bluuebunny/crossref_metadata_2025 huggingface dataset:
Crystal structure of heat‐labile enterotoxin from Escherichia coli with increased thermostability introduced by an engineered disulfide bond in the A subunit
Authors: Van Den Akker F. , Feil I. K. , Roach C. , Platas A. A. , Merritt E. A. , Hol Wim G. J.
Published: February 2009

Cholera toxin (CT) produced by Vibrio cholerae and heat‐labile enterotoxin (LT‐I), produced by enterotoxigenic Escherichia coli, are AB5 heterohexamers with an ADP‐ribosylating A subunit and a GM1 receptor binding B pentamer. These toxins are among the most potent mucosal adjuvants known and, hence, are of interest both for the development of anti‐diarrheal vaccines against cholera or enterotoxigenic Escherichia coli diarrhea and also for vaccines in general. However, the A subunits of CT and LT‐I are known to be relatively temperature sensitive. To improve the thermostability of LT‐I an additional disulfide bond was introduced in the A1 subunit by means of the double mutation N40C and G166C. The crystal structure of this double mutant of LT‐I has been determined to 2.0 Å resolution. The protein structure of the N40C/G166C double mutant is very similar to the native structure except for a few local shifts near the new disulfide bond. The introduction of this additional disulfide bond increases the thermal stability of the A subunit of LT‐I by 6°C. The enhancement in thermostability could make this disulfide bond variant of LT‐I of considerable interest for the design of enterotoxin‐based vaccines.
Expression, characterization, and crystallization of a member of the novel phospholipase D family of phosphodiesterases
Authors: Zhao Yi , Stuckey Jeanne A. , Lohse Daniel L. , Dixon Jack E.
Published: February 2009

A family of phospholipase D (PLD) proteins has recently been identified (Koonin, 1996; Ponting & Kerr, 1996) based upon amino acid sequence identity. This family includes human and plant PLDs, proteins encoded by open reading frames in pathogenic viruses and bacteria, as well as an endonuclease. The endo‐nuclease, known as Nuc, is encoded by the IncN plasmid, pKM101, present in Salmonella typhimurium. The recombinant Nuc protein has been expressed and purified from Escherichia coli. The amino‐terminal sequencing of the purified protein indicated that the mature protein started from the 23rd residue of the predicted sequence, suggesting that the protein is proteolytically processed during export to the periplasmic space. The recombinant enzyme was able to hydrolyze both double and single‐strand DNA and an artificial substrate, bis(4‐nitrophenyl) phosphate, which contains a phosphodiester bond. The enzyme activity was not inhibited in the presence of EDTA and was not regulated by divalent cations. The purified protein has been crystallized by hanging drop vapor diffusion methods, and those crystals diffract to 1.9 Å resolution.
Complete measurement of the pKavalues of the carboxyl and imidazole groups inBacillus circulansxylanase
Authors: Joshi Manish D. , Hedberg Anders , Mcintosh Lawrence P.
Published: February 2009

Electrostatic interactions in proteins can be dissected experimentally by determining the pKavalues of their constituent ionizable amino acids. To complement previous studies of the glutamic acid and histidine residues inBacillus circulansxylanase (BCX), we have used NMR methods to measure the pKas of the seven aspartic acids and the C‐terminus of this protein. The pKas of these carboxyls are all less than the corresponding values observed with random coil polypeptides, indicating that their ionization contributes favorably to the stability of the folded enzyme. In general, the aspartic acids with the most reduced pKas are those with limited exposure to the solvent and a high degree of conservation among homologous xylanases. Most dramatically, Asp 83 and Asp 101 have pKas < 2 and thus remain deprotonated in native BCX under all conditions examined. Asp 83 is completely buried, forming a strong salt bridge with Arg 136. In contrast, Asp 101 is located on the surface of the protein, stabilized in the deprotonated form by an extensive network of hydrogen bonds involving an internal water molecule and the neutral side‐chain and main‐chain atoms of Ser 100 and Thr 145. These data provide a complete experimental database for theoretical studies of the ionization behavior of BCX under acidic conditions.
HIV‐1 Nef protein: Purification, crystallizations, and preliminary X‐ray diffraction studies
Authors: Franken P. , Arold S. , Padilla A. , Hoh E , Strub M. P. , Boyer M. , Jullien M. , Dumas C. , Bodeus M. , Benarous R.
Published: February 2009

Human immunodeficiency virus Nef protein accelerates virulent progression of AIDS by its interaction with specific cellular proteins involved in cellular activation and signal transduction. Here we report the purification and crystallization of the conserved core of HTV‐1LAI Nef protein in the unliganded form and in complex with the wild‐type SH3 domain of the p59fyn protein‐tyrosine kinase. One‐dimensional NMR experiments show that full‐length protein and truncated fragment corresponding to the product of HIV‐1 protease cleavage have a well‐folded compact tertiary structure. The ligand‐free HIV‐1 Nefcore protein forms cubic crystals belonging to space group P23 with unit cell dimensions of a = b = c = 86.4 Å. The Nef‐Fyn SH3 cocrystals belong to the space group P6122 or its enantiomorph, P6522, with unit cell dimensions of a = b = 108.2 Å and c = 223.7 Å. Both crystal forms diffract to a resolution limit of 3.0 Å resolution using synchrotron radiation, and are thus suitable for X‐ray structure determination.
Refolding rate of stability‐enhanced cytochrome c is independent of thermodynamic driving force
Authors: McGee William A. , Nall Barry T.
Published: February 2009

N52I iso‐2 cytochrome c is a variant of yeast iso‐2 cytochrome c in which asparagine substitutes for isoleucine 52 in an alpha helical segment composed of residues 49–56. The N52I substitution results in a significant increase in both stability and cooperativity of equilibrium unfolding, and acts as a “global suppressor” of destabilizing mutations. The equilibrium m‐value for denaturant‐induced unfolding of N52I iso‐2 increases by 30%, a surprisingly large amount for a single residue substitution. The folding/unfolding kinetics for N52I iso‐2 have been measured by stopped‐flow mixing and by manual mixing, and are compared to the kinetics of folding/unfolding of wild‐type protein, iso‐2 cytochrome c. The results show that the observable folding rate and the guanidine hydrochloride dependence of the folding rate are the same for iso‐2 and N52I iso‐2, despite the greater thermodynamic stability of N52I iso‐2. Thus, there is no linear free‐energy relationship between mutation‐induced changes in stability and observable refolding rates. However, for N52I iso‐2 the unfolding rate is slower and the guanidine hydrochloride dependence of the unfolding rate is smaller than for iso‐2. The differences in the denaturant dependence of the unfolding rates suggest that the N52I substitution decreases the change in the solvent accessible hydrophobic surface between the native state and the transition state. Two aspects of the results are inconsistent with a two‐state folding/unfolding mechanism and imply the presence of folding intermediates: (1) observable refolding rate constants calculated from the two‐state mechanism by combining equilibrium data and unfolding rate measurements deviate from the observed refolding rate constants; (2) kinetically unresolved signal changes (“burst phase”) are observed for both N52I iso‐2 and iso‐2 refolding. The “burst phase” amplitude is larger for N52I iso‐2 than for iso‐2, suggesting that the intermediates formed during the “burst phase” are stabilized by the 52I substitution.
Motifs and structural fold of the cofactor binding site of human glutamate decarboxylase
Authors: Qu Kunbin , Martin David L. , Lawrence Charles E.
Published: February 2009

The pyridoxal‐P binding sites of the two isoforms of human glutamate decarboxylase (GAD65 and GAD67) were modeled by using PROBE (a recently developed algorithm for multiple sequence alignment and database searching) to align the primary sequence of GAD with pyridoxal‐P binding proteins of known structure. GAD's cofactor binding site is particularly interesting because GAD activity in the brain is controlled in part by a regulated interconversion of the apo‐ and holoenzymes. PROBE identified six motifs shared by the two GADs and four proteins of known structure: bacterial ornithine decarboxylase, dialkylglycine decarboxylase, aspartate aminotransferase, and tyrosine phenol‐lyase. Five of the motifs corresponded to the α/β elements and loops that form most of the conserved fold of the pyridoxal‐P binding cleft of the four enzymes of known structure; the sixth motif corresponded to a helical element of the small domain that closes when the substrate binds. Eight residues that interact with pyridoxal‐P and a ninth residue that lies at the interface of the large and small domains were also identified. Eleven additional conserved residues were identified and their functions were evaluated by examining the proteins of known structure. The key residues that interact directly with pyridoxal‐P were identical in ornithine decarboxylase and the two GADs, thus allowing us to make a specific structural prediction of the cofactor binding site of GAD. The strong conservation of the cofactor binding site in GAD indicates that the highly regulated transition between apo‐ and holoGAD is accomplished by modifications in this basic fold rather than through a novel folding pattern.
Crystal structures of the Klenow fragment of Thermus aquaticus DNA polymerase I complexed with deoxyribonucleoside triphosphates
Authors: Li Ying , Kong Yong , Korolev Sergey , Waksman Gabriel
Published: February 2009

The crystal structures of the Klenow fragment of the Thermus aquaticus DNA polymerase I (Klentaq 1) complexed with four deoxyribonucleoside triphosphates (dNTP) have been determined to 2.5 Å resolution. The dNTPs bind adjacent to the O helix of Klentaq 1. The triphosphate moieties are at nearly identical positions in all four complexes and are anchored by three positively charged residues, Arg659, Lys663, and Arg587, and by two polar residues, His639 and Gln613. The configuration of the base moieties in the Klentaq 1/dNTP complexes demonstrates variability suggesting that dNTP binding is primarily determined by recognition and binding of the phosphate moiety. However, when superimposed on the Taq polymerase/blunt end DNA complex structure (Eom et al., 1996), two of the dNTP/Klentaql structures demonstrate appropriate stacking of the nucleotide base with the 3′ end of the DNA primer strand, suggesting that at least in these two binary complexes, the observed dNTP conformations are functionally relevant.
The role of polar interactions in the molecular recognition of CD40L with its receptor CD40
Authors: Singh Juswinder , Garber Ellen , Vlijmen Herman Van , Karpusas Michael , Hsu Yen‐Ming , Zheng Zhongli , Thomas David , Naismith James H.
Published: February 2009

CD40 Ligand (CD40L) is transiently expressed on the surface of T‐cells and binds to CD40, which is expressed on the surface of B‐cells. This binding event leads to the differentiation, proliferation, and isotype switching of the B‐cells. The physiological importance of CD40L has been demonstrated by the fact that expression of defective CD40L protein causes an immunodeficiency state characterized by high IgM and low IgG serum levels, indicating faulty T‐cell dependent B‐cell activation. To understand the structural basis for CD40L/CD40 association, we have used a combination of molecular modeling, mutagenesis, and X‐ray crystallography. The structure of the extracellular region of CD40L was determined by protein crystallography, while the CD40 receptor was built using homology modeling based upon a novel alignment of the TNF receptor superfamily, and using the X‐ray structure of the TNF receptor as a template. The model shows that the interface of the complex is composed of charged residues, with CD40L presenting basic side chains (K143, R203, R207), and CD40 presenting acidic side chains (D84, El14, El17). These residues were studied experimentally through site‐directed mutagenesis, and also theoretically using electrostatic calculations with the program Delphi. The mutagenesis data explored the role of the charged residues in both CD40L and CD40 by switching to Ala (K143A, R203A, R207A of CD40L, and E74A, D84A, E114A, E117A of CD40), charge reversal (K143E, R203E, R207E of CD40L, and D84R, El 14R, El 17R of CD40), mutation to a polar residue (K143N, R207N, R207Q of CD40L, and D84N, El 17N of CD40), and for the basic side chains in CD40L, isosteric substitution to a hydrophobic side chain (R203M, R207M). All the charge‐reversal mutants and the majority of the Met and Ala substitutions led to loss of binding, suggesting that charged interactions stabilize the complex. This was supported by the Delphi calculations which confirmed that the CD40/CD40L residue pairs E74‐R203, D84‐R207, and El 17‐R207 had a net stabilizing effect on the complex. However, the substitution of hydrophilic side chains at several of the positions was tolerated, which suggests that although charged interactions stabilize the complex, charge per se is not crucial at all positions. Finally, we compared the electrostatic surface of TNF/TNFR with CD40L/CD40 and have identified a set of polar interactions surrounded by a wall of hydrophobic residues that appear to be similar but inverted between the two complexes.
Two “unrelated” families of ATP‐dependent enzymes share extensive structural similarities about their cofactor binding sites
Authors: Denessiouk Konstantin A. , Korpela Timo , Lehtonen Jukka V. , Johnson Mark S.
Published: February 2009

Two proteins, D‐alanine:D‐alanine ligase and cAMP‐dependent protein kinase, share a remarkable degree of structural convergence despite having different three‐dimensional folds and different enzymatic functions. Here we report that as many as 103 residues from 10 segments form two identical super‐secondary structures between which the cofactor ATP is bound. The cofactor, two bound metal cations, and several water molecules form a large network of electrostatic and hydrophobic interactions common to both enzymes, and these are mediated by the similar placement of equivalent amino acids within the common supersecondary structures.
Enhanced thermal stability of Clostridium beijerinckii alcohol dehydrogenase after strategic substitution of amino acid residues with prolines from the homologous thermophilic Thermoanaerobacter brockii alcohol dehydrogenase
Authors: Bogin Oren , Peretz Moshe , Hacham Yael , Burstein Yigal , Korkhin Yakov , Kalb(gilboa) A. Joseph , Frolow Felix
Published: February 2009

A comparison of the three‐dimensional structures of the closely related mesophilic Clostridium beijerinckii alcohol dehydrogenase (CBADH) and the hyperthermophilic Thermoanaerobacter brockii alcohol dehydrogenase (TBADH) suggested that extra proline residues in TBADH located in strategically important positions might contribute to the extreme thermal stability of TBADH. We used site‐directed mutagenesis to replace eight complementary residue positions in CBADH, one residue at a time, with proline. All eight single‐proline mutants and a double‐proline mutant of CBADH were enzymatically active. The critical sites for increasing thermostability parameters in CBADH were Leu‐316 and Ser‐24, and to a lesser degree, Ala‐347. Substituting proline for His‐222, Leu‐275, and Thr‐149, however, reduced thermal stability parameters. Our results show that the thermal stability of the mesophilic CBADH can be moderately enhanced by substituting proline at strategic positions analogous to nonconserved prolines in the homologous thermophilic TBADH. The proline residues that appear to be crucial for the increased thermal stability of CBADH are located at a β‐turn and a terminating external loop in the polypeptide chain. Positioning proline at the N‐caps of α‐helices in CBADH led to adverse effects on thermostability, whereas single‐proline mutations in other positions in the polypeptide had varying effects on thermal parameters. The finding presented here support the idea that at least two of the eight extra prolines in TBADH contribute to its thermal stability.
Reactivation of thermally inactivated pre‐β‐lactamase by DnaK, DnaJ, and GrpE
Authors: Mccarthy Diane , Kramer Gisela , Hardesty Boyd
Published: February 2009

To understand the role of the 23‐amino acid signal sequence in the folding and stability of β‐lactamase, the precursor and a mutantβ‐lactamase with a 19‐amino acid signal sequence deletion were synthesized in vitro using anEscherichia colicell‐free coupled transcription/translation system. Approximately 30% of the newly synthesized full‐length precursor and 60% of the deletion mutant polypeptides were terminated and released from the ribosomes as active enzyme. Activity of the pre‐β‐lactamase, but not the mutant, was unstable at 37 °C, suggesting that the signal sequence causes the enzyme to unfold. This inactivation was independent of ATP. Pre‐β‐lactamase activity was stabilized by lowering the temperature to 30 °C. Furthermore, addition of the molecular chaperones DnaK/J and GrpE, in the presence of ATP and Mg2+, restored the activity of the temperature‐inactivated precursor. The precursor formed a stable complex with DnaK and GrpE. Both ATP and DnaJ were required for recovery of enzymatic activity, indicating that DnaJ may bind transiently to the complex. These results suggest that the signal sequence of the pre‐β‐lactamase causes a temperature‐dependent unfolding of the synthesized enzyme and that DnaK/J and GrpE interact with unfolded pre‐β‐lactamase to promote refolding of the protein into its native, enzymatically active conformation.
Topology of sarcoplasmic reticulum Ca2+‐ATPase: An infrared study of thermal denaturation and limited proteolysis
Authors: Echabe Izaskun , Prado Adelina , Goñi Fé Lix M. , Arondo José Luis R. , Dornberger Utz
Published: February 2009

Sarcoplasmic reticulum Ca2+‐ATPase structure and organization in the membrane has been studied by infrared spectroscopy by decomposition of the amide I band. Besides the component bands assignable to secondary structure elements such as α‐helix, β‐sheet, etc…, two unusual bands, one at 1,645 cm −1 in H2O buffer and the other at 1,625 cm−1 in D2O buffer are present. By perturbing the protein using temperature and limited proteolysis, the band at 1,645 cm−1 is tentatively assigned to α‐helical segments located in the cytoplasmic domain and coupled to β‐sheet structure, whereas the band at 1,625 cm−1 arises probably from monomer‐monomer contacts in the native oligomeric protein. The secondary structure obtained is 33% α‐helical segments in the transmembrane plus stalk domain; 20% α‐helix and 22% β‐sheet in the cytoplasmic domain plus 19% turns and 6% unordered structure. Thermal unfolding of Ca2+‐ATPase is a complex process that cannot be described as a two‐state denaturation. The results obtained are compatible with the idea that the protein is an oligomer at room temperature. The loss of the 1,625 cm −1 band upon heating would be consistent with a disruption of the oligomers in a process that later gives rise to aggregates (appearance of the 1,618 cm −1 band). This picture would also be compatible with early results suggesting that processes governing Ca2+ accumulation and ATPase activity are uncoupled at temperatures above 37 °C, so that while ATPase activity proceeds at high rates, Ca2+ accumulation is inhibited.
The observation of chaperone‐ligand noncovalent complexes with electrospray ionization mass spectrometry
Authors: Bruce James E. , Liu Chuanliang , Smith Richard D. , Randall Linda L. , Smith Virginia F.
Published: February 2009

Fourier transform ion cyclotron resonance mass spectrometry (FTICR‐MS) was applied for the study of noncovalent chaperone SecB‐ligand complexes produced in solution and examined in the gas phase with the aid of electrospray ionization (ESI). Since chaperone proteins are believed to recognize and bind only with ligands with nonnative tertiary structure, this work required careful unfolding of the ligand and subsequent reaction with the intact chaperone (the noncovalent tetrameric protein, SecB). A high denaturant concentration was employed to produce nonnative structures of the OppA, and microdialysis of the resulting solutions containing the chaperone‐ligand complexes was carried out to rapidly remove the denaturant prior to analysis. Multistage mass spectrometry was essential to the successful study of these complexes since the initial mass spectra indicated extensive adduction that precluded mass measurements, even after microdialysis. However, low energy collisional activation of the ions in the FTICR trap proved useful for adduct removal, and careful control of excitation level preserved the intact complexes of interest, revealing a 1:1 SecB:OppA stoichiometry. To our knowledge, these results present the first direct observation of chaperone‐ligand noncovalent complexes and the highest molecular weight heterogeneous noncovalent complex observed to date by mass spectrometry. Furthermore, these results highlight the capabilities of FTICR for the study of such complex systems, and the development of a greater understanding of chaperone interactions in protein export.
Isolation and characterization of a DnaJ‐like protein in rats: The C‐terminal 10‐kDa domain of hsc70 is not essential for stimulating the ATP‐hydrolytic activity of hsc70 by a DnaJ‐like protein
Authors: Leng Chih‐Hsiang , Wang Chung , Brodsky Jeffrey L.
Published: February 2009

A DnaJ‐like protein, RDJ1, was isolated from a rat brain cDNA library. The protein is predicted to have 397 amino acid residues and shares 99% identity to that of HDJ2, a human DnaJ‐like protein. RDJ1 was also shown to rescue the temperature‐sensitive lethality of a strain containing a mutated cytosolic DnaJ in yeast, ydj1‐151. Fragments containing the J‐domain of RDJl either with or without the G/F motif were expressed in Escherichia coli. The purified proteins stimulated the ATPase activity of hsc70 and of the 60‐kDa N‐terminal fragment of hsc70. These results imply that RDJl can interact with the N‐terminal 60‐kDa fragment of hsc70 to activate ATP hydrolysis by hsc70.
Calorimetric analyses of the interaction between SecB and its ligands
Authors: Randall Linda L. , Topping Traci B. , Suciu Dominic , Hardy Simon J. S.
Published: February 2009

SecB is a chaperone in Escherichia coli dedicated to export of proteins from the cytoplasm to the periplasm and outer membrane. It functions to bind and deliver precursors of exported proteins to the translocation apparatus before they fold into their native structures, thus maintaining them in a competent state for translocation across the membrane. The natural ligands of SecB are precursor proteins containing leader sequences. There are numerous reports in the literature indicating that SecB does not specifically recognize the leader peptides. However, two published investigations have concluded that the leader peptide is the recognition element (Watanabe M, Blobel G. 1989. Cell 58:685‐705; Watanabe M, Blobel G. 1995. Proc Natl Acad Sci USA 92:10133‐10136). In this work we use titration calorimetry to show that SecB binds two physiological ligands, which contain leader sequences, with no higher affinity than the same molecules lacking their leader sequences. Indeed, for one ligand the presence of the leader sequence reduces the affinity. Therefore, it can be concluded that the leader sequence provides no positive contribution to the binding energy.
What should the Z‐score of native protein structures be?
Authors: Zhang Li , Skolnick Jeffrey
Published: February 2009

The Z‐score of a protein is defined as the energy separation between the native fold and the average of an ensemble of misfolds in the units of the standard deviation of the ensemble. The Z‐score is often used as a way of testing the knowledge‐based potentials for their ability to recognize the native fold from other alternatives. However, it is not known what range of values the Z‐scores should have if one had a correct potential. Here, we offer an estimate of Z‐scores extracted from calorimetric measurements of proteins. The energies obtained from these experimental data are compared with those from computer simulations of a lattice model protein. It is suggested that the Z‐scores calculated from different knowledge‐based potentials are generally too small in comparison with the experimental values.
The role of tyrosine 121 in cofactor binding of 5‐aminolevulinate synthase
Authors: Tan Dongwei , Barber Michael J. , Ferreira Gloria C.
Published: February 2009

5‐Aminolevulinate synthase (EC 2.3.1.37) is the first enzyme in the heme biosynthesis in nonplant eukaryotes and some prokaryotes. It functions as a homodimer and requires pyridoxal 5′‐phosphate as an essential cofactor. Tyr‐121 is a conserved residue in all known sequences of 5‐aminolevulinate synthases. Further, it corresponds to Tyr‐70 of Escherichia coli aspartate aminotransferase, which has been shown to interact with the cofactor and prevent the dissociation of the cofactor from the enzyme. To test whether Tyr‐121 is involved in cofactor binding in murine erythroid 5‐aminolevulinate synthase, Tyr‐121 of murine erythroid 5‐aminolevulinate synthase was substituted by Phe and His using site‐directed mutagenesis. The Y121F mutant retained 36% of the wild‐type activity and the Km value for substrate glycine increased 34‐fold, while the activity of the Y121H mutant decreased to 5% of the wild‐type activity and the Km value for glycine increased fivefold. The pKal values in the pH‐activity profiles of the wild‐type and mutant enzymes were 6.41, 6.54, and 6.65 for wild‐type, Y121F, and Y121H, respectively. The UV‐visible and CD spectra of Y121F and Y121H mutants were similar to those of the wild‐type with the exception of an absorption maximum shift (420 ∼ 395 nm) for the Y121F mutant in the visible spectrum region, suggesting that the cofactor binds the Y121F mutant enzyme in a more unrestrained manner. Y121F and Y121H mutant enzymes also exhibited lower affinity than the wild‐type for the cofactor, reflected in the Kd values for pyridoxal 5′‐phosphate (26.5, 6.75, and 1.78 μ. for Y121F, Y121H, and the wild‐type, respectively). Further, Y121F and Y121H proved less thermostable than the wild type. Taken together, these findings indicate that Tyr‐121 plays a critical role in cofactor binding of murine erythroid 5‐aminolevulinate synthase.
Mutational analysis of the active site of indoleglycerol phosphate synthase fromEscherichia coli
Authors: Darimont Beatrice , Stehlin Catherine , Szadkoski Halina , Kirscner Kasper
Published: February 2009

Indoleglycerol phosphate synthase catalyzes the ring closure of 1‐(2‐carboxyphenylamino)‐1‐deoxyribulose 5′‐phosphate to indoleglycerol phosphate, the fifth step in the pathway of tryptophan biosynthesis from chorismate. Because chemical synthesis of indole derivatives from arylamino ketones requires drastic solvent conditions, it is interesting by what mechanism the enzyme catalyzes the same condensation reaction. Seven invariant polar residues in the active site of the enzyme fromEscherichia colihave been mutated directly or randomly, to identify the catalytically essential ones. A strain ofE. colisuitable for selecting and classifying active mutants by functional complementation was constructed by precise deletion of thetrpC gene from the genome. Judged by growth rates of transformants on selective media, mutants with either S58 or S60 replaced by alanine were indistinguishable from the wild‐type, but R186 replaced by alanine was still partially active. Saturation random mutagenesis of individual codons showed that E53 was partially replaceable by aspartate and cysteine, whereas K114, E163, and N184 could not be replaced by any other residue. Partially active mutant proteins were purified and their steady‐state kinetic and inhibitor binding constants determined. Their relative catalytic efficiencies paralleled their relative complementation efficiencies. These results are compatible with the location of the essential residues in the active site of the enzyme and support a chemically plausible catalytic mechanism. It involves two enzyme‐bound intermediates and general acid‐base catalysis by K114 and E163 with the support of E53 and N184.
Characterization of Escherichia coli thioredoxin variants mimicking the active‐sites of other thiol/disulfide oxidoreductases
Authors: Mössner Ekkehard , Huber‐Wunderlich Martina , Glockshuber Rudi
Published: February 2009

Thiol/disulfide oxidoreductases like thioredoxin, glutaredoxin, DsbA, or protein disulfide isomerase (PDI) share the thioredoxin fold and a catalytic disulfide bond with the sequence Cys‐Xaa‐Xaa‐Cys (Xaa corresponds to any amino acid). Despite their structural similarities, the enzymes have very different redox properties, which is reflected by a 100,000‐fold difference in the equilibrium constant (Keq) with glutathione between the most oxidizing member, DsbA, and the most reducing member, thioredoxin. Here we present a systematic study on a series of variants of thioredoxin from Escherichia coli, in which the Xaa‐Xaa dipeptide was exchanged by that of glutaredoxin, PDI, and DsbA. Like the corresponding natural enzymes, all thioredoxin variants proved to be stronger oxidants than the wild‐type, with the order wild‐type < PDI‐type < DsbA‐type < glutaredoxin‐type. The most oxidizing, glutaredoxin‐like variant has a 420‐fold decreased value of Keq, corresponding to an increase in redox potential by 75 mV. While oxidized wild‐type thioredoxin is more stable than the reduced form (ΔΔGox/red = 16.9 kJ/mol), both redox forms have almost the same stability in the variants. The pH‐dependence of the reactivity with the alkylating agent iodoacetamide proved to be the best method to determine the pKa value of thioredoxin's nucleophilic active‐site thiol (Cys32). A pKa of 7.1 was measured for Cys32 in the reduced wild‐type. All variants showed a lowered pKa of Cys32, with the lowest value of 5.9 for the glutaredoxin‐like variant. A correlation of redox potential and the Cys32 pKa value could be established on a quantitative level. However, the predicted correlation between the measured ΔΔGox/red values and Cys32 pKa values was only qualitative.
The crystal structure of lactococcus lactis dihydroorotate dehydrogenase A complexed with the enzyme reaction product throws light on its enzymatic function
Authors: Rowland Paul , Larsen Sine , Björnberg Olof , Nielsen Finn S. , Jensen Kaj Frank
Published: February 2009

Dihydroorotate dehydrogenases (DHODs) catalyze the oxidation of (S)‐dihydroorotate to orotate, the fourth step and only redox reaction in the de novo biosynthesis of pyrimidine nucleotides. A description is given of the crystal structure of Lactococcus lactis dihydroorotate dehydrogenase A (DHODA) complexed with the product of the enzyme reaction orotate. The structure of the complex to 2.0 Å resolution has been compared with the structure of the native enzyme. The active site of DHODA is known to contain a water filled cavity buried beneath a highly conserved and flexible loop. In the complex the orotate displaces the water molecules from the active site and stacks above the DHODA flavin isoalloxazine ring, causing only small movements of the surrounding protein residues. The orotate is completely buried beneath the protein surface, and the orotate binding causes a significant reduction in the mobility of the active site loop. The orotate is bound by four conserved asparagine side chains (Asn 67, Asn 127, Asn 132, and Asn 193), the side chains of Lys 43 and Ser 194, and the main chain NH groups of Met 69, Gly 70, and Leu 71. Of these the Lys 43 side chain makes hydrogen bonds to both the flavin isoalloxazine ring and the carboxylate group of the orotate. Potential interactions with bound dihydroorotate are considered using the orotate complex as a basis for molecular modeling. The role of Cys 130 as the active site base is discussed, and the sequence conservation of the active site residues across the different families of DHODs is reviewed, along with implications for differences in substrate binding and in the catalytic mechanisms between these families.
Circular permutation of βB2‐crystallin changes the hierarchy of domain assembly
Authors: Wright G. , Basak A.K. , Wieligmann K. , Slingsby C. , Mayr E.‐M.
Published: February 2009

The βγ‐crystallins form a superfamily of eye lens proteins comprised of multiple Greek motifs that are symmetrically organized into domains and higher assemblies. In the βB2‐crystallin dimer each polypeptide folds into two similar domains that are related to monomeric γ‐crystallin by domain swapping. The crystal structure of the circularly permuted two‐domain βB2 polypeptide shows that permutation converts intermolecular domain pairing into intramolecular pairing. However, the dimeric permuted protein is, in fact, half a native tetramer. This result shows how the sequential order of domains in multi‐domain proteins can affect quaternary domain assembly.
Crystal structure of human cathepsin S
Authors: Mcgrath Mary E. , Palmer James T. , Brömme Ddeter , Somoza John R.
Published: February 2009

We have determined the 2.5 Å structure (Rcryst= 20.5%, Rfree = 28.5%) of a complex between human cathepsin S and the potent, irreversible inhibitor 4‐morpholinecarbonyl‐Phe‐hPhe‐vinyl sulfone‐phenyl. Noncrystallographic symmetry averaging and other density modification techniques were used to improve electron density maps which were nonoptimal due to systematically incomplete data. Methods that reduce the number of parameters were implemented for refinement. The refined structure shows cathepsin S to be similar to related cysteine proteases such as papain and cathepsins K and L. As expected, the covalently‐bound inhibitor is attached to the enzyme at Cys 25, and enzyme binding subsites S3‐S1' are occupied by the respective inhibitor substituents. A somewhat larger S2 pocket than what is found in similar enzymes is consistent with the broader specificity of cathepsin S at this site, while Lys 61 in the S3 site may offer opportunities for selective inhibition of this enzyme. The presence of Arg 137 in the S1' pocket, and proximal to Cys 25 may have implications not only for substrate specificity C‐terminal to the scissile bond, but also for catalysis.
Crystal structure of apo‐glycine N‐methyltransferase (GNMT)
Authors: Pattanayek R. , Newcomer M.E. , Wagner C.
Published: February 2009

The crystal structure of the recombinant apo‐form of glycine N‐methyltransferase (GNMT) has been determined at 2.5 Å resolution. GNMT is a tetrameric enzyme (monomer Mr = 32,423Da, 292 amino acids) that catalyzes the transfer of a methyl group from S‐adenosylmethionine (AdoMet) to glycine with the formation of S‐adenosylmethionine (AdoHcy) and sarcosine (N‐methylglycine). GNMT is a regulatory enzyme, which is inhibited by 5‐methyltetrahydrofolate pentaglutamate and believed to control the ratio of AdoMet to AdoHcy in tissues. The crystals belong to the orthorhombic space group P21212 (a = 85.39, b = 174.21, c = 44.71 Å) and contain one dimer per asymmetric unit. The AdoMet‐GNMT structure served as the starting model. The structure was refined to an R‐factor of 21.9%. Each monomer is a three‐domain structure with a large cavity enclosed by the three domains. The tetramer resembles a Square with a central Channel about which N‐terminal domains are intertwined. Only localized changes of the residues involved in the binding pocket are observed for the apo‐GNMT structure when compared to that determined in the presence of Substrate and Substrate analog.
The three‐dimensional structure of a helix‐less variant of intestinal fatty acid‐binding protein
Authors: Steele Ruth A. , Emmert Daniel A. , Kao Jeff , Hodson Michaele E. , Frieden Carl , Cistola David P.
Published: February 2009

Intestinal fatty acid‐binding protein (I‐FABP) is a cytosolic 15.1‐kDa protein that appears to function in the intracellular transport and metabolic trafficking of fatty acids. It binds a Single molecule of long‐chain fatty acid in an enclosed cavity surrounded by two five‐stranded antiparallel β‐sheets and a helix‐turn‐helix domain. To investigate the role of the helical domain, we engineered a variant of I‐FABP by deleting 17 contiguous residues and inserting a Ser‐Gly linker (Kim K et al., 1996, Biochemistry 35:7553‐7558). This variant, termed Δ17‐SG, was remarkably stable, exhibited a high β‐sheet content and was able to bind fatty acids with some features characteristic of the wild‐type protein. In the present study, we determined the structure of the Δ17‐SG/palmitate complex at atomic resolution using triple‐resonance 3D NMR methods. Sequence‐specific 1H, 13C, and 15N resonance assignments were established at pH 7.2 and 25 °C and used to define the consensus 1H/13C chemical shift‐derived secondary structure. Subsequently, an iterative protocol was used to identify 2,544 NOE‐derived interproton distance restraints and to calculate its tertiary structure using a unique distance geometry/simulated annealing algorithm. In spite of the sizable deletion, the Δ17‐SG structure exhibits a backbone conformation that is nearly superimposable with the β‐sheet domain of the wild‐type protein. The selective deletion of the α‐helical domain creates a very large opening that connects the interior ligand‐binding cavity with exterior solvent. Unlike wild‐type I‐FABP, fatty acid dissociation from Δ17‐SG is structurally and kinetically un‐impeded, and a protein conformational transition is not required. The Δ17‐SG variant of I‐FABP is the only wild‐type or engineered member of the intracellular lipid‐binding protein family whose structure lacks α‐helices. Thus, Δ17‐SG I‐FABP constitutes a unique model System for investigating the role of the helical domain in ligand‐protein recognition, protein stability and folding, lipid transfer mechanisms, and cellular function.
Comparative analysis of the noncollagenous NC1 domain of type IV collagen: Identification of structural features important for assembly, function, and pathogenesis
Authors: Netzer Kai‐Olaf , Suzuki Ko , Itoh Yoshifumi , Hudson Bolly G. , Khalifah Raja G.
Published: February 2009

Type IV collagen α1‐α6 chains have important roles in the assembly of basement membranes and are implicated in the pathogenesis of Goodpasture Syndrome, an autoimmune disorder, and Alport Syndrome, a hereditary renal disease. We report comparative sequence analyses and structural predictions of the noncollagenous C‐terminal globular NC1 domain (28 sequences). The inferred tree verified that type IV collagen sequences fall into two groups, α‐1‐like and α2‐like, and suggested that vertebrate α3/α4 sequences evolved before α1/α2 and α5/α6. About one fifth of NC1 residues were identified to confer either the α1 or α2 group‐specificity. These residues accumulate opposite Charge in subdomain B of α1 (positive) and α2 (negative) sequences and may play a role in the stoichiometric chain selection upon type IV collagen assembly. Neural network secondary structure prediction on multiple aligned sequences revealed a subdomain core structure consisting of six hydrophobic β‐strands and one short α‐helix with a significant hydrophobic moment. The existence of opposite charges in the a‐helices may carry implications for intersubdomain interactions. The results provide a rationale for defining the epitope that binds Goodpasture autoantibodies and a framework for understanding how certain NC1 mutations may lead to Alport Syndrome. A search algorithm, based entirely on amino acid properties, yielded a possible similarity of NC1 to tissue inhibitor of metalloproteinases (TIMP) and prompted an investigation of a possible functional relationship. The results indicate that NC1 preparations decrease the activity of matrix metalloproteinases 2 and 3 (MMP‐2, MMP‐3) toward a peptide Substrate, though not to [14C]‐gelatin. We suggest that an ancestral NC1 may have been incorporated into type IV collagen as an evolutionarily mobile domain carrying Proteinase inhibitor function.
Deciphering the structural code for proteins: Helical propensities in domain classes and statistical multiresidue information in α‐helices
Authors: Negrete Josée A. , Viñuales Yolanda , Palau Jaume
Published: February 2009

We made several statistical analyses in a large sample of nearly 4,000 helices (from 546 redundancy‐controlled PDB protein subunits), which give new insights into the helical properties of globular proteins. In a first experiment, the amino acid composition of the whole sample was compared with the composition of two helical sample subgroups (the “mainly‐α” and the “(α/β)8 barrel” domain classes); we reached the conclusion that composition‐based helical propensities for secondary structure prediction do not depend on the structural class.

Running a five‐residue window through the whole sample, the positional composition revealed that positive and negative residues are located throughout the helices and tend to neutralize the macrodipole effect. On this basis, we analyzed charged triplets using a running five‐residue window. The conclusion was that only mixed charged residues [positive (+) and negative (‐)] located at positions 1‐2‐5 and 1‐4‐5 are clearly favored. In these locations the most abundant are (–.+) and (‐.++), and this shows the existence of side chain microdipoles, which neutralize the large macrodipole of the helix.

We made a systematic statistical analysis of charged, dipolar, and hydrophobic + aromatic residues, which enabled us to work out rules that should be useful for modeling and design purposes.

Finally, we analyzed the relative abundance of all the different amphipathic double‐arcs that are present in helices formed by octapeptides (8) and nonapeptides (18). All of the double‐arcs that make up Schiffer and Edmundson's classical helical wheel are found in abundance in the sample.
Study of a noncovalent trp repressor: DNA operator complex by electrospray ionization time‐of‐flight mass spectrometry
Authors: Potter Noelle , Chernushevich Igor , Ens Werner , Standing Kenneth G. , Donald Lynda J. , Ayed Ayeda , Duckworth Harry W. , Arrowsmith Cheryl H.
Published: February 2009

Electrospray ionization time‐of‐flight mass spectrometry (ESI‐TOF MS) has been used to study noncovalent interactions between the trp apo‐repressor (TrpR), its co‐repressor tryptophan and its specific operator DNA. In 5 mM ammonium acetate, TrpR was detected as a partially unfolded monomer. In the presence of a 21‐base‐pair DNA possessing the two symmetrically arranged CTAG consensus sequences required for specific TrpR binding, a homodimer‐dsDNA complex with a 1:1 stoichiometry was observed. Co‐repressor was not needed for the complex to form under our experimental conditions. Collision induced dissociation (CID‐MS) revealed that this complex was very stable in the gas phase since dissociation was achieved only at energies that also broke covalent bonds. We saw no evidence for the presence of the six water molecules that mediate the interaction between the protein and the DNA in the crystal structure. To check the binding specificity of the TrpR for its target DNA, a competitive experiment was undertaken: the protein was mixed with an equimolar amount of three different DNAs in which the two CTAG sequences were separated by 2, 4, and 6 bp, respectively. Only the DNA with the correct consensus spacing of 4 bp was able to form stable interactions with TrpR. This experiment demonstrates the potential of ESI‐MS to test the sequence‐specificity of protein‐DNA complexes. The interactions between the TrpR‐DNA complex and 5‐methyl‐, L‐ and D‐tryptophan were also investigated. Two molecules of 5‐methyl‐ or L‐tryptophan were bound with high affinity to the TrpR‐DNA complex. On the other hand, D‐tryptophan appeared to bind to the complex with poor specificity and poor affinity.
Formation and properties of mixed disulfides between thioredoxin reductase from Escherichia coli and thioredoxin: Evidence that cysteine‐138 functions to initiate dithiol‐disulfide interchange and to accept the reducing equivalent from reduced flavin
Authors: Veine Donna M. , Mulrooney Scott B. , Wang Pan‐Fen , Williams Charles H.
Published: February 2009

Mutation of one of the cysteine residues in the redox active disulfide of thioredoxin reductase from Escherichia coli results in C135S with Cys138 remaining or C138S with Cys135 remaining. The expression system for the genes encoding thioredoxin reductase, wild‐type enzyme, C135S, and C138S has been re‐engineered to allow for greater yields of protein. Wild‐type enzyme and C135S were found to be as previously reported, whereas discrepancies were detected in the characteristics of C138S. It was shown that the original C138S was a heterogeneous mixture containing C138S and wild‐type enzyme and that enzyme obtained from the new expression system is the correct species. C138S obtained from the new expression system having 0.1% activity and 7% flavin fluorescence of wild‐type enzyme was used in this study. Reductive titrations show that, as expected, only 1 mol of sodium dithionite/mol of FAD is required to reduce C138S. The remaining thiol in C135S and C138S has been reacted with 5,5′‐dithiobis‐(2‐nitrobenzoic acid) to form mixed disulfides. The half time of the reaction was <5 s for Cys138 in C135S and approximately 300 s for Cys135 in C138S showing that Cys138 is much more reactive. The resulting mixed disulfides have been reacted with Cys32 in C35S mutant thioredoxin to form stable, covalent adducts C138S‐C35S and C135S‐C35S. The half times show that Cys138 is approximately fourfold more susceptible to attack by the nucleophile. These results suggest that Cysl38 may be the thiol initiating dithiol‐disulfide interchange between thioredoxin reductase and thioredoxin.
Crystal structures of the psychrophilic α‐amylase from Alteromonas haloplanctis in its native form and complexed with an inhibitor
Authors: Aghajari Nushin , Haser Richard , Feller Georges , Gerday Charles
Published: February 2009

On page 565 in Volume 7, Number 3, 1998, under Molecular fold and calcium binding site, the domain numbering was wrongly reported. The correct numbering is domain A: residues 1 to 86 and 147 to 356, and domain B: residues 87 to 146. Domain C was correctly cited and remains as reported.
Determination of the amino acid requirements for a protein hinge in triosephosphate isomerase
Authors: Sun Jeonghoon , Sampson Nicole S.
Published: February 2009

We have determined the sequence requirements for a protein hinge in triosephosphate isomerase. The codons encoding the hinge at the C‐terminus of the active‐site lid of triosephosphate isomerase were replaced with a genetic library of all possible 8, 000 amino acid combinations. The most active of these 8, 000 mutants were selected using in vivo complementation of a triosephosphate isomerase deficient strain ofE. coli, DF502. Approximately 3% of the mutants complement DF502 with an activity that is above 70% of wild‐type activity. The sequences of these hinge mutants reveal that the solutions to the hinge flexibility problem are varied. Moreover, these preferences are sequence dependent; that is, certain pairs occur frequently. They fall into six families of similar sequences. In addition to the hinge sequences expected on the basis of phylogenetic analysis, we selected three new families of 3‐amino‐acid hinges: X(A/S)(L/K/ M), X(aromatic/β‐branched)(L/K), and XP(S/N). The absence of these hinge families in the more than 60 known species of triosephosphate isomerase suggests that during evolution, not all of sequence space is sampled, perhaps because there is no neutral mutation pathway to access the other families.