Binding of amyloidogenic transthyretin to the plasma membrane alters membrane fluidity and induces neurotoxicity

Transthyretin (TTR) can deposit as amyloid in the peripheral nervous system and induce a peripheral neuropathy. We examined the mechanism of TTR amyloid neurotoxicity on SH-SY5Y neuroblastoma cells. Wild-type (WT) TTR and two amyloidogenic mutants (V30M and L55P) were expressed in Escherichia coli. Incubation (aging) of WT TTR at 37 degrees C for 1 week caused no significant aggregation. However, there was a significant increase in the extent of amyloid fibril formation after the amyloidogenic mutants had been aged. L55P TTR aggregated more readily than V30M TTR. Both amyloidogenic mutants were neurotoxic after aging. The order of neurotoxicity was as follows: L55P > V30M > WT. As binding of amyloid proteins to the plasma membrane may cause cytotoxicity, we studied the binding of TTR to a plasma membrane-enriched preparation from SH-SY5Y cells by surface plasmon resonance. All three forms bound to the plasma membrane through electrostatic interactions. The binding of the amyloidogenic mutants was increased by aging. The amount of binding correlated closely with the amount of aggregation and with the cytotoxicity of each form. As membrane fluidity can influence cell viability, we also examined the effect of TTR on membrane fluidity using a fluorescence anisotropy method. Binding of the amyloidogenic TTR mutants increased membrane fluidity, and once again, the order of potency was as follows: L55P > V30M > WT. These results demonstrate that TTR can bind to the plasma membrane and cause a change in membrane fluidity. Altered membrane fluidity may be the cause of the neurotoxicity.     

Effect of a specific hydrogen bond (N138ND2-Q106O) on conformational integrity, stability, and activity of staphylococcal nuclease

There are two hydrogen bonding interactions (N138ND2-Q106O and Y54OH-S141OG) between the C-terminal region and the main body of staphylococcal nuclease (SNase). To examine the role of these hydrogen bonds, SNase(141) and its three mutants, SNase(141)N138D, SNase(141)S141A, and SNase(141)N138D/S141A, were created. The N138D mutation has the N138ND2-Q106O interaction deleted and the S141A mutation has the Y54OH-S141OG and S141OG-N138O interactions deleted. The conformational features, stability, and activity of the proteins have been compared by using circular dichroism, intrinsic and ANS-binding fluorescence, GdnHCl-induced denaturation, and activity assay. The results clearly show that the N138D mutation significantly alters the secondary and tertiary structures of the protein, producing a partially unfolding state; in contrast, the S141A mutation has no such effect on structure. These results strongly suggest that the specific hydrogen bond, N138ND2-Q106O, plays an important role in maintaining the conformational integrity and stability of the nuclease.     

Crystal structure of tabtoxin resistance protein complexed with acetyl coenzyme A reveals the mechanism for beta-lactam acetylation

Tabtoxin resistance protein (TTR) is an enzyme that renders tabtoxin-producing pathogens, such as Pseudomonas syringae, tolerant to their own phytotoxins. Here, we report the crystal structure of TTR complexed with its natural cofactor, acetyl coenzyme A (AcCoA), to 1.55A resolution. The binary complex forms a characteristic "V" shape for substrate binding and contains the four motifs conserved in the GCN5-related N-acetyltransferase (GNAT) superfamily, which also includes the histone acetyltransferases (HATs). A single-step mechanism is proposed to explain the function of three conserved residues, Glu92, Asp130 and Tyr141, in catalyzing the acetyl group transfer to its substrate. We also report that TTR possesses HAT activity and suggest an evolutionary relationship between TTR and other GNAT members.     

Hypertrophy in skeletal myotubes induced by junctophilin-2 mutant, Y141H, involves an increase in store-operated Ca2+ entry via Orai1

Junctophilins (JPs) play an important role in the formation of junctional membrane complexes (JMC) in striated muscle by physically linking the transverse-tubule and sarcoplasmic reticulum (SR) membranes. Researchers have found five JP2 mutants in humans with hypertrophic cardiomyopathy. Among these, Y141H and S165F are associated with severely altered Ca(2+) signaling in cardiomyocytes. We previously reported that S165F also induced both hypertrophy and altered intracellular Ca(2+) signaling in mouse skeletal myotubes. In the present study, we attempted to identify the dominant-negative role(s) of Y141H in primary mouse skeletal myotubes. Consistent with S165F, Y141H led to hypertrophy and altered Ca(2+) signaling (a decrease in the gain of excitation-contraction coupling and an increase in the resting level of myoplasmic Ca(2+)). However, unlike S165F, neither ryanodine receptor 1-mediated Ca(2+) release from the SR nor the phosphorylation of the mutated JP2 by protein kinase C was related to the altered Ca(2+) signaling by Y141H. Instead, abnormal JMC and increased SOCE via Orai1 were found, suggesting that the hypertrophy caused by Y141H progressed differently from S165F. Therefore JP2 can be linked to skeletal muscle hypertrophy via various Ca(2+) signaling pathways, and SOCE could be one of the causes of altered Ca(2+) signaling observed in muscle hypertrophy.     

The influence of conserved aromatic residues in 3-hydroxy-3-methylglutaryl-CoA synthase

In order to evaluate the potential contribution of conserved aromatic residues to the hydrophobic active site of 3-hydroxy-3-methylglutaryl-CoA synthase, site-directed mutagenesis was employed to produce Y130L, Y163L, F204L, Y225L, Y346L, and Y376L proteins. Each mutant protein was expressed at levels comparable with wild-type enzyme and was isolated in highly purified form. Initial kinetic characterization indicated that F204L exhibits a substantial (>300-fold) decrease in catalytic rate (kcat). Upon modification with the mechanism-based inhibitor, 3-chloropropionyl-CoA, or in formation of a stable binary complex with acetoacetyl-CoA, F204L exhibits binding stoichiometries comparable with wild-type enzyme, suggesting substantial retention of active site integrity. Y130L and Y376L exhibit inflated values (80- and 40-fold, respectively) for the Km for acetyl-CoA in the acetyl-CoA hydrolysis partial reaction; these mutants also exhibit an order of magnitude decrease in kcat. Formation of the acetyl-S-enzyme reaction intermediate by Y130L, F204L, and Y376L proceeds slowly in comparison with wild-type enzyme. However, solvent exchange into the thioester carbonyl oxygen of these acetyl-S-enzyme intermediates is not slow in comparison with previous observations for D159A and D203A mutants, which also exhibit slow acetyl-S-enzyme formation. The magnitude of the differential isotope shift upon exchange of H218O into [13C]acetyl-S-enzyme suggests a polarization of the thioester carbonyl and a reduction in bond order. Such an effect may substantially contribute to the upfield 13C NMR shift observed for [13C]acetyl-S-enzyme. The influence on acetyl-S-enzyme formation, as well as observed kcat (F204L) and Km (Y130L; Y376L) effects, implicate these invariant residues as part of the catalytic site. Substitution of phenylalanine (Y130F, Y376F) instead of leucine at residues 130 and 376 diminishes the effects on catalytic rate and substrate affinity observed for Y130L and Y376L, underscoring the influence of aromatic side chains near the active site.     

Functional interaction between S1 and S4 segments in voltage-gated sodium channels revealed by human channelopathies

The p.I141V mutation of the voltage-gated sodium channel is associated with several clinical hyper-excitability phenotypes. To understand the structural bases of the p.I141V biophysical alterations, molecular dynamics simulations were performed. These simulations predicted that the p.I141V substitution induces the formation of a hydrogen bond between the Y168 residue of the S2 segment and the R225 residue of the S4 segment. We generated a p.I141V-Y168F double mutant for both the Na_v1.4 and Na_v1.5 channels. The double mutants demonstrated the abolition of the functional effects of the p.I141V mutation, consistent with the formation of a specific interaction between Y168-S2 and R225-S4. The single p.Y168F mutation, however, positively shifted the activation curve, suggesting a compensatory role of these residues on the stability of the voltage-sensing domain.     

Functional interaction between S1 and S4 segments in voltage-gated sodium channels revealed by human channelopathies

The p.I141V mutation of the voltage-gated sodium channel is associated with several clinical hyper-excitability phenotypes. To understand the structural bases of the p.I141V biophysical alterations, molecular dynamics simulations were performed. These simulations predicted that the p.I141V substitution induces the formation of a hydrogen bond between the Y168 residue of the S2 segment and the R225 residue of the S4 segment. We generated a p.I141V-Y168F double mutant for both the Na_v1.4 and Na_v1.5 channels. The double mutants demonstrated the abolition of the functional effects of the p.I141V mutation, consistent with the formation of a specific interaction between Y168-S2 and R225-S4. The single p.Y168F mutation, however, positively shifted the activation curve, suggesting a compensatory role of these residues on the stability of the voltage-sensing domain.     

Mutagenesis of Trp(54) and Trp(203) residues on Fibrobacter succinogenes 1,3-1,4-beta-D-glucanase significantly affects catalytic activities of the enzyme

The possible structural and catalytic functions of the nine tryptophan amino acid residues, including Trp(54), Trp(105), Trp(112), Trp(141), Trp(148), Trp(165), Trp(186), Trp(198), and Trp(203) in Fibrobacter succinogenes 1,3-1,4-beta-D-glucanase (Fs beta-glucanase), were characterized using site-directed mutagenesis, initial rate kinetics, fluorescence spectrometry, and structural modeling analysis. Kinetic studies showed that a 5-7-fold increase in K(m) value for lichenan was observed for W141F, W141H, and W203R mutant Fs beta-glucanases, and approximately 72-, 56-, 30-, 29.5-, 4.9-, and 4.3-fold decreases in k(cat) relative to that for the wild-type enzyme were observed for the W54F, W54Y, W141H, W203R, W141F, and W148F mutants, respectively. In contrast, W186F and W203F, unlike the other 12 mutants, exhibited a 1.4- and 4.2-fold increase in k(cat), respectively. W165F and W203R were the only two mutants that exhibited a 4-7-fold higher activity relative to the wild-type enzyme after they were incubated at pH 3.0 for 1 h. Fluorescence spectrometry indicated that all of the mutations on the nine tryptophan amino acid residues retained a folding similar to that of the wild-type enzyme. Structural modeling and kinetic studies suggest that Trp(54), Trp(141), Trp(148), and Trp(203) play important roles in maintaining structural integrity in the substrate-binding cleft and the catalytic efficiency of the enzyme.     

Unusual self-association properties of transthyretin Y114C related to familial amyloidotic polyneuropathy: effects on detection and quantification.

We performed biochemical and immunological examinations of heterozygotic carriers of the transthyretin (TTR) mutant Y114C associated with familial amyloidotic polyneuropathy (FAP). The total serum TTR levels in Y114C TTR carriers were extremely low when analyzed by single radial immunodiffusion (SRID), whereas by indirect enzyme-linked immunosorbent assay (ELISA) procedure, their total TTR concentrations were increased. Recombinant homozygotic Y114C TTR showed no immunoreactivity towards a TTR antibody when analyzed by SRID, whereas by the ELISA procedure presented the same degree of reactivity as that of normal TTR or isolated serum heterozygotic Y114C TTR. These results indicate that immunodifusion based techniques cannot properly determine TTR serum levels in Y114C carriers. Analyses of serum TTR of the Y114C TTR carriers by electrospray ionization mass spectrometry (ESI-MS) with the orifice corn voltage at 60 V revealed a small peak of the free Y114C TTR in addition to large TTR peaks of normal TTR. The levels of the free mutant TTR increased with the orifice corn voltage at 90 V. In contrast, increase in orifice voltage from 60 to 90 V produced a reduction in the level of normal TTR. The results suggest a different pattern of association between monomers in Y114C relative to normal TTR.     

Functional characterization of the mutations in Pepper mild mottle virus overcoming tomato tm‐1‐mediated resistance

In tomato plants, Pepper mild mottle virus (PMMoV) cannot replicate because the tm‐1 protein inhibits RNA replication. The resistance of tomato plants to PMMoV remains durable both in the field and under laboratory conditions. In this study, we constructed several mutant PMMoVs and analysed their abilities to replicate in tomato protoplasts and plants. We found that two mutants, PMMoV‐899R,F976Y and PMMoV‐899R,F976Y,D1098N, were able to replicate in tomato protoplasts, but only PMMoV‐899R,F976Y,D1098N was able to multiply in tomato plants. Further analysis showed that the D1098N mutation of the replication proteins weakened the inhibitory effect of the tm‐1 protein and enhanced the replication efficiency of PMMoV‐899R,F976Y,D1098N. We also observed that the infectivity of the viruses decreased in the order wild‐type PMMoV > PMMoV‐899R,F976Y > PMMoV‐899R,F976Y,D1098N in original host plants, pepper and tobacco plants. On the contrary, the single mutation D1098N abolished PMMoV replication in tobacco protoplasts. On the basis of these observations, it is likely that the deleterious side‐effects of mutations in replication proteins prevent the emergence of PMMoV mutants that can overcome tm‐1‐mediated resistance.     

Dynamics, stability and iron-binding activity of frataxin clinical mutants

Friedreich's ataxia results from a deficiency in the mitochondrial protein frataxin, which carries single point mutations in some patients. In the present study, we analysed the consequences of different disease-related mutations in vitro on the stability and dynamics of human frataxin. Two of the mutations, G130V and D122Y, were investigated for the first time. Analysis by CD spectroscopy demonstrated a substantial decrease in the thermodynamic stability of the variants during chemical and thermal unfolding (wild-type > W155R > I154F > D122Y > G130V), which was reversible in all cases. Protein dynamics was studied in detail and revealed that the mutants have distinct propensities towards aggregation. It was observed that the mutants have increased correlation times and different relative ratios between soluble and insoluble/aggregated protein. NMR showed that the clinical mutants retained a compact and relatively rigid globular core despite their decreased stabilities. Limited proteolysis assays coupled with LC-MS allowed the identification of particularly flexible regions in the mutants; interestingly, these regions included those involved in iron-binding. In agreement, the iron metallochaperone activity of the Friedreich's ataxia mutants was affected: some mutants precipitate upon iron binding (I154F and W155R) and others have a lower binding stoichiometry (G130V and D122Y). Our results suggest that, in heterozygous patients, the development of Friedreich's ataxia may result from a combination of reduced efficiency of protein folding and accelerated degradation in vivo, leading to lower than normal concentrations of frataxin. This hypothesis also suggests that, although quite different from other neurodegenerative diseases involving toxic aggregation, Friedreich's ataxia could also be linked to a process of protein misfolding due to specific destabilization of frataxin.     

Aspartic acid residues 72 and 75 and tyrosine-sulfate 73 of heparin cofactor II promote intramolecular interactions during glycosaminoglycan binding and thrombin inhibition

We used site-directed mutagenesis to investigate the role of Glu(69), Asp(70), Asp(71), Asp(72), Tyr-sulfate(73), and Asp(75) in the second acidic region (AR2) of the serpin heparin cofactor II (HCII) during formation of the thrombin.HCII complex with and without glycosaminoglycans. E69Q/D70N/D71N recombinant (r)HCII, D72N/Y73F/D75N rHCII, and E69Q/D70N/D71N/D72N/Y73F/D75N rHCII were prepared to localize acidic residues important for thrombin inhibition. Interestingly, D72N/Y73F/D75N rHCII had significantly enhanced thrombin inhibition without glycosaminoglycan (4-fold greater) and with heparin (6-fold greater), showing maximal activity at 2 microg/ml heparin compared with wild-type recombinant HCII (wt-rHCII) with maximal activity at 20 microg/ml heparin. The other rHCII mutants had lesser-enhanced activities, but they all eluted from heparin-Sepharose at significantly higher ionic strengths compared with wt-rHCII. Neutralizing and reversing the charge of Asp(72), Tyr-sulfate(73), and Asp(75) were done to characterize their individual contribution to HCII activity. Only Y73K rHCII and D75K rHCII have significantly increased heparin cofactor activity compared with wt-rHCII; however, all of the individual rHCII mutants required substantially less glycosaminoglycan at maximal inhibition than did wt-rHCII. Inhibition of either alpha-thrombin/hirugen or gamma(T)-thrombin (both with an altered anion-binding exosite-1) by the AR2 rHCII mutants was similar to wt-rHCII. D72N/Y73F/D75N rHCII and D75K rHCII were significantly more active than wt-rHCII in a plasma-based thrombin inhibition assay with glycosaminoglycans. These results indicate that improved thrombin inhibition in the AR2 HCII mutants is mediated by enhanced interactions between the acidic domain and anion-binding exosite-1 of thrombin and that AR2 may be a "molecular rheostat" to promote thrombin inhibition in the presence of glycosaminoglycans.     

Mixed regioselectivity in the Arg-177 mutants of Corynebacterium diphtheriae heme oxygenase as a consequence of in-plane heme disorder

It has been reported that the R183E and R183D mutants of rat heme oxygenase-1 (r-HO-1) produce approximately 30% delta-biliverdin [Zhou, H., et al. (2000) J. Am. Chem. Soc. 122, 8311-8312]. Two plausible mechanisms were proposed to explain the observations. (a) Electrostatic repulsion between E183 (D183) and one of the heme propionates forces the heme to rotate, thereby placing the delta-meso carbon in a position that is susceptible to oxidation. (b) Rearrangement of the distal pocket structure is triggered by the formation of a hydrogen bond between E183 (D183) and K179. A change in the pK(a) for the Fe(III)-H(2)O to Fe(III)-OH transition of the mutants was interpreted to be consistent with rearrangement of the hydrogen bond network in the distal pocket. The large similarities between the high-frequency portion of the (1)H NMR spectra corresponding to the wild type and R183E and R183D mutants were interpreted to indicate that the heme in the mutants is not rotated to a significant extent. We have re-examined this issue by studying the corresponding R177 mutants in heme oxygenase from Corynebacterium diphtheriae (cd-HO). Replacing R177 with E or D results in the formation of approximately 55% alpha- and 45% delta-biliverdin, whereas the R177A mutant retains alpha-regioselectivity. In addition, the K13N/Y130F/R177A triple mutant catalyzed the formation of 60% delta- and 40% alpha-biliverdin, while single mutants K13N and Y130F did not appreciably change the regioselectivity of the reaction. The pK(a) of the Fe(III)-H(2)O to Fe(III)-OH transition in wild-type cd-HO is 9.1, and those of the R177E, R177D, R177A, and K13N/Y130F/R177A mutants are 9.4, 9.5, 9.2, and 8.0, respectively. Thus, no obvious correlation exists between the changes in pK(a) and the altered regioselectivity. NMR spectroscopic studies conducted with the R177D and R177E mutants of cd-HO revealed the presence of three heme isomers: a major (M) and a minor (m) heme orientational isomer related by a 180 degrees rotation about the alpha-gamma meso axis and an alternative seating (m') which is related to m by an 85 degrees in-plane rotation of the macrocycle. The in-plane rotation of m to acquire conformation m' is triggered by electrostatic repulsion between the side chains of D or E at position 177 and heme propionate-6. As a consequence, the delta-meso carbon in m' is placed in the position occupied by the alpha-meso carbon in m, where it is susceptible to hydroxylation and subsequent formation of delta-biliverdin.     

Receptor binding,immune escape,and protein stability direct the natural selection of SARS-CoV-2 variants

Emergence of new severe acute respiratory syndrome coronavirus 2 variants has raised concerns related to the effectiveness of vaccines and antibody therapeutics developed against the unmutated wildtype virus. Here, we examined the effect of the 12 most commonly occurring mutations in the receptor-binding domain of the spike protein on its expression, stability, activity, and antibody escape potential. Stability was measured using thermal denaturation, and the activity and antibody escape potential were measured using isothermal titration calorimetry in terms of binding to the human angiotensin-converting enzyme 2 and to neutralizing human antibody CC12.1, respectively. Our results show that mutants differ in their expression levels. Of the eight best-expressed mutants, two (N501Y and K417T/E484K/N501Y) showed stronger affinity to angiotensin-converting enzyme 2 compared with the wildtype, whereas four (Y453F, S477N, T478I, and S494P) had similar affinity and two (K417N and E484K) had weaker affinity than the wildtype. Compared with the wildtype, four mutants (K417N, Y453F, N501Y, and K417T/E484K/N501Y) had weaker affinity for the CC12.1 antibody, whereas two (S477N and S494P) had similar affinity, and two (T478I and E484K) had stronger affinity than the wildtype. Mutants also differ in their thermal stability, with the two least stable mutants showing reduced expression. Taken together, these results indicate that multiple factors contribute toward the natural selection of variants, and all these factors need to be considered to understand the evolution of the virus. In addition, since not all variants can escape a given neutralizing antibody, antibodies to treat new variants can be chosen based on the specific mutations in that variant.     

Role of the aromatic residues in the near-amino terminal motif of vimentin in intermediate filament assembly in vitro

Type III and IV intermediate filament (IF) proteins share a conserved sequence motif of -Tyr-Arg-Arg-X-Phe- at the near-amino termini. To characterize significance of the aromatic residues in the motif, we prepared vimentin mutants in which Tyr-10 and Phe-14 are substituted with Asn and Ser (Vim[Y10N], Vim[F14S] and Vim[Y10N, F14S]), and examined assembly properties in vitro by electron microscopy and viscosity measurements. At 2 s after initiation of assembly reaction at pH 7.2 and 150 mM NaCl, all the vimentin mutants formed so-called unit-length filaments (ULFs) that were slightly larger than ULFs of wild-type vimentin. In following filament elongation, Vim[Y10N, F14S] and Vim[Y10N] performed longitudinal annealing of ULFs very rapidly and formed IFs within only 2.5 and 5 min, respectively, while Vim[F14S] and wild-type vimentin gave IFs by 40-60 min. The IFs of Vim[Y10N, F14S] and Vim[Y10N], however, tended to intertwine each other and formed bundles in parts of the specimens. The intertwinements decreased as the salt concentration decreased, and optimal salt concentration for the two mutants to form normal IFs was 50 mM. These results suggest that the aromatic residues, especially Tyr-10, in the motif have a role in controlling intermolecular interactions involved in IF assembly in vitro and suppress undesirable filament intertwinements at physiological ionic strength.     

Iron-binding process in the amino- and carboxyl-terminal lobes of ovotransferrin: quantitative studies utilizing single Fe3+-binding mutants

Iron-liganding-residue mutants of ovotransferrin, Y191F and Y524F, were investigated for their Fe(3+)-binding properties. The absorption spectrum and urea gel electrophoresis verified the single iron binding on the C- and N-lobes for Y191F and Y524F, respectively. A newly developed competitive Fe(3+)-binding analysis, in which equimolar Y191F and Y524F are mixed with less Fe(3+) than saturation, enabled us to quantitatively determine the lobe preference for initial iron entry as the ratio (alpha value) of N-lobe over C-lobe. The alpha value estimated on the basis of a kinetic model was highly dependent on pH; within a pH range from 6.5 to 9.0, alpha was increased from 2 to 5 on lowering pH with an apparent sigmoid curve. On differential scanning calorimetry, single thermal transition was observed around 61 degrees C for the apo forms of Y191F, Y524F, and wild-type ovotransferrin. The Fe(3+)-loaded mutants, however, showed dual transitions at 62.4 and 82.1 degrees C in Y191F and 66.4 and 76.0 degrees C in Y524F. According to the DeltaG(AB) value that is defined as the free energy change in a target lobe induced by the iron binding on the counter lobe, marked stabilization effects by interlobe interactions were found to be induced during the major iron-binding process: upon the primary N-lobe iron binding in the iron-free C-lobe (DeltaG(AB), -2.25 kcal/mol) and upon the secondary C-lobe iron binding in the monoferric N-lobe (DeltaG(AB), -6.45 kcal/mol).     

Grb2 mediates negative regulation of stem cell factor receptor/c-Kit signaling by recruitment of Cbl

Aberrant activation of c-Kit is involved in a number of human diseases including cancers and leukemias. Certain receptor tyrosine kinases, such as epidermal growth factor receptor, have been shown to indirectly recruit Cbl through the adapter protein Grb2, leading to receptor ubiquitination and degradation. In order to study the role of Grb2 in c-Kit degradation, a series of mutations of the Grb2 binding sites in c-Kit were generated (Y703F, Y936F, and Y703F/Y936F). Since other signal transduction molecules are also known to bind Y703 and Y936, the more selective asparagine-to-alanine (N-to-A) mutants N705A, N938A, and N705A/N938A were generated. We could clearly demonstrate that binding of Grb2 was dependent on intact phosphorylation sites Y703 and Y936. Furthermore, we could demonstrate the presence of Cbl in a complex with Grb2 and c-Kit. Thus, Grb2 is able to indirectly recruit Cbl to c-Kit. In the N-to-A mutants, Cbl phosphorylation was strongly reduced, which correlated with reduced ubiquitination of c-Kit as well as decreased internalization and degradation of the receptor. Taken together, we have demonstrated that, in addition to its role in positive signaling via the Ras/Erk pathway, Grb2 mediates c-Kit degradation through recruitment of Cbl to c-Kit, leading to ubiquitination of c-Kit followed by internalization and degradation.     

Farnesyl protein transferase: identification of K164 alpha and Y300 beta as catalytic residues by mutagenesis and kinetic studies.

Farnesyl protein transferase (FPT) is an alpha/beta heterodimeric zinc enzyme that catalyzes posttranslational farnesylation of many key cellular regulatory proteins, including oncogenic Ras. On the basis of the recently reported crystal structure of FPT complexed with a CVIM peptide and alpha-hydroxyfarnesylphosphonic acid, site-directed mutagenesis of the FPT active site was performed so key residues that are responsible for substrate binding and catalysis could be identified. Eight single mutants, including K164N alpha, Y166F alpha, Y166A alpha, Y200F alpha, H201A alpha, H248A beta, Y300F beta, and Y361F beta, and a double mutant, H248A beta/Y300F beta, were prepared. Steady-state kinetic analysis along with structural evidence indicated that residues Y200 alpha, H201 alpha, H248 beta, and Y361 beta are mainly involved in substrate binding. In addition, biochemical results confirm structural observations which show that residue Y166 alpha plays a key role in stabilizing the active site conformation of several FPT residues through cation-pi interactions. Two mutants, K164N alpha and Y300F beta, have moderately decreased catalytic constants (kcat). Pre-steady-state kinetic analysis of these mutants from rapid quench experiments showed that the chemical step rate constant was reduced by 41- and 30-fold, respectively. The product-releasing rate for each dropped approximately 10-fold. In pH-dependent kinetic studies, Y300F beta was observed to have both acidic and basic pKa values shifted 1 log unit from those of the wild-type enzyme, consistent with a possible role for Y300 beta as an acid-base catalyst. K164N alpha had a pKa shift from 6.0 to 5.3, which suggests it may function as a general acid. On the basis of these results along with structural evidence, a possible FPT reaction mechanism is proposed with both Y300 beta and K164 alpha playing key catalytic roles in enhancing the reactivity of the farnesyl diphosphate leaving group.