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E Rusinova

Publications and source records attributed to E Rusinova.

11 recordsLinked to original sources
PubMed2001

Multiple activation loop conformations and their regulatory properties in the insulin receptor's kinase domain.

Low catalytic efficiency of protein kinases often results from intrasteric inhibition caused by the activation loop blocking the active site. In the insulin receptor's kinase domain, Asp-1161 and Tyr-1162 in the peptide substrate-like sequence of the unphosphorylated activation loop can interact with four invariant residues in the active site: Lys-1085, Asp-1132, Arg-1136, and Gln-1208. Contributions of these six residues to intrasteric inhibition were tested by mutagenesis, and the unphosphorylated kinase domains were characterized. The mutations Q1208S, K1085N, and Y1162F each relieved intrasteric inhibition, increasing catalytic efficiency but without changing the rate-limiting step of the reaction. The mutants R1136Q and D1132N were virtually inactive. Steric accessibility of the active site was ranked by relative changes in iodide quenching of intrinsic fluorescence, and A-loop conformation was ranked by limited tryptic cleavage. Together these ranked the openness of the active site cleft as R1136Q approximately D1132N > or = D1161A > Y1162F approximately K1085N > Q1208S > or = wild-type. These findings demonstrate the importance of specific invariant residues for intrasteric inhibition and show that diverse activation loop conformations can produce similar steady-state kinetic properties. This suggests a broader range of regulatory properties for the activation loop than expected from a simple off-versus-on switch for kinase activation.

Adenosine Diphosphate↗
PubMed2001

Intrasteric inhibition of ATP binding is not required to prevent unregulated autophosphorylation or signaling by the insulin receptor.

Receptor tyrosine kinases may use intrasteric inhibition to suppress autophosphorylation prior to growth factor stimulation. To test this hypothesis we made an Asp1161Ala mutant in the activation loop that relieved intrasteric inhibition of the unphosphorylated insulin receptor (IR) and its recombinant cytoplasmic kinase domain (IRKD) without affecting the activated state. Solution studies with the unphosphorylated mutant IRKD demonstrated conformational changes and greater catalytic efficiency from a 10-fold increase in k(cat) and a 15-fold-lower K(m ATP) although K(m peptide) was unchanged. Kinetic parameters of the autophosphorylated mutant and wild-type kinase domains were virtually identical. The Asp1161Ala mutation increased the rate of in vitro autophosphorylation of the IRKD or IR at low ATP concentrations and in the absence of insulin. However, saturation with ATP (for the IRKD) or the presence of insulin (for the IR) yielded equivalent rates of autophosphorylation for mutant versus wild-type kinases. Despite a biochemically more active kinase domain, the mutant IR expressed in C2C12 myoblasts was not constitutively autophosphorylated. However, it displayed a 2.5-fold-lower 50% effective concentration for insulin stimulation of autophosphorylation and was dephosphorylated more slowly following withdrawal of insulin than wild-type IR. In tests of the regulation of the unphosphorylated basal state, these results demonstrate that neither intrasteric inhibition against ATP binding nor suppression of kinase activity is required to prevent premature autophosphorylation of the IR. Finally, the lower rate of dephosphorylation suggests invariant residues of the activation loop such as Asp1161 may function at multiple junctures in cellular regulation of receptor tyrosine kinases.

Adenosine Triphosphate↗
PubMed1999

Probing local environments of tryptophan residues in proteins: comparison of 19F nuclear magnetic resonance results with the intrinsic fluorescence of soluble human tissue factor.

19F nuclear magnetic resonance (19F NMR) of 5-fluorotryptophan (5F-Trp) and tryptophan (Trp) fluorescence both provide information about local environment and solvent exposure of Trp residues. To compare the information provided by these spectroscopies, the four Trp residues in recombinant soluble human tissue factor (sTF) were replaced with 5F-Trp. 19F NMR assignments for the 5F-Trp residues (14, 25, 45, and 158) were based on comparison of the wild-type protein spectrum with the spectra of three single Trp-to-Phe replacement mutants. Previously we showed from fluorescence and absorption difference spectra of mutant versus wild-type sTF that the side chains of Trpl4 and Trp25 are buried, whereas those of Trp45 and Trp158 are partially exposed to bulk solvent (Hasselbacher et al., Biophys J 1995;69:20-29). 19F NMR paramagnetic broadening and solvent-induced isotope-shift experiments show that position 5 of the indole ring of 5F-Trp158 is exposed, whereas that of 5F-Trp45 is essentially inaccessible. Although 5F-Trp incorporation had no discernable effect on the procoagulant cofactor activity of either the wild-type or mutant proteins, 19F NMR chemical shifts showed that the single-Trp mutations are accompanied by subtle changes in the local environments of 5F-Trp residues residing in the same structural domain.

Circular Dichroism↗
PubMed1998

Antiphospholipid antibodies accelerate plasma coagulation by inhibiting annexin-V binding to phospholipids: a "lupus procoagulant" phenomenon.

The antiphospholipid syndrome is a thrombophilic condition marked by antibodies that recognize anionic phospholipid-protein cofactor complexes. We recently reported that exposure to IgG fractions from antiphospholipid patients reduces the level of annexin-V, a phospholipid-binding anticoagulant protein, on cultured trophoblasts and endothelial cells and accelerates coagulation of plasma exposed to these cells. Therefore, we asked whether antiphospholipid antibodies might directly reduce annexin-V binding to noncellular phospholipid substrates. Using ellipsometry, we found that antiphospholipid IgGs reduce the quantity of annexin-V bound to phospholipid bilayers; this reduction is dependent on the presence of beta2-glycoprotein I. Also, exposure to plasmas containing antiphospholipid antibodies reduces annexin-V binding to phosphatidyl serine-coated microtiter plates, frozen thawed washed platelets, activated partial thromboplastin time (aPTT) reagent and prothrombin time reagent and reduces the anticoagulant effect of the protein. These studies show that antiphospholipid antibodies interfere with the binding of annexin-V to anionic phospholipid and with its anticoagulant activity. This acceleration of coagulation, due to reduced binding of annexin V, stands in marked contrast to the "lupus anticoagulant effect" previously described in these patients. These results are the first direct demonstration of the displacement of annexin-V and the consequent acceleration of coagulation on noncellular phospholipid surfaces by antiphospholipid antibodies.

Annexin A5↗
PubMed1997

Linkage between operator binding and dimer to octamer self-assembly of bacteriophage lambda cI repressor.

Cooperative binding of the bacteriophage lambda cI repressor dimer to specific sites of the phage operators OR and OL controls the developmental state of the phage. Cooperativity has long been thought to be mediated by self-assembly of repressor dimers to form tetramers which can bind simultaneously to adjacent operators. More recently, we demonstrated that when free repressor dimers self-associate in solution, tetramer is an intermediate in a concerted assembly reaction leading to octamer as the predominant higher order species [Senear, D. F., et al. (1993) Biochemistry 32, 6179-6189]. Even as a minority component in the assembly reaction, tetramer can account for pairwise cooperativity. In a similar manner, were it able to bind all three operators simultaneously, octamer could account for three-way cooperativity. In fact, based solely on repressor self-assembly, the naive prediction is that the repressor-OR interactions should be substantially more cooperative than they are. Evidently, there are unfavorable contributions to cooperativity from processes other than repressor self-assembly. Here, we focus on coupling between repressor self-association and operator binding as one possible unfavorable contribution to cooperativity. Sedimentation equilibrium analysis was used to compare the dimer-octamer association reactions of a repressor dimer-OR1 complex and free repressor dimer. Fluorescence anisotropy was used to investigate OR1 binding to free dimers and dimers assembled as higher order species. The results of these experiments indicate a significant and salt-dependent unfavorable contribution generated by such coupling. Since the oligonucleotides used in these experiments are the size of single operator sites, this coupling is mediated by the protein, not by the DNA. This mechanism does not account for an additional, salt-independent, unfavorable contribution which we presume is transmitted via the DNA. Thus, unfavorable contributions generated by structural transitions in both macromolecules serve to moderate the effect of self-association alone. We speculate that this is a general feature of cooperative protein-DNA interactions.

Bacteriophage lambda↗
PubMed1997

An aromatic stacking interaction between subunits helps mediate DNA sequence specificity: operator site discrimination by phage lambda cI repressor.

Sequence specific DNA binding by regulatory proteins provides the basis for regulation of initiation of transcription. A great deal of progress has been made toward understanding sequence specific recognition by individual protein subunits. An additional level of control that needs to be understood is that due to coupling between the subunits of oligomeric regulatory proteins. An example is the bacteriophage lambda cI repressor, a dimeric protein that regulates the lysogenic to lytic genetic switch of the phage. Two levels of specificity are critical to this regulation. First, like all transcriptional regulators, dimers distinguish operator from nonspecific DNA. Direct readout of the DNA sequence by the recognition helix is considered the well understood mechanism for this. However, differential affinity for O(R)1, O(R)2 and O(R)3 is equally critical to the switch because it mediates opposing regulation of divergent promoters. Site specificity at this second level is less well understood. Conformational adaptation by both the repressor and the different operators appears to be important. To evaluate how subunit-subunit interactions are involved in this process, we investigated the effects on both dimer stability and operator binding of amino acid substitutions at the contacts between the symmetrically related helices-5 in the dimer interface. Substitutions for Tyr88 alter dimer stability and greatly perturb differential operator affinity, but generally do not affect operator versus non-operator specificity. The pattern of these effects suggests that the geometry of the face-to-face aromatic stacking interaction between symmetrically related Tyr88 in each subunit, a group in the dimer interface but far removed from the DNA binding interface, plays a critical role in operator discrimination. Conformational changes in the tertiary structure of the subunits appears to be involved. By contrast, the significant effect of I84S substitution is to greatly decrease affinity for all three operators. Presumably, the altered packing of the dimer interface causes a quarternary structural change that moves the two helix-turn-helix motifs out of register with successive DNA major grooves.

Bacteriophage lambda↗
PubMed1995

5-Hydroxytryptophan: an absorption and fluorescence probe which is a conservative replacement for [A14 tyrosine] in insulin.

Use of insulin's intrinsic tyrosine absorption and fluorescence to monitor its interaction with the insulin receptor is limited because the spectral properties of the receptor tryptophan residues mask the spectral properties of the hormone tyrosine residues. We describe the synthesis of an insulin analog where A14 tyrosine is replaced by a tryptophan analog, 5-hydroxytryptophan. This insulin is spectrally enhanced since 5-hydroxytryptophan has an absorption band above 300 nm which is at lower energies than the absorption of tryptophan. Steady-state and time-resolved fluorescence parameters indicate that 5-hydroxytryptophan reports the same information about the environment of the A14 side chain as does the corresponding tryptophan-containing insulin. The synthetic hormone is a full agonist compared to porcine insulin, but has slightly reduced specific activity. Consequently, this spectrally enhanced insulin analog will be useful for hormone-receptor interaction studies since it can be observed by both absorption and fluorescence even in the presence of the tryptophan-containing receptor.

5-Hydroxytryptophan↗
PubMed1995

Environments of the four tryptophans in the extracellular domain of human tissue factor: comparison of results from absorption and fluorescence difference spectra of tryptophan replacement mutants with the crystal structure of the wild-type protein.

The local environments of the four tryptophan residues of the extracellular domain of human tissue factor (sTF) were assessed from difference absorption and fluorescence spectra. The difference spectra were derived by subtracting spectra from single Trp-to-Phe or Trp-to-Tyr replacement mutants from the corresponding spectrum of the wild-type protein. Each of the mutants was capable of enhancing the proteolytic activity of factor VIIa showing that the mutations did not introduce major structural changes, although the mutants were more susceptible to denaturation by guanidinium chloride. The difference spectra indicate that the Trp residues are buried to different extents within the protein matrix. This evaluation was compared with the x-ray crystal structure of sTF. There is excellent agreement between predictions from the difference spectra and the environments of the Trp residues observed in the x-ray crystal structure, demonstrating that difference absorption and particularly fluorescence spectra derived from functional single-Trp replacement mutants can be used to obtain information about the local environments of individual Trp residues in multi-tryptophan proteins.

Base Sequence↗
PubMed1993

The primary self-assembly reaction of bacteriophage lambda cI repressor dimers is to octamer.

Cooperative binding of the bacteriophage lambda cI repressor dimer to specific sites of the phage operators OR and OL controls the developmental state of the phage. It has long been believed that cooperativity is mediated by self-assembly of repressor dimers to form tetramers which can then bind simultaneously to adjacent operator sites. As a first step in defining the individual energy contributions to binding cooperativity, sedimentation equilibrium and steady-state fluorescence anisotropy methods have been used to study the higher order assembly reactions of the free repressor in solution. Wild-type repressor with 5-hydroxytryptophan (5-OHTrp) substituted for the native tryptophan [Ross et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 12023-12027] and two mutant repressor proteins that bind cooperatively to OR but have altered dimerization properties were also studied. We report here that the primary assembly mode of all four proteins is dimer to octamer. It is not dimer to tetramer as previously assumed. While tetramer does form as an assembly intermediate, dimer-octamer assembly is a concerted process so that tetramer is never a predominant species in solution. Sedimentation velocity experiments suggest that the octamer is highly asymmetric, consistent with an elongated shape. This conformation could allow octamers to bind simultaneously to all three operator sites at either OR or OL. Examination of tetramer and octamer concentrations suggests that both species could be involved in cooperative repressor-operator interactions. Our previous work used the unique spectral properties of 5-OHTrp to demonstrate that octamer binds single-operator DNA and is not dissociated to tetramer [Laue et al. (1993) Biochemistry 32, 2469-2472]. Taken together with the results presented here, octamers as well as tetramers must be considered in developing models to explain the cooperativity of lambda cI repressor binding to operator DNA.

5-Hydroxytryptophan↗
PubMed1992

Spectral enhancement of proteins: biological incorporation and fluorescence characterization of 5-hydroxytryptophan in bacteriophage lambda cI repressor.

We have used a tryptophan-requiring Escherichia coli auxotroph to replace the three tryptophan residues of lambda cI repressor with 5-hydroxy-L-tryptophan (5-OHTrp). By using a nonleaky promoter, we have achieved > 95% replacement of tryptophan in the repressor. We show that the absorbance and fluorescence properties of 5-OHTrp-lambda cI are clearly distinct from lambda cI repressor and that the fluorescence of 5-OHTrp-lambda cI repressor can be observed selectively in the presence of exogenous tryptophan. We also show that the 5-OHTrp-lambda cI repressor functional properties, as assessed by measurement of binding constants for self-association and for association to operator DNA, and structural properties, as assessed by fluorescence, are indistinguishable from the native repressor. Based on these results, we anticipate that the availability of spectrally enhanced proteins will significantly enhance the utility of both fluorescence and phosphorescence spectroscopies to study protein structure and function in complex interacting systems.

5-Hydroxytryptophan↗