Eur J Biochem 270, 2126–2136 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03562.x Tracking interactions that stabilize the dimer structure of starch phosphorylase from Corynebacterium callunae Roles of Arg234 and Arg242 revealed by sequence analysis and site-directed mutagenesis Richard Griessler1,2,3, Alexandra Schwarz1,3, Jan Mucha2 and Bernd Nidetzky1,3 Institute of Food Technology and 2Centre of Applied Genetics, University of Agricultural Sciences, Vienna, Austria; Institute of Biotechnology, Graz University of Technology, Austria Glycogen phosphorylases (GPs) constitute a family of widely spread catabolic a1,4-glucosyltransferases that are active as dimers of two identical, pyridoxal 5¢-phosphatecontaining subunits In GP from Corynebacterium callunae, physiological concentrations of phosphate are required to inhibit dissociation of protomers and cause a 100-fold increase in kinetic stability of the functional quarternary structure To examine interactions involved in this large stabilization, we have cloned and sequenced the coding gene and have expressed fully active C callunae GP in Escherichia coli By comparing multiple sequence alignment to structurefunction assignments for regulated and nonregulated GPs that are stable in the absence of phosphate, we have scrutinized the primary structure of C callunae enzyme for sequence changes possibly related to phosphate-dependent dimer stability Location of Arg234, Arg236, and Arg242 within the predicted subunit-to-subunit contact region made these residues primary candidates for site-directed muta- genesis Individual Arg fi Ala mutants were purified and characterized using time-dependent denaturation assays in urea and at 45 °C R234A and R242A are enzymatically active dimers and in the absence of added phosphate, they display a sixfold and fourfold greater kinetic stability of quarternary interactions than the wild-type, respectively The stabilization by 10 mM of phosphate was, however, up to 20-fold greater in the wild-type than in the two mutants The replacement of Arg236 by Ala was functionally silent under all conditions tested Arg234 and Arg242 thus partially destabilize the C callunae GP dimer structure, and phosphate binding causes a change of their tertiary or quartenary contacts, likely by an allosteric mechanism, which contributes to a reduced protomer dissociation rate Glycogen phosphorylases (GPs) catalyse degradation of glycogen and structurally related reserve polysaccharides in the cytosol to provide energy via the branch point metabolite a-D-glucose-1-phosphate All known GPs are functional homodimers composed of % 90-kDa subunits and require pyridoxal 5¢-phosphate (PLP) cofactor for activity [1–7] Although a very low basal activity may be present in the holoenzyme protomer, quarternary interactions clearly determine physiological levels of phosphorylase activity and are a prerequisite for the regulatory properties of eukaryotic GPs [8–10] Forces that stabilize the dimer structure of GP are therefore essential to optimal enzyme function under physiological boundary conditions GPs are a/b proteins that display a twodomain fold in which the N-terminal domain and the C-terminal domain are separated by a catalytic site cleft The structural elements that comprise the subunit–subunit interface are located in the N-terminal domain The dimer contact regions of regulated and nonregulated GPs share structural similiarity overall, but differ on the molecular level [3–7] Starch phosphorylase (StP) from the soil bacterium Corynebacterium callunae is a member of the GP family [11] Its activity is not under control of common allosteric effectors of mammalian GPs such as AMP or D-glucose6-phosphate The enzyme differs from structurally wellcharacterized GPs [3–7] because it requires physiological concentrations of phosphate (% mM) for stability of the functional oligomeric structure [12] Binding of phosphate to a protein site different from the site where the substrate phosphate binds causes apparent tightening of quarternary interactions present in StP and leads to a 100-fold increase in kinetic stability of the active dimer [12] The very low in-vitro lifetime of StP activity in the absence of phosphate (% 30 [12]) suggests that this stabilizing effect might be Correspondence to B Nidetzky, Institute of Biotechnology, Graz University of Technology, Petersgasse 12/I, A-8010 Graz, Austria Fax: + 43 316 873 8434, Tel.: + 43 316 873 8400; E-mail: bernd.nidetzky@tugraz.at Abbreviations: GP, glycogen phosphorylase; rmGP, rabbit muscle GP; MalP, maltodextrin phosphorylase; EcMalP, MalP from Escherichia coli; PLP, pyridoxal 5¢-phosphate; StP, starch phosphorylase Enzymes: a-glucan (glycogen, starch, maltodextrin) phosphorylase (1,4-a-D-glucan:phosphate a-D-glucosyltransferase) (EC 2.4.1.1) Note: The genomic sequence of C callunae that comprises the entire structural gene of starch phosphorylase is available under the GenBank accession number: AY102616 (Received 28 November 2002, accepted March 2003) Keywords: interface; oxyanion; phosphate; stabilization; subunit dissociation Ó FEBS 2003 Dimer stability of bacterial starch phosphorylase (Eur J Biochem 270) 2127 important in the physiology of C callunae In light of the fact that interactions apparently critical to a stable and active protein conformation converge at the dimer interface of GP, we considered oxyanion-dependent stability of the StP dimer to be a significant problem and hence worth examining We thus turned our attention to the primary structure of StP and report here the cloning, sequencing and heterologous expression in Escherichia coli of the gene encoding this enzyme In an effort to identify sequence changes relevant to GP dimer stability, we compared multiple sequence alignment with secondary structure assignments and dimer contacts in structurally characterized GPs [3–7,13–15] Through this process, the main element of the dimer interface in GPs, the so-called TOWER helix, was allocated to the linear sequence of StP In rabbit muscle GP (rmGP) and likewise other GPs [3–7,13–15], this helix forms intimate contacts with its counterpart helix on the opposite subunit Three arginine residues are located within the predicted TOWER helix of StP Because arginine are common components of interfaces of oligomeric proteins and frequently show interaction with oxyanion ligands such as phosphate or sulfate, the ÔTOWER argininesÕ of StP were selected as candidates to be replaced by alanine-scanning site-directed mutagenesis We have assessed the role of each arginine for oxyanion-dependent stability of the StP dimer Experimental procedures (GT)CT(AGT)GC-3¢; and reverse (p2), 5¢-GC(CT)TC (AGT)GGCA(AGT)(AC)AC(AGC)GT(AG)TGGTT(AGT) GT-3¢ Polymerase chain reactions (50 lL) were carried out with a Hybaid thermocycler (Thermo Life Sciences) and used 200 ng of chromosomal template DNA, 40 pmol sense and antisense primers, 150 lmol dNTPs in PCR buffer (Promega) PCRs consisted of 30 cycles of 30 s denaturation at 95 °C, followed by 20 s primer annealing at 55 °C and 40 s elongation at 72 °C The final extension step was carried out at 72 °C for The resulting PCR product was gel-purified and placed into a pUC 18 vector (Life Technologies) using SmaI cleavage and blunt-end cloning This recombinant vector was transferred by electroporation into competent cells of E coli DH5a (Stratagene), and after plasmid purification the insert was subjected to dideoxy sequencing with an ABI Prism 310 Genetic Analyzer (Applied Biosystems) using Universal Primer (Amersham Pharmacia Biotech) The PCR product was used as specific probe for Southern blot hybridization experiments Fragments of interest were cloned into pBluescript II SK(+/–) (Life Technologies) via BamHI and HindIII restriction sites and used to generate a partial genomic library of C callunae DNA Colony hybridization with the PCR probe was used to screen this library Positive clones were sequenced in both senses of the DNA at the VBC Genomics Sequencing Service Facility of the University of Vienna using the Ôprimer walkingÕ method Materials Construction of expression plasmids Natural StP from C callunae DSM 20145 was produced and purified by reported procedures [11] Materials and assays for measuring enzyme activities in the directions of a-glucan degradation and synthesis have been described elsewhere [11,12] Restriction endonucleases, T4DNA ligase and Taq DNA polymerase were obtained from Promega Pfu DNA polymerase, alkaline phosphatase, RNase, and positively charged nylon membranes were from Roche The expression vectors pQE 30 and pQE 70, the gel extraction kit Qiaex II, and the plasmid purification kit were from Qiagen The following primer set was designed to amplify the entire open reading frame of the StP gene by using PCR: Preparation of an oligonucleotide probe for the StP gene Chromosomal DNA from C callunae DSM 20145 was prepared by incubating approximately 200 mg of wet cell mass suspended in 10 mM Tris/HCl buffer, pH 7.6, containing mM EDTA and 15 mg lysozyme (Sigma) for h at 37 °C To this mixture were added mL of a solution of 0.4 M NaCl, 0.7% (w/v) SDS and mgỈmL)1 proteinase K (Sigma) in 10 mM Tris/HCl buffer, pH 8.2 After incubation at 50 °C for h, protein was precipitated with mL of M NaCl and removed by centrifugation at 13 000 g The DNA in the supernatant was precipitated with ethanol and purified by standard protocols [16] From a comparison of GP sequences in the GenBank database, two well-conserved peptides, GNGGLGRL (residues 131–138 in rmGP) and TNHTLMPEAL (residues 374–383 in rmGP), were chosen and reverse translated into a pair of degenerated PCR primers: forward (p1), 5¢-GG (ACT)AA(CT)GG(GCT)GGT(CT)T(AG)GG(ACT)CG pNter (5¢-CGCGCATGCAGCCCTGAAAAACAGCC-3¢) derived from the authentic N-terminus of StP [11], and pCter (5-ACGC-GTCGACCTACTTTTTAACAGCAG GAGTTG-3¢) where SphI and SalI restriction sites are underlined The 50-lL reaction mixture contained 200 ng of C callunae DNA, 50 pmol of pNter and pCter, 0.1 mM dNTPs, Pfu polymerase buffer, units of Pfu DNA polymerase and was subjected to 35 cycles of denaturation at 95 °C, annealing at 52 °C and elongation at 72 °C The resulting PCR product was blunt-end subcloned into a SmaI-digested pUC 19 vector, yielding pUC 19-StP pUC 19-StP was then digested with SphI and SalI and cloned in-frame into the SphI/SalI site of the pQE 30 vector to produce a fusion protein bearing an N-terminal metal affinity tag (RGSHHHHHHGSA) Competent cells of E coli XL1 Blue (Stratagene) were transformed with the pQE 30 vector containing the DNA insert (pQE 30-StP) In order to obtain nontagged recombinant StP, the StP gene was cloned in the SphI site of the expression vector pQE 70 The C-terminal His-tag provided by the plasmid was deleted by inserting a stop codon in the C-terminal primer The following primers were synthesized to amplify the open reading frame, pNter (5¢-CGCGCATGCCTGAAAAACAGCCACTCC-3¢), pCter (5¢-ACGGCATGCTTAAACAGCAGGAGTTGG-3¢), where restriction sites are underlined The resulting recombinant StP lacked Ser1 Ó FEBS 2003 2128 R Griessler et al (Eur J Biochem 270) Site-directed mutagenesis The single point mutations were introduced by the PCR-based overlap extension method [17] The following mutagenic oligonucleotide primers were used where the mismatched bases are underlined: 5¢-ATCGAAGCC GAGCGCGTTTCC-3¢ (R234A); 5¢-GAACGCGAGGCC GTTTCC-3¢ (R236A); and 5¢-GATATCTGCGCCGT GCTC-3¢ (R242A) A 1400-bp fragment of the StP gene, obtained by digestion of pQE 30-StP with SphI and Eco91I, was used as a template The flanking primers were pNter and pEco91I (5¢-CCAGATCGGTTACCCAATCATCGGAACCG-3¢) PCR conditions were as described above except for the annealing temperature which was 50 °C Plasmid mini-prep DNA was subjected to dideoxy sequencing to verify that the desired mutation had been introduced and that no misincorporation of nucleotides had occurred as a result of the DNA polymerase Each mutagenized fragment was then cloned into the residual pQE 30-StP vector Expression of the StP gene in Escherichia coli Cells of E coli XL1 Blue harbouring pQE 30-StP (or pQE 70-StP) were grown in media that contained tryptone (10 gỈL)1), yeast extract (5 gỈL)1), and NaCl (10 gỈL)1) and ampicillin (100 lgỈmL)1) After the optical density at 600 nm had reached a value of approximately 1, the initial temperature of 37 °C was reduced to 25 °C, and gene expression was induced with 0.5 mM of isopropyl thio-b-Dgalactoside for 12 h Cells were harvested by centrifugation (2000 g for 15 min) and diluted approximately twofold with 50 mM potassium phosphate buffer, pH 7.0 The suspension was passed three times through a 1-inch French pressure cell (Aminco), and cell debris was removed by centrifugation (10 000 g for 30 min) The resulting supernatant was used further Expression of the mutagenized StP genes, placed into the pQE 30 expression vector, was performed in exactly the same way as just described for the wild-type Purification and characterization of recombinant StP and mutants thereof Recombinant wild-type StP was purified by a reported protocol [18] The following procedure was used to purify His-tagged StP and mutants thereof The E coli cell extract (100 mg protein) was applied to a 10-mL copper-loaded chelating Sepharose fast flow resin column (Amersham Pharmacia Biotech; 16 mm diameter) equilibrated with a 50 mM triethanolamine buffer, pH 7.0, containing 20 mM of sodium sulfate Bound protein was eluted with a linear gradient from to 250 mM imidazole in the same buffer (pH 7.0) Fractions containing phosphorylase activity were pooled and brought to 65% saturation in ammonium sulfate The protein pellet obtained after centrifugation (10 000 g for 30 min) was dissolved in a small volume of 300 mM potassium phosphate buffer, pH 7.0, and incubated at 60 °C for 40 Note that heat treatment inactivates any remaining endogenous E coli maltodextrin phosphorylase [19] After centrifugation and concentration using 30-kDa Microsep tubes (Pall Filtron), further purification was carried out by size exclusion chromatography on Superose 12 Prep Grade (Amersham Pharmacia Biotech; 16 mm diameter, 140 mL) equilibrated with 50 mM phosphate buffer, pH 7.0, containing 0.2 M NaCl The methods used for the characterization of the activity and the stability of the recombinant enzymes were those described in detail elsewhere for natural StP [11,12,18] Unless mentioned otherwise, a continuous coupled enzyme assay at 30 °C was used to measure phosphorylase activity using as the substrate 30 gỈL)1 of maltodextrin (dextrin equivalent 19.4; Agrana [11]) dissolved in a 50 mM potassium phosphate buffer, pH 7.0, containing lM of glucose 1,6-bisphosphate, 2.5 mM of NAD+, rabbit muscle phosphoglucomutase (8 units; Boehringer), and glucose 6-phosphate dehydrogenase (3 units; Sigma) One unit of activity (1 U) refers to lmol NADH produced under the given conditions Binding of phosphate and sulfate to StP or mutants thereof was determined by using a previously reported procedure in which inhibition of quenching of cofactor fluorescence by iodide was measured [12] CD spectroscopic measurements were carried out as described recently [12] using protein solutions (0.1 mgỈmL)1 ± 10%) in a 20 mM Mops buffer, pH 7.0 CD data are expressed in terms of molar ellipticity Results Cloning and sequencing of the StP gene Using the PCR primers p1 and p2, a 710-bp fragment was amplified from chromosomal C callunae DNA, blunt-end cloned into pUC 18, and sequenced The sequence similarity search clearly indicated that this fragment was a part of a putative phosphorylase gene The [a-P32dCTP]-labeled PCR fragment was used as a probe for Southern blot hybridization to C callunae genomic DNA that had been exhaustively digested with different endonucleases A strong hybridization to a BamHI fragment of approximately 2.9 kb and a HindIII fragment of approximately 4.2 kb was found (Fig 1, panel A) These fragments were cloned into pBluescript II SK(+/–), and positive clones were identified by colony hybridization The sequence of the HindIII fragment comprised the entire StP gene except for 158 nucleotides corresponding to the N-terminal part of C callunae StP (Fig 1) The BamHI fragment included this part of the open reading frame, as shown in Fig (panel B) Further gene sequencing revealed the absence of another open reading frame % 1000 bp upstream of the start codon and % 2000 bp downstream of the stop codon of the StP gene The entire open reading frame for StP consisted of 2388 bp encoding a protein of 796 amino acids The calculated molecular mass of the StP subunit is 90 603 Da, in good agreement with the value of 88 000 obtained from protein characterization [11] Identification of dimer contact regions in StP from the structural alignment of StP with other GPs An alignment of the amino acid sequences of StP, rmGP and maltodextrin phosphorylase from Escherichia coli (EcMalP) is shown in Fig rmGP and EcMalP were chosen for structure-based sequence comparison because Ó FEBS 2003 Dimer stability of bacterial starch phosphorylase (Eur J Biochem 270) 2129 Fig Southern blot analysis for C callunae genomic DNA (A) and results of DNA library screening (B) using a 0.71 kb PCR probe for the StP gene Lanes 1–4 of the autoradiogram in panel A show the hybridization patterns of the 32P-labeled PCR probe with C callunae DNA (50 lg) digested for up to days with different endonucleases (10–20 U) as indicated DNA fragments were separated on a 0.8% agarose gel and after transfer to a nylon membrane (Roche) allowed to hybridize with the PCR probe overnight at 60 °C Arrows on the right and left of the blot indicate the sizes (in kb) of the main hybridizing DNA fragments of which the BamHI and HindIII fragments were cloned to give a partial genomic DNA library After screening using colony hybridization with the 710-bp PCR probe, positive clones were selected and the inserts sequenced The bottom of the figure (B) indicates the positions of BamHI and HindIII fragments, relative to the entire StP structural gene they represent prototypes of regulated and nonregulated GPs, respectively, and both have well-established structurefunction relationships StP is 41% identical to rmGP and 42% identical to EcMalP Figure maps structural and functional elements of rmGP [4] and EcMalP [3] onto the linear sequence of StP and thus measures the extent of conservation of the respective ÔsitesÕ in StP As in other GPs, the catalytic site and the PLP binding site of StP are virtually identical to the corresponding sites in rmGP and EcMalP By contrast, the regulatory sites of rmGP are almost completely lost in StP Interestingly, there is only small sequence identity between StP and EcMalP in the segments of the sequence that correspond to regulatory sites of rmGP Hudson et al [14] have classified dimer contacts in rmGP into three relatively independent networks of interacting groups The first two networks, often dubbed the cap¢a2-b7 interface, are mediated by residues in the ultimate N-terminal part of the rmGP protomers and are associated with control by allosteric effectors and covalent phosphorylation, as shown in Fig These dimer contact pairs of rmGP are conserved to a very low degree in EcMalP and likewise StP The third network constitutes the major dimer contact region in GP and involves the so-called TOWER (a7) helices (residues 266–277 in rmGP) and the subsequent gate loops of adjacent subunits While specific interactions between the subunits at the TOWER interface vary considerably among different GPs [3–7], the position of the a7 helix in the primary structures of rmGP, human liver GP, yeast GP, and EcMalP is very well conserved Therefore, the TOWER-GATE region could be easily assigned to the sequence of StP, as shown by underlining in Fig (StP residues 231–242) Considering the involvement of the TOWER interface of rmGP in signal transmission from regulatory sites into the active centre [4–6,15], it was interesting that residues in StP corresponding to the a7 helix of rmGP exhibited a higher degree of similarity to the mammalian enzyme than to EcMalP Furthermore, the occurrence of three arginine residues within the TOWER helix of StP at positions 234, 236 and 242 was interesting (Fig 2) Arginines are common components of protein interfaces and occur frequently at oxyanion-binding protein sites [20,21] Residues Arg234, Arg236, and Arg242 were thus targets for site-directed mutagenesis, and their PCRbased replacement by alanine was chosen to eliminate all electrostatic interactions at the respective position It is worth noting that Arg234 and Arg242 are positionally conserved in all mammalian a-glucan phosphorylases while the same positions show considerable variation in the related enzymes from bacteria, fungi and plants Expression of the wild-type and mutagenized StP genes in E coli, and purification and characterization of recombinant enzymes Following induction with isopropyl thio-b-D-galactoside using the conditions described in Experimental procedures, a specific phosphorylase activity of approximately 10 mg)1 (± 15% SD) was measured in cell extracts of E coli XL1 Blue cells transformed with either pQE 30-StP or pQE 70-StP Comparison of this figure with the known specific activity of 30 mg)1 for pure natural StP [11] shows that recombinant StP corresponded to % 30% of the total soluble E coli protein Recombinant wild-type and His-tagged StP, and likewise StP mutants were purified to apparent homogeneity and all were recovered in approximately 25 ± 5% yield Like native StP, His-tagged StP and all StP mutants contained 0.8–1.0 mol of PLP per mol of 90-kDa protomer, as expected if incorporation of cofactor during folding of the recombinant proteins had taken place correctly Circular dichroism (Fig 3) and Fourier-transform infrared spectra (not shown) of the purified recombinant proteins were recorded in an effort to identify alterations in structure, relative to the natural wild-type enzyme [12,18], as a result of recombinant protein production and mutagenesis There were no traceable differences between CD spectra of natural and recombinant StP (not shown) The CD spectrum of His-tagged StP and CD spectra of R234A and R242A mutants were not superimposable (Fig 3), but the overall picture is one of close structural similarity among the wild-type and the two mutants Therefore, site-specific replacements of Arg234 and Arg242 did not cause gross changes in the composition of secondary structural element in the two mutants, relative Ó FEBS 2003 2130 R Griessler et al (Eur J Biochem 270) Fig Comparison of the StP amino acid sequence with the sequences of EcMalP and rmGP The alignment was performed with the MEGALIGN program using CLUSTALW with standard settings Amino acids conserved are shaded in black Catalytic and regulatory sites of rmGP [14] are marked above the sequence, as follows: a, AMP-binding site; c, caffeine/ purine inhibitor site; g, active site residues; p, residues involved in covalent phosphorylation; s, glycogen storage sites; v, pyridoxal phosphate binding site Residues contributing to the dimer interface of rmGP are indicated using the letter, d The primary structure of StP is 41% and 42% identical to the sequences of rmGP and EcMalP, respectively, indicating overall conservation of the structural fold [3,4] The positions of the TOWER helices in EcMalP and rmGP are underlined by a thick line, and the mutations (to be reported later) are indicated by arrows Also, note that the natural enzyme isolated from C callunae [11] lacks Ser1 to the wild-type The specific activities of recombinant StP, His-tagged StP, and the R234A and R236A mutants were identical within the experimental error of ± 10% to the specific activity of StP isolated from C callunae [11] In the standard assay of phosphorylase activity (Experimental section), the R242A mutant displayed only 10% of wildtype activity However, under conditions of saturation in a-glucan substrate (30 gỈL)1 of maltodextrin) and phosphate (500 mM), the R242A mutant had a specific activity approximately 40% that of the wild-type (A discontinuous assay was used here because the high phosphate concentration interferes with coupled enzyme measurements [11]) The result reveals that maximum reaction rate and substrate affinity are both decreased in the R242A mutant, compared to the wild-type Although this implies that the replacement Arg242 by alanine is not without effect on steps involved in enzymic catalysis, we point out that in wild-type StP, loss of active site integrity and subunit dissociation occur as, clearly, kinetically uncoupled events at an elevated temperature [12] Therefore, the analysis of steady-state kinetic data for the R242A mutant and the wild-type must not be interpreted to weaken the comparative evaluation of stabilities of the same enzymes, which follows later The N-terminal His-tag causes formation of an active StP tetramer Preparations of His-tagged StP that were > 98% pure by the criterion of a single protein band in SDS/PAGE (not shown) eluted from a Superose 12 size exclusion column in Ó FEBS 2003 Dimer stability of bacterial starch phosphorylase (Eur J Biochem 270) 2131 Fig Comparison of CD spectra of wild-type StP (solid line), R234A mutant (dotted line), and R242A mutant (dashed line) Spectra were recorded in a 20 mM Mops buffer, pH 7.0, not containing oxyanion Note that the value of protein concentration (0.1 mgỈmL)1) contained 10% error and may be partly responsible for observed differences in molar ellipticity at 222 nm mutants gave elution profiles that were superimposable to that of the wild-type The StP tetramer was of interest because mammalian GP is known to form tetramers at high protein concentrations These tetramers are inactive but dissociate into active dimers when glycogen is present [4] To determine whether dissociation of the StP tetramer could be induced in the presence of substrate, we subjected the purified tetramer fraction to size exclusion chromatography (SEC) under conditions where the elution buffer contained a saturating concentration of maltohexaose (20 mM) or a-Dglucose-1-phosphate (20 mM) The tetramer was completely stable for the time of the experiment (% h) when one of the above ligands was present In marked contrast to observations made with His-tagged StP, the recombinant StP lacking the metal affinity fusion eluted as a single protein peak from the Superose 12 column Its estimated molecular mass was 180 kDa Automated Edman degradation of this recombinant StP yielded the sequence, ProGlu-Lys-Gln, for the N-terminal tetrapeptide of the recombinant wild-type, which is in accordance with the authentic N-terminal sequence of native StP [11] Therefore, the N-terminal metal affinity peptide appears to be responsible for the observed tetramer : dimer ratio of % 0.2 in Histagged StP, and likewise the Arg fi Ala mutants thereof The results suggest that if the occurrence of tetrameric and dimeric forms of His-tagged StP truly represents an altered oligomerization equilibrium, relative to wild-type StP, and is not an artifact of the protein folding process in the E coli cytosol, the conversion of the tetramer into its constituent dimers must take place at a slow rate The data suggest that the use of amino-terminal affinity tags may not be ideal for studies of GP structure However, we emphasize that dimer : tetramer heterogeneity of His-tagged wild-type StP was not changed in the His-tagged mutants and did thus not affect the conclusions of this work Determination of dissociation constants of binary enzyme–oxyanion complexes Fig Elution profile of purified recombinant His-tagged StP upon analytical gel filtration on a Superose 12 column Approximately 100 lg of protein in 100 lL triethanolamine buffer (50 mM, pH 7.0) were applied to the column (20 mL; 1.6 cm diameter) equilibrated with 50 mM potassium phosphate buffer, pH 7.0, containing 0.2 M NaCl Elution was carried out with the same phosphate buffer at a flow rate ă of 0.25 mLặmin)1 using an Aktaexplorer 100 system (Amersham Pharmacia Biotech) and detection at 280 nm Calibration of the sizing column was performed using appropriate protein standards of known molecular mass Apparent molecular masses of 180 kDa and 360 kDa were determined for the eluting protein fractions in this figure two fractions of well-defined apparent molecular masses, as shown in Fig 4: a major 180-kDa fraction corresponding to the dimer and containing approximately 85% of the total protein, and another fraction that accounted for the remainder protein and displayed a molecular mass of 360 kDa, as expected for a StP tetramer The minor protein fraction had the same specific enzyme activity as the dimeric wild-type A monomer fraction was not observed The StP Fluorescence titration assays [12] were carried out with Histagged StP and the R234A and R242A mutants and yielded dissociation constants for enzyme–sulfate (KdSO4) and enzyme–phosphate (KdPi) complexes These Kd values are summarized in Table The Kd values for the His-tagged wild-type enzyme agree closely with the corresponding values measured recently for native StP (KdSO4 ¼ 4; KdPi ¼ 16) [12] The data also reveal that the replacement of the guanidinium side chain of arginine by a methyl side chain of alanine in the R234A and R242A mutants caused only a small effect on the binding of sulfate An approximately 2.5-fold increase in KdSO4 was observed for the R234A mutant, compared to the wild-type The KdSO4 value for the R242A mutant was very similar to that of the wildtype These observations are not consistent with a scenario in which the original side chains of Arg234 and Arg242 participate in binding the sulfate dianion If these side chains provided direct interactions with sulfate, a much larger increase in KdSO4 would be expected for the mutants in comparison to wild-type We did not observe any significant inhibition of quenching of PLP fluorescence in the R234A and R242A mutants in the presence of phosphate at levels of 10 mM and 20 mM, relative to a control that did not Ó FEBS 2003 2132 R Griessler et al (Eur J Biochem 270) Table Comparison of stabilities of recombinant wild-type StP and two enzyme variants in urea and thermal denaturation experiments at pH 7.0 The experiments were carried out in 50 mM triethanolamine buffer, pH 7.0, and used 200 lgỈmL)1 of protein in each assay Other conditions and procedures were as reported previously [11,12,18] n.a., not applicable because no significant change in iodide quenching of cofactor fluorescence occurred in the presence of phosphate up to a concentration of 20 mM Cm at 30 °C (M)/t1/2 at 45 °C (min) Enzyme KdSO4 (mM)/KdPi (mM) No oxyanion added + sulfatea Wild-type 4.5 ± 0.5/18 ± 1.17 ± 0.03/3.2 ± 0.1 2.95 ± 0.10/stableb R234A R242A ± 3/n.a 3.8 ± 0.4/n.a 2.60 ± 0.06/20 ± 2.00 ± 0.02/12 ± 0.5 4.45 ± 0.05/stableb 2.93 ± 0.02/stableb + phosphatea 5.2 (3.45 3.55 2.27 ± ± ± ± 0.2c/stableb,c 0.03d/stableb,d) 0.02d/stableb,d 0.02d/43 ± 5d a Potassium phosphate and ammonium sulfate were used Unless indicated, the oxyanion concentrations matched the respective Kd values It was shown in separate control experiments that the cation, K+ or NH4+, had no influence on stabilities of wild-type StP and mutants thereof b Being stable means that no significant inactivation occurred during a 0.5-h long incubation at 45 °C c,d Data obtained in the presence of c 20 mM and d 10 mM phosphate contain the oxyanion This could result if the site-directed replacement of Arg234 and Arg242 strongly weakened binding of phosphate or if it altered the conformational change in response to phosphate binding Considering that values of KdSO4 are not very sensitive to the mutations, the latter interpretation would seem to be more likely, but the relatively high KdPi value for the wild-type prevents any firm conclusion on the mutants Stability of recombinant wild-type StP and TOWER helix mutants thereof as revealed in urea and thermal denaturation experiments To compare the stabilities of StP and Arg fi Ala mutants thereof, we carried out urea denaturation assays in which protein concentration and incubation time were constant parameters, and [urea] was varied in steps of 0.25 M between 0.0 and 6.0 M The chosen assay monitors enzyme inactivation that is completely irreversible and thus provides a measure of the kinetic stability of the respective enzyme under the conditions used For each protein, the dependence of percentage of remaining enzyme activity on [urea] was analyzed under conditions in which either no oxyanion was present, or phosphate or sulfate was added in a concentration corresponding approximately to the dissociation constant (Kd) of the respective binary enzyme– phosphate or enzyme–sulfate complex at 30 °C (Table 1) Saturation in oxyanion was not attempted to avoid possible interferences of stability measurements by a lyotropic anion effect in the presence of high concentrations of phosphate or sulfate Using nonlinear least squares regression analysis with the SIGMAPLOT 2000 programme (SPSS Inc.), data were fitted to Eqn (1), which describes a sigmoidal decrease of enzyme activity (EA) with increasing concentration of denaturant, EA (urea) ¼ a=ẵ1 ỵ expbẵurea cị 1ị where a, b, and c are parameters (which are not derived from any formal mechanism of denaturation of StP) The apparent denaturation midpoint (Cm) is calculated by using Eqn (1) and the respective parameter estimates, and corresponds to the urea concentration where half the original enzyme activity has been lost Cm values for wild-type StP and two Arg fi Ala mutants thereof are summarized in Table Results for the R236A mutant are not shown in the Table because the stabilities of this mutant and the wild-type were identical within limits of experimental error (DCm % ± 0.15 M) under all conditions examined The Cm values in Table reveal large stabilizing effects of the Arg fi Ala replacements at positions 234 and 242 for conditions in which no oxyanion was present Note that values of the parameter b, which is a measure of the slope of the decrease in EA as [urea] increases, showed little variation in dependence of the enzyme or the reaction conditions and were in the range )0.43 to )0.49 The extra stability brought about by the mutations is reflected by significant shifts of the Cm values for the mutants, relative to that for the wild-type, to higher urea concentrations by % M or greater The stabilization of wild-type StP by a half-saturating concentration of phosphate can be expressed quantitatively by a dramatic up-shift in Cm value by 4.0 M, compared to the control reaction lacking phosphate The observed increase in Cm value effected by a sulfate level matching KdSO4 was 1.2 M, suggesting that under the conditions used, the StP–sulfate complex displays a much smaller kinetic stability than the StP–phosphate complex The DCm-values for the wild-type serve as a frame of reference for analyzing the stabilities of the mutants Taking into account the large stabilization of wild-type StP by bound phosphate, it was unfortunate that KdPi values were not accessible for the R234A and R242A mutants and so defined conditions with regard to saturation in oxyanion were possible only for sulfate Irrespective of the added oxyanion, observed DCm-values for the R242A mutant were smaller than corresponding values for the wild-type (Table 1) Considering that sulfate binding takes place with almost identical affinities in wildtype StP and the R242A mutant and assuming that this reflects similar sulfate binding modes in both proteins, the results show that the binding event as such is not sufficient for sulfate to induce a large stabilization, which in turn is mirrored in the value of DCm It is important to recognize therefore that denaturation midpoints in the presence of half-saturating levels of sulfate were identical within the experimental error for the wild-type and the R242A mutant The simplest explanation of this finding is that Ó FEBS 2003 Dimer stability of bacterial starch phosphorylase (Eur J Biochem 270) 2133 the enzyme-sulfate complexes of wild-type and R242A mutant share similar kinetic stabilities; and that the Arg fi Ala replacement at position 242 offsets the stabilizing effect of sulfate binding in the wild-type to the extent that this mutation stabilizes the enzyme when no sulfate is present (Fig 5B) Interestingly therefore bound sulfate stabilized the R234A mutant and the wild-type equally Hence, although Arg234 is clearly destabilizing in unligated StP, site-directed mutagenesis of the side chain of Arg234 into the methyl side chain of alanine did not diminish the stabilizing effect of sulfate binding in comparison to wildtype, as it was observed for the R242A mutant This result is interesting because it leads to a different interpretation of the role of Arg234 and Arg242 for oxyanion-dependent stability of StP The stabilization brought about by the presence of 10 mM of phosphate was substantially smaller for the R234A mutant (DCm %1 M) than the wild-type (DCm %2.3 M) Even in the absence of a KdPi value for R234A (and likewise R242A), the comparison at a fixed phosphate level is relevant It shows that site-specific replacement in each mutant either decreases the affinity for phosphate, relative to the wild-type, or lowers the kinetic stability of the mutant-phosphate complex, relative to the same wild-type complex Figure illustrates this point by comparing the dependence of DCm-values on the concentrations of phosphate and sulfate for wild-type StP and the R242A mutant The results show a marked preference for stabilization by sulfate over stabilization by phosphate in the mutant, which is clearly different to what was observed for the wild-type Note that the separation of the parallel lines in panel B of Fig corresponds to the difference in Cm-values for the R242A mutant and the wild-type under conditions in which no sulfate was added The data in Fig can be used to roughly estimate the apparent half-saturation constants (app K) for the stabilization of the wild-type (app KSO4 %17 mM; app KPi %28 mM) and the R242A mutant (app KSO4 %12 mM; app KPi % 130 mM) Thermal stabilities of wild-type and Arg fi Ala mutants were determined at 45 °C and are shown in Table In the absence of added oxyanion, the R242A and R234A mutants were 3.8- and 6.2-fold more stable than the wild-type, respectively No significant inactivation of StP and the two enzyme variants was seen over an incubation time of 30 in the presence of sulfate concentration matching KdSO4 In the presence of 10 mM phosphate, wild-type and the R234A mutant were stable while the R242A mutant displayed significant loss of activity Discussion Fig Stabilization of wild-type StP and the R242A mutant by phosphate (A) and sulfate (B) against urea denaturation Results show DCmvalues, which report the difference between Cm at the shown oxyanion concentration and the Cm measured in buffer lacking oxyanion The data are presented as a double reciprocal plot to emphasize the saturatable dependence of DCm on [oxyanion] However, extrapolation to infinite [oxyanion] must be made with caution (hence, the broken lines) because of the additional lyotropic anion effect Also note that in panel A, lines not have identical intercept values Experiments were carried out at 30 °C in 50 mM triethanolamine buffer, pH 7.0, using conditions reported in the text The goal of the present paper was to advance the relationships between structure and oxyanion-dependent stability of StP from Corynebacterium callunae Cloning, sequencing, and heterologous expression of the gene encoding StP were essential requirements for the utilization of site-directed mutagenesis to examine the functional roles of potentially important amino acid residues that were identifiable through analysis of the StP primary structure The results have revealed clearly that Arg234 and Arg242 of the TOWER interface region of StP partially destabilize the dimer structure of the unligated enzyme so that loss of these residues in the Arg fi Ala mutants leads to significantly higher kinetic stability Phosphate binding appears to cause a change in interactions of these arginines, most probably by an allosteric mechanism as discussed below, contributing to the observed stabilization An unexpected finding was that replacements of Arg242 and Arg234 induced a large apparent preference for sulfate over phosphate with regard to the stabilizing effect 2134 R Griessler et al (Eur J Biochem 270) Relationships between StP structure and oxyaniondependent kinetic stability Previous studies have shown that subunit dissociation occurs as an early step during denaturation of StP at elevated temperatures (30 °C) [12] or in urea (R Griessler, & B Nidetzky, unpublished observations) Under conditions of dilute protein and in the absence of free PLP, loss of oligomer structure is accompanied by immediate release of cofactor from the StP subunit Therefore, it is not detectably reversible on the time scale of the assay for phosphorylase activity (% 1–2 min) [12] Measurement of irreversible inactivation of StP can thus serve as a useful reporter of the protomer dissociation event It would seem likely therefore that observed changes in Cm and t1/2-values for irreversible inactivation in urea and at elevated temperatures, brought about by site-specific amino acid replacements in the dimer contact region of StP and likewise, oxyanion bound at the enzyme oxyanion site, result from altered kinetic barriers for subunit dissociation, relative to unliganded wild-type StP We stress, however, that based on the available data, it is not possible to rule out completely a contribution of thermodynamic effects to the measured kinetic stabilities Arginine residues are known for their prevalence in both intra- and inter-chain interfaces [22,23] where the charged guanidinium group is often involved in formation of strong intermolecular hydrogen bonds Such non-covalent interactions have been hypothesized to stabilize multidomain and oligomeric proteins by strengthening either the network of interfacial contacts or the tertiary bonds that prevail in the segment of the interface Site directed mutagenesis has been used, in a few instances though, to verify the role of arginines as stabilizing elements of dimer contact regions [24,25] Therefore, irrespective of the exact orientation of Arg234, Arg236, and Arg242 at the TOWER interface region of StP, it was unexpected that two out of three enzyme variants harboring the Arg fi Ala substitution exhibited a considerably greater kinetic stability in thermal and urea denaturation studies than the wild-type Hydrogen-bonding or other electrostatic interactions involving the ÔTOWER argininesÕ are obviously not optimized for kinetic stability In this scenario, oxyanions could have a stabilizing effect if their binding was capable of either decreasing nonfavorable contacts between protomers or increasing the favorable ones This could occur by various mechanisms, but likely an allosteric one in which oxyanion binding affects the tertiary and/or quarternary interactions involving Arg234 and Arg242 thus leading to a greater stability For the interpretation of the kinetic stabilities of the R234A and R242A mutants, it is most useful to first consider the effects of phosphate and sulfate on conformation and stability of wild-type StP It was shown here that under conditions of half-saturation in oxyanion, phosphate stabilizes the wild-type much more efficiently against denaturation by urea than sulfate, the difference in DCmvalue (which is the increase in Cm compared to the unliganded enzyme when oxyanion is present) being as large as 2.2 M A greater stability of the enzyme-phosphate complex than the enzyme–sulfate complex correlates well with a greater compactness of the former complex, as revealed recently by comparing iodide quenching of Ó FEBS 2003 cofactor fluorescence in StP saturated with phosphate and sulfate [12] The results for wild-type StP imply that a conformational change in protein structure accompanies the oxyanion-binding event and is required for kinetic stability The extent of the structural rearrangement is larger for a phosphate than a sulfate ligand, suggesting that more binding energy from the StP–oxyanion interaction can be translated into a stabilized protein conformation when phosphate is bound The comparison of Kd values for enzyme-sulfate complexes of wild-type and the two Arg fi Ala mutants reveals that a direct participation of the side chains of Arg234 or Arg242 in binding of sulfate is not likely However, both arginines, clearly, take part in the just described oxyaniondependent conformational relay of wild-type StP, and analysis of R234A and R242A mutants serves to emphasize the differential effect of bound phosphate and sulfate in the wild-type In both mutants, however, mainly R242A, phosphate has lost much of the stabilizing potential originally present in the wild-type Expressed as the ratio of DCm (M) and [phosphate] (M), the phosphate-specific stabilization is % 30 for the wild-type, but only % 10 and % for the R234A and R242A mutant, respectively The situation is different for sulfate, which stabilizes wild-type StP and the R234A mutant to approximately the same extent In the R242A mutant, the stabilizing effect of the Arg fi Ala replacement in the unligated protein is offset by the smaller stabilization when sulfate is bound, compared to wild-type StP In conclusion, these data can be summarized to yield the following hypothetical model of the stabilization of StP by oxyanions Phosphate binding at an allosteric site, perhaps within the subunit, leads to propagation of a conformational change into the dimer contact region of the protein Arg242 is a key residue implicated in this structural rearrangement and may even have an active role in relaying the phosphate-dependent and to a lesser extent though, the sulfate-dependent conformational switches Arg234 appears to be part of the relay when phosphate is bound, but not when sulfate is bound Comparison of StP with rmGP and other a-glucan phosphorylases Structure-function studies of rmGP are highly relevant for the interpretation of results for StP First of all, a dissociative mechanism of thermal denaturation of rmGP, similar to that proposed for StP, has been reported recently [26] A major difference between rmGP and StP, however, pertains to the moderate effect that oxyanions have on rmGP stability [26] Secondly, Fletterick and coworkers have mutated TOWER helix residues of rmGP, among them Arg277, which is the rmGP counterpart of Arg242, into alanine and characterized the variant enzymes structurally and with respect to allosteric activation by AMP [27] Their conclusion from a detailed comparison of intersubunit contacts in X-ray structures of wild-type rmGP and R277A was that the Arg fi Ala replacement would destabilize significantly the quaternary interactions originally present in the muscle enzyme Keeping in mind the limitations of using irreversible inactivation as a measure of global protein stability, our results then suggest that Arg242 in StP must participate in interactions clearly different from those of the Ó FEBS 2003 Dimer stability of bacterial starch phosphorylase (Eur J Biochem 270) 2135 corresponding residue in rmGP Interestingly, mutating Arg242 and Arg277 had similar effects on the catalytic competence of StP and rmGP, respectively, resulting in each case, in a significant decrease in specific activity, compared to the wild-type level The side chains of Arg269 and Arg277 make direct hydrogen bonds across the dimer interface of activated rmGP with side chains of Asn250¢ and Asn270¢, respectively, on the adjacent subunit Each of the two asparagines of rmGP is replaced positionally by a glutamate in StP Considerations of charge and packing arrangements suggest that if Arg234 and Arg242 were truly involved in inter-subunit interactions analogous to those seen in rmGP structures, bonding across the interface of StP should be stronger, compared to rmGP, which is unlikely in light of the experimental evidence for StP Another interesting difference between StP and rmGP revealed by structurebased sequence comparison pertains to contacts of Arg277 (and likewise Arg242) within the subunit In rmGP, this arginine forms a charged hydrogen bond with the carboxylate group of Glu162 [4,27] In StP, Glu162 is positionally replaced by an arginine (Arg142), and this is a likely reason for different atomic environments of Arg242 in StP and Arg277 in rmGP Multiple alignment of the first one-hundred a-glucan phosphorylase sequences identified through screening of the nonredundant data bases with the StP primary structure using the BLAST program (not shown) revealed an interesting conservation pattern for positions equivalent to Arg142 and Arg242 of StP In all but two cases, namely a-glucan phosphorylases from Corynebacterium glutamicum (Q8NQW4) and Fusobacterium nucleatum (Q8RF61), the pair of arginine residues found in StP is not observed A pair of amino acids with oppositely charged side chains, glutamate (or aspartate) at position 142 and arginine (or lysine) at position 242, occurs most frequently in the aligned sequences Several other pairwise combinations of amino acids are possible, but bulk and charge at a certain position appear not to be conserved across all organisms and cell types To give two examples for structurally characterized enzymes, EcMalP has a glutamine-lysine pair whereas Saccharomyces cerevisiae glycogen phosphorylase has a glutamate-alanine pair However, an interesting generalization is that enzymes from (hyper)thermophilic bacteria and archaea contain a conserved pair of lysine (position 142) and glutamic acid (position 242) It would be interesting therefore to examine if positional charge reversal for extremophilic structures, compared to most other a-glucan phosphorylase sequences including the mammalian ones, is related to increased stability [28–30] Furthermore, our comparisons show that if an arginine residue occurs at position 142 in a-glucan phosphorylases, position 242 is generally taken by an alanine, serine, or threonine These residues whose side chains are uncharged and sterically less demanding than the side chain of arginine may be primed to avoid unfavorable (destabilizing) interactions with or relayed to counterpart Arg142 This interpretation of the sequence changes among aligned a-glucan phosphorylases is in excellent agreement with the observed kinetic stability of R242A mutant of StP, compared to wild-type enzyme It also provides a rational for an allosteric mechanism of stabilization of StP by oxyanion binding In light of the fact that Arg242 is conserved in all mammalian GPs and considering that the R242A mutant shows only 40% of wild-type activity, it seems probable that Arg242 has been selected in StP for so far unknown reasons of enzyme function Acknowledgements Financial support from the Austrian Science Funds (P-15118-MOB to B.N.) is gratefully acknowledged References Palm, D., Klein, H.W., Schinzel, R., Buehner, M & Helmreich, E.J (1990) The role of pyridoxal 5¢-phosphate in glycogen phosphorylase catalysis Biochemistry 29, 1099–1107 Schinzel, R & Nidetzky, B (1999) Bacterial a-glucan phosphorylases FEMS Microbiol Lett 171, 73–79 Watson, K.A., Schinzel, R., Palm, D & Johnson, L.N (1997) The crystal structure of Escherichia coli maltodextrin phosphorylase provides an explanation for the activity without control in this basic archetype of a phosphorylase EMBO J 16, 1–14 Johnson, L.N (1992) Glycogen phosphorylase: control by 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Gosslar, U., Kriegshaeuser, G & Liebl, W (1998) Isolation and analysis of genes for amylolytic enzymes of hyperthermophilic bacterium Thermotoga maritima FEMS Microbiol Lett 158, 9–15 Supplementary Material The following material is available from http://www blackwellpublishing.com/products/journals/suppmat/EJB/ EJB3562/EJB3562sm.htm ... through analysis of the StP primary structure The results have revealed clearly that Arg234 and Arg242 of the TOWER interface region of StP partially destabilize the dimer structure of the unligated... Nidetzky, B (2000) Thermal denaturation pathway of starch phosphorylase from Corynebacterium callunae: oxyanion binding provides the glue that efficiently stabilizes the dimer structure of the protein... elements of rmGP [4] and EcMalP [3] onto the linear sequence of StP and thus measures the extent of conservation of the respective ÔsitesÕ in StP As in other GPs, the catalytic site and the PLP