The A391E mutation enhances FGFR3 activation in the absence of ligand

The A391E mutation in the transmembrane domain of fibroblast growth factor receptor 3 leads to aberrant development of the cranium. It has been hypothesized that the mutant glutamic acid stabilizes the dimeric receptor due to hydrogen bonding and enhances its ligand-independent activation. We previously tested this hypothesis in lipid bilayers and showed that the mutation stabilizes the isolated transmembrane domain dimer by -1.3°kcal/mol. Here we further test the hypothesis, by investigating the effect of the A391E mutation on the activation of full-length fibroblast growth factor receptor 3 in human embryonic kidney 293T cells in the absence of ligand. We find that the mutation enhances the ligand-independent activation propensity of the receptor by -1.7°kcal/mol. This value is consistent with the observed strength of hydrogen bonds in membranes, and supports the above hypothesis.     

FGFR3 dimer stabilization due to a single amino acid pathogenic mutation

Mutations in the transmembrane (TM) domains of receptor tyrosine kinases (RTKs) have been implicated in the induction of pathological phenotypes. These mutations are believed to stabilize the RTK dimers, and thus promote unregulated signaling. However, the energetics behind the pathology induction has not been determined. An example of a TM domain pathogenic mutation is the Ala391-->Glu mutation in fibroblast growth factor receptor 3 (FGFR3), linked to Crouzon syndrome with acanthosis nigricans, as well as to bladder cancer. Here, we determine the free energy of dimerization of wild-type and mutant FGFR3 TM domain in lipid bilayers using F?rster resonance energy transfer, and we show that hydrogen bonding between Glu391 and the adjacent helix in the dimer is a feasible mechanism for dimer stabilization. The measured change in the free energy of dimerization due to the Ala391-->Glu pathogenic mutation is -1.3 kcal/mol, consistent with previous reports of hydrogen bond strengths in proteins. This is the first quantitative measurement of mutant RTK stabilization in a membrane environment. We show that this seemingly modest value can lead to a large increase in dimer fraction and thus profoundly affect RTK-mediated signal transduction.     

Effects of the oncogenic V(664)E mutation on membrane insertion, structure, and sequence-dependent interactions of the Neu transmembrane domain in micelles and model membranes: an integrated biophysical and simulation study

Receptor tyrosine kinases bind ligands such as cytokines, hormones, and growth factors and regulate key cellular processes, including cell division. They are also implicated in the development of many types of cancer. One such example is the Neu receptor tyrosine kinase found in rats (homologous to the human ErbB2 protein), which can undergo a valine to glutamic acid (V(664)E) mutation at the center of its α-helical transmembrane domain. This substitution results in receptor activation and oncogenesis. The molecular basis of this dramatic change in behavior upon introduction of the V(664)E mutation has been difficult to pin down, with conflicting results reported in the literature. Here we report the first quantitative, thermodynamic analysis of dimerization and biophysical characterization of the rat Neu transmembrane domain and several mutants in a range of chemical environments. These data have allowed us to identify the effects of the V(664)E mutation in the isolated TM domain with respect to protein-protein and protein-lipid interactions, membrane insertion, and secondary structure. We also report the results from a 100 ns atomistic molecular dynamics simulation of the Neu transmembrane domain in a model membrane bilayer (dipalmitoylphosphatidylcholine). The results from simulation and experiment are in close agreement and suggest that, in the model systems investigated, the V(664)E mutation leads to a weakening of the TM dimer and a change in sequence-dependent interactions. These results are contrary to recent results obtained in mammalian membranes, and the implications of this are discussed.     

The intrinsic energy of the gating isomerization of a neuromuscular acetylcholine receptor channel

Nicotinic acetylcholine receptor (AChR) channels at neuromuscular synapses rarely open in the absence of agonists, but many different mutations increase the unliganded gating equilibrium constant (E0) to generate AChRs that are active constitutively. We measured E0 for two different sets of mutant combinations and by extrapolation estimated E0 for wild-type AChRs. The estimates were 7.6 and 7.8×10(-7) in adult-type mouse AChRs (-100 mV at 23°C). The values are in excellent agreement with one obtained previously by using a completely different method (6.5×10(-7), from monoliganded gating). E0 decreases with depolarization to the same extent as does the diliganded gating equilibrium constant, e-fold with ～60 mV. We estimate that at -100 mV the intrinsic energy of the unliganded gating isomerization is +8.4 kcal/mol (35 kJ/mol), and that in the absence of a membrane potential, the intrinsic chemical energy of this global conformational change is +9.4 kcal/mol (39 kJ/mol). Na+ and K+ in the extracellular solution have no measureable effect on E0, which suggests that unliganded gating occurs with only water occupying the transmitter binding sites. The results are discussed with regard to the energy changes in receptor activation and the competitive antagonism of ions in agonist binding.     

Transmembrane interactions in the activation of the Neu receptor tyrosine kinase

The Neu receptor tyrosine kinase is constitutively activated by a single amino acid change in the transmembrane domain of the receptor. The mutation of Val664 to glutamate or glutamine induces receptor dimerization and autophosphorylation of the receptor's intracellular kinase domain. The ability of this single mutation to activate the receptor is sequence-dependent, suggesting that specific helix-helix interactions stabilize the transmembrane dimer. We have determined the local secondary structure and interhelical contacts in the region of position 664 in peptide models of the activated receptor using solid-state rotational resonance and rotational echo double-resonance (REDOR) NMR methods. Intrahelical (13)C rotational resonance distance measurements were made between 1-(13)C-Thr662 and 2-(13)C-Gly665 on peptides corresponding to the wild-type Neu and activated Neu transmembrane sequences containing valine and glutamate at position 664, respectively. We observed similar internuclear distances (4.5 +/- 0.2 A) in both Neu and Neu*, indicating that the region near residue 664 is helical and is not influenced by mutation. Interhelical (15)N...(13)C REDOR measurements between Gln664 side chains on opposing helices were not consistent with hydrogen bonding between the side chain functional groups. However, interhelical rotational resonance measurements between 1-(13)C-Glu664 and 2-(13)C-Gly665 and between 1-(13)C-Gly665 and 2-(13)C-Gly665 demonstrated close contacts (4.3-4.5 A) consistent with the packing of Gly665 in the Neu* dimer interface. These measurements provide structural constraints for modeling the transmembrane dimer and define the rotational orientation of the transmembrane helices in the activated receptor.     

Influenza viral hemagglutinin complicated shape is advantageous to its binding affinity for sialosaccharide receptor

Do the complexity and the bulkiness of a protein affect the affinity between protein and ligand? We attempted to investigate this problem by using ab initio fragment molecular orbital (FMO) method to calculate the binding energy between human influenza viral hemagglutinin (HA) and human oligo-saccharide receptor. We compared the binding energies of 4 different sizes of human A virus HA H3 subtype complexed with human receptor Neu5Ac(alpha2-6)Gal as a model. The full shape receptor binding domain complexed with Neu5Ac(alpha2-6)Gal had the highest binding energy 170.3kcal/mol at the FMO-HF/STO-3G level, which was 52.3kcal/mol higher than that of the smallest domain-receptor complex. These data provide the consideration of the backyard bulkiness beyond the binding site of protein to the protein-ligand stability.     

FGFR3 Transmembrane Domain Interactions Persist in the Presence of Its Extracellular Domain

Isolated receptor tyrosine kinase transmembrane (TM) domains have been shown to form sequence-specific dimers in membranes. Yet, it is not clear whether studies of isolated TM domains yield knowledge that is relevant to full-length receptors or whether the large glycosylated extracellular domains alter the interactions between the TM helices. Here, we address this question by quantifying the effect of the pathogenic A391E TM domain mutation on the stability of the fibroblast growth factor receptor 3 dimer in the presence of the extracellular domain and comparing these results to the case of the isolated TM fibroblast growth factor receptor 3 domains. We perform the measurements in plasma membrane-derived vesicles using a Förster-resonance-energy-transfer-based method. The effect of the mutation on dimer stability in both cases is the same (∼−1.5 kcal/mol), suggesting that the interactions observed in simple TM-peptide model systems are relevant in a biological context.     

Activation of Neu (ErbB-2) Mediated by Disulfide Bond-Induced Dimerization Reveals a Receptor Tyrosine Kinase Dimer Interface

Receptor dimerization is a crucial intermediate step in activation of signaling by receptor tyrosine kinases (RTKs). However, dimerization of the RTK Neu (also designated ErbB-2, HER-2, and p185^neu), while necessary, is not sufficient for signaling. Earlier work in our laboratory had shown that introduction of an ectopic cysteine into the Neu juxtamembrane domain induces Neu dimerization but not signaling. Since Neu signaling does require dimerization, we hypothesized that there are additional constraints that govern signaling ability. With the importance of the interreceptor cross-phosphorylation reaction, a likely constraint was the relative geometry of receptors within the dimer. We have tested this possibility by constructing a consecutive series of cysteine substitutions in the Neu juxtamembrane domain in order to force dimerization along a series of interreceptor faces. Within the group that dimerized constitutively, a subset had transforming activity. The substitutions in this subset all mapped to the same face of a predicted alpha helix, the most likely conformation for the intramembrane domain. Furthermore, this face of interaction aligns with the projected Neu* V664E substitution and with a predicted amphipathic interface in the Neu juxtamembrane domain. We propose that these results identify an RTK dimer interface and that dimerization of this RTK induces an extended contact between juxtamembrane and intramembrane alpha helices.     

Neu1 sialidase and matrix metalloproteinase-9 cross-talk is essential for Toll-like receptor activation and cellular signaling

The signaling pathways of mammalian Toll-like receptors (TLRs) are well characterized, but the precise mechanism(s) by which TLRs are activated upon ligand binding remains poorly defined. Recently, we reported a novel membrane sialidase-controlling mechanism that depends on ligand binding to its TLR to induce mammalian neuraminidase-1 (Neu1) activity, to influence receptor desialylation, and subsequently to induce TLR receptor activation and the production of nitric oxide and proinflammatory cytokines in dendritic and macrophage cells. The α-2,3-sialyl residue of TLR was identified as the specific target for hydrolysis by Neu1. Here, we report a membrane signaling paradigm initiated by endotoxin lipopolysaccharide (LPS) binding to TLR4 to potentiate G protein-coupled receptor (GPCR) signaling via membrane Gα(i) subunit proteins and matrix metalloproteinase-9 (MMP9) activation to induce Neu1. Central to this process is that a Neu1-MMP9 complex is bound to TLR4 on the cell surface of naive macrophage cells. Specific inhibition of MMP9 and GPCR Gα(i)-signaling proteins blocks LPS-induced Neu1 activity and NFκB activation. Silencing MMP9 mRNA using lentivirus MMP9 shRNA transduction or siRNA transfection of macrophage cells and MMP9 knock-out primary macrophage cells significantly reduced Neu1 activity and NFκB activation associated with LPS-treated cells. These findings uncover a molecular organizational signaling platform of a novel Neu1 and MMP9 cross-talk in alliance with TLR4 on the cell surface that is essential for ligand activation of TLRs and subsequent cellular signaling.     

Regulated coupling of the Neu receptor to phosphatidylinositol 3'-kinase and its release by oncogenic activation.

The neu protooncogene encodes a tyrosine kinase receptor that is involved in the regulation of normal growth and malignant transformation. To circumvent the use of the incompletely characterized ligand of Neu, we constructed a chimeric protein composed of the ligand-binding domain of the epidermal growth factor receptor and the transmembrane and cytoplasmic portions of Neu. By expressing this Neu-epidermal growth factor receptor chimera (termed NEC), we found that following stimulation by the heterologous ligand, the tyrosine kinase of Neu became associated with a phosphatidylinositol (PI) kinase activity. The association was dependent on the concentration of the ligand and was almost maximal within 30 s after ligand binding. The lipid kinase was identified as a type I PI 3'-kinase on the basis of its inhibition by Nonidet P-40 and high pressure liquid chromatography of the phosphorylated product. To confirm the identification of PI 3'-kinase as an effector of Neu, we raised antibodies to the alpha-isoform of the regulatory subunit of PI 3'-kinase (p85). Using these antibodies, it was possible to directly demonstrate ligand-dependent formation of a tyrosine-phosphorylated complex of NEC and PI 3'-kinase. Apparently, both PI 3'-kinase and phospholipase C gamma, another substrate of the Neu kinase, simultaneously associated with the same activated NEC molecule. Nevertheless, immunofluorescence localization of PI 3'-kinase revealed no significant cellular redistribution of the enzyme after activation of the Neu kinase. Interestingly, PI 3'-kinase was localized primarily to the cell nucleus and to confined regions of the plasma membrane. Analysis of mutants of the Neu protein indicated that the oncogenic point-mutated Neu (Glu664) was permanently coupled to PI 3'-kinase; but two nontransforming versions of the oncoprotein, a kinase-defective protein and a carboxyl-terminally deleted Neu, were devoid of the constitutive association with PI 3'-kinase. Hence, we concluded that phosphatidylinositol 3'-kinase is a physiological substrate of the Neu receptor, but the regulation of this coupling is released upon oncogenic activation.     

The Pathogenic A391E Mutation in FGFR3 Induces a Structural Change in the Transmembrane Domain Dimer

Fibroblast growth factor receptor 3 (FGFR3) is a single-pass membrane protein and a member of the receptor tyrosine kinase family of proteins that is involved in the regulation of skeletal growth and development. FGFR3 has three distinct domains: the ligand binding extracellular domain, the cytosolic kinase domain and the transmembrane domain (TMD). Previous work with the isolated FGFR3 TMD has shown that it has the ability to dimerize. Clinical and genetic studies have also correlated mutations in the TMD with a variety of skeletal and cranial dysplasias and cancer. Although the structures of the extracellular and cytosolic domains of FGFR3 have been solved, the structure of the TMD dimer is still unknown. Furthermore, very little is known regarding the effects of pathogenic mutations on the TMD dimer structure. We, therefore, carried out ToxR activity assays to determine the role of the SmXXXSm motif in the dimerization of the FGFR3 TMD. This motif has been shown to drive the association of many transmembrane proteins. Our results indicate that the interaction between wild-type FGFR3 TMDs is not mediated by two adjacent SmXXXSm motifs. In contrast, studies using the TMD carrying the pathogenic A391E mutation suggest that the motifs play a role in the dimerization of the mutant TMD. Based on these observations, here we report a new mechanistic model in which the pathogenic A391E mutation induces a structural change that leads to the formation of a more stable dimer.     

Interactions of the acid and base catalysts on staphylococcal nuclease as studied in a double mutant.

The mechanism of the phosphodiesterase reaction catalyzed by staphylococcal nuclease is believed to involve concerted general acid-base catalysis by Arg-87 and Glu-43. The mutual interactions of Arg-87 and Glu-43 were investigated by comparing kinetic and thermodynamic properties of the single mutant enzymes E43S (Glu-43 to Ser) and R87G (Arg-87 to Gly) with those of the double mutant, E43S + R87G, in which both the basic and acidic functions have been inactivated. Denaturation studies with guanidinium chloride, CD, and 600-MHz 1D and 2D proton NMR spectra, indicate all enzyme forms to be predominantly folded in absence of the denaturant and reveal small antagonistic effects of the E43S and R87G mutations on the stability and structure of the wild-type enzyme. The free energies of binding of the divalent cation activator Ca2+, the inhibitor Mn2+, and the substrate analogue 3',5'-pdTp show simple additive effects of the two mutations in the double mutant, indicating that Arg-87 and Glu-43 act independently to facilitate the binding of divalent cations and of 3',5'-pdTP by the wild-type enzyme. The free energies of binding of the substrate, 5'-pdTdA, both in binary E-S and in active ternary E-Ca(2+)-S complexes, show synergistic effects of the two mutations, suggesting that Arg-87 and Glu-43 interact anticooperatively in binding the substrate, possibly straining the substrate by 1.6 kcal/mol in the wild-type enzyme. The large free energy barriers to Vmax introduced by the R87G mutation (delta G1 = 6.5 kcal/mol) and by the E43S mutation (delta G2 = 5.0 kcal/mol) are partially additive in the double mutant (delta G1+2 = 8.1 kcal/mol). These partially additive effects on Vmax are most simply explained by a cooperative component to transition state binding by Arg-87 and Glu-43 of -3.4 kcal/mol. The combination of anticooperative, cooperative, and noncooperative effects of Arg-87 and Glu-43 together lower the kinetic barrier to catalysis by 8.1 kcal/mol.     

X-ray crystallography and QM/MM investigation on the oligosaccharide synthesis mechanism of rice BGlu1 glycosynthases

Nucleophile mutants of retaining β-glycosidase can act as glycosynthases to efficiently catalyze the synthesis of oligosaccharides. Previous studies proved that rice BGlu1 mutants E386G, E386S and E386A catalyze the oligosaccharide synthesis with different rates. The E386G mutant gave the fastest transglucosylation rate, which was approximately 3- and 19-fold faster than those of E386S and E386A. To account for the differences of their activities, in this paper, the X-ray crystal structures of BGlu1 mutants E386S and E386A were solved and compared with that of E386G mutant. However, they show quite similar active sites, which implies that their activities cannot be elucidated from the crystal structures alone. Therefore, a combined quantum mechanical/molecular mechanical (QM/MM) calculations were further performed. Our calculations reveal that the catalytic reaction follows a single-step mechanism, i.e., the extraction of proton by the acid/base, E176, and the formation of glycosidic bond are concerted. The energy barriers are calculated to be 19.9, 21.5 and 21.9 kcal/mol for the mutants of E386G, E386S and E386A, respectively, which is consistent with the order of their experimental relative activities. But based on the calculated activation energies, 1.1 kcal/mol energy difference may translate to nearly 100 fold rate difference. Although the rate limiting step in these mutants has not been established, considering the size of the product and the nature of the active site, it is likely that the product release, rather than chemistry, is rate limiting in these oligosaccharides synthesis catalyzed by BGlu1 mutants.     

Temperature dependence of kinetic interactions between progesterone and the uterine cytoplasmic receptor

The temperature dependence of the rates of dissociation and association for progesterone-receptor interactions was measured over the temperature range of 0–20°C. The dissociation process is biphasic indicating that either two forms of receptor are present or that the binding of progesterone to the receptor is a concatenated reaction.The enthalpy of activation for the dissociation of progesterone from the receptor is about 26–28 kcal/mol and the entropic energy of activation is about ?5 kcal/mol. The enthalpy of activation for the association of these molecules is about 3 kcal/mol and the entropic energy of activation is about 6 kcal/mol. These data are consistent with a model of progesterone binding to the receptor that includes hydrogen bonds between each of the two ketone groups and hydrogen donors on the receptor protein and involves van der Waals' interactions, due to the close proximity of the receptor binding site to a large fraction of the progesterone surface.     

Transmembrane helix packing of ErbB/Neu receptor in membrane environment: a molecular dynamics study

Dimerization or oligomerization of the ErbB/Neu receptors are necessary but not sufficient for initiation of receptor signaling. The two intracellular domains must be properly oriented for the juxtaposition of the kinase domains allowing trans-phosphorylation. This suggests that the transmembrane (TM) domain acts as a guide for defining the proper orientation of the intracellular domains. Two structural models, with the two helices either in left-handed or in right-handed coiling have been proposed as the TM domain structure of the active receptor. Because experimental data do not distinguish clearly helix-helix packing, molecular dynamics (MD) simulations are used to investigate the energetic factors that drive Neu TM-TM interactions of the wild and the oncogenic receptor (Val664/Glu mutation) in DMPC or in POPC environments. MD results indicate that helix-lipid interactions in the bilayer core are extremely similar in the two environments and raise the role of the juxtamembrane residues in helix insertion and helix-helix packing. The TM domain shows a greater propensity to adopt a left-handed structure in DMPC, with helices in optimal position for strong inter-helical Hbonds induced by the Glu mutation. In POPC, the right-handed structure is preferentially formed with the participation of water in inter-helical Hbonds. The two structural arrangements of the Neu(TM) helices both with GG4 residue motif in close contact at the interface are permissible in the membrane environment. According to the hypothesis of a monomer-dimer equilibrium of the proteins it is likely that the bilayer imposes structural constraints that favor dimerization-competent structure responsible of the proper topology necessary for receptor activation.     

Comparison of proto-oncogenic and mutant forms of the transmembrane region of the Neu receptor in TFE

A single mutation within the transmembrane region of the Neu receptor (Val664-->Glu) is known to enhance tyrosine kinase activity, by promoting receptor dimerization. In order to gain insight into potential structural changes that arise as a result of the mutation, peptides corresponding to the complete transmembrane domain of proto-oncogenic and mutant forms of Neu have been studied by 1H nuclear magnetic resonance in the solvent trifluoroethanol (TFE). The chemical shifts are similar for both forms of the peptide, with the exception of amide residues close to the mutation site. Both peptides adopt a helical conformation, with a distinct bend one turn downstream of the mutation site. This deformation gives rise to several nuclear Overhauser effects, the majority of which were detected in both peptides, that are atypical for a straight canonical alpha-helix. Our data in this solvent do not support a conformational change in the transmembrane domain of monomeric Neu as a result of the mutation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicates that proto-oncogenic Neu peptides have a higher propensity to oligomerize in the solvent TFE than the Glu664 oncogenic form.     

Rational design of a lipase to accommodate catalysis of Baeyer–Villiger oxidation with hydrogen peroxide

The mechanism and potential energy surface for the Baeyer-Villiger oxidation of acetone with hydrogen peroxide catalyzed by a Ser105-Ala mutant of Candida antarctica Lipase B has been determined using ab initio and density functional theories. Initial substrate binding has been studied using an automated docking procedure and molecular dynamics simulations. Substrates were found to bind to the active site of the mutant. The activation energy for the first step of the reaction, the nucleophilic attack of hydrogen peroxide on the carbonyl carbon of hydrogen peroxide, was calculated to be 4.4 kcal x mol(-1) at the B3LYP/6-31+G* level. The second step, involving the migration of the alkyl group, was found to be the rate-determining step with a computed activation energy of 19.9 kcal x mol(-1) relative the reactant complex. Both steps were found to be lowered considerably in the reaction catalyzed by the mutated lipase, compared to the uncatalyzed reaction. The first step was lowered by 36.0 kcal x mol(-1) and the second step by 19.5 kcal x mol(-1). The second step of the reaction, the rearrangement step, has a high barrier of 27.7 kcal x mol(-1) relative to the Criegee intermediate. This could lead to an accumulation of the intermediate. It is not clear whether this result is an artifact of the computational procedure, or an indication that further mutations of the active site are required. Figure Second TS (18TS) in the Baeyer-Villiger oxidation in a mutant of CALB. Distances in A     

Strong dimerization of wild-type ErbB2/Neu transmembrane domain and the oncogenic Val664Glu mutant in mammalian plasma membranes

Here, we study the homodimerization of the transmembrane domain of Neu, as well as an oncogenic mutant (V664E), in vesicles derived from the plasma membrane of mammalian cells. For the characterization, we use a Förster resonance energy transfer (FRET)-based method termed Quantitative Imaging-FRET (QI-FRET), which yields the donor and acceptor concentrations in addition to the FRET efficiencies in individual plasma membrane-derived vesicles. Our results demonstrate that both the wild-type and the mutant are 100% dimeric, suggesting that the Neu TM helix dimerizes more efficiently than other RTK TM domains in mammalian membranes. Furthermore, the data suggest that the V664E mutation causes a very small, but statistically significant change in dimer structure. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.