bluuebunny/crossref_metadata_2025 huggingface dataset:
A theoretical study of the active sites of papain and S195C rat trypsin: Implications for the low reactivity of mutant serine proteinases
Authors: Beveridge Allan J.
Published: February 2009

The serine and cysteine proteinases represent two important classes of enzymes that use a catalytic triad to hydrolyze peptides and esters. The active site of the serine proteinases consists of three key residues, Asp … His … Ser. The hydroxyl group of serine functions as a nucleophile and the imidazole ring of histidine functions as a general acid/general base during catalysis. Similarly, the active site of the cysteine proteinases also involves three key residues: Asn, His, and Cys. The active site of the cysteine proteinases is generally believed to exist as a zwitterion (Asn … His+ … Cys−) with the thiolate anion of the cysteine functioning as a nucleophile during the initial stages of catalysis. Curiously, the mutant serine proteinases, thiol subtilisin and thiol trypsin, which have the hybrid Asp … His … Cys triad, are almost catalytically inert. In this study, ab initio Hartree‐Fock calculations have been performed on the active sites of papain and the mutant serine proteinase S195C rat trypsin. These calculations predict that the active site of papain exists predominately as a zwitterion (Cys− … His+ … Asn). However, similar calculations on S195C rat trypsin demonstrate that the thiol mutant is unable to form a reactive thiolate anion prior to catalysis. Furthermore, structural comparisons between native papain and S195C rat trypsin have demonstrated that the spatial juxtapositions of the triad residues have been inverted in the serine and cysteine proteinases and, on this basis, I argue that it is impossible to convert a serine proteinase to a cysteine proteinase by site‐directed mutagenesis.
Differences in hydropathic properties of ligand binding at four independent sites in wheat germ agglutinin‐oligosaccharide crystal complexes
Authors: Wright Christine Schubert , Kellogg Glen E.
Published: February 2009

The binding interactions of N‐acetyl‐D‐neuraminic acid and N,N' diacetyl‐chitobiose (GlcNAc‐β‐1,4‐GIcNAc), observed in crystal complexes of wheat germ agglutinin (WGA) at four independent sites/monomer, were analyzed and compared with the modeling program HINT (Hydropathic INTeractions). This empirical method allows assessment of relative ligand binding strength and is particularly applicable to cases of weak binding where experimental data is absent. Although the four WGA binding sites are interrelated by a fourfold sequence repeat (eight sites/dimer), similarity extends only to the presence of an aromatic amino acid‐rich pocket and a conserved serine. Strong binding requires additional interactions from a contacting domain in the second subunit. Ligand positions were either derived from crystal structures and further optimized by modeling and molecular mechanics, or from comparative modeling. Analysis of the overall HINT binding scores for the two types of ligands are consistent with the presence of two high‐affinity and two low‐affinity sites per monomer. Identity of these sites correlates well with crystal structure occupancies. The high‐affinity sites are roughly equivalent, as predicted from solution binding studies. Binding scores for the low‐affinity sites are weaker by at least a factor of two. Quantitative estimates for polar, nonpolar, and ionic interactions revealed that H‐bonding makes the largest contribution to complex stabilization in the seven bound configurations, consistent with published thermodynamic data. Although the observed nonpolar interactions are small, they may play a critical role in orienting the ligand optimally.
Crystal structures of the active site mutant (Arg‐243 → Ala) in the T and R allosteric states of pig kidney fructose‐1,6‐bisphosphatase expressed in escherichia coli
Authors: Stec Boguslaw , Abraham Reimar , Giroux Eugene , Kantrowitz Evan R.
Published: February 2009

The active site of pig kidney fructose‐l,6‐bisphosphatase (EC 3.1.3.11) is shared between subunits, Arg‐243 of one chain interacting with fructose‐1,6‐bisphosphate or fructose‐2,6‐bisphosphate in the active site of an adjacent chain. In this study, we present the X‐ray structures of the mutant version of the enzyme with Arg‐243 replaced by alanine, crystallized in both T and R allosteric states. Kinetic characteristics of the altered enzyme showed the magnesium binding and inhibition by AMP differed slightly; affinity for the substrate fructose‐1,6‐bisphosphate was reduced 10‐fold and affinity for the inhibitor fructose‐2,6‐bisphosphate was reduced 1,000‐fold (Giroux E, Williams MK, Kantrowitz ER, 1994, J Biol Chem 269:31404–31409). The X‐ray structures show no major changes in the organization of the active site compared with wild‐type enzyme, and the structures confirm predictions of molecular dynamics simulations involving Lys‐269 and Lys‐274. Comparison of two independent models of the T form structures have revealed small but significant changes in the conformation of the bound AMP molecules and small reorganization of the active site correlated with the presence of the inhibitor. The differences in kinetic properties of the mutant enzyme indicate the key importance of Arg‐243 in the function of fructose‐1,6‐bisphosphatase. Calculations using the X‐ray structures of the Arg‐243 Ala enzyme suggest that the role of Arg‐243 in the wild‐type enzyme is predominantly electrostatic in nature.
Contribution of the tyrosines to the structure and function of the human U1A N‐terminal RNA binding domain
Authors: Kranz James K. , Lu Jirong , Hall Kathleen B.
Published: February 2009

RNA binding domains (RBDs) are members of a large family of proteins that share minimal sequence conservation but adopt an αβ sandwich global fold. Defining the contributions of specific amino acids to RBD structure and RNA binding is critical to understanding the functions of these proteins. In these experiments with the human U1A N‐terminal RNA binding domain (RBD1), the contributions from each of its four tyrosines to protein structure, stability, and RNA binding were measured. Each tyrosine was substituted with phenylalanine and one other selected residue, and the resulting proteins were characterized by chemical denaturation to measure their unfolding free energy, by binding free energies to the wild‐type RNA hairpin, and by 19F NMR to probe for structural changes. Features of the protein identified in these experiments include a possible tyrosine/lysine contact in an α‐helix, which may be an example of an energetically favorable aromatic/amino side chain interaction. One long loop of the protein, which shows unusual 15N backbone and tyrosine side‐chain dynamics, is implicated in protein:protein association. The diverse interactions of the four tyrosine residues in the organization of RBD1 illustrate how each member of this family of proteins will have unique molecular details that contribute to function.
Preparation and properties of pure, full‐length Ic1R protein of escherichia coli. Use of time‐of‐flight mass spectrometry to investigate the problems encountered
Authors: Donald Lynda J. , Chernushevich Igor V. , Zhou Jie , Verentchikov Anatoli , Poppe‐Schriemer Nancy , Hosfield David J. , Westmore John B. , Ens Werner , Duckworth Harry W. , Standing Kenneth G.
Published: February 2009

IclR protein, the repressor of the aceBAK operon of Escherichia coli, has been examined by time‐of‐flight mass spectrometry, with ionization by matrix assisted laser desorption or by electrospray. The purified protein was found to have a smaller mass than that predicted from the base sequence of the cloned icl R gene. Additional measurements were made on mixtures of peptides derived from IclR by treatment with trypsin and cyanogen bromide. They showed that the amino acid sequence is that predicted from the gene sequence, except that the protein has suffered truncation by removal of the N‐terminal eight or, in some cases, nine amino acid residues. The peptide bond whose hydrolysis would remove eight residues is a typical target for the E. coli protease OmpT. We find that, by taking precautions to minimize OmpT proteolysis, or by eliminating it through mutation of the host strain, we can isolate full‐length IclR protein (lacking only the N‐terminal methionine residue). Full‐length IclR is a much better DNA‐binding protein than the truncated versions: it binds the aceBAK operator sequence 44‐fold more tightly, presumably because of additional contacts that the N‐terminal residues make with the DNA. Our experience thus demonstrates the advantages of using mass spectrometry to characterize newly purified proteins produced from cloned genes, especially where proteolysis or other covalent modification is a concern. This technique gives mass spectra from complex peptide mixtures that can be analyzed completely, without any fractionation of the mixtures, by reference to the amino acid sequence inferred from the base sequence of the cloned gene.
Crystal structure of a biologically inactive mutant of toxic shock syndrome toxin‐1 at 2.5 A resolution
Authors: Papageorgiou Anastassios C. , Quinn Conrad P. , Beer David , Brehm Rossalyn D. , Tranter Howard S. , Bonventre Peter F. , Acharya K.Ravi
Published: February 2009

Toxic shock syndrome toxin‐1 (TSST‐1) is one of a family of staphylococcal exotoxins recognized as microbial superantigens. The toxin plays a dominant role in the genesis of toxic shock in humans through a massive activation of the immune system. This potentially lethal illness occurs as a result of the interaction of TSST‐1 with a significant proportion of the T‐cell repertoire. TSST‐1, like other superantigens, can bind directly to class II major histocompatibility (MHC class II) molecules prior to its interaction with entire families of Vβ chains of the T‐cell receptor (TCR). The three‐dimensional structure of a mutant (His‐135‐Ala) TSST‐1 was compared with the structure of the native (wild‐type) TSST‐1 at 2.5 Å resolution. The replacement of His 135 of TSST‐1 with an Ala residue results in the loss of T‐cell mitogenicity and toxicity in experimental animals. This residue, postulated to be directly involved in the toxin‐TCR interactions, is located on the major helix α2, which forms the backbone of the molecule and is exposed to the solvent. In the molecular structure of the mutant toxin, the helix α2 remains unaltered, but the His to Ala modification causes perturbations on the neighboring helix α1 by disrupting helix‐helix interactions. Thus, the effects on TCR binding of the His 135 residue could actually be mediated, wholly or in part, by the α1 helix.
Structural characterization of the tumor suppressor p16, an ankyrin‐like repeat protein
Authors: Boice Judith A. , Fairman Robert
Published: February 2009

The p16 protein has been identified as a tumor suppressor that functions by inhibiting the cyclin‐dependent kinases CDK4 and CDK6. Deletions or point mutations in the p16 gene have been found in a number of human cancers, emphasizing its importance in regulating cell cycle progression. Inhibition by p16 occurs through protein‐protein interactions with its targets. This is not surprising, since p16 is thought to contain ankyrin‐like repeats, motifs implicated in protein‐protein interactions. Our goal was to identify structural characteristics of p16 not only as an important step towards understanding CDK4 inhibition but also to explore the role of ankyrin repeats in the p16 structure, as no detailed structure of any protein containing these motifs has been reported. We have expressed, refolded, and purified p16 from E. coli and have shown it to be functionally active by specific binding to CDK4. Analytical ultracentrifugation has shown that p16 weakly self‐associates to form dimers with a Kd = 270 μM. The CD spectrum indicates that the protein is composed of 33% α‐helix, 22% β‐sheet, 19% β‐turn, and 27% other (which includes aromatic and random coil contributions). Further CD experiments suggest that p16 exhibits low structural stability with a ΔG of ‐2.3 kcal/mol. This weak stability is a consequence of a highly dynamic structure as measured by ANS‐binding, NMR hydrogen‐deuterium exchange, and fluorescence. It is possible that a well‐defined tertiary structure is imparted upon the binding of p16 to CDK4.
An optimized g‐tensor for rhodobacter capsulatus cytochrome c2 in solution: A structural comparison of the reduced and oxidized states
Authors: Zhao Dezheng , Mackenzie Neil E. , Hutton Harold M. , Cusanovich Michael A.
Published: February 2009

The optimized g‐tensor parameters for the oxidized form of Rhodobacter capsulatus cytochrome c2 in solution were obtained using a set (50) of backbone amide protons. Dipolar shifts for more than 500 individual protons of R. capsulatus cytochrome c2 have been calculated by using the optimized g‐tensor and the X‐ray crystallographic coordinates of the reduced form of R. capsulatus cytochrome c2. The calculated results for dipolar shifts are compared with the observed paramagnetic shifts. The calculated and the observed data are in good agreement throughout the entire protein, but there are significant differences between calculated and experimental results localized to the regions in the immediate vicinity of the heme ligand and the region of the front crevice of the protein (residues 44–50, 53–57, and 61–68). The results not only indicate that the overall solution structures are very similar in both the reduced and oxidized states, but that these structures in solution are similar to the crystal structure. However, there are small structural changes near the heme and the rearrangement of certain residues that result in changes in their hydrogen bonding concomitant with the change in the oxidation states; this was also evident in the data for the NH exchange rate measurements for R. capsulatus cytochrome c2.
Denaturants can accelerate folding rates in a class of globular proteins
Authors: Camacho Carlos J. , Thirumalai D.
Published: February 2009

We present a lattice Monte Carlo study to examine the effect of denaturants on the folding rates of simplified models of proteins. The two‐dimensional model is made from a three‐letter code mimicking the presence of hydrophobic, hydrophilic, and cysteine residues. We show that the rate of folding is maximum when the effective hydrophobic interaction ϵH is approximately equal to the free energy gain ϵS upon forming disulfide bonds. In the range 1 ≤ ϵH/ϵS ≤ 3, multiple paths that connect several intermediates to the native state lead to fast folding. It is shown that at a fixed temperature and ϵS the folding rate increases as ϵH decreases. An approximate model is used to show that ϵH should decrease as a function of the concentration of denaturants such as urea or guanidine hydrochloride. Our simulation results, in conjunction with this model, are used to show that increasing the concentration of denaturants can lead to an increase in folding rates. This occurs because denaturants can destabilize the intermediates without significantly altering the energy of the native conformation. Our findings are compared with experiments on the effects of denaturants on the refolding of bovine pancreatic trypsin inhibitor and ribonuclease T1. We also argue that the phenomenon of denaturant‐enhanced folding of proteins should be general.
Identification of protein kinase A phosphorylation sites on NBD1 and R domains of CFTR using electrospray mass spectrometry with selective phosphate ion monitoring
Authors: Townsend R. Reid , Lipniunas Peter H. , Tulk Barry M. , Verkman A.S.
Published: February 2009

HPLC‐electrospray mass spectrometry was used to identify the phosphorylated sites on a bacterially expressed cystic fibrosis transmembrane conductance regulator (CFTR) fragment containing the first nucleotide binding domain (NBD1) and the regulatory domain (R). Tryptic digests of NBD1‐R (CFTR residues 404–830) were analyzed after protein kinase A (PKA) treatment for all possible peptides and phosphopeptides (a total of 118 species) containing Ser residues within “high‐probability” PKA consensus sequences: R‐R/K‐X‐S/T, R‐X‐X‐S/T, and R‐X‐S/T. Three criteria were used to assign phosphorylated sites: (1) an 80‐Da increase in the predicted average molecular weight of the tryptic peptides; (2) co‐elution with the PO3− ion induced by stepped energy collision; and (3) the relative elution positions of the phosphorylated and unmodified peptides. Ser residues within the eight dibasic sites in the NBD1 and R domains (positions 422, 660, 700, 712, 737, 768, 795, and 813) were phosphorylated, a pattern similar to that observed for full‐length CFTR. The serine at position 753, which in CFTR is phosphorylated in vivo, was not phosphorylated. The remaining potential PKA sites, Ser489, Ser519, Ser557, Ser670, and Thr788, were not phosphorylated. The “low‐probability” PKA sites (those not containing an Arg residue) were not phosphorylated. The results suggest that isolated domains of CFTR developed useful models for investigating the biochemical and structural effects of phosphorylation within CFTR. The mass spectrometry approach in this study should prove useful for defining phosphorylation sites of CFTR in vitro and in vivo.
High‐ and low‐temperature unfolding of human high‐density apolipoprotein A‐2
Authors: Gursky Olga , Atkinson David
Published: February 2009

Human plasma apolipoprotein A‐2 (apoA‐2) is the second major protein of the high‐density lipoproteins that mediate the transport and metabolism of cholesterol. Using CD spectroscopy and differential scanning calorimetry, we demonstrate that the structure of lipid‐free apoA‐2 in neutral low‐salt solutions is most stable at ∼25 °C and unfolds reversibly both upon heating and cooling from 25 °C. High‐temperature unfolding of apoA‐2, monitored by far‐UV CD, extends from 25–85 °C with midpoint Th = 56 ± 2 °C and vant Hoff's enthalpy ΔH(Th) = 17 ± 2 kcal/mol that is substantially lower than the expected enthalpy of melting of the α‐helical structure. This suggests low‐cooperativity apoA‐2 unfolding. The apparent free energy of apoA‐2 stabilization inferred from the CD analysis of the thermal unfolding, ΔGapp(25°) = 0.82 ± 0.15 kcal/mol, agrees with the value determined from chemical denaturation. Enhanced low‐temperature stability of apoA‐2 observed upon increase in Na2HPO4 concentration from 0.3 mM to 50 mM or addition of 10% glycerol may be linked to reduced water activity. The close proximity of the heat and cold unfolding transitions, that is consistent with low ΔGAPP (25°), indicates that lipid‐free apoA‐2 has a substantial hydrophobic core but is only marginally stable under near‐physiological solvent conditions. This suggests that in vivo apoA‐2 transfer is unlikely to proceed via the lipid‐free state. Low ΔH(Th) and low apparent ΔCp ∼ 0.52 kcal/mol‐K inferred from the far‐UV CD analysis of apoA‐2 unfolding, and absence of tertiary packing interactions involving Tyr groups suggested by near‐UV CD, are consistent with a molten globular‐like state of lipid‐free apoA‐2.
Binding sites for blood coagulation factor Xa and protein S involving residues 493–506 in factor Va
Authors: Heeb Mary J. , Kojima Yumi , Hackeng Tilman M. , Griffin John H.
Published: February 2009

Inactivation due to cleavage of Factor Va (FVa) at Arg 506 by activated protein C (APC) helps to downregulate blood coagulation. To identify potential functional roles of amino acids near Arg 506, synthetic overlapping pentadecapeptides comprising FVa heavy chain residues 481–525 were tested for their ability to inhibit prothrombin activation by prothrombinase complexes [Factor Xa (FXa):FVa:phospholipids:Ca2+]. The most potent inhibition was observed for peptide VP493 (residues 493–506), with 50% inhibition at 2.5 μM. VP493 also inhibited FXa in plasma in FXa‐1‐stage clotting assays by 50% at 3 μM. When the C‐terminal carboxamide group of VP493 was replaced by a carboxyl group, most prothrombinase inhibitory activity was lost. VP493 preincubated with FXa inhibited prothrombinase with a pattern of mixed inhibition. Homologous peptides from Factor VIII sequences did not inhibit prothrombinase. Affinity‐purified antibodies to VP493 inhibited prothrombinase activity and prolonged FXa‐1‐stage clotting times. VP493 also blocked the ability of protein S to inhibit prothrombinase independently of APC. Immobilized VP493 bound specifically with similar affinity to both FXa and protein S (Kd ∼ 40 nM), but did not measurably bind prothrombin or APC. These studies suggest that FVa residues 493–506 contribute to binding sites for both FXa and protein S, providing a rationale for the ability of protein S to negate the protective effect of FXa toward APC cleavage of FVa. Possible loss of this FVa binding site for FXa due to cleavage at Arg 506 by APC may help explain why this cleavage causes 40% decrease in FVa activity and facilitates inactivation of FVa.
Cation selective promotion of tubulin polymerization by alkali metal chlorides
Authors: Wolff J. , Sackett Dan L. , Knipling Leslie
Published: February 2009

A role for charge‐based interactions in protein stability at the monomer or dimer level is well known. We show here that such interactions can also be important for the higher‐order structures of microtubule assembly. Alkali metal chlorides increase the rate of polymerization of pure tubulin driven by either taxol or dimethyl sulfoxide. The effect is cation selective, exhibiting a sequence Na+> K+> Li+> Cs+, with optimal concentrations for Na+at ∽ 160 mM. Hofmeister anion effects are additive with these rate stimulations. Sodium is less potent than guanidinium ion stimulation reported previously, but produces a larger fraction of normal microtubules. Alkali metal cations lower the critical concentration by a factor of ∽2, produce cold reversible polymers whose formation is sensitive to podophyllotoxin inhibition, increase the fraction of polymers present as microtubules from ∽0.9 to 0.99, and reverse or prevent urea‐induced depolymerization of microtubules. In the presence of microtubule‐associated proteins, the promotion of polymerization is no longer cation selective. In the polymerization of tubulin S, in which the acidic C termini of both monomers have been cleaved, the cation enhancement is markedly decreased, although selectivity persists.

Because the selectivity sequence is similar to that of the coil/helix transition of polyglutamic acid, we suggest that a major part, although not all, of the cation selective enhancement of polymerization results from shielding of the glutamate‐rich C termini of the tubulin monomers.
Fluorescence energy transfer measurement of distances between ligand binding sites of tubulin and its implication for protein‐protein interaction
Authors: Bhattacharya Anusree , Roy Siddhartha , Bhattacharyya Bhabatarak
Published: February 2009

9‐(Dicyanovinyl) julolidine (DCVJ) is a fluorescent probe, which binds to a unique site on the tubulin dimer and exhibits different properties that are dependent upon its oligomeric state (Kung & Reed, 1989). DCVJ binds to tubulin, the tubulin‐colchicine complex, and the tubulin‐ruthenium red complex equally well, but binds tighter to the ANS‐tubulin complex than to tubulin alone. The energy transfer studies indicate a small amount of energy transfer with colchicine, but a significant energy transfer with ANS. It was shown previously that ruthenium red binds near the C‐terminal tail region of the α‐subunit. Ruthenium red causes major quenching of fluorescence of the tubulin‐DCVJ complex, suggesting proximity of binding sites.

The derived distances are consistent with DCVJ binding near the αβ interface, but on the opposite face of the colchicine binding site. Location of the binding site correlates with the observed effect of a different polymerized state of tubulin on the DCVJ spectroscopic properties. The effect of dimer‐dimer association on DCVJ binding, at high protein concentrations (Kung & Reed, 1989), suggests that such an association may occur through lateral contacts of the elongated tubulin dimer, at least in a significant fraction of the cases. Transmission of ANS‐induced conformational change to the DCVJ binding site, which is near important dimer‐dimer contact sites, makes it possible that such conformational changes may be responsible for polymerization inhibition by anilino‐naphthalene sulfonates.
Extended repertoire of permissible peptide ligands for HLA‐B*2702
Authors: Raghavan Malini , Lebrón José A. , Johnson Jennifer L. , Bjorkman Pamela J.
Published: February 2009

Recognition of self peptides bound to the class I major histocompatibility complex molecule HLA‐B27 is thought to trigger proliferation of autoreactive T cells and result in autoimmune arthritic diseases. Previous work from other laboratories established that a predominant feature of endogenous peptides eluted from purified B27 is an arginine at position 2. We studied the binding of peptides containing both natural and unnatural amino acids by the subtype HLA‐B*2702, with the goal of gaining insight into peptide binding by this B27 subtype that is associated with susceptibility to arthritic disease. A soluble form of B*2702 was depleted of endogeneous peptides. We tested the binding of peptides substituted with cysteine, homocysteine, or an α‐amino‐ϵ‐mercapto hexanoic acid side chain (Amh) instead of the naturally occurring arginine at position 2, to determine whether the peptide sulfhydryl residue could be covalently linked to cysteine 67 in the B*2702 binding cleft. Although none of the altered peptide sequences bound covalently to B*2702, the affinities of the homocysteine‐ and Amh‐substituted peptides were close to that of the native peptide sequence. Substitutions at position 2 with other side chains, such as glutamine and methionine, also resulted in peptides that bound with only slightly reduced affinity. These results demonstrate that peptide side chains other than arginine at position 2 can be accomodated within the B*2702 peptide binding site with only minor reductions in affinity. This extended repertoire of permissible B27‐binding peptides should be taken into account for a consideration of disease‐associated peptide sequences.
Water mediated protein‐DNA interactions: The relationship of thermodynamics to structural detail
Authors: Morton Craig J. , Ladbury John E.
Published: February 2009

The elucidation of a relationship between the thermodynamic parameters and the structural changes accompanying bio‐molecular interactions could lead to predictive algorithms. For example, based on some knowledge of the structure of a target molecule the affinities of ligands could be determined with obvious implications for the pharmaceutical industry. In attempting to relate the thermodynamic and structural changes on formation of a protein‐DNA complex, the correlation between change in heat capacity and burial of surface area has proved successful. However, this correlation appears to break down when water molecules are included in the binding interface. Here we present data that support the hypothesis that bound water molecules have to be considered as contributing to the change in heat capacity and could, thus, be used in ligand design.
Crystallization and preliminary X‐ray analysis of the monomeric Cu, Zn superoxide dismutase from Escherichia coli
Authors: Battistoni Andrea , Folcarelli Silvia , Rotilio Giuseppe , Desideri Alessandro , Capasso Clemente , Pesce Alessandra , Bolognesi Martino
Published: February 2009

The Cu, Zn superoxide dismutase (Cu, Zn SOD) originally isolated from the periplasmic space of Escherichia coli has been cloned and overexpressed in the E. coli strain BMH 71/18. The protein has been purified as a single component of 17,000 Da, corresponding to one subunit of the common dimeric eukaryotic Cu, Zn SODs. Large crystals of the purified protein have been grown in the presence of polyethylene glycol 4,000 at pH 8.5; the crystals belong to the monoclinic space group P21, with unit cell constants α = 33.1 Å, b = 52.6 Å, c = 43.3 Å, β = 111.4 degrees. One SOD subunit is contained in the asymmetric unit, yielding a Vm value of 2.1 Å3/Da; the crystals diffract X‐rays beyond 2.0 Å resolution.
Crystallization and preliminary X‐ray analysis of aldehyde dehydrogenase from Vibrio harveyi
Authors: Croteau Nathalie , Vedadi Masoud , Delarge Marc , Meighen Edward , Vrielink Alice , Abu‐Abed Mona , Howell P. Lynne
Published: February 2009

Aldehyde dehydrogenase from Vibrio harveyi catalyzes the oxidation of long‐chain aliphatic aldehydes to acids. The enzyme is unique among the family of aldehyde dehydrogenases in that it exhibits much higher specificity for the cofactor NADP+ than for NAD+. The sequence of this form of the enzyme varies significantly from the NAD+ dependent forms, suggesting differences in the three‐dimensional structure that may be correlated to cofactor specificity. Crystals of the enzyme have been grown both in the presence and absence of NADP+ using the hanging drop vapor diffusion technique. In order to improve crystal size and quality, iterative seeding techniques were employed. The crystals belong to space group P21, with unit cell dimensions a = 79.4 Å, b = 131.1 Å, c = 92.2 Å, and β = 92.4°. Freezing the crystal to 100K has enabled a complete set of data to be collected using a rotating anode source (λ. = 1.5418 Å). The crystals diffract to a minimum d‐spacing of 2.6 Å resolution. Based on density calculations, two homodimers of molecular weight 110 kDa are estimated to be present in the asymmetric unit. Self‐rotation functions show the presence of 3 noncrystallographic twofold symmetry axes.
High‐resolution X‐ray structure of UDP‐galactose 4‐epimerase complexed with UDP‐phenol
Authors: Thoden James B. , Frey Perry A. , Holden Hazel M.
Published: February 2009

UDP‐galactose 4‐epimerase from Escherichia coli catalyzes the interconversion of UDP‐glucose and UDP‐galactose. In recent years, the enzyme has been the subject of intensive investigation due in part to its ability to facilitate nonstereospecific hydride transfer between β‐NADH and a 4‐keto hexopyranose intermediate. The first molecular model of the epimerase from E. coli was solved to 2.5 Å resolution with crystals grown in the presence of a substrate analogue. UDP‐phenol (Bauer AJ, Rayment I, Frey PA, Holden HM, 1992, Proteins Struct Funct Genet 12:372–381). There were concerns at the time that the inhibitor did not adequately mimic the sugar moiety of a true substrate. Here we describe the high‐resolution X‐ray crystal structure of the ternary complex of UDP‐galactose 4‐epimerase with NADH and UDP‐phenol. The model was refined to 1.8 Å resolution with a final overall R‐factor of 18.6%. This high‐resolution structural analysis demonstrates that the original concerns were unfounded and that, in fact, UDP‐phenol and UDP‐glucose bind similarly. The carboxamide groups of the dinucleotides, in both subunits, are displaced significantly from the planes of the nicotinamide rings by hydrogen bonding interactions with Ser 124 and Tyr 149. UDP‐galactose 4‐epimerase belongs to a family of enzymes known as the short‐chain dehydrogenases, which contain a characteristic Tyr‐Lys couple thought to be important for catalysis. The epimerase/NADH/UDP‐phenol model presented here represents a well‐defined ternary complex for this family of proteins and, as such, provides important information regarding the possible role of the Tyr‐Lys couple in the reaction mechanism.
A missing link in cupredoxins: Crystal structure of cucumber stellacyanin at 1.6 Å resolution
Authors: Hart P. John , Eisenberg David , Nersissian Aram M. , Valentine Joan Selverstone , Herrmann Reinhold G. , Nalbandyan Robert M.
Published: February 2009

Stellacyanins are blue (type I) copper glycoproteins that differ from other members of the cupredoxin family in their spectroscopic and electron transfer properties. Until now, stellacyanins have eluded structure determination. Here we report the three‐dimensional crystal structure of the 109 amino acid, non‐glycosylated copper binding domain of recombinant cucumber stellacyanin refined to 1.6 Å resolution. The crystallographic R‐value for all 18,488 reflections (σ > 0) between 50–1.6 Å is 0.195. The overall fold is organized in two β‐sheets, both with four β‐strands. Two α‐helices are found in loop regions between β‐strands. The β‐sheets form a β‐sandwich similar to those found in other cupredoxins, but some features differ from proteins such as plastocyanin and azurin in that the β‐barrel is more flattened, there is an extra N‐terminal α‐helix, and the copper binding site is much more solvent accessible. The presence of a disulfide bond at the copper binding end of the protein confirms that cucumber stellacyanin has a phytocyanin‐like fold. The ligands to copper are two histidines, one cysteine, and one glutamine, the latter replacing the methionine typically found in mononuclear blue copper proteins. The Cu‐Gln bond is one of the shortest axial ligand bond distances observed to date in structurally characterized type I copper proteins. The characteristic spectroscopic properties and electron transfer reactivity of stellacyanin, which differ significantly from those of other well‐characterized cupredoxins, can be explained by its more exposed copper site, its distinctive amino acid ligand composition, and its nearly tetrahedral ligand geometry. Surface features on the cucumber stellacyanin molecule that could be involved in interactions with putative redox partners are discussed.
Cloning, expression, and spectroscopic characterization of Cucumis sativus stellacyanin in its nonglycosylated form
Authors: Nersissian Aram M. , Valentine Joan Selverstone , Mehrabian Zara B. , Nalbandyan Robert M. , Hart P. John , Fraczkiewicz Grazyna , Czernuszewicz Roman S. , Bender Christopher J. , Peisach Jack , Herrmann Reinhold G.
Published: February 2009

The cDNA encoding the 182 amino acid long precursor stellacyanin from Cucumis sativus was isolated and characterized. The protein precursor consists of four sequence domains: I, a 23 amino acid hydrophobic N‐terminal signal peptide with features characteristic of secretory proteins; II, a 109 amino acid copper‐binding domain; III, a 26 amino acid hydroxyproline‐ and serine‐rich peptide characteristic of motifs found in the extensin family, extracellular structural glycoproteins found in plant cell walls; and IV, a 22 amino acid hydrophobic extension. Maturation of the protein involves posttranslational processing of domains I and IV. The copper‐binding domain (domain II), which shares high sequence identity with other stellacyanins, has been expressed without its carbohydrate attachment sites, refolded from the Escherichia coli inclusion bodies, purified, and characterized by electronic absorption, EPR, ESEEM, and RR spectroscopy. Its spectroscopic properties are nearly identical to those of stellacyanin from the Japanese lacquer tree Rhus vernicifera, the most extensively studied and best characterized stellacyanin, indicating that this domain folds correctly, even in the absence of its carbohydrate moiety. The presence of a hydroxyproline‐ and serine‐rich domain III suggests that stellacyanin may have a function other than that of a diffusible electron transfer protein, conceivably participating in redox reactions localized at the plant cell wall, which are known to occur in response to wounding or infection of the plant.
Probing the structure and mobility of Pseudomonas aeruginosa azurin by circular dichroism and dynamic fluorescence anisotropy
Authors: Mei G. , Agró A. Finazzi , Rosato N. , Gilardi G. , Venanzi M. , Canters G.W.
Published: February 2009

The UV dynamic fluorescence and CD of several Pseudomonas aeruginosa azurins bearing single amino acid mutation have been studied. Two classes of mutants were examined. In the first class, two hydrophobic residues in the core of the protein, Ile 7 and Phe 110, nearest to the azurin single tryptophan Trp 48, were substituted by a serine (mutants I7S and F110S). In the second class, two residues in the outer sphere of the copper ligand field were changed, obtaining the following mutants: M44K, H35F, H35L, and H35Q. All these proteins showed two fluorescence lifetimes in the copper‐containing form, but only one in the copper‐free form. The lifetime of the latter derivatives was different from either those of the metal‐bound samples, definitely ruling out the presence of apo‐like species in the holo protein. Copper‐free I7S and F110S showed a more complex fluorescence decay profile requiring a distribution of lifetimes rather than a single lifetime. Holo F110S was also better fitted, in the limit of confidence, with two distributions rather than a pair of lifetimes. Time‐resolved anisotropy of these two mutants as well as of wild‐type (wt) protein showed two components (rotational times for wt ≤ 200 ps and 7 ns, respectively). These components were not affected significantly by copper removal in the case of wt protein. Instead, the short rotational component of the mutants dropped dramatically to values near zero, indicating a much greater mobility of the tryptophanyl residue in the mutant apo azurins. These data were supported by CD measurements showing a small effect of the copper presence in the region below 250 nm, i.e., in the secondary structure, but almost a collapse of the aromatic asymmetry at 270–295 nm related to a relaxation of the structural constraint around the tryptophan. Altogether these data show that copper does not play a structural role in wt azurin, whereas it is crucial in the stabilization of I7S and F110S mutants. Furthermore, although the metal site geometry is rigidly kept in wt apo‐azurin, it regains the native form only in the presence of the metal in the “core” mutants. This finding is important for the theory of entatic states in metalloproteins (Williams RJP, 1995, Eur J Biochem 234:363–381).
The allosteric activator ATP induces a substrate‐dependent alteration of the quaternary structure of a mutant aspartate transcarbamoylase impaired in active site closure
Authors: Baker Darren P. , Kantrowitz Evan R. , Vachette Patrice , Fetler Luc
Published: February 2009

Aspartate transcarbamoylase from Escherichia coli shows homotropic cooperativity for aspartate as well as hetero‐tropic regulation by nucleotides. Structurally, it consists of two trimeric catalytic subunits and three dimeric regulatory subunits, each chain being comprised of two domains. Glu‐50 and Ser‐171 are involved in stabilizing the closed conformation of the catalytic chain. Replacement of Glu‐50 or Ser‐171 by Ala in the holoenzyme has been shown previously to result in marked decreases in the maximal observed specific activity, homotropic cooperativity, and affinity for aspartate (Dembowski NJ, Newton CJ, Kantrowitz ER, 1990, Biochemistry 29:3716–3723; Newton CJ, Kantrowitz ER, 1990, Biochemistry 29:1444–1451). We have constructed a double mutant enzyme combining both mutations. The resulting Glu‐50/Ser‐171 &RR; Ala enzyme is 9‐fold less active than the Ser‐171 &RR; Ala enzyme, 69‐fold less active than the Glu‐50 &RR; Ala enzyme, and shows 1.3‐fold and 1.6‐fold increases in the [S]0.5Asp as compared to the Ser‐171 &RR; Ala and Glu‐50 &RR; Ala enzymes, respectively. However, the double mutant enzyme exhibits some enhancement of homotropic cooperativity with respect to aspartate, relative to the single mutant enzymes. At subsaturating concentrations of aspartate, the Glu‐50/Ser‐171 &RR; Ala enzyme is activated less by ATP than either the Glu‐50 &RR; Ala or Ser‐171 &RR; Ala enzyme, whereas CTP inhibition is intermediate between that of the two single mutants. As opposed to the wild‐type enzyme, the Glu‐50/Ser‐171 &RR; Ala enzyme is activated by ATP and inhibited by CTP at saturating concentrations of aspartate. Structural analysis of the Ser‐171 &RR; Ala and Glu‐50/Ser‐171 &RR; Ala enzymes by solution X‐ray scattering indicates that both mutants exist in the same T quaternary structure as the wild‐type enzyme in the absence of ligands, and in the same R quaternary structure in the presence of saturating N‐(phosphonoacetyl)‐L‐aspartate. However, saturating concentrations of carbamoyl phosphate and succinate are unable to convert a significant fraction of either mutant enzyme population to the R quaternary structure, as has been observed previously for the Glu‐50 &RR; Ala enzyme. The curves for both the Ser‐171 &RR; Ala and Glu‐50/Ser‐171 &RR; Ala enzymes obtained in the presence of substoichiometric amounts of PALA are linear combinations of the two extreme T and R states. The structural consequences of nucleotide binding to these two enzymes were also investigated. Most surprisingly, the direction and amplitude of the effect of ATP upon the double mutant enzyme were shown to vary depending upon the substrate analogue used.
Evidence for an active T‐state pig kidney fructose 1,6‐bisphosphatase: Interface residue Lys‐42 is important for allosteric inhibition and AMP cooperativity
Authors: Lu Guqiang , Stec Boguslaw , Giroux Eugene L. , Kantrowitz Evan R.
Published: February 2009

During the R &RR; T transition in the tetrameric pig kidney fructose‐1,6‐bisphosphatase (Fru‐1,6‐P2ase, EC 3.1.3.11) a major change in the quaternary structure of the enzyme occurs that is induced by the binding of the allosteric inhibitor AMP (Ke HM, Liang JY, Zhang Y, Lipscomb WN, 1991, Biochemistry 30:4412–4420). The change in quaternary structure involving the rotation of the upper dimer by 17° relative to the lower dimer is coupled to a series of structural changes on the secondary and tertiary levels. The structural data indicate that Lys‐42 is involved in a complex set of intersubunit interactions across the dimer‐dimer interface with residues of the 190′s loop, a loop located at the pivot of the allosteric rotation. In order to test the function of Lys‐42, we have replaced it with alanine using site‐specific mutagenesis. The kcat and Km values for Lys‐42 &RR; Ala Fru‐1,6‐P2ase were 11 s−1 and 3.3 μM, respectively, resulting in a mutant enzyme that was slightly less efficient catalytically than the normal pig kidney enzyme. Although the Lys‐42 &RR; Ala Fru‐1,6‐P2ase was similar kinetically in terms of Km and kcat the response to inhibition by AMP was significantly different than that of the normal pig kidney enzyme. Not only was AMP inhibition no longer cooperative, but also it occurred in two stages, corresponding to high‐ and low‐affinity binding sites. Saturation of the high‐affinity sites only reduced the activity by 30%, compared to 100% for the wild‐type enzyme. In order to determine in what structural state the enzyme was after saturation of the high‐affinity sites, the Lys‐42 &RR; Ala enzyme was crystallized in the presence of Mn2+, fructose‐6‐phosphate (Fru‐6‐P), and 100 μM AMP and the data collected to 2.3 Å resolution. The X‐ray structure showed the T state with AMP binding with full occupancy to the four regulatory sites and the inhibitor Fru‐6‐P bound at the active sites. The results reported here suggest that, in the normal pig kidney enzyme, the interactions between Lys‐42 and residues of the 190′s loop, are important for propagation of AMP cooperativity to the adjacent subunit across the dimer‐dimer interface as opposed to the monomer‐monomer interface, and suggest that AMP cooperativity is necessary for full allosteric inhibition by AMP.
Perturbations of the denatured state ensemble: Modeling their effects on protein stability and folding kinetics
Authors: Wrabl James O. , Shortle David
Published: February 2009

By considering the denatured state of a protein as an ensemble of conformations with varying numbers of sequence‐specific interactions, the effects on stability, folding kinetics, and aggregation of perturbing these interactions can be predicted from changes in the molecular partition function. From general considerations, the following conclusions are drawn: (1) A perturbation that enhances a native interaction in denatured state conformations always increases the stability of the native state. (2) A perturbation that promotes a non‐native interaction in the denatured state always decreases the stability of the native state. (3) A change in the denatured state ensemble can alter the kinetics of aggregation and folding. (4) The loss (or increase) in stability accompanying two mutations, each of which lowers (or raises) the free energy of the denatured state, will be less than the sum of the effects of the single mutations, except in cases where both mutations affect the same set of partially folded conformations. By modeling the denatured state as the ensemble of all non‐native conformations of hydrophobic‐polar (HP) chains configured on a square lattice, it can be shown that the stabilization obtained from enhancement of native interactions derives in large measure from the avoidance of non‐native interactions in the D state. In addition, the kinetic effects of fixing single native contacts in the denatured state or imposing linear gradients in the HH contact probabilities are found, for some sequences, to significantly enhance the efficiency of folding by a simple hydrophobic zippering algorithm. Again, the dominant mechanism appears to be avoidance of non‐native interactions. These results suggest stabilization of native interactions and imposition of gradients in the stability of local structure are two plausible mechanisms involving the denatured state that could play a role in the evolution of protein folding and stability.
Novel domains in NADPH oxidase subunits, sorting nexins, and PtdIns 3‐kinases: Binding partners of SH3 domains?
Authors: Ponting Christopher P.
Published: February 2009

Two SH3 domain‐containing cytosolic components of the NADPH oxidase, p47phox and p40phox, are shown by analyses of their sequences to contain single copies of a novel class of domain, the PX (phox) domain. Homologous domains are demonstrated to be present in the Cpk class of phosphatidylinositol 3‐kinase, S. cerevisiae Bem1p, and S. pombe Scd2, and a large family of human sorting nexin 1 (SNX1) homologues. The majority of these domains contains a polyproline motif, typical of SH3 domain‐binding proteins. Two further findings are reported. A third NADPH oxidase subunit, p67phox, is shown to contain four tetratricopeptide repeats (TPRs) within its N‐terminal RaclGTP‐binding region, and a 28 residue motif in p40phox is demonstrated to be present in protein kinase C isoforms τ/λ and ζ, and in three ZZ domain‐containing proteins.
Kinetic analysis of ligand binding to interleukin‐2 receptor complexes created on an optical biosensor surface
Authors: Myszka David G. , Arulanantham Peter R. , Sana Theodore , Wu Zining , Morton Thomas A. , Ciardelli Thomas L.
Published: February 2009

The interleukin‐2 receptor (IL‐2R) is composed of at least three cell surface subunits, IL‐2Rα, IL‐2Rβ, and IL‐2Rγc. On activated T‐cells, the α‐ and β‐subunits exist as a preformed heterodimer that simultaneously captures the IL‐2 ligand as the initial event in formation of the signaling complex. We used BIAcore to compare the binding of IL‐2 to biosensor surfaces containing either the α‐subunit, the β‐subunit, or both subunits together. The receptor ectodomains were immobilized in an oriented fashion on the dextran matrix through unique solvent‐exposed thiols. Equilibrium analysis of the binding data established IL‐2 dissociation constants for the individual α‐ and β‐subunits of 37 and 480 nM, respectively. Surfaces with both subunits immobilized, however, contained a receptor site of much higher affinity, suggesting the ligand was bound in a ternary complex with the α‐ and β‐subunits, similar to that reported for the pseudo‐high‐affinity receptor on cells. Because the binding responses had the additional complexity of being mass transport limited, obtaining accurate estimates for the kinetic rate constants required global fitting of the data sets from multiple surface densities of the receptors. A detailed kinetic analysis indicated that the higher‐affinity binding sites detected on surfaces containing both α‐ and β‐subunits resulted from capture of IL‐2 by a preformed complex of these subunits. Therefore, the biosensor analysis closely mimicked the recognition properties reported for these subunits on the cell surface, providing a convenient and powerful tool to assess the structure‐function relationships of this and other multiple subunit receptor systems.