Abstract The pupal cuticle protein from Aedes aegypti (AaPC) inhibits dengue virus (DENV) infection; however, the underlying mechanism of this inhibition remains unknown. Here, we report that AaPC is an intrinsically disordered protein and interacts with domain I/II of the DENV envelope protein via residues Asp59, Asp61, Glu71, Asp73, Ser75, and Asp80. AaPC can directly bind to and cause the aggregation of DENV, which in turn blocks virus infection during the virus‐cell fusion stage. AaPC may also influence viral recognition and attachment by interacting with human immune receptors DC‐SIGN and CD4. These findings enhance our understanding of the role of AaPC in mitigating viral infection and suggest that AaPC is a potential target for developing inhibitors or antibodies to control dengue virus infection.
Glutamate receptor interacting protein 1 (GRIP1) is a scaffold protein composed of seven PDZ (Postsynaptic synaptic density-95/Discs large/Zona occludens-1) domains. The protein plays important roles in the synaptic targeting of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. The interaction between GRIP1 PDZ7 and a Ras guanine nucleotide exchange factor, GRASP-1, regulates synaptic distribution of AMPA receptors. Here, we describe the three-dimensional structure of GRIP1 PDZ7 determined by NMR spectroscopy. GRIP1 PDZ7 contains a closed carboxyl group-binding pocket and a narrow αB/βB-groove that is not likely to bind to classical PDZ ligands. Unexpectedly, GRIP1 PDZ7 contains a large solvent-exposed hydrophobic surface at a site distinct from the conventional ligand-binding αB/βB-groove. NMR titration experiments show that GRIP1 PDZ7 binds to GRASP-1 via this hydrophobic surface. Our data uncover a novel PDZ domain-mediated protein interaction mode that may be responsible for multimerization of other PDZ domain-containing scaffold proteins. Glutamate receptor interacting protein 1 (GRIP1) is a scaffold protein composed of seven PDZ (Postsynaptic synaptic density-95/Discs large/Zona occludens-1) domains. The protein plays important roles in the synaptic targeting of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. The interaction between GRIP1 PDZ7 and a Ras guanine nucleotide exchange factor, GRASP-1, regulates synaptic distribution of AMPA receptors. Here, we describe the three-dimensional structure of GRIP1 PDZ7 determined by NMR spectroscopy. GRIP1 PDZ7 contains a closed carboxyl group-binding pocket and a narrow αB/βB-groove that is not likely to bind to classical PDZ ligands. Unexpectedly, GRIP1 PDZ7 contains a large solvent-exposed hydrophobic surface at a site distinct from the conventional ligand-binding αB/βB-groove. NMR titration experiments show that GRIP1 PDZ7 binds to GRASP-1 via this hydrophobic surface. Our data uncover a novel PDZ domain-mediated protein interaction mode that may be responsible for multimerization of other PDZ domain-containing scaffold proteins. Postsynaptic synaptic density-95/Discs large/Zona occludens-1 (PDZ) 1The abbreviations used are: PDZ, Postsynaptic density-95/Discs large/Zona occludens-1; PSD-95, postsynaptic density-95; GRIP, glutamate receptor-interacting protein 1; NOE, nuclear Overhauser effect. 1The abbreviations used are: PDZ, Postsynaptic density-95/Discs large/Zona occludens-1; PSD-95, postsynaptic density-95; GRIP, glutamate receptor-interacting protein 1; NOE, nuclear Overhauser effect. domains are among the most abundant protein-interacting modules in the genomes of metazoans (1Schultz J. Copley R.R. Doerks T. Ponting C.P. Bork P. Nucleic Acids Res. 2000; 28: 231-234Google Scholar). PDZ domain containing proteins have been shown to play important roles in diverse cellular processes including receptor/ion channel clustering, signaling complex assembly, and protein targeting (2Craven S.E. Bredt D.S. Cell. 1998; 93: 495-498Google Scholar, 3Harris B.Z. Lim W.A. J. Cell Sci. 2001; 114: 3219-3231Google Scholar, 4Sheng M. Sala C. Annu. Rev. Neurosci. 2001; 24: 1-29Google Scholar). A typical PDZ domain contains ∼90 amino acid residues folded into a compact globular structure comprising a six-stranded β-barrel flanked by two α-helices (5Morais Cabral J.H. Petosa C. Sutcliffe M.J. Raza S. Byron O. Poy F. Marfatia S.M. Chishti A.H. Liddington R.C. Nature. 1996; 382: 649-652Google Scholar, 6Doyle D.A. Lee A. Lewis J. Kim E. Sheng M. MacKinnon R. Cell. 1996; 85: 1067-1076Google Scholar). The majority of PDZ domain-mediated interactions involves the binding of short peptide fragments located at the extreme carboxyl termini of target proteins to grooves formed by the second α-helix (αB) and the second β-strand (βB) of the PDZ domains (6Doyle D.A. Lee A. Lewis J. Kim E. Sheng M. MacKinnon R. Cell. 1996; 85: 1067-1076Google Scholar, 7Schultz J. Hoffmuller U. Krause G. Ashurst J. Macias M.J. Schmieder P. Schneider-Mergener J. Oschkinat H. Nat. Struct. Biol. 1998; 5: 19-24Google Scholar, 8Daniels D.L. Cohen A.R. Anderson J.M. Brunger A.T. Nat. Struct. Biol. 1998; 5: 317-325Google Scholar, 9Tochio H. Zhang Q. Mandal P., Li, M. Zhang M. Nat. Struct. Biol. 1999; 6: 417-421Google Scholar). The carboxyl peptide augments the βB strand in an anti-parallel fashion. Amino acid residues at the 0 and −2 positions of the carboxyl peptide play dominant roles in the peptide binding to a cognate PDZ domain, although residues at the −1 and −3 positions and those at further upstream positions also contribute to the binding (10Songyang Z. Fanning A.S., Fu, C., Xu, J. Marfatia S.M. Chishti A.H. Crompton A. Chan A.C. Anderson J.M. Cantley L.C. Science. 1997; 275: 73-77Google Scholar). In addition to binding to carboxyl peptides, PDZ domains can also use the αB/βB-target-binding groove to interact with internal short peptide fragments with defined structures (9Tochio H. Zhang Q. Mandal P., Li, M. Zhang M. Nat. Struct. Biol. 1999; 6: 417-421Google Scholar, 11Hillier B.J. Christopherson K.S. Prehoda K.E. Bredt D.S. Lim W.A. Science. 1999; 284: 812-815Google Scholar, 12Tochio H. Mok Y.K. Zhang Q. Kan H.M. Bredt D.S. Zhang M. J. Mol. Biol. 2000; 303: 359-370Google Scholar). Glutamate receptor-interacting protein 1 (GRIP1) contains seven PDZ domains and interacts through PDZ4 and PDZ5 with the COOH-terminal "ESVKI" sequence of the GluR2/3 subunits of AMPA receptors (13Dong H. O'Brien R.J. Fung E.T. Lanahan A.A. Worley P.F. Huganir R.L. Nature. 1997; 386: 279-284Google Scholar,14Wyszynski M. Valtschanoff J.G. Naisbitt S. Dunah A.W. Kim E. Standaert D.G. Weinberg R. Sheng M. J. Neurosci. 1999; 19: 6528-6537Google Scholar). GRIP1 has several closely related gene products, GRIP2 and AMPA receptor binding protein (ABP), and they are all able to bind to AMPA receptors (15Srivastava S. Osten P. Vilim F.S. Khatri L. Inman G. States B. Daly C. DeSouza S. Abagyan R. Valtschanoff J.G. Weinberg R.J. Ziff E.B. Neuron. 1998; 21: 581-591Google Scholar, 16Dong H. Zhang P. Song I. Petralia R.S. Liao D. Huganir R.L. J. Neurosci. 1999; 19: 6930-6941Google Scholar). In addition, the GRIP family proteins can interact through PDZ6 with EphB2/EphA7 receptor tyrosine kinase and the ephrinB1 ligand (17Bruckner K. Pablo Labrador J. Scheiffele P. Herb A. Seeburg P.H. Klein R. Neuron. 1999; 22: 511-524Google Scholar, 18Lin D. Gish G.D. Songyang Z. Pawson T. J. Biol. Chem. 1999; 274: 3726-3733Google Scholar, 19Torres R. Firestein B.L. Dong H. Staudinger J. Olson E.N. Huganir R.L. Bredt D.S. Gale N.W. Yancopoulos G.D. Neuron. 1998; 21: 1453-1463Google Scholar). Genetic studies have shown thatGRIP1 −/− mice are embryonic lethal, and the mutant embryos develop abnormalities in the dermo-epidermal junction (20Bladt F. Tafuri A. Gelkop S. Langille L. Pawson T. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 6816-6821Google Scholar). GRIP1 additionally interacts through its PDZ7 with GRASP-1, a Ras guanine-nucleotide exchange factor that regulates the synaptic distribution of AMPA receptors (21Ye B. Liao D. Zhang X. Zhang P. Dong H. Huganir R.L. Neuron. 2000; 26: 603-617Google Scholar). The interaction between GRIP1 PDZ7 and GRASP-1 is rather unusual, as GRASP-1 does not contain obvious PDZ-interacting sequence at its carboxyl terminus. Biochemical analysis has indicated that a 100-residue fragment located at the COOH-terminal end of GRASP-1 is responsible for the interaction of the protein with GRIP1 (21Ye B. Liao D. Zhang X. Zhang P. Dong H. Huganir R.L. Neuron. 2000; 26: 603-617Google Scholar). The solution structure of the PDZ7 of GRIP1 determined in this study shows that the conventional target-binding groove formed by the αB helix and the βB strand adopts a closed conformation that is not likely to be capable of interacting with carboxyl peptides. Unexpectedly, GRIP1 PDZ7 contains a solvent-exposed hydrophobic surface formed by the βE strand, the αB helix, and the loop connecting the two secondary structural elements. We show that GRIP1 PDZ7 interacts with GRASP-1 via this hydrophobic surface. The data presented in this study uncover a novel mode of PDZ domain-mediated protein-protein interaction. The seventh PDZ domain of rat GRIP1 (amino acid residues 980–1070) and the COOH-terminal 100-residue fragment of rat GRASP-1 were polymerase chain reaction-amplified from the respective full-length cDNAs of the proteins with specific primers. The amplified DNA fragments were inserted into a modified version of pET32a (Novagen) in which the DNA sequences encoding the S-tag and thioredoxin were removed. The resulting recombinant plasmid harboring the respective target genes were individually transformed intoEscherichia coli BL21(DE3) host cells for large scale protein productions. Uniformly 15N- and15N/13C-labeled proteins were prepared by growing bacteria in M9 minimal medium using15NH4Cl (1 g/liter) and13C6-glucose (1 g/liter) as stable isotope sources. Recombinant GRIP1 PDZ7 and GRASP-1 fragment were purified by a combination of nickel-nitrilotriacetic acid affinity column (Qiagen) followed by gel filtration chromatography. The purified recombinant proteins contained an NH2-terminal His tag carried over from the cloning vector. Four NMR samples were prepared for structural determination of GRIP1 PDZ7 with a protein concentration of ∼1.0 mm (unlabeled PDZ7 in 99.9% D2O,15N-labeled protein in 90% H2O/10% D2O, two 15N/13C-labeled samples in 99.9% D2O and in 90% H2O/10% D2O). The protein was dissolved in 100 mmpotassium phosphate buffer at pH 6.5 (direct meter reading). The15N-labeled sample in 90% H2O/10% D2O was also used for backbone relaxation data measurements. All of the NMR experiments were carried out at 25 °C on Varian Inova 500 and 750 spectrometers equipped with 5 mm z-shielded gradient triple resonance probes. NMR spectra were processed with the nmrPipe software package (22Delaglio F. Grzesiek S. Vuister G.W. Zhu G. Pfeifer J. Bax A. J. Biomol. NMR. 1995; 6: 277-293Google Scholar) and analyzed using PIPP (23Garrett D.S. Powers R. Gronenborn A.M. Clore G.M. J. Magn. Reson. 1991; 95: 214-220Google Scholar) and Sparky (www.cgl.ucsf.edu/home/sparky/). Sequential backbone resonance assignments of the protein were obtained by standard heteronuclear correlation experiments including HNCO, HNCACB, and CBCA(CO)NH and confirmed by a three-dimensional 15N-separated NOESY experiment (24Bax A. Grzesiek S. Acc. Chem. Res. 1993; 26: 131-138Google Scholar, 25Kay L.E. Gardner K.H. Curr. Opin. Struct. Biol. 1997; 7: 722-731Google Scholar). The non-aromatic non-exchangeable side-chain assignments were obtained using an HCCH-TOCSY experiment. The side chains of the aromatics were assigned by 1The abbreviations used are: PDZ, Postsynaptic density-95/Discs large/Zona occludens-1; PSD-95, postsynaptic density-95; GRIP, glutamate receptor-interacting protein 1; NOE, nuclear Overhauser effect.H two-dimensional TOCSY and NOESY experiments with an unlabeled protein sample in D2O (26Wüthrich K. NMR of Proteins and Nucleic Acids. John Wiley & Sons, Inc., New York1986Google Scholar). The −NH2 side chains of Asn and Gln residues were assigned by a three-dimensional 15N-separated NOESY experiment with the 15N-labeled protein dissolved in H2O. The pulse sequences used to obtain the 15N-longitudinal relaxation times, T1, the 15N spin-lattice relaxation times, T2, and the 1The abbreviations used are: PDZ, Postsynaptic density-95/Discs large/Zona occludens-1; PSD-95, postsynaptic density-95; GRIP, glutamate receptor-interacting protein 1; NOE, nuclear Overhauser effect.H-15N steady-state NOE values were described previously (27Farrow N.A. Muhandiram R. Singer A.U. Pascal S.M. Kay C.M. Gish G. Shoelson S.E. Pawson T. Forman-Kay J.D. Kay L.E. Biochemistry. 1994; 33: 5984-6003Google Scholar). All of the data were processed using nmrPipe and nmrDraw software (22Delaglio F. Grzesiek S. Vuister G.W. Zhu G. Pfeifer J. Bax A. J. Biomol. NMR. 1995; 6: 277-293Google Scholar), and the peak intensities were characterized as peak heights using the Sparky software. The relaxation data were fitted using the Modelfree 4.0 program (provided by A.G. Palmer). Approximate interproton distance restraints were derived from three NOESY spectra (a 1The abbreviations used are: PDZ, Postsynaptic density-95/Discs large/Zona occludens-1; PSD-95, postsynaptic density-95; GRIP, glutamate receptor-interacting protein 1; NOE, nuclear Overhauser effect.H two-dimensional homonuclear NOESY, a 15N-separated NOESY, and a 13C-separated NOESY). NOEs were grouped into three distance ranges of 1.8–2.7 Å (1.8–2.9 Å for NOEs involving NH protons), 1.8–3.3 Å (1.8–3.5 Å for NOEs involving NH protons), and 1.8–5.0 Å, respectively, corresponding to strong, medium, and weak NOEs. Hydrogen-bonding restraints (two per hydrogen bond wherer NH–O = 1.8–2.2 Å andr N–O = 2.2–3.3 Å) were generated from the standard secondary structure of the protein based on the NOE patterns and backbone secondary chemical shifts. Backbone dihedral angle restraints (φ and ψ angles) were derived from3 J HNα coupling constants measured using an HNHA experiment and the backbone chemical shift analysis program TALOS (28Cornilescu G. Delaglio F. Bax A. J. Biomol. NMR. 1999; 13: 289-302Google Scholar). Structures were calculated using the program CNS (29Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sec. D. 1998; 54: 905-921Google Scholar). The coordinates of the structures of GRIP1 PDZ7 have been deposited in the Protein Data Bank (PDB code 1M5Z), and the chemical shift assignments of PDZ7 have been deposited in the BioMagResBank (accession number 5499). To investigate the mechanism of the interaction between GRIP1 and GRASP-1, we determined the three-dimensional structure of GRIP1 PDZ7 by NMR spectroscopy. A total of 1826 experimentally derived distance and torsion angle restraints allowed us to determine the three-dimensional structure of PDZ7 with high precision (Table I). Fig.1 A shows a stereoview of the best-fit superposition of a family of 20 structures of PDZ7. As expected, PDZ7 adopts a typical PDZ domain conformation consisting of six β-strands (from βA to βF) and two α-helices (αA and αB) (Fig. 1 B). A comparison of the structures of PDZ7 and PDZ2 of PSD-95 using the respective secondary structural elements shows a root mean square difference of 1.57 Å.Table IStructural statistics for the family of 20 structuresaNone of the structures exhibits distance violations greater than 0.3 Å or dihedral angle violations greater than 4°.Distance restraintsIntraresidue (i–j = 0)596Sequential (‖i–j‖ = 1)376Medium range (2 < ‖i–j‖ < 4)225Long range (‖i–j‖ > 5)447Hydrogen bonds56Total1700Dihedral angle restraintsΦ52Ψ523 J HNαcoupling constants22Total126Mean r.m.s. deviations from the experimental restraintsDistance (Å)0.013 ± 0.001Dihedral angle (Å)0.228 ± 0.0323-bond J coupling (Å)0.468 ± 0.041Mean r.m.s. deviations from idealized covalent geometryBond (Å)0.002 ± 0.000Angle (Å)0.355 ± 0.010Improper (Å)0.186 ± 0.011Mean energies (kcal mol−1)EnoebThe final values of the square-well NOE and dihedral angle potentials were calculated with force constants of 50 kcal mol−1 Å−2 and 200 kcal mol−1rad−2, respectively.23.38 ± 3.55EcdihbThe final values of the square-well NOE and dihedral angle potentials were calculated with force constants of 50 kcal mol−1 Å−2 and 200 kcal mol−1rad−2, respectively.0.33 ± 0.09EL–J−260.60 ± 14.76Ramachandran plotcThe program Procheck (38) was used to assess the overall quality of the structures.Residues 980–1070% residues in the most favorable regions75.9additional allowed regions19.5generously allowed regions3.9Atomic r.m.s. differences (Å)dThe precision of the atomic coordinates is defined as the average r.m.s. difference between the 20 final structures and the mean coordinates of the protein.(Residues 985–992, 1002–1007, 1012–1018, 1023–1027, 1034–1038, 1041–1042, 1048–1056, 1061–1068 in protein)Backbone heavy atoms (N, Cα, and C′)0.31Heavy atoms0.81a None of the structures exhibits distance violations greater than 0.3 Å or dihedral angle violations greater than 4°.b The final values of the square-well NOE and dihedral angle potentials were calculated with force constants of 50 kcal mol−1 Å−2 and 200 kcal mol−1rad−2, respectively.c The program Procheck (38Laskowski R.A. Rullmannn J.A. MacArthur M.W. Kaptein R. Thornton J.M. J. Biomol. NMR. 1996; 8: 477-486Google Scholar) was used to assess the overall quality of the structures.d The precision of the atomic coordinates is defined as the average r.m.s. difference between the 20 final structures and the mean coordinates of the protein. Open table in a new tab The amino acid sequence analysis reveals that PDZ7 from the GRIP family proteins (GRIP1/2 and ABP) have very high sequence identities with one another. The amino acid sequence of PDZ7 is also highly conserved throughout the evolution (Fig.2). In contrast, the rest of PDZ domains in the data base have rather limited amino acid sequence identities with that of PDZ7 (the PDZ1 of Calliphora vicinaInaD has the highest sequence identity with GRIP1 PDZ7 of 37%). We further note that the amino acid residue in the first residue of the αB helix (αB1) of GRIP PDZ7 is an absolutely conserved Cys residue (Cys-1048). A data base search revealed that no other known PDZ domains contain a Cys residue in the αB1 position (1Schultz J. Copley R.R. Doerks T. Ponting C.P. Bork P. Nucleic Acids Res. 2000; 28: 231-234Google Scholar). The amino acid residue in the αB1 position is known to play a critical role in recognizing the −2 residue in target carboxyl peptides (6Doyle D.A. Lee A. Lewis J. Kim E. Sheng M. MacKinnon R. Cell. 1996; 85: 1067-1076Google Scholar, 10Songyang Z. Fanning A.S., Fu, C., Xu, J. Marfatia S.M. Chishti A.H. Crompton A. Chan A.C. Anderson J.M. Cantley L.C. Science. 1997; 275: 73-77Google Scholar). We analyzed the structural features of this residue as well as the αB/βB-groove in detail, hoping to gain insight into the potential target-binding mechanism of this PDZ domain. Fig.3 A compares the conformation of the αB/βB-grooves of PDZ7 and PSD-95 PDZ2. The side chain of Cys-1048 in GRIP1 PDZ7 is packed with hydrophobic residues from both αB and βB (see an example of NOE contacts between Cys-1048 β-protons and side chains of Val-1004, Val-1013, and Val-1052 in Fig.4 A). Because of the relatively small size of Cys-1048, the NH2-terminal end of αB in PDZ7 is slightly closer to βB with respect to the αB helix in PSD-95 PDZ2. In addition, the whole αB-helix rotates ∼18° anti-clockwise toward βB (Fig. 3 A), resulting in a significantly smaller αB/βB-groove. The size of the αB/βB-groove in PDZ7 is not likely to accommodate a target carboxyl peptide, which is supposed to assume a β-strand conformation when binding to the PDZ domain. Additionally, the conformation of the carboxyl group-binding pocket of PDZ7 is not suited to interact with negatively charged carboxyl groups in the target peptides. The βA/βB-loop of PDZ7 (corresponding to the carboxyl group-binding loop of conventional PDZ domains) contains two additional residues (Fig. 2). The conformation of this loop is largely defined (Fig.1 A) by a number of NOE contacts between amino acid residues from this loop with those from the COOH-terminal end of the αB helix (see Fig. 4 B for an example). The limited conformational freedom of the βA/βB-loop is also supported by the order parameter values (S2) of the backbone amides in this region measured from the NMR relaxation experiments (Fig. 4 C). Fig. 3,B and C, compares the surface structures of the αB/βB-grooves of PDZ7 and PSD-95 PDZ2. Because of the packing of the βA/βB-loop with the αB helix, PDZ7 does not have a hydrophobic pocket to accommodate the side chain of the target peptide residue at the 0 position (Fig. 3 B). Additionally, a highly conserved positively charged residue that functions to stabilize the negative charge of the carboxyl group of target peptide (Lys-233 in PSD-95 PDZ2) (Fig. 3 C) is missing in PDZ7. Instead, several negatively charged residues (Glu-998, Asp-999, and Glu-1057) surround the remaining small hydrophobic surface in PDZ7 (Fig. 3 B). Taken together, we conclude that the GRIP1 PDZ7 αB/βB-groove adopts a closed conformation. We predict that GRIP1 PDZ7 is not likely to be able to interact with carboxyl peptides using its αB/βB-groove. Consistent with our prediction, no proteins with classical PDZ-binding carboxyl termini were found to interact with GRIP1 PDZ7 in a recent yeast two-hybrid screening (21Ye B. Liao D. Zhang X. Zhang P. Dong H. Huganir R.L. Neuron. 2000; 26: 603-617Google Scholar). 2R. Huganir, personal communication. However, we cannot rule out the possibility that GRIP1 PDZ7 may undergo a large conformational change in the αB/βB region, rendering an interaction of the domain with carboxyl peptides.Figure 3The GRIP1 PDZ7 contains a closed αB/βB-groove. A, comparison of the αB/βB-groove conformation of GRIP1 PDZ7 to that of PSD-95 PDZ2. In this comparison, the two PDZ domains were superimposed with each other using their respective βB-strand. The amino acid residue in the αB1 position of each PDZ domain is drawn in explicit atom representations. B and C, surface representation of the αB/βB-grooves of GRIP1 PDZ7 (B) and PSD-95 PDZ2 (C). In this presentation, the hydrophobic amino acid residues are drawn in yellow, the positively charged residues in are shown in blue, the negatively charged residues are shown in red, and the uncharged polar residues are shown in gray. The orientations of the molecules in B and C are the same as shown in A. The surface diagram is generated using the program GRASP (37Nicholls A. GRASP: Graphical Representation and Analysis of Surface Properties. Columbia University, New York1992Google Scholar).View Large Image Figure ViewerDownload (PPT)Figure 4Characterization of theαB/βB-groove. A, representative NOE contacts between Cys-1048 in the αB1 with the residues from βB and αB. The data is taken from a three-dimensional13C-separated NOESY spectrum of the protein at the Cβ chemical shift of Cys-1048. B, an 15N-NOESY strip showing NOE contacts between the backbone amide proton of Phe-1000 in the DFGF-loop with Ile-1055 at the end of αB of PDZ7.C, backbone amide-order parameters (S2) of GRIP1 PDZ7 determined by NMR relaxation experiments. The S2values were derived from T1, T2, and1H-15N NOE values of GRIP1 PDZ7 measured on field strength of 500 MHz. For easy comparison, the secondary structure of the protein is included in the figure.View Large Image Figure ViewerDownload (PPT) Structural analysis shows that the space between the backbones of αB and βE of PDZ7 is larger than that of PSD-95 PDZ2 (as well as other PDZ domains with known structures) (Fig.5 A). A prominent hydrophobic surface formed by amino acid residues from βE to αB can be observed in PDZ7 (Fig. 5 B). We term this hydrophobic surface as the βE/αB-surface. The entire αB helix of GRIP PDZ7 is composed of hydrophobic amino acid residues, and it contributes four residues (Cys-1049, Leu-1050, Pro-1053, and Leu-1054) to the βE/αB-hydrophobic surface (Figs. 2 and 5 B). Furthermore, the amino acid residues forming the βE/αB-hydrophobic surface are highly conserved in both GRIP1 and GRIP2 (Fig. 2). Such hydrophobic surface is not observed in other PDZ domains including PSD-95 PDZ2 (Fig. 5 C). It is possible that GRIP1 PDZ7 may use the βE/αB-hydrophobic surface rather than the αB/βB-groove to interact with its binding partner(s). The binding of GRASP-1 to GRIP1 PDZ requires a ∼100-residue fragment at the COOH-terminal end of GRASP-1, and this 100-residue PDZ7-binding domain of GRASP-1 contains neither a classical PDZ-binding motif at its carboxyl terminus nor a PDZ-like fold (21Ye B. Liao D. Zhang X. Zhang P. Dong H. Huganir R.L. Neuron. 2000; 26: 603-617Google Scholar). Preliminary NMR and circular dichroism spectroscopic studies of the purified COOH-terminal 100-residue PDZ7-binding domain of GRASP-1 indicated an α-helix-rich structure, and the domain forms a large molecular multimer in solution (data not shown). We titrated15N-labeled PDZ7 with the unlabeled PDZ7-binding domain of GRASP-1. The addition of substoichiometric amounts of GRASP-1 resulted in chemical shift as well as peak intensity changes of a number of backbone amides of PDZ7 (Fig.6 A), confirming that GRASP-1 indeed interacts with GRIP1 via direct binding to its PDZ7 domain (21Ye B. Liao D. Zhang X. Zhang P. Dong H. Huganir R.L. Neuron. 2000; 26: 603-617Google Scholar). The data in Fig. 6 B summarizes the backbone amide chemical shift changes of PDZ7 induced by GRASP-1 binding. The amino acid residues at the COOH-terminal end of βD, the whole βE as well as part of αB facing the βD/βE-strands, experience the largest amplitude of GRASP-1-induced chemical shift changes. The region that shows obvious GRASP-1-induced chemical shift changes closely overlaps with the βE/αB-hydrophobic surface shown in Fig. 5 B, indicating that GRASP-1 binds to PDZ7 via the distinct βE/αB-hydrophobic surface. In contrast, we do not observe any GRASP-1-induced chemical shift changes of the residues in the "DFGF-loop" (the conventional carboxyl group-binding motif) as well as those in the entire βB-strand (Fig. 6). In addition, no chemical shift changes could be detected for the Cys-1048 in the αB1 position of PDZ7 upon the addition of GRASP-1. Taken together, the chemical shift perturbation data indicate that the conventional target-binding groove (i.e. the βB/αB-groove) is not involved in GRASP-1 binding. Chemical shift perturbation data also indicate that GRASP-1 binding induces localized conformational changes to PDZ7 (in the βE/αB-region). To our knowledge, the data presented here demonstrate for the first time that a PDZ domain can interact with its target via a site distinct from the well characterized target-binding βB/αB-groove. PDZ domain-mediated dimerization/multimerization is a common feature for a number of PDZ domain proteins. It is possible that a PDZ domain may dimerize and/or multimerize via the βE/αB-hydrophobic surface seen in GRIP1 PDZ7 (30Zhang Q. Fan J.S. Zhang M. J. Biol. Chem. 2001; 276: 43216-43220Google Scholar, 31Xu X.Z. Choudhury A., Li, X. Montell C. J. Cell Biol. 1998; 142: 545-555Google Scholar, 32Fouassier L. Duan C. Feranchak A.P. Yun C.H. Sutherland E. Simon F. Fitz J.G. Doctor R.B. Hepatology. 2001; 33: 166-176Google Scholar, 33Lau A.G. Hall R.A. Biochemistry. 2001; 40: 8572-8580Google Scholar). Further work is ongoing to directly prove or refute whether some of the above mentioned PDZ domain multimerizations are indeed mediated via the βE/αB-hydrophobic surface seen in GRIP1 PDZ7. In addition, it will be interesting to experimentally test whether other atypical PDZ domain-mediated interactions such as CLP-36 PDZ binding to spectrin-like repeats of α-actinin (39Vallenius T. Luukko K. Makela T.P. J. Biol. Chem. 2000; 275: 11100-11105Google Scholar) are mediated via the βE/αB-hydrophobic surface. In summary, the three-dimensional structure of GRIP1 PDZ7 determined in this work shows that the PDZ domain has a deformed βB/αB-groove that is not likely to be capable of binding to carboxyl peptide ligands. Instead, GRIP1 PDZ7 contains a distinct hydrophobic surface largely composed of the residues from the βE to αB regions of the protein. Unexpectedly, GRIP1 PDZ7 binds to its target GRASP-1 via the βE/αB-hydrophobic surface instead of the conventional target-binding βB/αB-groove. The novel protein-binding surface revealed in this work may help us to understand the molecular basis of frequently observed PDZ domain-mediated multimerization of multimodular scaffold proteins. We thank Drs. Richard Huganir and Morgan Sheng for providing cDNA constructs of GRASP-1 and GRIP1 and Dr. Virginia Unkefer for critical reading of the paper.
A new 11(15→1)-abeotaxane diterpene, 7,9-dideacetyltaxayuntin (1), and 11 known taxane diterpenes were isolated from the bark of Taxus yunnanensis. Their structures were determined primarily on the basis of analysis of their 1H NMR, 13C NMR, DEPT,1H−1H COSY, HMQC, HMBC, and mass spectra.
The fast-inactivation process in the hERG channel can be affected by mutations in the pore or S6 domain, similar to the C-type inactivation in the Shaker channel. However, differences in the kinetics and voltage dependence of inactivation between these two channels suggest that different structural determinants may be involved. To explore this possibility, we mutated a serine in the outer mouth region of hERG (S631) to residues of different physicochemical properties and compared the resulting changes in the channel's inactivation process with those resulting from mutations of an equivalent position in the Shaker channel (T449). The most dramatic differences are seen when this position is occupied by a charged residue: S631K and S631E disrupted C-type inactivation in hERG, whereas T449K and T449E facilitate C-type inactivation in Shaker. S631K and S631E also disrupted the K selectivity of hERG pore, a change not seen in T449K or T449E of Shaker. To further study why there are such differences, we replaced S631 with cysteine. This allowed us to manipulate the properties of thiol groups at position 631 and correlate side-chain properties here with changes in channel function. S631C behaved like the wild-type channel when the thiol groups were in the reduced state. Oxidizing thiol groups with H2O2 or modifying them with MTSET or MTSES disrupted C-type inactivation and K selectivity, similar to the phenotype of S631K and S631E. The same thiol-modifying maneuvers did not affect the wild-type channel function. Our results suggest differences in the outer mouth structure between hERG and Shaker, and we propose a "molecular spring" hypothesis to explain these differences.
A computer model is developed for the characterization of Ca/sup 2+/-induced calcium release (CICR) from the sarcoplasmic reticulum (SR) in the rat ventricular cell. The fluid compartment model is configured to describe the trigger Ca/sup 2+/ influx (I/sub Ca,L/) through the membrane of the sarcolemma (SL); the diffusion of Ca/sup 2+/ throughout a small cleft space, which is located between the SL and the junctional sarcoplasmic reticulum (jSR); and a distribution of five ryanodine (Ry)-sensitive Ca/sup 2+/ release channels called the "Ca/sup 2+/-release complex". Each Ry-receptor controlled channel (or RyR channel) is characterized by a 4-state Markovian kinetic scheme. Two Ca/sup 2+/ ions are required to bind to RyR for channel activation, and one Ca/sup 2+/ ion is required for channel inactivation. The model provides both sufficient Ca/sup 2+/-release gain and graded release behavior. The complete model is used to simulate the whole-cell Ca/sup 2+/ transient data, evoked in a voltage clamp test. We also studied the linear relationship between the rising rate of the Ca/sup 2+/ transients and the peak trigger I/sub Ca,L/. The model results suggest that this relationship is the indirect result of 2/sup nd/-order RyR activation dynamics, filtered by fluorescent indicator dye.
Early afterdepolarization (EAD) was studied in isolated ventricular myocytes of guinea pig heart. Under K(+)-free's treatment, most of the myocytes showed hyperpolarization in resting potential and the duration of action potential was prolonged, eventually leading to the appearance of EAD with the second plateau of -76 +/- 3 mV. TTX (10 microM) and verapamil (10 microM) or normal Tyrode's solution abolished the EAD. The background I-V curve showed inward rectifying with a crossover in the level of -80 to -30 mV and the reversal potential shifted from -80 to -120 mV when normal Tyrode's solution was changed to K(+)-free solution. The changes of I-V relationship of inward current IK) were similar to the background ones except without crossover. The delayed rectifier current (IK) was inhibited significantly under K(+)-free treatment. Low K+ (2.7 mM) superfusion was able to induce EAD in only a few cases (4/15). Adding Cs+ (5.0 mM) into low K+ solution, EAD was induced in almost every case. The background I-V curve was inhibited slightly under low K+ superfusion, but was inhibited significantly with a remarkable crossover under low K+ and Cs+ treatment. The changes of I-V curve IK1 under low K+ or low K+ and Cs+ treatment were similar to the changes of background ones. There were no significant changes in the IK under low K+ superfusion while a remarkable inhibition occurred under low K+ and Cs+ treatment. It was suggested that both IK1 and IK were involved in the induction of EAD under K(+)-free or Cs+ treatment in guinea pig ventricular myocytes.
A novel approach is proposed for suppression of diagonal peaks in 15N- and 13C-edited NOESY spectroscopy based on subtracting a spectrum with only diagonal peaks from the conventional NOESY spectrum. This method can be applied to most heteronuclear-edited NOESY experiments. It is far more sensitive than the TROSY-based approach for biomolecules with little TROSY effect, and nearly complete suppression of diagonal peaks can be achieved. The method has been demonstrated on samples of 15N-labeled calmodulin (17 kDa), 13C-labeled DdCAD-1 (24 kDa), and 13C-labeled hemoglobin (65 kDa), showing that cross-peaks very close to diagonals can be assigned reliably based on the difference spectra.
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