Conformational changes accompany phosphorylation of the epidermal growth factor receptor C-terminal domain

The precise regulation of epidermal growth factor receptor (EGFR) signaling is crucial to its function in cellular growth control. Various studies have suggested that the C-terminal phosphorylation domain, itself a substrate for the EGFR kinase activity, exerts a regulatory influence upon it, although the molecular mechanism for this regulation is unknown. The fluorescence resonance energy transfer (FRET) technique was employed to examine how C-terminal domain conformational changes in the context of receptor activation and autophosphorylation might regulate EGFR enzymatic activity. A novel FRET reporter system was devised in which recombinant purified EGFR intracellular domain (ICD) proteins of varying C-terminal lengths were site-specifically labeled at their extreme C termini with blue fluorescent protein (BFP) and a fluorescent nucleotide analog, 2'(3')-O-(2,4,6-trinitrophenyl)-adenosine 5'-triphosphate (TNP-ATP), binding at their active sites. This novel BFP/TNP-ATP FRET pair demonstrated efficient energy transfer as evidenced by appreciable BFP-donor quenching by bound TNP-ATP. In particular, a marked reduction in energy transfer was observed for the full-length BFP-labeled EGFR-ICD protein upon phosphorylation, likely reflecting its movement away from the active site. The estimated distances from the BFP module to the TNP-ATP-occupied active site for the full-length and C-terminally truncated proteins also reveal the possible folding geometry of this domain with respect to the kinase core. The present studies demonstrate the first use of BFP/TNP-ATP as a FRET reporter system. Furthermore, the results described here provide biophysical evidence for phosphorylation-dependent conformational changes in the C-terminal phosphorylation domain and its likely interaction with the kinase core.     

Conformational regulation of the EGFR kinase core by the juxtamembrane and C-terminal tail: a molecular dynamics study

The catalytic domain of epidermal growth factor receptor (EGFR) is activated by dimerization, which requires allosteric coupling between distal dimerization and catalytic sites. Although crystal structures of EGFR kinases, solved in various conformational states, have provided important insights into EGFR activation by dimerization, the atomic details of how dimerization signals are dynamically coupled to catalytic regions of the kinase core are not fully understood. In this study, we have performed unrestrained and targeted molecular dynamics simulations on the active and inactive states of EGFR, followed by principal component analysis on the simulated trajectories, to identify correlated motions in the EGFR kinase domain upon dimerization. Our analysis reveals that the conformational changes associated with the catalytic functions of the kinase core are highly correlated with motions in the juxtamembrane (JM) and C-terminal tail, two flexible structural elements that play an active role in EGFR kinase activation and dimerization. In particular, the opening and closing of the ATP binding lobe relative to the substrate binding lobe is highly correlated with motions in the JM and C-terminal tail, suggesting that ATP and substrate binding can be coordinated with dimerization through conformational changes in the JM and C-terminal tail. Our study pinpoints key residues involved in this conformational coupling, and provides new insights into the role of the JM and C-terminal tail segments in EGFR kinase functions.     

Expression of epidermal growth factor receptor sequences as E. coli fusion proteins: applications in the study of tyrosine kinase function

To investigate the functions of key domains of the epidermal growth factor receptor (EGFR), various EGFR-derived peptide sequences were expressed in Escherichia coli as glutathione S-transferase (GST) fusion proteins. The purified fusion proteins (GST-TK0-8) were tested as substrates for the tyrosine kinase activities of the EGFR and c-src. Both the GST-TK4 fusion protein, which contains the major C-terminal tyrosine autophosphorylation sites of the EGFR, and GST-TK7, which contains the connecting sequence between the EGFR kinase domain and the C-terminal autophosphorylation domain, were strongly phosphorylated by the EGFR and c-src. Hence the candidate tyrosine phosphorylation sites present in the connecting sequences of the EGFR, as well as the known autophosphorylation sites of the EGFR, can be phosphorylated by the two tyrosine kinases. The protein GST-TK7 was phosphorylated by c-src with a KM of 5-10 microM, which indicated a potential interaction between the connecting segment of the EGFR and the c-src kinase. The GST fusion proteins were also used to map the sites recognized by two anti-EGFR monoclonal antibodies and a polyclonal serum raised against an EGFR tyrosine kinase domain fragment. The recognition site of one monoclonal antibody was determined to be in a short sequence surrounding tyr1068, a primary site of autophosphorylation in the C-terminal domain of the receptor. The anti-peptide polyclonal serum recognized only sequences in the GST-TK7 fusion protein, and hence binds to the connecting sequence between the kinase core and the C-terminal domain. These antibodies will therefore be useful reagents for studying the function of two key structural elements of the EGFR tyrosine kinase. The GST-TK fusion proteins should have many other applications in the study of EGFR catalysis and mitogenic signalling.     

Phosphorylation of the 27-kDa gap junction protein by protein kinase C in vitro and in rat hepatocytes

We previously demonstrated that the 27-kDa major component protein in rat liver gap junctions was phosphorylated by protein kinase C in vitro (Takeda, A. et al. (1987) FEBS Lett. 210, 169-172). In this study, we examined this further and examined the phosphorylation of the 27-kDa gap junction protein in rat hepatocytes by metabolically labeling cells with [32P]orthophosphate and using a monoclonal antibody to immunoprecipitate the protein. The in vitro phosphorylation was inhibited by monoclonal antibodies recognizing the carboxyl- (C-)terminal domain of the 27-kDa protein. Protease digestion analysis revealed that phosphorylation occurred at the C-terminal domain. In rat hepatocytes, the phorbol esters, 12-O-tetradecanoylphorbol-13-acetate and phorbol-12,13-dibutyrate, stimulated the 27-kDa protein phosphorylation, whereas 4 alpha-phorbol-12,13-didecanoate did not. 1-Oleoyl-2-acetyl-sn-glycerol also stimulated the 27-kDa protein phosphorylation. In addition, norepinephrine stimulated the phosphorylation and pretreatment of hepatocytes with staurosporine, a potent inhibitor of protein kinase C, inhibited this stimulatory effect of norepinephrine. Both in vitro and in hepatocytes, analysis of chemical cleavage of the 27-kDa phosphoprotein revealed that phosphorylation occurred mainly at a 10-kDa fragment which the antibodies recognized. These results indicate that protein kinase C phosphorylates the 27-kDa gap junction protein, not only in vitro but also in hepatocytes, at the C-terminal domain of the protein.     

An allosteric mechanism for activation of the kinase domain of epidermal growth factor receptor

The mechanism by which the epidermal growth factor receptor (EGFR) is activated upon dimerization has eluded definition. We find that the EGFR kinase domain can be activated by increasing its local concentration or by mutating a leucine (L834R) in the activation loop, the phosphorylation of which is not required for activation. This suggests that the kinase domain is intrinsically autoinhibited, and an intermolecular interaction promotes its activation. Using further mutational analysis and crystallography we demonstrate that the autoinhibited conformation of the EGFR kinase domain resembles that of Src and cyclin-dependent kinases (CDKs). EGFR activation results from the formation of an asymmetric dimer in which the C-terminal lobe of one kinase domain plays a role analogous to that of cyclin in activated CDK/cyclin complexes. The CDK/cyclin-like complex formed by two kinase domains thus explains the activation of EGFR-family receptors by homo- or heterodimerization.     

Autophosphorylation of EGFR at Y954 Facilitated Homodimerization and Enhanced Downstream Signals

Asymmetric dimer formation of epidermal growth factor receptor (EGFR) is crucial for EGF-induced receptor activation. Even though autophosphorylation is important for activation, its role remains elusive in the context of regulating dimers. In this study, employing overlapping time series analysis to raster image correlation spectroscopy (RICS), we observed time-dependent transient dynamics of EGFR dimerization and found EGFR kinase activity to be essential for dimerization. As a result of which, we hypothesized that phosphorylation could influence dimerization. Evaluating this point, we observed that one of the tyrosine residues (Y954) located in the C-terminal lobe of the activator kinase domain was important to potentiate dimerization. Functional imaging to monitor Ca²⁺ and ERK signals revealed a significant role of Y954 in influencing downstream signaling cascade. Crucial for stabilization of EGFR asymmetric dimer is a “latch” formed between kinase domains of the binding partners. Because Y954 is positioned adjacent to the latch binding region on the kinase domain, we propose that phosphorylation strengthened the latch interaction. On the contrary, we identified that threonine phosphorylation (T669) in the latch domain negatively regulated EGFR dimerization and the downstream signals. Overall, we have delineated the previously anonymous role of phosphorylation at the latch interface of kinase domains in regulating EGFR dimerization.     

Structural investigations of chromatin core protein by nuclear magnetic resonance

A complex derived from chromatin containing one molecule of each of histones H2A, H2B, H3, and H4, termed core protein, was studied by 13C and 1H nuclear magnetic resonance. 13C line widths, when analyzed and compared with those of native and thermally unfolded representative globular proteins, showed that regions of the core protein possess considerable mobility. Studies of Calpha and Cbeta line widths, and Calpha spin-spin relaxation times, show that this mobility arises from sections of random-coil polypeptide. It is argued that these regions are N-terminal "tails", attached to C-terminal globular polypeptides. The 270-MHz 1H nuclear magnetic resonance spectrum shows numerous ring current shifted resonances, indicating that the C-terminal globular domain has a precise tertiary structure. The globular domain most likely forms the histone "core" of the chromatin monomer particle, whilst the basic tails probably wind around the grooves of the double helix, enabling the basic side chains to interact with the DNA phosphate groups. Some biological implications of this model are considered.     

Protein kinase Cepsilon may act as EGF-inducible scaffold protein for phospholipase Cgamma1

Phospholipase Cgamma1 (PLCgamma1) represents a major downstream signalling component of the epidermal growth factor (EGF) receptor (EGFR) and is activated by tyrosine phosphorylation. Here we show for the first time that cellular knockdown of protein kinase Cepsilon (PKCepsilon) leads to decreased activation of PLCgamma1 by EGF and that EGF induces tyrosine phosphorylation of PKCepsilon as well as association of PKCepsilon with both EGFR and PLCgamma1. Using several mutants, co-immunoprecipitation and phosphopeptide-based pull-down experiments we found that in dependency on c-Src and EGF-stimulation PKCepsilon may bind to the c-Src-specific phosphorylation site pY845-EGFR. Furthermore, we identified a single tyrosine residue, PKCepsilon-Y573, within a consensus binding sequence of the C-terminal SH2 domain of PLCgamma1 which is critical for both tyrosine phosphorylation of PKCepsilon and its association with PLCgamma1. Thus, in particular cells and independent of the kinase activity PKCepsilon may form a signalling module with EGFR and PLCgamma1. Thereby the tyrosine phosphorylation of PLCgamma1 via the EGFR may be facilitated. This is a novel function of PKCepsilon upstream of PLCgamma1 and a novel paradigm for the EGF-induced formation of multi-protein complexes.     

Phosphorylation of the Core Protein of Hepatitis B Virus by a 46-Kilodalton Serine Kinase

Core protein is the major component of the core particle (nucleocapsid) of human hepatitis B virus. Core particles and core proteins are involved in a number of important functions in the replication cycle of the virus, including RNA packaging, DNA synthesis, and recognition of viral envelope proteins. Core protein is a phosphoprotein with most, if not all, of the phosphorylation on C-terminal serine residues. In this study, we identified a serine kinase activity from the ribosome-associated protein fraction of cytoplasm that could specifically bind and phosphorylate the C-terminal portion of recombinant core protein. This kinase is referred to as core-associated kinase (CAK). CAK could be inhibited by the kinase inhibitors heparin and manganese ions but not by spermidine, DRB, H89, or H7, indicating that CAK is distinct from protein kinase A and protein kinase C. CAK could be partially purified by heparin-Sepharose CL-6B and phosphocellulose P11 columns. By using a far-Western assay, three specific proteins, of 46, 35, and 13 kDa, were shown to interact with the C-terminal part of the core protein. These three proteins were present only in the eluted fractions that contains the CAK activity. An in-gel kinase assay showed that a 46-kDa kinase in the same fraction could bind and phosphorylate the C-terminal part of the recombinant core protein. These results indicate that this 46-kDa kinase is most probably CAK. A similar 46-kDa kinase, which exhibits the same profile of sensitivity to kinase inhibitors as that of CAK, is present in both purified intracellular core particles and extracellular 42-nm virions, suggesting that CAK is a candidate for the core particle-associated kinase.     

Backbone dynamics of calmodulin studied by 15N relaxation using inverse detected two-dimensional NMR spectroscopy: the central helix is flexible.

The backbone dynamics of Ca(2+)-saturated recombinant Drosophila calmodulin has been studied by 15N longitudinal and transverse relaxation experiments, combined with 15N(1H) NOE measurements. Results indicate a high degree of mobility near the middle of the central helix of calmodulin, from residue K77 through S81, with order parameters (S2) in the 0.5-0.6 range. The anisotropy observed in the motion of the two globular calmodulin domains is much smaller than expected on the basis of hydrodynamic calculations for a rigid dumbbell type structure. This indicates that, for the purposes of 15N relaxation, the tumbling of the N-terminal (L4-K77) and C-terminal (E82-S147) lobes of calmodulin is effectively independent. A slightly shorter motional correlation time (tau c approximately 6.3 ns) is obtained for the C-terminal domain compared to the N-terminal domain (tau c approximately 7.1 ns), in agreement with the smaller size of the C-terminal domain. A high degree of mobility, with order parameters of approximately 0.5, is also observed in the loop that connects the first with the second EF-hand type calcium binding domain and in the loop connecting the third and fourth calcium binding domain.     

Molecular modeling of the chromatosome particle

In an effort to understand the role of the linker histone in chromatin folding, its structure and location in the nucleosome has been studied by molecular modeling methods. The structure of the globular domain of the rat histone H1d, a highly conserved part of the linker histone, built by homology modeling methods, revealed a three-helical bundle fold that could be described as a helix–turn–helix variant with its characteristic properties of binding to DNA at the major groove. Using the information of its preferential binding to four-way Holliday junction (HJ) DNA, a model of the domain complexed to HJ was built, which was subsequently used to position the globular domain onto the nucleosome. The model revealed that the primary binding site of the domain interacts with the extra 20 bp of DNA of the entering duplex at the major groove while the secondary binding site interacts with the minor groove of the central gyre of the DNA superhelix of the nucleosomal core. The positioning of the globular domain served as an anchor to locate the C-terminal domain onto the nucleosome to obtain the structure of the chromatosome particle. The resulting structure had a stem-like appearance, resembling that observed by electron microscopic studies. The C-terminal domain which adopts a high mobility group (HMG)-box-like fold, has the ability to bend DNA, causing DNA condensation or compaction. It was observed that the three S/TPKK motifs in the C-terminal domain interact with the exiting duplex, thus defining the path of linker DNA in the chromatin fiber. This study has provided an insight into the probable individual roles of globular and the C-terminal domains of histone H1 in chromatin organization.     

Rotational dynamics of the single tryptophan of porcine pancreatic phospholipase A2, its zymogen, and an enzyme/micelle complex. A steady-state and time-resolved anisotropy study

The rotational dynamics of the single tryptophan of porcine pancreatic phospholipase A2 and its zymogen (prophospholipase A2) have been studied by polarized fluorescence using steady-state and time-resolved single-photon counting techniques. The motion of Trp-3 in phospholipase A2 consists of a rapid subnanosecond wobble of the indole ring with an amplitude of about +/- 20 degrees accompanied by slower isotropic rotation of the entire protein. The rotational correlation times for overall particle rotational diffusion are consistent with conventional hydrodynamic theory. When phospholipase A2 binds to micelles of n-hexadecylphosphocholine, the amplitude of the fast ring rotation decreases. The whole particle rotational correlation time of the enzyme/micelle complex is smaller than the minimum value calculated from hydrodynamic theory. A similar result is obtained for the micelle itself by using the lipophilic probe transparinaric acid. These low values for the particle correlation times can be understood by postulating that an isotropic motion of the fluorophore in the small detergent particles contributes to the angular reorientation of the fluorophore. The internal reorientational motion of the tryptophan in the zymogen, prophospholipase A2, is of larger amplitude than that observed for the enzyme; specifically, the proenzyme exhibits a motion with a significant amplitude on the nanosecond time scale. This additional freedom of motion is attributed to segmental mobility of the N-terminal residues of prophospholipase A2. This demonstrates that this region of the protein is flexible in the zymogen but not in the processed enzyme. The implications of these findings for the mechanism of surface activation of phospholipase A2 are discussed by analogy with a trypsinogen-trypsin activation model.     

Phosphorylation of high-Mr caldesmon by protein kinase C modulates the regulatory function of this protein on the interaction between actin and myosin

High-Mr caldesmon, which is involved in smooth muscle contraction, was phosphorylated by protein kinase C. By chymotryptic digestion, actin- and calmodulin-binding assays and immunoprecipitation with the antibody to the C-terminal 35-kDa fragment, we have identified that all phosphate groups are incorporated exclusively into this fragment, which is the functional domain for binding actin and calmodulin. Phosphorylation of high-Mr caldesmon and its C-terminal 35-kDa fragment reduced their binding abilities to both F-actin and calmodulin. Further, their inhibitory effects on the actin-activated ATPase activity of gizzard myosin were also reversed in proportion to the degree of phosphorylation. These results suggest that phosphorylation of high-Mr caldesmon by protein kinase C, which is restricted within the C-terminal 35-kDa domain, results in the modulation of its activity in the smooth muscle actin--myosin interaction.     

Canstatin inhibits Akt activation and induces Fas-dependent apoptosis in endothelial cells

Canstatin, a 24-kDa peptide derived from the C-terminal globular non-collagenous (NC1) domain of the alpha2 chain of type IV collagen, was previously shown to induce apoptosis in cultured endothelial cells and to inhibit angiogenesis in vitro and in vivo. In this report, we demonstrate that canstatin inhibits the phosphorylation of Akt, focal adhesion kinase, mammalian target of rapamycin, eukaryotic initiation factor-4E-binding protein-1, and ribosomal S6 kinase in cultured human umbilical vein endothelial cells. It also induces Fas ligand expression, activates procaspases 8 and 9 cleavage, reduces mitochondrial membrane potential, and increases cell death (as determined by propidium iodide staining). Canstatin-induced activation of procaspases 8 and 9 as well as the induced reduction in mitochondrial membrane potential and cell viability were attenuated by the forced expression of FLICE-inhibitory protein. Canstatin-induced procaspase 8 activation and cell death were also inhibited by a neutralizing anti-Fas antibody. Collectively, these data indicate that canstatin-induced apoptosis is associated with phosphatidylinositol 3-kinase/Akt inhibition and is dependent upon signaling events transduced through membrane death receptors.     

The 3D solution structure of the C-terminal region of Ku86 (Ku86CTR)

In eukaryotes the non-homologous end-joining repair of double strand breaks in DNA is executed by a series of proteins that bring about the synapsis, preparation and ligation of the broken DNA ends. The mechanism of this process appears to be initiated by the obligate heterodimer (Ku70/Ku86) protein complex Ku that has affinity for DNA ends. Ku then recruits the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). The three-dimensional structures of the major part of the Ku heterodimer, representing the DNA-binding core, both free and bound to DNA are known from X-ray crystallography. However, these structures lack a region of ca 190 residues from the C-terminal region (CTR) of the Ku86 subunit (also known as Lupus Ku autoantigen p86, Ku80, or XRCC5) that includes the extreme C-terminal tail that is reported to be sufficient for DNA-PKcs-binding. We have examined the structural characteristics of the Ku86CTR protein expressed in bacteria. By deletion mutagenesis and heteronuclear NMR spectroscopy we localised a globular domain consisting of residues 592-709. Constructs comprising additional residues either to the N-terminal side (residues 543-709), or the C-terminal side (residues 592-732), which includes the putative DNA-PKcs-binding motif, yielded NMR spectra consistent with these extra regions lacking ordered structure. The three-dimensional solution structure of the core globular domain of the C-terminal region of Ku86 (Ku86CTR(592-709)) has been determined using heteronuclear NMR spectroscopy and dynamical simulated annealing using structural restraints from nuclear Overhauser effect spectroscopy, and scalar and residual dipolar couplings. The polypeptide fold comprises six regions of alpha-helical secondary structure that has an overall superhelical topology remotely homologous to the MIF4G homology domain of the human nuclear cap binding protein 80 kDa subunit and the VHS domain of the Drosophila protein Hrs, though strict analysis of the structures suggests that these domains are not functionally related. Two prominent hydrophobic pockets in the gap between helices alpha2 and alpha4 suggest a potential ligand-binding characteristic for this globular domain.     

Identification of SRPK1 and SRPK2 as the major cellular protein kinases phosphorylating hepatitis B virus core protein

Phosphorylation of hepatitis B virus (HBV) core protein has recently been shown to be a prerequisite for pregenomic RNA encapsidation into viral capsids, but the host cell kinases mediating this essential step of the HBV replication cycle have not been identified. We detected two kinases of 95 and 115 kDa in HuH-7 total cell lysates which interacted specifically with the HBV core protein and phosphorylated its arginine-rich C-terminal domain. The 95-kDa kinase was purified and characterized as SR protein-specific kinase 1 (SRPK1) by mass spectrometry. Based on this finding, the 115-kDa kinase could be identified as the related kinase SRPK2 by immunoblot analysis. In vitro, both SRPKs phosphorylated HBV core protein on the same serine residues which are found to be phosphorylated in vivo. Moreover, the major cellular HBV core kinase activity detected in the total cell lysate showed biochemical properties identical to those of SRPK1 and SRPK2, as examined by measuring binding to a panel of chromatography media. We also clearly demonstrate that neither the cyclin-dependent kinases Cdc2 and Cdk2 nor protein kinase C, previously implicated in HBV core protein phosphorylation, can account for the HBV core protein kinase activity. We conclude that both SRPK1 and SRPK2 are most likely the cellular protein kinases mediating HBV core protein phosphorylation during viral infection and therefore represent important host cell targets for therapeutic intervention in HBV infection.     

Coarse-Grained Molecular Simulation of Epidermal Growth Factor Receptor Protein Tyrosine Kinase Multi-Site Self-Phosphorylation

Upon the ligand-dependent dimerization of the epidermal growth factor receptor (EGFR), the intrinsic protein tyrosine kinase (PTK) activity of one receptor monomer is activated, and the dimeric receptor undergoes self-phosphorylation at any of eight candidate phosphorylation sites (P-sites) in either of the two C-terminal (CT) domains. While the structures of the extracellular ligand binding and intracellular PTK domains are known, that of the ∼225-amino acid CT domain is not, presumably because it is disordered. Receptor phosphorylation on CT domain P-sites is critical in signaling because of the binding of specific signaling effector molecules to individual phosphorylated P-sites. To investigate how the combination of conventional substrate recognition and the unique topological factors involved in the CT domain self-phosphorylation reaction lead to selectivity in P-site phosphorylation, we performed coarse-grained molecular simulations of the P-site/catalytic site binding reactions that precede EGFR self-phosphorylation events. Our results indicate that self-phosphorylation of the dimeric EGFR, although generally believed to occur in trans, may well occur with a similar efficiency in cis, with the P-sites of both receptor monomers being phosphorylated to a similar extent. An exception was the case of the most kinase-proximal P-site-992, the catalytic site binding of which occurred exclusively in cis via an intramolecular reaction. We discovered that the in cis interaction of P-site-992 with the catalytic site was facilitated by a cleft between the N-terminal and C-terminal lobes of the PTK domain that allows the short CT domain sequence tethering P-site-992 to the PTK core to reach the catalytic site. Our work provides several new mechanistic insights into the EGFR self-phosphorylation reaction, and demonstrates the potential of coarse-grained molecular simulation approaches for investigating the complexities of self-phosphorylation in molecules such as EGFR (HER/ErbB) family receptors and growth factor receptor PTKs in general.     

Fluorescence spectroscopy as a probe of the effect of phosphorylation at serine 40 of tyrosine hydroxylase on the conformation of its regulatory domain

Phosphorylation of Ser40 in the regulatory domain of tyrosine hydroxylase activates the enzyme by increasing the rate constant for dissociation of inhibitory catecholamines from the active site by 3 orders of magnitude. To probe the changes in the structure of the N-terminal domain upon phosphorylation, individual phenylalanine residues at positions 14, 34, and 74 were replaced with tryptophan in a form of the protein in which the endogenous tryptophans had all been mutated to phenylalanine (W(3)F TyrH). The steady-state fluorescence anisotropy of F74W W(3)F TyrH was unaffected by phosphorylation, but the anisotropies of both F14W and F34W W(3)F TyrH increased significantly upon phosphorylation. The fluorescence of the single tryptophan residue at position 74 was less readily quenched by acrylamide than those at the other two positions; fluorescence increased the rate constant for quenching of the residues at positions 14 and 34 but did not affect that for the residue at position 74. Frequency domain analyses were consistent with phosphorylation having no effect on the amplitude of the rotational motion of the indole ring at position 74, resulting in a small increase in the rotational motion of the residue at position 14 and resulting in a larger increase in the rotational motion of the residue at position 34. These results are consistent with the local environment at position 74 being unaffected by phosphorylation, that at position 34 becoming much more flexible upon phosphorylation, and that at position 14 becoming slightly more flexible upon phosphorylation. The results support a model in which phosphorylation at Ser40 at the N-terminus of the regulatory domain causes a conformational change to a more open conformation in which the N-terminus of the protein no longer inhibits dissociation of a bound catecholamine from the active site.     

Solution structure of the dimeric SAM domain of MAPKKK Ste11 and its interactions with the adaptor protein Ste50 from the budding yeast: implications for Ste11 activation and signal transmission through the Ste50-Ste11 complex

Ste11, a homologue of mammalian MAPKKKs, together with its binding partner Ste50 works in a number of MAPK signaling pathways of Saccharomyces cerevisiae. Ste11/Ste50 binding is mediated by their sterile alpha motifs or SAM domains, of which homologues are also found in many other intracellular signaling and regulatory proteins. Here, we present the solution structure of the SAM domain or residues D37-R104 of Ste11 and its interactions with the cognate SAM domain-containing region of Ste50, residues M27-Q131. NMR pulse-field-gradient (PFG) and rotational correlation time measurements (tauc) establish that the Ste11 SAM domain exists predominantly as a symmetric dimer in solution. The solution structure of the dimeric Ste11 SAM domain consists of five well-defined helices per monomer packed into a compact globular structure. The dimeric structure of the SAM domain is maintained by a novel dimer interface involving interactions between a number of hydrophobic residues situated on helix 4 and at the beginning of the C-terminal long helix (helix 5). The dimer structure may also be stabilized by potential salt bridge interactions across the interface. NMR H/2H exchange experiments showed that binding of the Ste50 SAM to the Ste11 SAM very likely involves the positively charged extreme C-terminal region as well as exposed hydrophobic patches of the dimeric Ste11 SAM domain. The dimeric structure of the Ste11 SAM and its interactions with the Ste50 SAM may have important roles in the regulation and activation of the Ste11 kinase and signal transmission and amplifications through the Ste50-Ste11 complex.     

Dynamics of the core tryptophan during the formation of a productive molten globule intermediate of barstar

The evolution of the nanosecond dynamics of the core tryptophan, Trp53, of barstar has been monitored during the induction of collapse and structure formation in the denatured D form at pH 12, by addition of increasing concentrations of the stabilizing salt Na(2)SO(4). Time-resolved fluorescence methods have been used to monitor the dynamics of Trp53 in the intermediates that are populated during the salt-induced transition of the D form to the molten globule B form. The D form approximates a random coil and displays two rotational correlation times. A long rotational correlation time of 2.54 ns originates from segmental mobility, and a short correlation time of 0.26 ns originates from independent motion of the tryptophan side chain. Upon addition of approximately 0.1 M Na(2)SO(4), the long rotational correlation time increases to approximately 6.4 ns, as the chain collapses and the segmental motions merge to reflect the global tumbling motion of a pre-molten globule P form. The P form exists as an expanded form with approximately 30% greater volume than the native (N) state. The persistence of an approximately 50% contribution to anisotropy decay by the short rotational correlation time suggests that the core of the P form is highly molten and permits free rotation of the Trp side chain. With increasing salt concentrations, tight core packing is achieved before secondary and tertiary structure formation is complete, an observation which agrees well with earlier kinetic folding studies. Thus, the equilibrium model developed here for describing the formation of structure during folding faithfully captures snapshots of transient kinetic intermediates observed on the folding pathway of barstar. A comparison of the refolding kinetics at pH 7, when refolding is initiated from the D, P, and B forms, suggests that formation of a collapsed state with a rigid core and approximately 30% secondary and tertiary structure, which presumably defines a coarse native-like topology, constitutes the intrinsic barrier in the folding of barstar.