FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1973603507> ?p ?o ?g. }

• W1973603507 endingPage "13029" @default.
• W1973603507 startingPage "13022" @default.
• W1973603507 abstract "Syndecan-4, a transmembrane heparan sulfate proteoglycan, is a coreceptor with integrins in cell adhesion. It has been suggested to form a ternary signaling complex with protein kinase Cα and phosphatidylinositol 4,5-bisphosphate (PIP2). Syndecans each have a unique, central, and variable (V) region in their cytoplasmic domains, and that of syndecan-4 is critical to its interaction with protein kinase C and PIP2. Two oligopeptides corresponding to the variable region (4V) and whole domain (4L) of syndecan-4 cytoplasmic domain were synthesized for nuclear magnetic resonance (NMR) studies. Data from NMR and circular dichroism indicate that the cytoplasmic domain undergoes a conformational transition and forms a symmetric dimer in the presence of phospholipid activator PIP2. The solution conformations of both free and PIP2-complexed 4V have been determined by two-dimensional NMR spectroscopy and dynamical simulated annealing calculations. The 4V peptide in the presence of PIP2 formed a compact dimer with two twisted strands packed parallel to each other and the exposed surface of the dimer consisted of highly charged and polar residues. The overall three-dimensional structure in solution exhibits a twisted clamp shape having a cavity in the center of dimeric interface. In addition, it has been observed that the syndecan-4V strongly interacts not only with fatty acyl groups but also the anionic head group of PIP2. These findings reveal that PIP2 promotes oligomerization of syndecan-4 cytoplasmic domain for transmembrane signaling and cell-matrix adhesion. Syndecan-4, a transmembrane heparan sulfate proteoglycan, is a coreceptor with integrins in cell adhesion. It has been suggested to form a ternary signaling complex with protein kinase Cα and phosphatidylinositol 4,5-bisphosphate (PIP2). Syndecans each have a unique, central, and variable (V) region in their cytoplasmic domains, and that of syndecan-4 is critical to its interaction with protein kinase C and PIP2. Two oligopeptides corresponding to the variable region (4V) and whole domain (4L) of syndecan-4 cytoplasmic domain were synthesized for nuclear magnetic resonance (NMR) studies. Data from NMR and circular dichroism indicate that the cytoplasmic domain undergoes a conformational transition and forms a symmetric dimer in the presence of phospholipid activator PIP2. The solution conformations of both free and PIP2-complexed 4V have been determined by two-dimensional NMR spectroscopy and dynamical simulated annealing calculations. The 4V peptide in the presence of PIP2 formed a compact dimer with two twisted strands packed parallel to each other and the exposed surface of the dimer consisted of highly charged and polar residues. The overall three-dimensional structure in solution exhibits a twisted clamp shape having a cavity in the center of dimeric interface. In addition, it has been observed that the syndecan-4V strongly interacts not only with fatty acyl groups but also the anionic head group of PIP2. These findings reveal that PIP2 promotes oligomerization of syndecan-4 cytoplasmic domain for transmembrane signaling and cell-matrix adhesion. Cell adhesion mediated by cell surface receptors triggers signal transduction cascades. Integrins, which are well characterized transmembrane receptors, are known to be involved in several cell regulatory mechanism including cell proliferation, morphology, and motility. During cell-matrix interaction, integrin clustering activates tyrosine kinases (1Miyamoto S. Teramoto H. Coso O.A. Gutkind J.S. Burbelo D.D. Akiyama S.K. Yamada K.M. J. Cell Biol. 1995; 131: 791-805Crossref PubMed Scopus (1106) Google Scholar, 2Clark E.A. Brugge J.S. Science. 1995; 268: 233-239Crossref PubMed Scopus (2822) Google Scholar, 3Burridge K. Chrzanowska-Wodnicka M. Annu. Rev. Cell. Dev. Biol. 1996; 12: 463-518Crossref PubMed Scopus (1663) Google Scholar). Adhesion of several cell types to extracellular matrix molecules such as fibronectin involves both integrin- and heparin-binding domains, where assays were performed in the absence of serum and protein synthesis (3Burridge K. Chrzanowska-Wodnicka M. Annu. Rev. Cell. Dev. Biol. 1996; 12: 463-518Crossref PubMed Scopus (1663) Google Scholar, 4Burridge K. Turner C.E. Romer L.H. J. Cell Biol. 1992; 119: 893-903Crossref PubMed Scopus (1185) Google Scholar, 5Beyth R.C. Culp L.A. Exp. Cell Res. 1984; 155: 537-548Crossref PubMed Scopus (32) Google Scholar, 6Streeter H.B. Rees D.A. J. Cell Biol. 1987; 105: 507-515Crossref PubMed Scopus (67) Google Scholar, 7Woods A. Couchman J.R. J. Cell Sci. 1992; 101: 277-290Crossref PubMed Google Scholar, 8Woods A. Couchman J.R. Adv. Exp. Med. Biol. 1992; 313: 87-96Crossref PubMed Google Scholar, 9Woods A. Couchman J.R. Johansson S. Hook M. EMBO J. 1986; 5: 665-670Crossref PubMed Scopus (322) Google Scholar, 10Woods A. McCarthy J.B. Furcht L.T. Couchman J.R. Mol. Biol. Cell. 1993; 4: 605-613Crossref PubMed Scopus (182) Google Scholar, 11Huhtala P. Humphries M.J. McCarthy J.B. Tremble P.M. Werb Z. Damsky C.H. J. Cell Biol. 1995; 129: 867-879Crossref PubMed Scopus (369) Google Scholar). The specific interactions of the cytoplasmic domain of each integrin member are critical to trigger the downstream signal cascades. Although the functions of these interactions are not clear, some kinases such as focal adhesion kinase and integrin-linked kinase, and structural proteins such as α-actinin, talin, and filamin, are known to interact with the cytoplasmic domain of β1integrin subunit. The four mammalian syndecans are type I glycoproteins that form dimers or higher order multimers. Oligomerization apparently involves their highly homologous transmembrane domains as well as regions of their cytoplasmic domains (12Oh E.-S. Woods A. Couchman J.R. J. Biol. Chem. 1997; 272: 11805-11811Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 13Oh E.-S. Woods A. Couchman J.R. J. Biol. Chem. 1997; 272: 8133-8136Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, 14Carey D.J. Biochem. J. 1997; 327: 1-16Crossref PubMed Scopus (606) Google Scholar). The central region of each syndecan cytoplasmic domain is unique, and has been termed the variable (V) 1The abbreviations used are: V, variable; C, constant; PIP2, phosphatidylinositol 4,5-bisphosphate; PKC, protein kinase C; NOESY, nuclear Overhauser effect spectroscopy; NOE, nuclear Overhauser effect; TOCSY, total correlation spectroscopy; DQF, double quantum filtered; COSY, correlated spectroscopy; SA, simulated annealing; cPKC, classical PKC. region, flanked N- and C-terminally by constant (C1 and C2) regions (12Oh E.-S. Woods A. Couchman J.R. J. Biol. Chem. 1997; 272: 11805-11811Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 13Oh E.-S. Woods A. Couchman J.R. J. Biol. Chem. 1997; 272: 8133-8136Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). Syndecan-4 is the only family member that is a widespread focal adhesion component, potentially functioning as a coreceptor in integrin-mediated adhesion. It readily forms oligomers that may be required for its function (12Oh E.-S. Woods A. Couchman J.R. J. Biol. Chem. 1997; 272: 11805-11811Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 13Oh E.-S. Woods A. Couchman J.R. J. Biol. Chem. 1997; 272: 8133-8136Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). Phosphatidylinositol 4,5-bisphosphate (PIP2) plays important roles in signal transduction, since PIP2 is hydrolyzed by phospholipase Cγ to generate two intracellular messengers: inositol 1,4,5-triphosphate, which mobilizes Ca2+, and diacylglycerol, which is a physiological activator of protein kinase C (PKC) (15Asaoka Y. Nakamura S.-I. Yoshida K. Nishizuka Y. Trends Biochem. Sci. 1992; 17: 414-417Abstract Full Text PDF PubMed Scopus (365) Google Scholar, 16Hunter T. Karin M. Cell. 1992; 70: 375-387Abstract Full Text PDF PubMed Scopus (1120) Google Scholar, 17Liou H.-C. Baltimore D. Curr. Opin. Cell Biol. 1993; 5: 477-487Crossref PubMed Scopus (518) Google Scholar, 18Nishizuka Y. Science. 1992; 258: 607-614Crossref PubMed Scopus (4231) Google Scholar, 19Sternweis P.C. Smrcka A.V. Trends Biochem. Sci. 1992; 17: 502-506Abstract Full Text PDF PubMed Scopus (175) Google Scholar). However, in addition to its function as precursor for other second messenger, PIP2may also act as a second messenger by directly or indirectly regulating several proteins including PKC, phospholipase Cδ, phospholipase Cγ, G protein-coupled receptor kinase, and phosphatidylinositol 3-kinase. PIP2 binds the cytoskeletal proteins gelsolin, profilin, and vinculin and regulates their interaction with actin (20Sun H. Lin K. Yin H.L. J. Cell Biol. 1997; 138: 811-820Crossref PubMed Scopus (85) Google Scholar, 21Flanagan L.A. Cunninghanm C.C. Chen J. Prestwich G.D. Kosik K.S. Jaumey P.A. Biophys. J. 1997; 73: 1440-1447Abstract Full Text PDF PubMed Scopus (75) Google Scholar, 22Sakisaka T. Itoh T. Miura K. Takenawa T. Mol. Cell. Biol. 1997; 17: 3841-3849Crossref PubMed Scopus (147) Google Scholar, 23De Corte V. Gettemans J. Vandekerckhove J. FEBS Lett. 1997; 410: 191-196Crossref PubMed Scopus (74) Google Scholar). Recently, some β1integrin cytoplasmic domains have been demonstrated to affect phosphatidylinositol 4,5-kinase, possibly regulating PIP2synthesis in vivo (24Hinchlitte K.A. Irvine R.F. Divecha N. EMBO J. 1996; 15: 6516-6524Crossref PubMed Scopus (63) Google Scholar, 25Gironcel D. Racaud-Sultan C. Dayrastre B. Haricot M. Borchert G. Kieffer N. Breton M. Chap H. FEBS Lett. 1996; 389: 253-256Crossref PubMed Scopus (29) Google Scholar). Indeed, it is known that PIP2 is accumulated in the cytoplasmic face of plasma membrane during cell adhesion and it may be regulated by G-proteins such as Rac and Rho, which may activate phosphatidylinositol 4,5-kinase (26Tolias K.F. Cantley L.C. Carpenter C.L. J. Biol. Chem. 1995; 270: 17656-17659Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar, 27Ren X.-D. Bokoch G.M. Traynor-Kaplan A. Jenkins G.H. Anderson R.A. Schwartz M.A. Mol. Biol. Cell. 1996; 7: 435-442Crossref PubMed Scopus (199) Google Scholar). PIP2 is also known to be involved in focal adhesion and stress fiber formation (3Burridge K. Chrzanowska-Wodnicka M. Annu. Rev. Cell. Dev. Biol. 1996; 12: 463-518Crossref PubMed Scopus (1663) Google Scholar), probably through its interactions with focal adhesion components such as vinculin, α-actinin, and syndecan-4. Focal adhesion formation is dependent not only on integrins, but syndecans (most likely syndecan-4), which may act as co-receptors (2Clark E.A. Brugge J.S. Science. 1995; 268: 233-239Crossref PubMed Scopus (2822) Google Scholar,8Woods A. Couchman J.R. Adv. Exp. Med. Biol. 1992; 313: 87-96Crossref PubMed Google Scholar, 9Woods A. Couchman J.R. Johansson S. Hook M. EMBO J. 1986; 5: 665-670Crossref PubMed Scopus (322) Google Scholar). It has been previously shown that syndecan and PKC are involved in the focal adhesion formation, and syndecan-4 regulates the distribution and activity of classical PKC (cPKC). In addition, it has been suggested the interaction of syndecan-4 cytoplasmic domain and cPKC depends on cPKC activity. Recently, we have reported that PIP2 interacts specifically with the cytoplasmic domain of syndecan-4, and together with PIP2, syndecan-4 cytoplasmic domain potentiated PKC activity (28Oh E.S. Woods A. Lim S.-T. Theibert A.W. Couchman J.R. J. Biol. Chem. 1998; 273: 10624-10629Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). It was also shown that PIP2 regulates the interaction of the cytoplasmic domain of syndecan-4 with PKCα and potentiation activity of syndecan-4 cytoplasmic domain on PKCα (28Oh E.S. Woods A. Lim S.-T. Theibert A.W. Couchman J.R. J. Biol. Chem. 1998; 273: 10624-10629Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). As we suggested previously, syndecan-4 and PIP2 together with PKC may form a ternary complex. PKC is activated through this complex, which may then phosphorylate cytoplasmic proteins involved in focal adhesion formation. Here, we present the solution structures of syndecan-4 cytoplasmic core domain both with and without activator PIP2 by two-dimensional nuclear magnetic resonance spectroscopy and dynamical simulated annealing calculations. Peptides of both the whole domain with RMKKKDEGSYDLGKKPIYKKAPTNEFYA sequence corresponding to residues 170–197 (4L) and the variable region with LGKKPIYKKA corresponding to residues 181–190 (4V) of the chicken syndecan-4 cytoplasmic domain were synthesized using an improved version of the solid phase method on a model 431A peptide synthesizer (Applied Biosystems Inc.). The peptide sequences are identical to those of human and rat syndecan-4 cytoplasmic domain. Peptides were purified by reversed-phase liquid chromatography using a Vydac 218TP152050 C18 column on a Waters Delta Prep 4000 system. Purification was achieved by equilibrating the column with 0.1% trifluoroacetic acid in water and developing with a linear gradient of acetonitrile. The free peptide samples for NMR measurements were prepared by dissolving in 90% H2O, 10% D2O or 99.9% D2O solution at pH value of 7.4 with 50 mmsodium phosphate buffer. The final peptide concentration was 2–4 mm in 0.5 ml of buffer solution. PIP2 was purchased from Sigma. For NMR measurements of PIP2-peptide complex, 10–50 μl of concentrated PIP2 solution was titrated to free peptide up to a maximum ratio of 1:1 (PIP2:peptide). The final pH value of 7.0 was adjusted by adding 0.1 n HCl solution before NMR experiments. CD data were collected on a Jasco J-715 spectropolarimeter using 1.0-mm path length cells with scan speed of 500 mm/min, 1-nm bandwidth, and 32 accumulations. Both free and PIP2-complexed samples of 4L and 4V peptides were prepared as volumes of 8 μm in H2O solution, and CD data were collected for temperatures ranges of 20–70 °C. A standard noise reduction procedure was used to prepare final spectrum. All NMR experiments were performed on a Bruker DMX600 spectrometer in quadrature detection mode equipped with an SGI INDY computer. All data were collected at either 10 °C or 20 °C, and the strong solvent resonance was suppressed by water-gated pulse sequence combined with pulsed-field gradient pulses. The temperature was calibrated using a methanol standard (29Van Geet A.L. Anal. Chem. 1970; 42: 679-680Crossref Scopus (944) Google Scholar, 30Bax A. Davis D.G. J. Magn. Reson. 1985; 65: 355-360Google Scholar). A series of one-dimensional NMR spectra were recorded for both 4V and 4L peptide with concentration ranges of 0–2 mm PIP2. All of the two-dimensional NMR measurements were performed on both free and peptide-PIP2 (2:1) samples based on results from PIP2 titration analysis. Mixing times of 100, 200, 300, 400, 500, and 800 ms were used in collecting NOE spectra for both free and PIP2 complex samples. Total correlation spectroscopy (TOCSY) data were also recorded in both H2O and D2O solutions with a mixing time of 78 ms using MLEV17 spin lock pulses(30). Double quantum-filtered (DQF) COSY spectra (31Rance M. Soerensen O.W. Bodehausen G. Wagner G. Ernst R.R. Wuthrich K. Biochem. Biophys. Res. Commun. 1983; 117: 479-485Crossref PubMed Scopus (2596) Google Scholar) were collected in H2O solution. All data were recorded in the phase-sensitive mode using the time proportional phase incrementation method (32Wuthrich K. Billeter M. Braun W. J. Mol. Biol. 1983; 169: 949-961Crossref PubMed Scopus (1007) Google Scholar) with 2048 data points in the acquisition domain and 512 or 256 in the time domain. Two-dimensional NOESY (33Macura S. Ernst R.R. Mol. Phys. 1980; 41: 95Crossref Scopus (1583) Google Scholar) experiments were also performed to identify slowly exchanging amide hydrogens on a freshly prepared D2O solution after lyophilization of an H2O sample. Small flexible linear peptides such as 4V have relatively short correlation times compared with globular proteins. As a result, longer mixing times or rotating frame Overhauser effect spin lock times are typically needed before NOE or rotating frame Overhauser effect cross-peaks begin to build up in the spectrum. From the NOESY growth curve, we have ascertained that spin-diffusion effects are not significant even for 600–800-ms mixing times. The interesting cross-peaks observed in the NOESY spectra are due to direct effects since no other cross-peaks from protons that could mediate the intensity through spin diffusion were observed in the spectra. Long mixing times in the range of 400–800 ms have typically been used for small peptides. All NMR data were processed using nmrPipe (Biosym/Molecular Simulations Inc.) or Bruker XWIN-NMR (Bruker Instruments) software on SGI Indigo2 workstation. Prior to Fourier transformation in thet 1 dimension (34Otting G. Widmer H. Wagner G. Wuthrich K. Magn. Reson. 1988; 66: 187-193Google Scholar), the first row was half-weighted to suppress t 1 ridges. The DQF-COSY data were processed to 8192 × 1024 data matrices to obtain a maximum digital resolution for coupling constant measurements. The proton chemical shifts were referenced with internal sodium 4,4-dimethyl-4-silapentane 1-sulfonate. All calculations were performed for syndecan-4V using XPLOR 3.1 (Biosym/Molecular Simulations, Inc) on an SGI Indigo2 workstation using the topology and parameter files of topallhdg.pro and parallhdg.pro. Monomer 4V structures were generated with random backbone dihedral angles and used as starting structures for simulated annealing (SA) calculations (35Nilges M. Proteins. 1993; 17: 297-309Crossref PubMed Scopus (308) Google Scholar, 36Nilges M. Clore G.M. Gronenborn A.M. FEBS Lett. 1988; 229: 317-324Crossref PubMed Scopus (772) Google Scholar, 37Nilges M. Clore G.M. Gronenborn A.M. FEBS Lett. 1988; 239: 129-136Crossref PubMed Scopus (523) Google Scholar, 38Nilges M. Gronenborn A.M. Brunger A.T. Clore G.M. Protein Eng. 1988; 2: 27-38Crossref PubMed Scopus (516) Google Scholar). The procedure by Nilges (35Nilges M. Proteins. 1993; 17: 297-309Crossref PubMed Scopus (308) Google Scholar) was also used for symmetric dimer generations with minor modifications described by Leeet al. (39Lee W. Harvey T.S. Yin Y. Yau P. Litchfield D. Arrowsmith C.H. Nat. Struct. Biol. 1994; 1: 877-890Crossref PubMed Scopus (234) Google Scholar) as follows. Symmetric dimers were generated by duplications of the random coordinates. The combined use of XPLOR noncrystallographic symmetry and symmetry pseudo-NOE term were served to satisfy monomer symmetry as described by Nilges (35Nilges M. Proteins. 1993; 17: 297-309Crossref PubMed Scopus (308) Google Scholar). The modeling protocol used by us consists of two separate stages: (i) initial and extensive regulation and (ii) simulated annealing and refinement. Regulation procedures were cycled twice to satisfy both experimental constraints and noncrystallographic symmetry parameters, followed by simulated annealing and refinement. The potential energy function consisted of covalent, repulsion, NOE, and torsional angle terms. The target function forms of NOE and torsional angles are the same as used by Driscoll et al. (40Driscoll P.C. Gronenborn A.M. Beress L. Clore G.M. Biochemistry. 1989; 28: 2188-2198Crossref PubMed Scopus (131) Google Scholar). A total of 116 distance restraints and 16 torsional angle constraints were used for structural calculations. All NOEs were classified and converted to distance constraints as strong (1.8–2.7 Å), medium (1.8–3.3 Å), and weak (1.8–5.0 Å) based on their intensities of NOESY spectrum. Corrections for pseudoatom representations were used for non-stereo specifically assigned methylene, methyl group, and tyrosine ring protons (32Wuthrich K. Billeter M. Braun W. J. Mol. Biol. 1983; 169: 949-961Crossref PubMed Scopus (1007) Google Scholar). Dihedral angle restraints were derived from measured3JHN-Hα coupling constants in DQF-COSY spectra in H2O solution (41Wagner G. Braun W. Havel T.F. Schaumann T. Go N. Wuthrich K. J. Mol. Biol. 1987; 196: 611-639Crossref PubMed Scopus (635) Google Scholar, 42Clore G.M. Gronenborn A.M. Nilges M. Ryan C.A. Biochemistry. 1987; 26: 8012-8023Crossref PubMed Scopus (264) Google Scholar). The structure of 4V·PIP2 complex was generated with Insight II (Biosym/Molecular Simulation Inc.) based on intermolecular NOE data of two-dimensional NOESY spectrum. The CD spectra of the 4V peptide (sequence LGKKPIYKKA) at pH 7.4 and 25 °C were consistent with a random coil characteristics (43Yang J.T. Wu C.-S.C. Martinez H.M. Methods Enzymol. 1986; 130: 208-269Crossref PubMed Scopus (1739) Google Scholar). However, the CD spectra obtained from 4V peptide complexed with PIP2were different from that of peptide alone, having a small positive mean residue ellipticity at 222 nm. The same spectral changes of 4L peptide were observed in the presence of PIP2. The CD data suggested that the structural transitions of both peptides were occurred in the presence of PIP2. Fig.1 shows that the positive ellipticity intensity of 4V·PIP2 complex at 222 nm decreased gradually at temperature ranges of 20–60 °C, indicating a slow process of dissociation. The one-dimensional proton NMR spectra of both 4V and 4L peptides are shown in Fig. 2. The spectra have demonstrated that both peptides produced considerable changes in the whole peptide resonance as well as in linewidths of resonance throughout PIP2 titration. In particular, 4L peptide experiences a severe aggregation at ratio of 8:6 (4V:PIP2) (Fig. 2,B, d). There are only single glycine, tyrosine, and alanine residues in the 4V peptide sequence. Glycine was easily identified by the distinctive fingerprint pattern in the H2O COSY spectra, and the lone alanine residue was identified from connectivities in TOCSY spectra by its characteristic methyl resonance. One AMX spin system from the tyrosine residue was easily identified in TOCSY spectra. These preliminary resonance assignments served as starting points for the sequence-specific assignment procedure (44Wuthrich K. NMR of Proteins and Nucleic Acids. John Wiley & Sons, New York1986Crossref Google Scholar). Sequential resonance assignments were made based on two-dimensional TOCSY and NOESY spectra in 90% H2O, 10% D2O solution. Sequential assignments for the backbone protons were completed by following dαN connectivities from NH-CαH COSY cross-peaks of previously identified amino acids. The side-chain proton chemical shifts were completed by TOCSY connectivities. The resonances of PIP2 were identified from the previously published chemical shifts (45Reid D.G. Gajjar K. J. Biol. Chem. 1987; 262: 7967-7972Abstract Full Text PDF PubMed Google Scholar) and PIP2titration procedure (Fig. 2). Some of the PIP2 resonance signals in 4V·PIP2 complex were assigned from proton one-dimensional spectrum, followed by PIP2 titration to 4V. Since most of PIP2 resonances were severely overlapped with peptide signals, PIP2 chemical shifts were identified from all assigned peptide resonances in two-dimensional TOCSY and NOESY spectra. All these sequential and medium NOE connectivities for both free and complex are summarized in Fig.3. Most of the NOEs shown in Fig. 3 were measured at mixing times of 100–400 ms. For 4L peptide, since the backbone NMR resonances were severely overlapped due to 7 lysine residues in the sequence, not all NOEs from residues involved in the variable domain could be assigned completely.Figure 3Summary of the NOE connectivities identified in both free (A) and 4V·PIP2 (B) complex (peptide:PIP2 = 2:1) at 20 °C, pH 7.0 (complex) and pH 7.4 (free), respectively. Mixing times of 100–400 ms were used, and observed NOE intensities are classified by the thickness of the lines.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The proton chemical shifts for the individual residues in free 4V peptide are in excellent agreement with the corresponding random coil values (46Wishart D.S. Sykes B.D. Richards F.M. J. Mol. Biol. 1991; 222: 311-333Crossref PubMed Scopus (1791) Google Scholar). As expected for a highly flexible peptide, NH-CαH coupling constants for all the residues are of the order of 6.5–8.0 Hz, suggesting retention of significant conformational flexibility. In addition, the sequential NOE intensities displayed a standard pattern of the preferred random coil conformation of the peptide. However, when peptide was titrated with PIP2, both the chemical shifts and NOE patterns exhibited a significant change in the entire peptide sequence indicating a structural transition (Figs. 3 and 4). In addition, a number of intermolecular NOEs, which are NOEs between peptide and PIP2 and intersubunit NOEs on the 4V dimer, were observed. The aliphatic proton resonance region of the two-dimensional NOESY spectrum of 4V·PIP2 is shown in Fig. 5, displaying both intersubunit and intermolecular NOEs in the complex.Figure 5600-MHz two-dimensional NOESY proton spectrum with mixing time of 400 ms in 90% H2O/10% D2O solution at 20 °C and pH value of 7.0. The spectrum are labeled with some of NOEs including intersubunit NOEs (marked as *) as well as intermolecular NOEs between PIP2 and 4V peptide.View Large Image Figure ViewerDownload Hi-res image Download (PPT) It is of interest to compare the proton chemical shift differences between free and PIP2complex 4V. Fig. 4 shows that, whereas proton chemical shifts of the side chains showed not much changes, those of the backbone NH and CαH changed dramatically in N-terminal as well as C-terminal regions. The side-chain chemical shifts, especially Pro185 and Ile186 residues in the center of peptide, exhibited significant changes. These interesting results indicate that the 4V dimer has extensive intermolecular contacts not only at both peptide terminus but also in the middle of the peptide (Fig. 4). In addition, these results strongly suggest that, while the side-chain atoms would have close intersubunit contacts with each other in the center of peptide, the backbone atoms do so at peptide termini. The solution structures for the 4V dimer were generated using the experimental constraints described above. A total of 90 starting substructures were used in the initial stage. After two cycles of regulation protocol, 60 structures that show no constraint violations greater than 0.5 Å for distances and 5° for torsional angles were identified. Among 60 structures, the 14 lowest energy structures (〈SA〉kr) were selected for detailed analysis (Table I). The structures are well defined with a root-mean-square deviation between backbone atom coordinates of 1.05 Å for all residues. The average structure was generated from the geometrical average from 14 structure coordinates and was subjected to restrained energy minimization to correct bond length and angle distorsions. This average structure exhibited 0.59-Å root-mean-square deviation for backbone atoms with respect to 14 〈SA〉kr structures. A best fit superposition of all final structures and the backbone conformation for average restrained energy minimized structure (〈SA¯〉kr) are shown in Fig.6. The atomic average root-mean-square deviations of the final structures with respect to average restrained energy minimized structure are shown in Fig.7. A Ramachandran plot (Fig.8) indicates that φ,ψ values of all 14 final NMR structures are distributed properly in energetically acceptable regions (47Morris A.L. MacArthur M.W. Hutchison E.G. Thornton J.M. Proteins. 1992; 12: 345-364Crossref PubMed Scopus (1419) Google Scholar).Table IStructural statistics for the 14 final simulated annealing (SA) structures of the dimeric 4VStatistics〈SA〉k〈SA¯〉krA. Root-mean-square deviations from experimental distance restraints (Å) All (116)0.03490.0399 Sequential (‖i−j‖ = 1) (32)0.03910.0227 Intraresidue (80)0.03410.0355 Interresidue (36)0.03530.0483B. Energies E total (kcal mol−1)77.2055.61 E NOE(all) (kcal mol−1)7.198.65 E tor (kcal mol−1)48.1229.16 E repel (kcal mol−1)11.578.35 E ncs (kcal mol−1)0.190.004C. Deviations from idealized covalent geometry Bonds (Å)0.0032830.0029 Angles (degree)0.6731140.5002 Impropers (degree)0.4741570.3530 Open table in a new tab Figure 7Distribution of the average atomic root-mean-square deviations with respect to average structure of the backbone (A) and side-chain (B) atoms in the 14 final simulated annealing structures for the 4V dimer.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 8Distribution of all (φ,ψ) values for the final 14 simulated annealing structures of the 4V in 4V·PIP2 complex.The glycine residues are represented by open circles.View Large Image Figure ViewerDownload Hi-res image Download (PPT) An inspection of the average energy-minimized structure in Fig.6 B clearly shows that major driving forces for dimer formation could be originated from hydrophobic interactions among side-chain atoms. 4V peptide in 4V·PIP2 complex forms a compact symmetric dimer with two extended strands twisted parallel each other. This compactness explains why 4V dimer dissociates slowly during the subunit exchange experiment as shown in Fig. 1. The exposed surface of the 4V consisted of very polar residues with basic side chains. Interestingly, the Pro185 residue that resides in the center of the cytoplasmic core has been shown to play a key role for twisted dimeric formation. No standard regular secondary structural elements were observed, as shown in Fig. 1. However, each monomer conformation exhibits similar to that of extended form with both N and C termini with kink. Strikingly, the overall three-dimensional structure of the 4V in solution demonstrates a twisted clamp shape having a cavity in the center of dimeric twist (Fig.6 C). A number of NOE contacts between 4V and PIP2 have been observed, which are NOEs between Pro185 ring protons of 4V and head group protons of PIP2, Ile186CδH3 methyl group of 4V and fatty acyl group of PIP2, and CγH2 of Lys188 and olefinic protons of PIP2. These NOEs strongly suggest that both anionic head group and fatty acyl side chain of PIP2 have close contacts with center of the peptide. Table II summarizes NOEs between 4V and PIP2. Especially, a possible hydrogen bond between phosphatidylinositol 4-phosphate oxygen and Lys188εNH3+ of 4V was observed from modeling structure based on NMR data. Fig. 9 shows the averaged energy-minimized solution structure of 4V complexed with PIP2. The 4V dimer grips PIP2 having interactions with both head group and fatty acyl chains of PIP2. The solution structure clearly reveals that side chains of cytoplasmic core residues play an important role for PIP2 binding.Table IIIntermolecular NOEs between 4V peptide and PIP2PIP24V2-aNOEs are classified as s (strong), m (medium), and w (weak) at mixing times of 100–400 ms at 25 °C. The numbering of PIP2 is based on IUPAC nomenclature.R1-C5,6,8,9HPro185CβH2 (m)Ile186CδH3 (w)Lys188CγH2 (s)R1-C7HPro185 CβH2(m)Pro185 CδH2 (w)Ile186 CδH3 (w)Ring-1′/5′/6′Ile186 CδH3(m)2-a NOEs are classified as s (strong), m (medium), and w (weak) at mixing times of 100–400 ms at 25 °C. The numbering of PIP2 is based on IUPAC nomenclature. Open table in a new tab It has been suggested that the syndecan core proteins, which are highly conserved among the syndecan family, exhibit a propensity to form non-covalently linked dimers and higher order oligomers (12Oh E.-S. Woods A. Couchman J.R. J. Biol. Chem. 1997; 272: 11805-11811Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 13Oh E.-S. Woods A. Couchman J.R. J. Biol. Chem. 1997; 272: 8133-8136Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, 14Carey D.J. Biochem. J. 1997; 327: 1-16Crossref PubMed Scopus (606) Google Scholar). It is also well known that oligomer formation is essential in protein kinase C interaction and activation (12Oh E.-S. Woods A. Couchman J.R. J. Biol. Chem. 1997; 272: 11805-11811Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). Recently, we have reported that SDS-resistant multimerization of syndecan-4 core protein correlates with PKC regulatory activity and variable region unique to syndecan-4 (4V) could potentiate phospholipid-induced activation of PKCαβγ (12Oh E.-S. Woods A. Couchman J.R. J. Biol. Chem. 1997; 272: 11805-11811Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 13Oh E.-S. Woods A. Couchman J.R. J. Biol. Chem. 1997; 272: 8133-8136Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). In addition, it has been shown that a central penta-peptide KPIYK sequence is necessary for multimerization of syndecan-4 core protein, resulting in both direct activation of PKCαβγ and phospholipid-induced activation (12Oh E.-S. Woods A. Couchman J.R. J. Biol. Chem. 1997; 272: 11805-11811Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 13Oh E.-S. Woods A. Couchman J.R. J. Biol. Chem. 1997; 272: 8133-8136Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). Further, 4V or 4L peptides in the presence of PIP2 strongly activate PKCα (28Oh E.S. Woods A. Lim S.-T. Theibert A.W. Couchman J.R. J. Biol. Chem. 1998; 273: 10624-10629Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). The data from NMR and gel filtration clearly support the hypothesis that PIP2 induces dimerization of both 4V and 4L peptide, and the induced dimer of 4V forms a compact structure bound to PIP2 (Fig. 2). We have recently reported that the peptide of whole cytoplasmic domain (4L) displayed less tendency to oligomerize than 4V in the absence of PIP2, resulting reduced activity of 4L. This could be due to highly basic residues of the conserved (C1) region, which lies between the V region of the cytoplasmic domain and the membrane. These residues may decrease the self-association of the 4L peptide. However, very recently, we have shown that both 4V and 4L peptides exhibited essentially the same activity with respect to PIP2-mediated activation of PKC (28Oh E.S. Woods A. Lim S.-T. Theibert A.W. Couchman J.R. J. Biol. Chem. 1998; 273: 10624-10629Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). Therefore, we concluded that the solution structure of 4L in the presence of PIP2 should resemble that of PIP2-4V complex; in other words, the molecular topology of 4L and 4V in the presence of PIP2 must be similar. Major interaction of 4V peptide with PIP2 occurs between aliphatic side chains of peptide and whole area of PIP2. No close contacts of peptide backbone to PIP2 has been observed. The other finding is that, even though PIP2 promotes association of 4V initially, further driving or stabilizing forces could be peptide-peptide interactions. The NMR structure clearly shows that peptide backbones of each monomer consists of intertwined circular shape and side chains provide additional stabilizing forces by hydrophobic interactions from an outward position. Surprisingly, two extended monomers form a quite stable twisted dimeric conformation, even if there is no hydrogen bonds between monomers. Since the calculated 14 structures (〈SA〉kr) based on NMR constraints exhibited a good convergence, we conclude that the 4V dimer has a stable and unique conformation in solution state in the presence of PIP2. This was also confirmed by melting experimental data based on circular dichroism (Fig. 1) and NMR linewidth analysis at temperature ranges of 20–70 °C (data not shown). We have recently demonstrated that 4V peptides of altered sequence have much reduced ability to regulate PKCα activity (12Oh E.-S. Woods A. Couchman J.R. J. Biol. Chem. 1997; 272: 11805-11811Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 13Oh E.-S. Woods A. Couchman J.R. J. Biol. Chem. 1997; 272: 8133-8136Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, 28Oh E.S. Woods A. Lim S.-T. Theibert A.W. Couchman J.R. J. Biol. Chem. 1998; 273: 10624-10629Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). It was suggested that substitution of Lys184, Lys188, Pro185, and Ile186 compromised both oligomerization and cPKC activation. The data here support this hypothesis, showing that all these residues take part in monomer-monomer interactions. Our previous data also showed that, while replacement of Tyr187 with phenylalanine had a profound effect on PKC activation, it had the least effect on dimer formation. This is in very good agreement with solution structure analysis. Tyr187 appears not to be involved in oligomerization of 4V, and we hypothesize that since its side chain is mostly solvent-exposed, the hydroxyl group may be critical for contact with, and activation of, PKC. Structural studies of the 4V·PIP2·PKCα ternary complex will provide this important information. We thank TMSI Korea for the use of the molecular simulation programs (Molecular Modeling Tools, Molecular Simulations Inc.)." @default.
• W1973603507 created "2016-06-24" @default.
• W1973603507 creator A5035485431 @default.
• W1973603507 creator A5042988495 @default.
• W1973603507 creator A5046605351 @default.
• W1973603507 creator A5051382096 @default.
• W1973603507 creator A5073284312 @default.
• W1973603507 date "1998-05-01" @default.
• W1973603507 modified "2023-09-29" @default.
• W1973603507 title "Solution Structure of a Syndecan-4 Cytoplasmic Domain and Its Interaction with Phosphatidylinositol 4,5-Bisphosphate" @default.
• W1973603507 cites W1548025383 @default.
• W1973603507 cites W1569544376 @default.
• W1973603507 cites W1593567129 @default.
• W1973603507 cites W1853807720 @default.
• W1973603507 cites W189749484 @default.
• W1973603507 cites W1931472297 @default.
• W1973603507 cites W1964659291 @default.
• W1973603507 cites W1968434409 @default.
• W1973603507 cites W1971809347 @default.
• W1973603507 cites W1978743038 @default.
• W1973603507 cites W1991708828 @default.
• W1973603507 cites W1993844842 @default.
• W1973603507 cites W2008608024 @default.
• W1973603507 cites W2011009086 @default.
• W1973603507 cites W2024422510 @default.
• W1973603507 cites W2031983592 @default.
• W1973603507 cites W2035095021 @default.
• W1973603507 cites W2036510848 @default.
• W1973603507 cites W2036620431 @default.
• W1973603507 cites W2037304739 @default.
• W1973603507 cites W2041661092 @default.
• W1973603507 cites W2044815019 @default.
• W1973603507 cites W2045950270 @default.
• W1973603507 cites W2060538012 @default.
• W1973603507 cites W2062980512 @default.
• W1973603507 cites W2063982283 @default.
• W1973603507 cites W2066367871 @default.
• W1973603507 cites W2072871114 @default.
• W1973603507 cites W2078133735 @default.
• W1973603507 cites W2082797919 @default.
• W1973603507 cites W2086524338 @default.
• W1973603507 cites W2093059615 @default.
• W1973603507 cites W2098349937 @default.
• W1973603507 cites W2102598791 @default.
• W1973603507 cites W2109922359 @default.
• W1973603507 cites W2135096442 @default.
• W1973603507 cites W2139783945 @default.
• W1973603507 cites W2140969448 @default.
• W1973603507 cites W2148849095 @default.
• W1973603507 cites W2157165549 @default.
• W1973603507 cites W2166928150 @default.
• W1973603507 cites W2167956058 @default.
• W1973603507 cites W2289547287 @default.
• W1973603507 cites W34966227 @default.
• W1973603507 doi "https://doi.org/10.1074/jbc.273.21.13022" @default.
• W1973603507 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/9582338" @default.
• W1973603507 hasPublicationYear "1998" @default.
• W1973603507 type Work @default.
• W1973603507 sameAs 1973603507 @default.
• W1973603507 citedByCount "87" @default.
• W1973603507 countsByYear W19736035072012 @default.
• W1973603507 countsByYear W19736035072013 @default.
• W1973603507 countsByYear W19736035072014 @default.
• W1973603507 countsByYear W19736035072015 @default.
• W1973603507 countsByYear W19736035072016 @default.
• W1973603507 countsByYear W19736035072017 @default.
• W1973603507 countsByYear W19736035072018 @default.
• W1973603507 countsByYear W19736035072019 @default.
• W1973603507 countsByYear W19736035072020 @default.
• W1973603507 countsByYear W19736035072021 @default.
• W1973603507 countsByYear W19736035072022 @default.
• W1973603507 countsByYear W19736035072023 @default.
• W1973603507 crossrefType "journal-article" @default.
• W1973603507 hasAuthorship W1973603507A5035485431 @default.
• W1973603507 hasAuthorship W1973603507A5042988495 @default.
• W1973603507 hasAuthorship W1973603507A5046605351 @default.
• W1973603507 hasAuthorship W1973603507A5051382096 @default.
• W1973603507 hasAuthorship W1973603507A5073284312 @default.
• W1973603507 hasBestOaLocation W19736035071 @default.
• W1973603507 hasConcept C134306372 @default.
• W1973603507 hasConcept C1491633281 @default.
• W1973603507 hasConcept C185592680 @default.
• W1973603507 hasConcept C190062978 @default.
• W1973603507 hasConcept C2777853560 @default.
• W1973603507 hasConcept C2780610907 @default.
• W1973603507 hasConcept C33923547 @default.
• W1973603507 hasConcept C36503486 @default.
• W1973603507 hasConcept C49658373 @default.
• W1973603507 hasConcept C55493867 @default.
• W1973603507 hasConcept C62478195 @default.
• W1973603507 hasConcept C65001120 @default.
• W1973603507 hasConcept C70721500 @default.
• W1973603507 hasConcept C86803240 @default.
• W1973603507 hasConcept C95444343 @default.
• W1973603507 hasConceptScore W1973603507C134306372 @default.
• W1973603507 hasConceptScore W1973603507C1491633281 @default.
• W1973603507 hasConceptScore W1973603507C185592680 @default.
• W1973603507 hasConceptScore W1973603507C190062978 @default.

• next