Geometry of interaction of metal ions with histidine residues in protein structures

An analysis of the geometry and the orientation of metal ionsbound to histidine residues in proteins is presented. Cationsare found to lie in the imidazole plane along the lone pairon the nitrogen atom. Out of the two tautomeric forms of theimidazole ring, the NE2-protonated form is normally preferred.However, when bound to a metal ion the ND1-protonated form ispredominant and NE2 is the ligand atom. When the metal coordinationis through ND1, steric interactions shift the side chain torsionalangle, X₂ from its preferred value of 90 or 270. The orientationof histidine residues is usually stabilized through hydrogenbonding; ND1-protonated form of a helical residue can form ahydrogen bond with the carbonyl oxygen atom in the precedingturn of the helix. A considerable number of ligands are foundin helices and ß-sheets. A helical residue hound toa heme group is usually found near the C-terminus of the helix.Two ligand groups four residues apart in a helix, or two residuesapart in a ß-strand are used in many proteins to bindmetal ions.     

Predicted ligand interactions for 3',5'-cyclic nucleotide-gated channel binding sites: comparison of retina and olfactory binding site models

Cyclic nucleotide-gated channels (CNGC) open in response tothe binding of 3',5'-cyclic nucleotides. Members of the CNGCfamily vary as much as 100-fold in their ability to respondto cAMP and cGMP. Molecular models of the nucleotide bindingdomains of the bovine retina and catfish and rat olfactory CNGCswere built from the crystal structure of cAMP bound to catabolitegene activator protein (CAP) with AMMP, a program for molecularmechanics and dynamics. The nucleotide conformation can be predictedfrom the number of strong and weak interactions between thepurine ring and the binding site. The amino acids predictedto be important for determining the nucleotide affinity andspecificity are residues 61, 83 (mediated through a water molecule),119 and 127 (CAP sequence numbers) which interact with the purinering. These residues also dictate the conformation of the ligandin the binding pocket cGMP is preferentially bound in the synconformation in bovine retina, bovine olfactory and rat olfactoryCNGCs due to Thr83, while either conformation can bind in catfisholfactory CNGC. cAMP is predicted to bind either in syn or anticonformation, depending on the interaction with residue 119:the anti conformation is preferentially bound in olfactory CNGCs.     

Modular mutagenesis of a TEM-barrel enzyme: the crystal structure of a chimeric E.coli TIM having the eighth {beta}{alpha}-unit replaced by the equivalent unit of chicken TIM

Abstract The crystal structure of a hybrid Escherichia coli triosephosphateisomerase (TIM) has been determined at 2.8 Å resolution.The hybrid TIM (ETIM8CHI) was constructed by replacing the eighthß-unit of E.coli TIM with the equivalent unit of chickenTIM. This replacement involves 10 sequence changes. One of thechanges concerns the mutation of a buried alanine (Ala232 instrand 8) into a phenylalanine. The ETIM8CHI structure showsthat the A232F sequence change can be incorporated by a side-chainrotation of Phe224 (in helix 7). No cavities or strained dihedralsare observed in ETIM8CHI in the region near position 232, whichis in agreement with the observation that ETIM8CHI and E.coliTIM have similar stabilities. The largest CA (C-alpha atom)movements, 3 Å, are seen for the C-terminal end of helix8 (associated with the outward rotation of Phe224) and for theresidues in the loop after helix 1 (associated with sequencechanges in helix 8). From the structure it is not clear whythe k_cat of ETIM8CHI is 10 times lower than in wild type E.coliTIM     

Structural characterization by computer experiments of the lipid-free LDL-receptor-binding domain of apolipoprotein E

The structure and dynamics of the lipid-free LDL-receptor-bindingdomain of apolipoprotein E (apoE-RBD) has been investigatedby Molecular Dynamics Simulations. ApoE-RBD in its monomericlipid-free form is a singular four-helix bundle made up of fourelongated amphipathic helices. Analysis of one 1.5 ns moleculardynamics trajectory of apoE-RBD performed in water indicatesthat the lipid-free domain adopts a structure that exhibitscharacteristics found in native proteins: it has very stablehelices and presents a compact structure. Yet its interior exhibitsa larger number of transient atomic-size cavities relative tothat found in other proteins of similar size and its apolarside chains are more mobile. The latter features distinguishthe elongated four-helix bundle as a slightly disordered structure,which shows a structural likeness with some de novo designedfour-helix bundle proteins and shares with the latter a leucine-richresidue composition. We anticipate that these unique propertiescompared with other native helix bundles may be related to thepostulated ability of apoE-RBD to undergo an opening of itsbundle upon interaction with phospholipids. The distributionof empty cavities computed along the trajectory in the interfaceregions between the different pairs of helices reveals thatthe tertiary contacts in one of the interfaces are weaker suggestingthat this particular interface could be more easily rupturedupon lipid association.     

3-D structure of the D153G mutant of Escherichia coli alkaline phosphatase: an enzyme with weaker magnesium binding and increased catalytic activity

The substitution of aspartate at position 153 in Escherichiacoli alkaline phosphatase by glycine results in a mutant enzymewith 5-fold higher catalytic activity (k_cat but no change inKm at pH 8.0 in 50 mM Tris-HCl. The increased k_cat is achievedby a faster release of the phosphate product as a result ofthe lower phosphate affinity. The mutation also affects Mg²⁺binding, resulting in an enzyme with lower metal affinity. The3-D X-ray structure of the D153G mutant has been refined at2.5 Å to a crystallographic Rfactor of 16.2%. An analysisof this structure has revealed that the decreased phosphateaffinity is caused by an apparent increase in flexibility ofthe guanidinium side chain of Argl66 involved in phosphate binding.The mutation of Aspl53 to Gly also affects the position of thewater ligands of Mg²⁺, and the loop Glnl52–Thrl55 is shiftedby 0.3 Å away from the active site. The weaker Mg²⁺ bindingof the mutant compared with the wild type is caused by an alteredcoordination sphere in the proximity of the Mg²⁺ ion, and alsoby the loss of an electrostatic interaction (Mg²⁺.COO^-Aspl53)in the mutant Its ligands W454 and W455 and hydroxyl of Thrl55,involved in the octahedral coordination of the Mg²⁺ ion, arefurther apart in the mutant compared with the wild-type     

An approach to protein homology modelling based on an ensemble of NMR structures: application to the Sox-5 HMG-box protein

A new approach has been developed to reduce multiple proteinstructures obtained from NMR structure analysis to a smallernumber of representative structures which still reflect thestructural diversity of the data sets. The method, based onthe clustering of similar structures, has been tested in thehomology model building of the structure of Sox-5, a sequence-specificDNA-binding protein belonging to the high mobility group (HMG)nuclear proteins family. Sox (SRY box) genes are the autosomalgenes related to the sex-determining SRY, Y chromosomal gene.The Sox-5 protein, encoded by one of the SRY-related genes,displays a 29% sequence identity with the HMG1 B-box domainwhose structure, determined previously by NMR, has been usedin our study to predict the structure of Sox-5. Two independentensembles of HMG1 structures, each represented by closely relatedcoordinate sets, were used. Nine representative structures forHMG1 were subsequently selected as starting points for the modellingof Sox-5. The model of the protein shows close similarity tothe HMG1 fold, with differences at the secondary structure levellocated mainly in a-helices 1 and 3. A left-handed, three residueper turn polyproline II helix, forming a conserved polyprolineII/-helix supersecondary motif, was identified in the N-terminalregion of Sox-5 and other HMG boxes.     

Intrahelical side chain-side chain contacts: the consequences of restricted rotameric states and implications for helix engineering and design

Intrahelical side chain–side chain (sc–sc) interactionsare assumed to play a crucial role in the formation and stabilityof -helices, yet it was found that only 37.2% of all helicalresidues are involved in such close contacts, assuming a specificminimum contact distance. The majority (58.0%) of these weredetected between residues with amino acid sequence spacing i,i + 4. The low frequency of intrahelical sc–sc contactswith sequence separations i, i + 1 and ii, i + 3, each observedwith only about one-third of the i, i + 4 counts, can be directlyand generally attributed to the absence of the g^- conformationin helices for the dihedral angle X₁- However, if it was assumedthat each side chain may maximally make only one sc–sccontact, as most commonly observed, the percentage of contactingpairs increased relative to the maximum possible pairs for agiven sequence spacing by a factor of {small tilde}4, e.g. from20.9 to 81.7% for i, i + 4 contacts. Stereochemical reasonsare also given for the observation that i, i + 3 contacts arecomposed largely of ion or polar pairs, while hydrophobic residuesdominate the i, i + 4 contacts. No significantly increased densityof intrahelical sc–sc contacts with increasing helix lengthwas found. Although there were generally fewer intrahelicalcontacts between buried helical residues when more contactswere made to the tertiary protein environment, the number ofintrahelical contacts did not increase with increasing solventexposure of the helices. Implications for helix design and thepacking of helices are discussed.     

The evolution of sugar isomerases

L-Arabinosc isomerase (EC 5.3.1.4 [EC] ) catalyzes the isomerizationof L-arabinose to L-ribulose. Here we report on the purification,kinetic mechanism and chemical mechanism of L-arabinose isomerasefrom Escherichia coli. The enzyme catalyzes the isomerizationof L-arabinose to L-ribulose by a proton transfer mechanism,in contrast to xylose isomerase which uses a hydride transfermechanism to perform a similar isomerization. Arabinose isomeraseactivity is metal dependent, although the enzyme can catalyzethe exchange of the proton attached to carbon 2 of arabinosewith the solvent in the absence of metal ion. Manganese(II)is the only metal ion which renders the enzyme active for theisomerization reaction. Arabinose isomerase has high substratespecificity for L-arabinose. The difference in chemical mechanismbetween xylose isomerase and arabinose isomerase suggests thatthese enzymes are not related by convergent evolution. Thiswork also suggests that unless convergent evolution has beendemonstrated, the mechanism of one enzyme may not give any insightinto the mechanism of a second enzyme catalyzing the same reaction     

The structure of E.coli soluble inorganic pyrophosphatase at 2.7 A resolution

Hie structure of E.coli soluble inorganic pyrophosphatase hasbeen refined at 2.7 resolution to an R-factor of 20.9. Theoverall fold of the molecule is essentially the same as yeastpyrophosphatase, except that yeast pyrophosphatase is longerat both the N- and C-termini. Escherichia coli pyrophosphataseis a mixed +ß protein with a complicated topology.The active site cavity, which is also very similar to the yeastenzyme, is formed by seven ß-strands and an -helixand has a rather asymmetric distribution of charged residues.Our structure-based alignment extends and improves upon earliersequence alignment studies; it shows that probably no more than14, not 15–17 charged and polar residues are part of theconserved enzyme mechanism of pyrophosphatases. Six of theseconserved residues, at the bottom of the active site cavity,form a tight group centred on Asp70 and probably bind the twoessential Mg⁺ ions. The others, more spreadout and more positivelycharged, presumably bind substrate. Escherichia coli pyrophosphatasehas an extra aspartate residue in the active site cavity, whichmay explain why the two enzymes bind divalent cation differently.Based on the structure, we have identified a sequence motifthat seems to occur only in soluble inorganic pyrophosphatases.     

Mutant forms of {beta}-galactosidase with an altered requirement for magnesium ions

By random approaches we have previously isolated many variantsof Escherichia coli ß-galactosidase within a shortcontiguous tract near the N-terminus (residues 8–12 ofwildtype enzyme), some of which have increased stability towardsheat and denaturants. The activity of these mutants was originallyanalysed and quantitated in situ in activity gels without theaddition of magnesium ions to the buffer system. We now showthat the improved stability is only observable under such conditionsof limiting magnesium ion concentrations or in the presenceof appropriate concentrations of a metal chelator. In the presenceof EDTA, purified preparations of one of these mutant enzymeswere much more resistant to denaturants than wild-type, butthis differential was completely nullified in the presence of1 mM Mg²⁺. However, the stability of this mutant enzyme in EDTAwas lower than that shown by it, or the wild-type enzyme, inthe presence of magnesium ions. In addition, certain alterationswithin another N-terminal tract (residues 27–31 of wild-type)resulted in enzymes with greater dependence on Mg²⁺ than naturalß-galactosidase. We conclude that a small number ofresidue changes in a large protein can profoundly modulate therequirement for metal ion stabilization, allowing partial abrogationof this need in certain cases. Thus, some enzymes which requiredivalent metal ions for structural purposes only may be engineeredtowards metal independence.     

Replacing the glutamate ligand in the structural zinc site of Sulfolobus solfataricus alcohol dehydrogenase with a cysteine decreases thermostability

The alcohol dehydrogenase gene from the thermophilic archaeumSulfolobus solfataricus has been subcloned and expressed inEscherichia coli under the control of the T7 inducible promoter.Therecombinant protein shows properties analogous to those of thenative enzyme, including thermostability, despite the fact thatE.coli does not post-translationally modify two lysine residueswhich are N--methylated in the native enzyme. We constructeda 3-D model of the S.solfataricus alcohol dehydrogenase usingthe known structure of its isozyme from horse liver as a template.Our analysis of the structural zinc binding site suggested thatthis site is present andfunctional in the S.solfataricus enzymeand that a glutamate ligand can contribute to thermostabilityby influencing electrostatic interactions around the metal centre.To investigate thishypothesis, we constructed, expressed andcharacterized a mutant where the glutamate is replaced by acysteine, thus restoring the zinc binding site of mesophilicalcohol dehydrogenases. Themutant shows the same activity buta reduced thermostability with respect to the wild-type recombinantprotein, as suggested by our model.     

Molecular modeling of coiled-coil {alpha}-tropomyosin: analysis of staggered and in register helix--helix interactions

In register and staggered models of tropomyosin coiled-coilwere built from X-ray C coordinates and refined via moleculardynamics. The two models show similar structural features withthe X-ray structure of GCN4 leucine zipper. Empirical energeticmethods used to compare the in register and staggered modelsindicate that both are equally probable. The two models havesimilar profiles of solvation free energy of folding for residuesat positions a and d of the repeating heptad, indicating thatresidues at these positions are as well buried in an in registerstructure as in a staggered one. Neither the in register northe 14 residues staggered structure can be ruled out based onhydrophobic or e–g' (g–e') electrostatic interactionswhich are not able to distinguish between the two models andare therefore not selective. However, the eg–b'c' electrostaticinteractions, although smaller in magnitude, are in favor ofthe in register model. Furthermore, analysis of hydrophobicand electrostatic interactions along the tropomyosin sequenceshows that bulky residues in positions a and d prevent the formationof inter-chain salt bridges.     

Stereochemistry of the N-glycosylation sites in glycoproteins

The stereochemical features displayed by the N-glycosidic linkagein crystalline N-linked glycoproteins are analyzed. From thestatistical analysis of 44 different glycosylation sites belongingto 26 glycoproteins of the Brookhaven Protein Data Bank, a meanstandard geometry for the GlcNAc moiety, along with a rationalizationof its confornia-tional behavior, can be proposed. As for theglycopeptide linkage, the distribution of observed conformationshas been analyzed on the basis of molecular mechanics calculations.The rotamer distribution of the Asn side chains conforms tothat observed on non-glycosylated structures, and it agreeswith the pattern of flexible conformations gathered from NMRmeasurements. In characterizing the protein-glycan interactions,some hydrogen bonds occur. Stacking between the amphiphilicmoiety of the glycan and some surrounding aromatic, or at leasthydrophobic, amino acid residues is also found. When lookingat the secondary structure of the glycosylated peptide, only25% of the glycosylation sites correspond to situations whereAsn is located at the top of a ß-turn. Other typesof secondary structure exist which fulfil the spatial requirementof having the glycan exposed at the surface of the protein.These data can be compared with the most recent studies on thepeptide conformation which would be required for glycosylation.     

Ligand-induced conformational changes in wild-type and mutant yeast pyruvate kinase

A mutant form of pyruvate kinase in which serine 384 has beenmutated to proline has been engineered in the yeast Saccharomycescerevisiae. Residue 384 is located in a helix in a subunit interfaceof the tetrameric enzyme, and the mutation was anticipated toalter the conformation of the helix and hence destabilize theinterface. Previous results indicate that the mutant favoursthe T quarternary conformation over the R conformation, andthis is confirmed by the results presented here. Addition ofphosphoenol-pyruvate (PEP), ADP and fructose-1,6-bisphosphate(Fru 1,6-P₂) singly to the wild-type and mutant enzymes resultsin a significant quenching of tryptophan fluorescence (12–44%),and for Fru-1,6-P₂, a red shift of 15 nm in the emission maximum.Fluorescence titration experiments showed that PEP, ADP andFru-1,6-P₂ induce conformations which have similar ligand-bindingproperties in the wild-type and mutant enzymes. However, theFru-1,6-P₂ induced conformation is demonstrably different fromthose induced by either ADP or PEP. The enzymes differ in theirsusceptibility to trypsin digestion and N-ethylmaleimide inhibition.The thermal stability of the enzyme is unaltered by the mutantion.Far-UV CD spectra show that both enzymes adopt a similar overallsencondary structure in solution. Taken together, the resultssuggest that the Ser384-Pro mutaion causes the enzyme to adopta diffenrent tertiary and/or quaternary structure from the wild-typeenzyme and affects the type and extent of the conformationalchanges induced in the enzyme upon ligand binding. A simplifiedminimal reaction mechanism is proposed in which the R and Tstates differ in both affinity and K_cat. Thus, in terms of themodels of cooperativity and allsoteric interaction, pyruvatekinase is both a K and a V system.     

Quantitative analysis of protein far UV circular dichroism spectra by neural networks

A new method based on neural network theory is presented toanalyze and quantify the information content of far UV circulardichroism spectra. Using a backpropagation network model witha single hidden layer between input and output, it was possibleto deduce five different secondary structure fractions (helix,parallel and antiparallel ß-sheet, ß-turnand random coil) with satisfactory correlations between calculatedand measured secondary structure data. We demonstrate that foreach wavelength interval a specific network is suitable. Theremaining discrepancy between the secondary structure data fromneural network prediction and crystallography may be attributedto errors in the determination of protein concentration andrandom noise in the CD signal, as indicated by simulations.     

Engineering the aggregation properties of dodecameric glutamine synthetase: a single amino acid substitution controls 'salting out'

Escherichia coli glutamine synthetase (GS) is a dodecamer ofidentical subunits which are arranged as two face-to-face hexamericrings. In the presence of 10% ammonium sulfate, wild type GSexhibits a pH-dependent ‘salting out’ with a pK_aof 4.51. Electron micrographs indicate that the pH-dependentaggregation corresponds to a highly specific self-assembly ofGS tubules, which result from stacking of individual dodecamers.This stacking of dodecamers is similar to the metal ion-inducedGS tubule formation previously described. Site-directed mutagenesisexperimentsindicate that the N-terminal helix of each subunit is involvedin the salting out reaction, as it is in the metal-induced stacking.A single substitution of alanine for His4 completely abolishesthe (NH₄₂SO₄-induced aggregation. However, the H4C mutant proteindoes nearly completely precipitate under the same salting outconditions. Mutations at other residues within the helix haveno effect on the stacking reaction. Differential catalyticactivityof unadenylylated GS versus adenylylated GS has been used todetermine whether wild type dodecamers ‘complement’the H4A mutant in the stacking reaction. The complementationexperiments indicate that His4 residues on bothsides of thedodecamer-dodecamer interfaces are not absolutely required forsalting out, although the wild type dodecamers clearly stackpreferentially with other wild type dodecamers. Approximately20% of the protein precipitated fromthe mixtures containingthe wild type GS and the H4A mutant is the mutant. The implicationsof these results for protein engineering are discussed.     

Theoretical examination of the mechanism of aldose-ketose isomerization

Two mechanisms for an aldose–ketose isomerization havebeen examined using high level ab initio and semiempirical molecularorbital methods. The proton transfer pathway via an enediolintermediate is shown to be favored in the absence of a metalion, while the hydride transfer pathway becomes favored in thepresence of a metal ion. Our calculations explain why the protontransfer pathway is operative in most aldose–ketose isomerizationreactions. These calculations also provide further support forthe previously proposed metal ion-mediated hydride transfermechanism for xylose isomerase.     

Modeling of transmembrane seven helix bundles

Transmembrane seven helix bundles form a large family of membraneinserted receptors and are responsible for a wide range of biologicalfunctions. Experimental data suggest that their overall structureis similar to bacteriorhodopsin. We describe here a new approachfor the modeling of transmembrane seven helix bundles basedon statistically derived environmental preference parameterscombined with experimentally determined features of the receptors.The method was used to create a model for the human ß₂-adrenoreceptor.This model is physically plausible, is in reasonable agreementwith experimental data and may be helpful in planning new receptorengineering experiments.     

Cellulose-binding domains: potential for purification of complex proteins

The endoglucanase CenA and the exoglucanase Cex from Cellulomonasfimi each contain a discrete cellulose-binding domain (CBD),at the amino-terminus or carboxyl-terminus respectively. Thegene fragment encoding the CBD can be fused to the gene of aprotein of interest. Using this approach hybrid proteins canbe engineered which bind reversibly to cellulose and exhibitthe biological activity of the protein partner. Alkaline phosphatase(PhoA) from Escherichia coli, and a ß-glucosidase(Abg) from an Agrobacterium sp. are dimeric proteins. The fusionpolypeptides CenA-PhoA and Abg-CBC_cex are sensitive to proteolysisat the junctions between the fusion partners. Proteolysis resultsin a mixture of homo- and heterodimers; these bind to celluloseif one or both of the monomers carry a CBD, e.g. CenA-PhoA/CenA-PhoAand CenA-PhoA/PhoA. CBD fusion polypeptides could be used inthis way to purify polypeptides which associate with the fusionpartner.     

The cystine knot of a squash-type protease inhibitor as a structural scaffold for Escherichia coli cell surface display of conformationally constrained peptides

The Ecballium elaterium trypsin inhibitor II (EETI-II), a memberof the squash family of protease inhibitors, is composed of28 amino acid residues and is a potent inhibitor of trypsin.Its compact structure is defined by a triple-stranded antiparallelß-sheet, which is held together by three intramoleculardisulfide bonds forming a cystine knot. In order to explorethe potential of the EETI-II peptide to serve as a structuralscaffold for the presentation of randomized oligopeptides, weconstructed two EETI-II derivatives, where the six-residue inhibitorloop was replaced by a 13-residue epitope of Sendai virus L-proteinand by a 17-residue epitope from human bone Gla-protein. EETI-IIand derived variants were produced via fusion to maltose bindingprotein MalE. By secretion of the fusion into the periplasmicspace, fully oxidized and correctly folded EETI-II was obtainedin high yield. EETI-II and derived variants could be presentedon the Escherichia coli outer membrane by fusion to truncatedLpp'–OmpA', which comprises the first nine residues ofmature lipoprotein plus the membrane spanning ß-strandfrom residues 46–66 of OmpA protein. Gene expression wasunder control of the strong and tightly regulated tetA promoter/operator.Cell viability was found to be drastically reduced by high levelexpression of Lpp'–OmpA'–EETI-II fusion protein.To restore cell viability, net accumulation of fusion proteinin the outer membrane was reduced to a tolerable level by introductionof an amber codon at position 9 of the lpp' sequence and utilizingan amber suppressor strain as expression host. Cells expressingEETI-II variants containing an epitope were shown to be surfacelabeled with the respective monoclonal antibody by indirectimmunofluorescence corroborating the cell surface exposure ofthe epitope sequences embedded in the EETI-II cystine knot scaffold.Cells displaying a particular epitope sequence could be enriched10⁷-fold by combining magnetic cell sorting with fluorescence-activatedcell sorting. These results demonstrate that E.coli cell surfacedisplay of conformationally constrained peptides tethered tothe EETI-II cystine knot scaffold has the potential to becomean effective technique for the rapid isolation of small peptidemolecules from combinatorial libraries that bind with high affinityto acceptor molecules.