and the second nucleotide. The m 7G base is sandwiched between the two aromatic side-chains of Trp102 and Trp56. The two (m 7G)NH–O (Glu103 carboxy group) hydrogen bonds stabilize the stacking interaction. The basic residues of Arg157 and Lys162 and water molecules construct a binding pocket for the triphosphate moiety, where a universal hydrogen-bonding network is formed. The flexible C-terminal loop region unobserved in the m 7GTP complex was clearly observed in the m 7GpppA complex, as a result of the fixation of this loop by the interaction with the adenosine moiety, indicating the function of this loop as a receiving pocket for the second nucleotide. On the other hand, MD simulation in an aqueous solution system revealed that the cap-binding pocket, especially its C-terminal loop structure, is flexible in the cap-free eIF4E, and the entrance of the cap-binding pocket becomes narrow, although the depth is relatively unchanged. SDS-PAGE analyses showed that this structural instability is highly related to the fast degradation of cap-free eIF4E, compared with cap-bound or 4E-BP/cap-bound eIF4E, indicating the conferment of structural stability of eIF4E by the binary or ternary complex formation. MD simulation of m 7GpppA-bound Ser209-phosphorylated eIF4E showed that the size of the cap-binding entrance is dependent on the ionization state in the Ser209 phosphorylation, which is associated with the regulatory function through the switching on/off of eIF4E phosphorylation.

Analysis of a Plasmodium falciparum EBA-175 peptide with high binding capacity to erythrocytes and their analogues using 1H NMR

A 175-erythrocyte-binding protein (EBA-175) conserved high-activity binding peptide (HABP), called 1783 (nonimmunogenic, nonprotective against Plasmodium falciparum malaria), was analyzed for antigenic and protective activity in Aotus monkeys, together with several of its analogues. 1 H NMR studies of peptides 17912, 14016, and 22814 allowed their structure to be related to their biological function. These peptides showed helical regions having differences in their position and length. Nonimmunogenic, nonprotective peptides 1783 and 17912 showed an extensive helical region, while the 22814 immunogenic protective peptide’s α-helix was found in the N-terminal region. This suggests that the more flexible C-terminal region will allow better interaction between these peptides and immune system molecules as well as relating these peptides’ three-dimensional structure to their immunogenicity and protective activity, thus leading to a more rational development of the new malaria multicomponent vaccine.

Crystal structures of 7-methylguanosine 5'-triphosphate (m(7)GTP)- and P(1)-7-methylguanosine-P(3)-adenosine-5',5'-triphosphate (m(7)GpppA)-bound human full-length eukaryotic initiation factor 4E: biological importance of the C-terminal flexible region.

The crystal structures of the full-length human eukaryotic initiation factor (eIF) 4E complexed with two mRNA cap analogues [7-methylguanosine 5'-triphosphate (m(7)GTP) and P(1)-7-methylguanosine-P(3)-adenosine-5',5'-triphosphate (m(7)GpppA)] were determined at 2.0 A resolution (where 1 A=0.1 nm). The flexibility of the C-terminal loop region of eIF4E complexed with m(7)GTP was significantly reduced when complexed with m(7)GpppA, suggesting the importance of the second nucleotide in the mRNA cap structure for the biological function of eIF4E, especially the fixation and orientation of the C-terminal loop region, including the eIF4E phosphorylation residue. The present results provide the structural basis for the biological function of both N- and C-terminal mobile regions of eIF4E in translation initiation, especially the regulatory function through the switch-on/off of eIF4E-binding protein-eIF4E phosphorylation.

Crystal structures of 7-methylguanosine 5′-triphosphate (m7GTP)- and P1-7-methylguanosine-P3-adenosine-5′,5′-triphosphate (m7GpppA)-bound human full-length eukaryotic initiation factor 4E: biological importance of the C-terminal flexible region

The crystal structures of the full-length human eukaryotic initiation factor (eIF) 4E complexed with two mRNA cap analogues [7-methylguanosine 5′-triphosphate (m7GTP) and P1-7-methylguanosine-P3-adenosine-5′,5′-triphosphate (m7GpppA)] were determined at 2.0Å resolution (where 1Å = 0.1nm). The flexibility of the C-terminal loop region of eIF4E complexed with m7GTP was significantly reduced when complexed with m7GpppA, suggesting the importance of the second nucleotide in the mRNA cap structure for the biological function of eIF4E, especially the fixation and orientation of the C-terminal loop region, including the eIF4E phosphorylation residue. The present results provide the structural basis for the biological function of both N- and C-terminal mobile regions of eIF4E in translation initiation, especially the regulatory function through the switch-on/off of eIF4E-binding protein—eIF4E phosphorylation.

The Flexible C-Terminal Region of Aspergillus terreus Blasticidin S Deaminase: Identification of Its Functional Roles with Deletion Enzymes

Although the four polypeptides of blasticidin S (BS) deaminase (BSD) are packed rather tightly coordinated to the “structural and catalytic” zinc atom of each subunit, the C-terminal region of the enzyme was suggested to be somewhat molten and flexible [M. Kimura, S. Sekido, Y. Isogai, and I. Yamaguchi (2000) J. Biochem. 127, 955–963]. To understand roles of this flexible region, we constructed five C-terminal deletion variants of BSD (each successively deleted from the C-terminal end up to five residues) and analyzed their biochemical properties focusing on the structure and activity of the enzyme. BSD and all of the deletion mutants showed the unique rigid conformation (e.g., characterized by their stabilities in SDS solution) and high levels of resistance against protease digestions. Furthermore, both the wild-type and deletion apoenzymes exhibited similar physical properties in thermodynamic refolding into the stable tetramer conformation. However, these small C-terminal deletions exerted deleterious effects on the catalytic efficiency of the enzyme as indicated by their strongly reduced kcat/Km value. Judging from the altered kinetic parameters and unaltered structural properties of the deletion variants, these C-terminal residues appear to be directly involved in enzyme–substrate interaction. In this short flexible region, Tyr-126, Trp-128, and Gly-130 were the key residues. Most notably, removal of Gly-130 markedly increased Km for BS without affecting its kcat value. These results indicate that the flexible C-terminal region is important for catalytic function and that a single Gly residue at the C-terminal end of BSD contributes significantly in facilitating access of a substrate to the active site.

The structure of human liver fructose-1,6-bisphosphate aldolase.

The X-ray crystallographic structure of the human liver isozyme of fructose-1,6-bisphosphate aldolase has been determined by molecular replacement using a tetramer of the human muscle isozyme as a search model. The liver aldolase (B isozyme) crystallized in space group C2, with unit-cell parameters a = 291.1, b = 489.8, c = 103.4 A, alpha = 90, beta = 103.6, gamma = 90 degrees. These large unit-cell parameters result from the presence of 18 subunits in the asymmetric unit: four catalytic tetramers and a dimer from a fifth tetramer positioned on the twofold crystallographic axis. This structure provides further insight into the factors affecting isozyme specificity. It reveals small differences in secondary structure that occur in regions previously determined to be isozyme specific. Two of these regions are at the solvent-exposed enzyme surface away from the active site of the enzyme. The most significant changes are in the flexible C-terminal region of the enzyme, where there is an insertion of an extra alpha-helix. Point mutations of the human liver aldolase are responsible for the disease hereditary fructose intolerance. Sequence information is projected onto the new crystal structure in order to indicate how these mutations bring about reduced enzyme activity and affect structural stability.