Family of G protein {alpha} chains: amphipathic analysis and predicted structure of functional domains

The G proteins transduce hormonal and other signals into regulationof enzymes such as adenylyl cyclase and retinal cGMP phosphodiesterase.Each G protein contains an subunit that binds and hydrolyzesguanine nucleotides and interacts with ß subunitsand specific receptor and effector proteins. Amphipathic andsecondary structure analysis of the primary sequences of fivedifferent chains (bovine _s, _t1 and _t2, mouse _i, and rat _o)predicted the secondary structure of a composite chain (_avg).The chains contain four short regions of sequence homologousto regions in the GDP binding domain of bacterial elongationfactor Tu (EF-Tu). Similarities between the predicted secondarystructures of these regions in _avg and the known secondary structureof EF-Tu allowed us to construct a three-dimensional model ofthe GDP binding domain of _avg. Identification of the GDP bindingdomain of _avg defined three additional domains in the compositepolypeptide. The first includes the amino terminal 41 residuesof _avg, with a predicted am phipathic helical structure; thisdomain may control binding of the chains to the ßcomplex. The second domain, containing predicted ßstrands and helices, several of which are strongly amphipathic,probably contains sequences responsible for interaction of chains with effector enzymes. The predicted structure of thethird domain, containing the carhoxy terminal 100 amino acids,is predominantly ß sheet with an amphipathic helixat the carboxy terminus. We propose that this domain is reponsiblefor receptor binding. Our model should help direct further experimentsinto the structure and function of the G protein chain.     

Cellular inhibition of protein tyrosine phosphatase 1B by uncharged thioxothiazolidinone derivatives

As important regulators of cellular signal transduction, members of the protein tyrosine phosphatase (PTP) family are considered to be promising drug targets. However, to date, the most effective in vitro PTP inhibitors have tended to be highly charged, thus limiting cellular permeability. Here, we have identified an uncharged thioxothiazolidinone derivative (compound 1), as a competitive inhibitor of a subset of PTPs. Compound 1 effectively inhibited protein tyrosine phosphatase 1B (PTP1B) in two cell-based systems: it sensitized wild-type, but not PTP1B-null fibroblasts to insulin stimulation and prevented PTP1B-dependent dephosphorylation of the FLT3-ITD receptor tyrosine kinase. We have also tested a series of derivatives in vitro against PTP1B and proposed a model of the PTP1B-inhibitor interaction. These compounds should be useful in the elucidation of cellular PTP function and could represent a starting point for development of therapeutic PTP inhibitors.     

The role of tyrosine residues in the DNA-binding site of the Pf1 gene 5 protein

The 144 amino acid gene 5 protein of bacteriophage Pf1 bindstightly and cooperatively to single-stranded DNA during replicationof the phage genome. It has been suggested that aromatic aminoacid side chains are important for this interaction, probablythrough base stacking with the DNA. We have analysed the accessibilityof tyrosine residues in the DNA—protein complex, and theirimportance to the DNA-binding activity of the protein, by chemicalmodification and protection experiments using tetranitromethane.Tyrosines 21, 30 and 55 are surface accessible in the free proteinbut are protected from modification in the complex with phageDNA. Moreover, modification of these residues in the free proteinabolishes the ability to bind to DNA or oligonucleotides, asjudged by fluorescence spectroscopy and gel retardation analysis.Modification of the protein also results in the formation ofan intersubunit covalent cross-link between Tyr55 and Phe76,suggesting that Phe76 is located within the DNA-binding cleftof the protein. It is proposed that residues 17–34 ofthe Pf1 gene 5 protein form a beta-hairpin analogous to the‘DNA-binding wing’ of the fd and Ike gene 5 proteins.We suggest the existence of a single-stranded DNA binding motif,in which Tyr30 of the Pf1 protein is equivalent to the functionallyimportant Tyr26 of the fd gene 5 protein.     

Phosphopeptide ligands of the SHP-1 N-SH2 domain: effects on binding and stimulation of phosphatase activity

Src homology 2 (SH2)-domain-mediated interactions with phosphotyrosine (pY)-containing ligands are critical for the regulation of SHP-1 phosphatase activity. Peptides based on a binding site from receptor tyrosine kinase Ros (EGLN-pY2267-MVL, 1) have recently been shown to bind to the SHP-1 N-terminal SH2 domain (N-SH2) with considerably high affinity. In addition, two peptides cyclized between positions -1 and +2 relative to pY (EGLc[K(COCH(2)NH)pYMX]L-NH(2), 2: X=D, 3: X=E) bound to the N-SH2 domain, but did not activate the enzyme and even partially prevented stimulation of SHP-1 activity by the physiological ligand 1. These findings prompted us to further examine the determinants for optimal binding to the N-SH2 domain and for the stimulation and inhibition of SHP-1 activity. Herein we demonstrate that combining the preferred residues in both pY+1 (such as Phe or norleucine, Nle) and pY+3 (such as homophenylalanine, Hfe) leads to highly efficient activating ligands of SHP-1. Particularly in the context of the cyclic peptides 7 (EGLc[K(COCH(2)NH)pYFD]Hfe-NH(2)) and 8 (EGLc[K(COCH(2)NH)pYNleD]HfeL-NH(2)), the incorporation of these residues resulted in high-affinity ligands with a significantly increased ability to stimulate SHP-1 activity. We suggest that different binding modes (according to consensus sequences class I and II) are responsible for obtaining either activating (7 and 8) or nonactivating (2 and 3) ligands. Peptides such as 7 and 8 that bind in the extended fashion of the type II mode activate the phosphatase through complete filling of the cavity for pY+3. In contrast, peptides such as 2 and 3 that bind in the class I mode do not activate the enzyme because they allow more conformational space at pY+3. Therefore, their binding does not force the conformational transition necessary to trigger the dissociation of N-SH2 and the catalytic domain.     

Single antibody domains as small recognition units: design and in vitro antigen selection of camelized, human VH domains with improved protein stability

Folding stabilities of camelized human antibody VH domains werestudied through the determination of their melting points inthermodenaturation experiments. The melting point of a VH domainoriginating from a synthetic library of human VHs, which hadbeen optimized for the use as small recognition units throughthe mimicking of camelid antibody heavy chains occurring naturallywithout light chain, was 56.6C compared with 71.2C of theoriginal human VH. Its stability was improved (melting point61.6C) through three mutations to mimic camelid VHs even further:Va137 was replaced by phenylalanine and two cysteines were introducedat positions 33 and 100b. The resulting VH folded properly andformed a second intradomain disulphide between the extra cysteines.The new mutations were then built constitutively into a phage-displayVH library, from which antigen-specific VHs were selected. Twowere analysed for stability with melting points of 72.6 and75.3C. Thus secondary camelization enabled the isolation ofVHs with improved folding stabilities exceeding even that ofthe original human VH. This indicates an effect on folding stabilityfor some mutations specific in the light chain lacking camelidheavy chains.     

Three-dimensional structure of protein C inhibitor predicted from structure of {alpha}1-antitrypsin with computer graphics

The three-dimensional structure of a proteolytically modifiedprotein C inhibitor, a member of the serine protease inhibitorsuperfamily, was constructed with computer graphics based onits amino acid sequence homology with that of the modified 1-antitrypsinwhose structure had been elucidated by X-ray crystallography.The intact form of protein C inhibitor was predicted with an-carbon model based on its hydrophilicity and hydrogen bondpattern. Furthermore, a model of its interaction with activatedprotein C was constructed based on the structure of the complexbetween trypsin and its inhibitor, which had been determinedby X-ray crystallography.     

Biochemical properties of the kringle 2 and protease domains are maintained in the refolded t-PA deletion variant BM 06.022

BM 06.022 is a t-PA deletion variant which comprises the kringle2 and the protease domain. Production of BM 06.022 in Escherichiacoli leads to the formation of inactive inclusion bodies, whichhave to be refolded by an in vitro refolding process to achieveactivity and proper structure of the domains. We analysed thebiochemical properties of BM 06.022 to obtain some informationabout the structure of kringle 2 and the protease as comparedwith the structure of these domains in the intact t-PA molecule.The kinetic analysis of the amidolytic activity of BM 06.022and CHO-t-PA yielded similar values for k_cat (13.9 s^-1and 11.4s^-1for the single chain forms and 33.9 s^-1and 27.1 s^-1for thetwo chain forms of BM 06.022 and CHO-t-PA, respectively) andfor k_m, (2.5 mM and 2.1 mM for the single chain forms and 0.5mM and 0.3 mM for the two chain forms of BM 06.022 and CHO-t-PA,respectively). BM 06.022 and CHO-t-PA have the same plasminogenolyticactivity in the absence of CNBr fragments of fibrinogen. However,BM 06.022 has a lower plasminogenolytic activity in the presenceof CNBr fragments of fibrinogen and a lower affinity to fibrinas compared with CHO-t-PA. The affinity of BM 06.022 for fibrinis completely suppressed by 0.3 mM eaminocaproic acid, whilethe intact t-PA has a residual affinity of 30%. The dissociationconstants for the interaction with the lysine analogue e-aminocaprokacid are 0.10 mM and 0.09 mM for BM 06.022 and the intact t-PA,respectively. Furthermore, BM 06.022 and CHO-t-PA are inhibitedby PAI-1 in a similar manner     

Hydrophobicity engineering of cholera toxin A1 subunit in the strong adjuvant fusion protein CTA1-DD

Protein engineering of the cholera toxin A1 subunit (CTA1) fusedto a dimer of the Ig-binding D-region of Staphylococcus aureusprotein A (DD) was employed to investigate the effect of specificamino acid changes on solubility, stability, enzymatic activityand capacity to act as an adjuvant in vivo. A series of CTA1-DDanalogues were selected by a rational modeling approach, inwhich surface-exposed hydrophobic amino acids of CTA1 were exchangedfor hydrophilic counterparts modeled for best structural fit.Of six different mutants initially produced, two analogues,CTA1Phe132Ser-DD and CTA1Pro185Gln-DD, were demonstrated tohave 50 and 70% increased solubility, respectively, at neutralpH. The double mutant CTA1Phe132Ser/Pro185Gln-DD was at leastthreefold more soluble, demonstrating an additive effect ofthe two mutations. Only the Phe132Ser analogue retained fullbiological activity and stability compared with the native CTA1-DDfusion protein. Two mutants, Pro185Gln and Phe31His mutations,exhibited unaltered ADP-ribosyltransferase activity in vitro,but demonstrated markedly reduced adjuvant function. Since thePro185 and Phe31 amino acids are located in close vicinity onthe distal side of the molecule relative to the enzymaticallyactive cleft, it is conceivable that this region is involvedin mediating a biological function, separate from the enzymaticactivity but intrinsic to the adjuvant activity of CTA1.     

The mechanism of sequence non-specific DNA binding of HMG1/2-box B in HMG1 with DNA

The DNA binding mechanism of box B in HMG1, a member of thesequence non-specific DNA binding HMG1/2-box family of proteins,has been examined by both mutation analyses and molecular modelingtechniques. Substitution of the residue 102F, which is characteristicallyexposed to solvent, with a small hydrophobic amino acid affectedits DNA binding activity. However, no additional effect wasobserved by the further mutation of flanking 101F. Moleculardynamics simulation and modeling studies revealed that 102Fintercalates into DNA base-pairs, being supported by the flanking101F. The mutants with a small hydrophobic residue at position102 tolerated the substitution for 101F because the side chainat position 102 is too short to intercalate. Thus the intercalationof 102F and the positive effect of the flanking 101F residueare important for the sequence non-specific DNA binding of theHMG1/2-box. The conserved basic residues of 95K, 96R and 109Rwere also examined for their roles in DNA binding. These residuesinteracted with DNA mainly by electrostatic interaction andmaintained the location of the box on the DNA, which prescribedthe intercalation of 102F. The DNA intercalation by HMG1 consistsof an ingenious mechanism which brings DNA conformational changesnecessary for biological functions.     

Development of tumor targeting anti-MUC-1 multimer: effects of di-scFv unpaired cysteine location on PEGylation and tumor binding

MUC1 mucin expressed in epithelial cancer, such as prostate and breast, is aberrantly glycosylated providing unique targets for imaging and therapy. In order to create a broadly applicable construct to target these unique epitopes on metastatic cancer, we selected an antibody fragment (scFv) that binds both synthetic MUC1 core peptide and epithelial cancer cell-expressed MUC1, and developed a recombinant bivalent molecule (di-scFv). Genetically engineered modifications of the di-scFv were constructed to create five molecular versions, each having a free cysteine (di-scFv-c) at different locations for site-specific conjugation. The effects of the engineered cysteine in the varied sites were studied relative to tumor binding and polyethylene glycol-maleimide (PEG-Mal) conjugation (PEGylation). Escherichia coli production as well as binding to MUC1 core peptide, human tumor cell lines and human tumor biopsies, were comparable. However, the location of the engineered cysteine in these di-scFv-c did influence PEGylation efficiency of this free thiol; higher PEGylation efficiency occurred with this cysteine in the inter-scFv linkage. Di-scFv-c PEG, with the cysteine engineered after the fifth amino acid in the linker, was used as an example to demonstrate comparable antigen-binding to non-PEGylated di-scFv-c. In summary, novel anti-MUC1 di-scFv-c molecules can be efficiently produced, purified and conjugated by site-specific PEGylation without loss of immunoreactivity, thus providing flexible multidentate constructs for cancer-targeted imaging and therapy.     

Hydrophobic effects on protein/nucleic acid interaction: enhancement of substrate binding by mutating tyrosine 45 to tryptophan in ribonuclease Tl

Hydrophobic effects on binding of ribonuclease Tl to guaninebases of several ribonucleotides have been proved by mutatinga hydrophobic residue at the recognition site and by measuringthe effect on binding. Mutation of a hydrophobic surface residueto a more hydrophobic residue (Tyr45 – Trp) enhances thebinding to ribonucleotides, including mononucleotide inhibitorand product, and a synthetic substrate-analog trinudeotide aswell as the binding to dinucleotide substrates and RNA. Enhancementson binding to non-substrate ribonucleotides by the mutationhave been observed with free energy changes ranging from –2.2 to – 3 .9 kJ/mol. These changes are in good agreementwith that of substrate binding, –2.3 kJ/mol, which iscalculated from Michaelis constants obtained from kinetic studies.It is shown, by comparing the observed and calculated changesin binding free energy with differences in the observed transferfree energy changes of the amino acid side chains from organicsolvents to water, that the enhancement observed on guaninebinding comes from the difference in the hydrophobic effectsof the side chains of tyrosine and tryptophan. Furthermore,a linear relationship between nucleolytic activities and hydrophobicityof the residues (Ala, Phe, Tyr, Trp) at position 45 is observed.The mutation could not change substantially the base specificityof RNase Tl, which exhibits a prime requirement for guaninebases of substrates.     

Influenza virus M2 protein: a molecular modelling study of the ion channel

The influenza A M₂ protein forms cation-selective ion channelswhich are blocked by the anti-influenza drug amantadine. A molecularmodel of the M₂ channel is presented in which a bundle of fourparallel M₂ transbilayer helices surrounds a central ion-permeablepore. Analysis of helix amphipathicity was used to aid determinationof the orientation of the helices about their long axes. Thehelices are tilted such that the N-terminal mouth of the poreis wider than the C-terminal mouth. The channel is lined byresidues V27, S31 and I42. Residues D24 and D44 are locatedat opposite mouths of the pore, which is narrowest in the vicinityof I42. Energy profiles for interaction of the channel withNa⁺, amantadine-H⁺ and cyclopentylamine-H⁺ are evaluated. Theinteraction profile for Na⁺ exhibits three minima, one at eachmouth of the pore, and one in the region of residue S31. Theamantadine-H⁺ profile exhibits a minimum close to S31 and abarrier near residue I42. This provides a molecular model foramantadine-H⁺ block of M₂ channels. The profile for cyclopentylamine-H⁺does not exhibit such a barrier. It is predicted that cyclopentyl-amine-H⁺will not act as an M₂ channel blocker.     

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.     

Normal-mode analysis suggests important flexibility between the two N-terminal domains of CD4 and supports the hypothesis of a conformational change in CD4 upon HIV binding

Human CD4 is the receptor for human immunodeficiency virus (HTV).It is well established that the first domain of CD4 binds withhigh affinity to gp120, an envelope protein of HIV, but it hasalso been demonstrated that amino acids located in its seconddomain, within or close to residues 120–127 or 163–166(lying 15 Å away from the binding site), play a role invirus infectivity. We show here that these two stretches ofamino acids happen to be important for the largest amplitudemotion obtained with the normal-mode theory for the two N-terminaldomains of human CD4: an overall rigid-body displacement ofone domain with respect to the other. Such a ‘hinge-bending’motion is unexpected since these two domains were found by crystallographersto be tightly abutting. On the other hand, since for severalproteins the hinge-bending motion experimentally observed uponligand binding was found to be similar to the largest amplitudemotion obtained with the normal-mode theory for these proteins,our results suggest that CD4 may undergo such a kind of conformationalchange upon HTV binding.     

Structural interpretation of site-directed mutagenesis and specificity of the catalytic subunit of protein kinase CK2 using comparative modelling

The catalytic subunit of protein kinase casein kinase 2 (CK2),which has specificity for both ATP and GTP, shows significantamino acid sequence similarity to the cyclin-dependent kinase2 (CDK2). We constructed site-directed mutants of CK2 and useda three-dimensional model to investigate the basis for the dualspecificity. Introduction of Phe and Gly at positions 50 and51, in order to restore the pattern of the glycine-rich motif,did not seriously affect the specificity for ATP or GTP. Weshow that the dual specificity probably originates from theloop situated around the position His115 to Asp120 (HVNNTD).The insertion of a residue in this loop in CK2 subunits, comparedwith CDK2 and other kinases, might orient the backbone to interactwith the base A and G; this insertion is conserved in all knownCK2. The mutant N118, the design of which was based on the modelling,showed reduced affinity for GTP as predicted from the model.Other mutants were intended to probe the integrity of the catalyticloop, alter the polarity of a buried residue and explore theimportance of the carboxy terminus. Introduction of Arg to replaceAsn189, which is mapped on the activation loop, results in amutant with decreased k_cat, possibly as a result of disruptionof the interaction between this residue and basic residues inthe vicinity. Truncation at position 331 eliminates the last60 residues of the subunit and this mutant has a reduced catalyticefficiency compared with the wild-type. Catalytic efficiencyis restored in the truncation mutant by the replacement of apotentially buried Glu at position 252 by Lys, probably owingto a higher stability resulting from the formation of a saltbridge between Lys252 and Asp208.     

Cloning of rabbit interleukin-1{beta}: differential evolution of IL-1{alpha} and IL-1{beta} proteins

We have cloned the rabbit IL-1ß cDNA, which encodesa 268 amino acid precursor similar in length to other sequencedIL-1 precursors. Comparison of all published IL-1 and IL-1ßsequences respectively indicates that the IL-1 gene family isevolving faster than the IL-1ß family, and that thetwo genes diverged –270 million years ago. Surprisingly,there are differences in the regions preferentially conservedwithin the two families. The IL-1 family is most conserved atthe amino terminus whereas the IL-1ß family is mostconserved in the carboxy-terminal half. This is despite thefact that the carboxy-terminal half encodes the active portionof both molecules and would be expected to adopt a similar ß-sheetstructure in IL-1 as in the published X-ray structure of matureIL-1ß. These findings suggest that differences inthe function and properties of the IL-1 and IL-1ßprecursor molecules may have been conserved. These differencesmay therefore provide an explanation for the existence of twoIL-1 molecules.     

Site-directed mutagenesis and X-ray crystallography of two phospholipase A2 mutants: Y52F and Y73F

Tyr52 and Tyr73 are conserved amino acid residues throughoutall vertebrate phospholipases A₂. They are part of an extendedhydrogen bonding system that links the N-terminal -NH⁺₃ -groupto the catalytic residues His48 and Asp99. These tyrosines werereplaced by phenylalanines in a porcine pancreatic phospholipaseA₂ mutant, in which residues 62–66 had been deleted (62–66PLA₂).The mutations did not affect the catalytic properties of theenzyme, nor the folding kinetics. The stability against denaturatlonby guanidine hydrochloride was decreased, however. To analysehow the enzyme compensates for the loss of the tyrosine hydroxylgroup, the X-ray structures of the Y52F and AY73F mutants weredetermined. After crystallographic refinement the final crystallographicR-factors were 18.1% for the %Y52F mutant (data between 7 and2.3 Å resolution) and 19.1% for the Y73F mutant (databetween 7 and 2.4 Å resolution). No conformational changesoccurred in the mutants compared with the 62–66PLA₂, butan empty cavity formed at the site of the hydroxyl group ofthe former tyrosine. In both mutants the Asp99 side chain losesone of its hydrogen bonds and this might explain the observeddestabilization.     

Engineering the C-region of human insulin-like growth factor-1: implications for receptor binding

Recombinant wild-type human IGF-1 and a C-region mutant in whichresidues 28–37 have been replaced by a 4-glycine bridge(4-Gly IGF-1) were secreted and purified from yeast. An IGF-1analogue in which residues 29–41 of the C-region havebeen deleted (mini IGF-1) was created by site-directed mutagenesisand also expressed. All three proteins adopted the insulin-foldas determined by circular dichroism. The significantly raisedexpression levels of mini IGF-1 allowed the recording of two-dimensionalNMR spectra. The affinity of 4-Gly IGF-1 for the IGF-1 receptorwas {small tilde}100-fold lower than that of wild-type IGF-1and the affinity for the insulin receptor was {small tilde}10-foldlower. Mini IGF-1 showed no affinity for either receptor. Notonly does the C-region of IGF-1 contribute directly to thefree energy of binding to the IGF-1 receptor, but also the absenceof flexibility in this region eliminates binding altogether.As postulated for the binding of insulin to its own receptor,it is proposed that binding of IGF-1 to the IGF-1 receptor alsoinvolves a conformational change in which the C-terminal B-regionresidues detach from the body of the molecule to expose theunderlying A-region residues.     

A mutational analysis of receptor binding sites of interleukin-1{beta}:differences in binding of human interleukin-1{beta} muteins to human and mouse receptors

The 3-D crystal structure of interleukin-1ß(IL-1ß)has been used to define its receptor binding surface by mutationalanalysis. The surface of IL-1ß was probed by site-directedmutagenesis. A total of 27 different IL-1ß muteinswere constructed, purified and analyzed. Receptor binding measurementson mouse and human cell lines were performed to identify receptoraffinities. IL-1ß muteins with modified receptor affinitywere evaluated for structural integrity by CD spectroscopy orX-ray crystallography. Changes in six surface loops, as wellas in the C- and N-termini, yielded muteins with lower bindingaffinities. Two muteins with intact binding affinities showed10- to 100-fold reduced biological activity. The surface regioninvolved in receptor binding constitutes a discontinuous areaof 1000 Å2 formed by discontinuous polypeptide chain stretches.Based on these results, a subdivision into two distinct localareas is proposed. Differences in receptor binding affinitiesfor human and mouse receptors have been observed for some muteins,but not for wild-type IL-1ß. This is the first timea difference in binding affinity of IL-1ß muteinsto human and mouse receptors has been demonstrated