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• W2109623198 abstract "The minus-ended microtubule motor cytoplasmic dynein contains a number of low molecular weight light chains including the 14-kDa Tctex-1. The assembly of Tctex-1 in the dynein complex and its function are largely unknown. Using partially deuterated, 15N,13C-labeled protein samples and transverse relaxation-optimized NMR spectroscopic techniques, the secondary structure and overall topology of Tctex-1 were determined based on the backbone nuclear Overhauser effect pattern and the chemical shift values of the protein. The data showed that Tctex-1 adopts a structure remarkably similar to that of the 8-kDa light chain of the motor complex (DLC8), although the two light chains share no amino acid sequence homology. We further demonstrated that Tctex-1 binds directly to the intermediate chain (DIC) of dynein. The Tctex-1 binding site on DIC was mapped to a 19-residue fragment immediately following the second alternative splicing site of DIC. Titration of Tctex-1 with a peptide derived from DIC, which contains a consensus sequence R/KR/KXXR/K found in various Tctex-1 target proteins, indicated that Tctex-1 binds to its targets in a manner similar to that of DLC8. The experimental results presented in this study suggest that Tctex-1 is likely to be a specific cargo adaptor for the dynein motor complex. The minus-ended microtubule motor cytoplasmic dynein contains a number of low molecular weight light chains including the 14-kDa Tctex-1. The assembly of Tctex-1 in the dynein complex and its function are largely unknown. Using partially deuterated, 15N,13C-labeled protein samples and transverse relaxation-optimized NMR spectroscopic techniques, the secondary structure and overall topology of Tctex-1 were determined based on the backbone nuclear Overhauser effect pattern and the chemical shift values of the protein. The data showed that Tctex-1 adopts a structure remarkably similar to that of the 8-kDa light chain of the motor complex (DLC8), although the two light chains share no amino acid sequence homology. We further demonstrated that Tctex-1 binds directly to the intermediate chain (DIC) of dynein. The Tctex-1 binding site on DIC was mapped to a 19-residue fragment immediately following the second alternative splicing site of DIC. Titration of Tctex-1 with a peptide derived from DIC, which contains a consensus sequence R/KR/KXXR/K found in various Tctex-1 target proteins, indicated that Tctex-1 binds to its targets in a manner similar to that of DLC8. The experimental results presented in this study suggest that Tctex-1 is likely to be a specific cargo adaptor for the dynein motor complex. Tctex-1 (t-complextestis-expressed-1) was originally identified as a multigene family that maps to the t-complex, a large region of mouse chromosome 17 (known as t-haplotypes) containing four nonoverlapping inversions that suppress recombination (1Lader E. Ha H.S. O'Neill M. Artzt K. Bennett D. Cell.. 1989; 58: 969-979Google Scholar). Male +/t-heterozygotes transmit the mutant chromosome at >99% frequency to their progeny, a non-Mendelian phenomenon known as transmission ratio distortion, and malet/t-homozygotes are completely sterile. The aberrant expression of Tctex-1 in the t-haplotype mice (4-fold overexpressed in +/t and 8-fold in t/t) was suggested to be functionally related to sterility and transmission ratio distortion (1Lader E. Ha H.S. O'Neill M. Artzt K. Bennett D. Cell.. 1989; 58: 969-979Google Scholar). This Tctex-1-related meiotic drive hypothesis was supported by the finding that Tctex-1 is a light chain of cytoplasmic as well as flagellar inner arm dynein complexes (2King S.M. Dillman I.I.I., J.F. Benashski S.E. Lye R.J. Patel-King R.S. Pfister K.K. J. Biol. Chem... 1996; 271: 32281-32287Google Scholar, 3Harrison A. Olds-Clarke P. King S.M. J. Cell Biol... 1998; 140: 1137-1147Google Scholar). Cytoplasmic dynein is a microtubule-based molecular motor involved in various intracelluar motile events including retrograde vesicle transport, axonal transport, mitotic spindle positioning, and nuclear migration (4King S.M. Biochim. Biophys. Acta.. 2000; 1496: 60-75Google Scholar, 5Karki S. Holzbaur E.L. Curr. Opin. Cell Biol... 1999; 11: 45-53Google Scholar, 6Hirokawa N. Science.. 1998; 279: 519-526Google Scholar, 7Vallee R.B. Sheetz M.P. Science.. 1996; 271: 1539-1544Google Scholar). The dynein motor is a multicomponent protein and contains two heavy chains (∼530 kDa), two intermediate chains (DIC1; ∼74 kDa), four light intermediate chains (∼50–60 kDa), and several light chains (DLC; 8, 14, and 22 kDa) (4King S.M. Biochim. Biophys. Acta.. 2000; 1496: 60-75Google Scholar). Dynein heavy chains directly attach the dynein complex to microtubules and contain ATPase activity, which is required for force generation of the motor. DIC is involved in linking the motor to vesicle-based cargoes by mediating the interaction between dynein and dynactin (8Vaughan K.T. Vallee R.B. J. Cell Biol... 1995; 131: 1507-1516Google Scholar, 9Ma S. Trivinos-Lagos L. Graf R. Chisholm R.L. J. Cell Biol... 1999; 147: 1261-1273Google Scholar, 10Susalka S.J. Hancock W.O. Pfister K.K. Biochim. Biophys. Acta.. 2000; 1496: 76-88Google Scholar). The functions of other dynein subunits are largely unknown because of the complexity of the motor complex. In addition to functioning as a stoichiometric subunit of the cytoplasmic dynein complex (2King S.M. Dillman I.I.I., J.F. Benashski S.E. Lye R.J. Patel-King R.S. Pfister K.K. J. Biol. Chem... 1996; 271: 32281-32287Google Scholar, 11King S.M. Barbarese E. Dillman I.I.I., J.F. Benashski S.E. Do K.T. Patel-King R.S. Pfister K.K. Biochemistry.. 1998; 37: 15033-15041Google Scholar), Tctex-1 was also found to interact with a number of cellular proteins of diverse function. Tctex-1 interacts with the N-terminal region of Doc2, and the interaction between these two proteins was suggested to be involved in the dynein-mediated vesicle transport (12Nagano F. Orita S. Sasaki T. Naito A. Sakaguchi G. Maeda M. Watanabe T. Kominami E. Uchiyama Y. Takai Y. J. Biol. Chem... 1998; 273: 30065-30068Google Scholar). Tctex-1 was also shown to interact with a 19-residue fragment located at the extreme N terminus of p59fyn Src family tyrosine protein kinase (13Campbell K.S. Cooper S. Dessing M. Yates S. Buder A. J. Immunol... 1998; 161: 1728-1737Google Scholar). Colocalization of Tctex-1 and Fyn at the cleavage furrow and mitotic spindles in T cell hybridomas undergoing cytokinesis points to possible roles of dynein in cell cycle control. Interaction of a lymphocyte surface glycoprotein CD5 with Tctex-1 was suggested to be linked to internalization of CD5 (14Bauch A. Campbell K.S. Reth M. Eur. J. Immunol... 1998; 28: 2167-2177Google Scholar). Additionally, Tctex-1 was recently reported to interact directly with the cytoplasmic tail of rhodopsin. This interaction was suggested to be responsible for the transport of rhodopsin-laden vesicles across the inner segment to the base of the connecting cilium (15Tai A.W. Chuang J.-Z. Bode C. Wolfrum U. Sung C.-H. Cell.. 1999; 97: 877-887Google Scholar). The discovery of a large number of functionally unrelated Tctex-1-binding proteins suggests that Tctex-1 is likely to function as an adaptor serving to link specific cargoes to the dynein motor. Because of limited biochemical and structural characterization of Tctex-1, the molecular mechanisms governing the interactions between the protein and its binding partners are unknown. In this work, we performed a detailed structural characterization of Tctex-1 by NMR spectroscopic techniques. Using purified recombinant proteins, we showed that Tctex-1 binds directly to the intermediate chain of cytoplasmic dynein, and the Tctex-1 binding site was mapped to a short stretch of amino acid residues in the N-terminal region of DIC. We further demonstrate that Tctex-1 shares remarkable structural and target binding similarities with DLC8, although the two light chains share no amino acid sequence homology. The cDNA encoding mouse Tctex-1 was generously provided by Prof. Yoshimi Takai. The Tctex-1 expression plasmid was constructed by inserting polymerase chain reaction-amplified Tctex-1 gene fragment into the NdeI and BamHI sites of the pET3a vector (Novagen). The full-length mouse DIC gene was constructed by assembling three overlapping genomic clones as described in our earlier work (31Lo K.W.-H. Naisbitt S. Fan J.-S. Sheng M. Zhang M. J. Biol. Chem.. 2001; 276: 14059-14066Google Scholar). The N-terminal region of DIC (amino acids 1–213) was inserted into theBamHI and EcoRI sites of the pGEX-4T-1 vector (Amersham Pharmacia Biotech) for expression of a GST fusion protein. Various truncation, deletion, and point mutations of this DIC fragment were constructed using standard polymerase chain reaction and cloning techniques. The C-terminal WD repeats (amino acids 214–628) were constructed as an N-terminal His-tagged protein (31Lo K.W.-H. Naisbitt S. Fan J.-S. Sheng M. Zhang M. J. Biol. Chem.. 2001; 276: 14059-14066Google Scholar). Tctex-1 was expressed by transforming the pET3a vector containing the Tctex-1 gene intoEscherichia coli BL21(DE3) host cells. A single colony ofE. coli cells harboring the expression plasmid was inoculated into 50 ml of LB with 100 μg/ml ampicillin (LBA). The cell culture was incubated overnight at 37 °C and then inoculated into 1 liter of fresh LBA medium. Tctex-1 expression was induced by the addition of isopropyl-1-thio-β-d-galactopyranoside when the A600 of the culture reached ∼0.6. Pelleted cells from 2 liters of culture were resuspended in 50 ml of buffer A (50 mm Tris-HCl, pH 7.9, 5 mmβ-mercaptoethanol, and 1 mm EDTA) containing 0.1 mm phenylmethylsulfonyl fluoride and lysed by sonication. The lysate was centrifuged at 18,000 rpm (Sorvall SS34 rotor) for 30 min at 4 °C, and the supernatant was loaded onto a 50-ml DEAE-Sepharose Fast Flow column (Amersham Pharmacia Biotech). The column was washed with buffer A until the A280reading was steady, and the column was then eluted with 200 ml of buffer A with a linear NaCl gradient of 0–0.3 m. Fractions containing Tctex-1 were pooled and concentrated to about 8 ml before loading onto a Sephacryl-100 (Amersham Pharmacia Biotech) gel filtration column preequilibrated with buffer A containing 0.5m NaCl. The eluted fractions containing Tctex-1 were pooled and dialyzed against buffer A before loading onto a Mono Q HR 10/10 column (Amersham Pharmacia Biotech). The Mono Q column was washed with buffer A, and Tctex-1 was eluted using 70 ml of the same buffer with an NaCl gradient of 0–0.35 m. The purified Tctex-1 was dialyzed against buffer A and stored at −80 °C. Various forms of GST-fused DIC mutant proteins were expressed inE. coli cells. In a typical purification procedure, cell pellet from 1 liter of culture was resuspended in 25 ml of PBS buffer (140 mm NaCl, 2.7 mm KCl, 10 mmNa2HPO4, and 1.8 mmKH2PO4) containing 0.1 mmphenylmethylsulfonyl fluoride. The GST fusion proteins were purified following the instructions of the manufacturer (Amersham Pharmacia Biotech). The purified proteins were dialyzed extensively against PBS prior to binding experiments. All NMR samples were prepared by concentrating purified Tctex-1 using a Centriprep-3 (Amicon) ultrafiltration device. The protein was exchanged into the desired NMR sample buffer (50 mm Tris-d11, pH 7.0, 2 mm dithiothreitol-d10, and 10% D2O), with or without 0.4 m KCl, using ultrafiltration. Preparation of the partially deuterated Tctex-1 followed a protocol described previously with slight modifications (16Rosen M.K. Gardner K.H. Willis R.C. Parris W.E. Pawson T. Kay L.E. J. Mol. Biol... 1996; 263: 627-636Google Scholar). Briefly, a single colony of E. coli cells harboring the Tctex-1 expression plasmid was inoculated into 50 ml of LBA and incubated overnight at 37 °C. About 4 ml of this overnight culture was inoculated into 200 ml of15N/H2O/[13C]glucose (for2H,15N,13C triple labeling) or15N/H2O/[12C]glucose (for2H,15N double labeling) M9 medium with an initial A600 value of ∼0.04. The cells were incubated at 37 °C until the A600 value reached ∼0.5. The cells were spun down at 4 °C and then gently resuspended into 200 ml of 15N, 80% D2O, [13C]glucose or 15N, 80% D2O, [12C]glucose M9 medium. The suspension was diluted five times into 800 ml of 15N, 80% D2O, [13C]glucose or 15N, 80% D2O, [12C]glucose M9 medium to give anA600 value of ∼0.1. The cultures were incubated at 37 °C until the A600 reading reached ∼0.5, and Tctex-1 expression was induced by the addition of isopropyl-1-thio-β-d-galactopyranoside to a final concentration of 0.5 mm. The bacterial culture was incubated overnight at 16 °C before the cells were harvested. All NMR experiments were performed on a four-channel Varian Inova 750-MHz spectrometer equipped with az-axis pulsed field gradient unit and an actively shielded triple resonance probe. Sequential backbone assignment of Tctex-1 was completed using three pairs of TROSY-enhanced triple resonance experiments (17Pervushin K. Braun D. Fernandez C. Wuthrich K. J. Biomol. NMR.. 2000; 17: 195-202Google Scholar), namely HN(CA)CB and HN(COCA)CB; HNCA and HN(CO)CA; HNCO and HN(CA)CO (18Kay L.E. Gardner K.H. Curr. Opin. Struct. Biol... 1997; 7: 722-731Google Scholar, 19Kay L.E. Biochem. Cell Biol... 1997; 75: 1-15Google Scholar, 20Clore G.M. Gronenborn A.M. Trends Biotechnol... 1998; 16: 22-34Google Scholar) on an 15N,13C uniformly, 80% 2H-partially labeled Tctex-1 sample. An1H-15N HSQC-NOESY experiment (21Zhang O. Forman-Kay J.D. Shortle D. Kay L.E. J. Biomol. NMR.. 1997; 9: 181-200Google Scholar) recorded on an 15N-lableled Tctex-1 sample with a mixing time of 90 ms was used to confirm the sequential assignment. Backbone HN-HN NOEs were obtained using a 1H-15N HSQC-NOESY-HSQC experiment (22Ikura M. Bax A. Clore G.M. Gronenborn A.M. J. Am. Chem. Soc... 1990; 112: 9020-9022Google Scholar) recorded on an15N-uniformly, 80% 2H-partially labeled Tctex-1 sample with a mixing time of 150 ms. The protein concentrations of all NMR samples were ∼1.0 mm. For each pull-down assay, 15 μl of a 75% slurry of GST-Sepharose 4B was first washed three times with 0.5 ml of PBS for equilibration. About 0.1 mg of the GST fusion proteins (250 μl) was added, and the suspension was agitated at 4 °C for 1 h. The GST-protein-loaded beads were washed four times with 0.5 ml of PBS to remove any unbound protein. A total of 100 μl of Tctex-1 (35 μm) solution was added, and the suspension was agitated at 4 °C for an additional 2 h. The beads were then washed four times with 0.5 ml of PBS to remove any unbound Tctex-1 and subsequently boiled with 20 μl of 2 × SDS-polyacrylamide gel sample buffer. The intensities of the Tctex-1 bands on the SDS-polyacrylamide gel were used to judge the strength of the interaction between Tctex-1 and various GST-DIC fusion proteins. Competition between DLC8 and Tctex-1 for DIC binding was examined by adding 100-μl aliquots of DLC8 solutions of increasing concentration to suspensions of Tctex-1 premixed with GST-DIC-loaded GSH-Sepharose beads (at a 1:1 molar ratio of Tctex-1 to GST-DIC). The mixtures were agitated for an additional 2 h, and the beads were subsequently washed four times with 0.5 ml of PBS. The relative amounts of Tctex-1 and DLC8 pelleted by the GSH-Sepharose beads were used to judge possible competition between Tctex-1 and DLC8 for DIC binding. To uncover the molecular basis of Tctex-1's cellular function, we characterized the structure of the protein in solution by NMR spectroscopic techniques. An efficient Tctex-1 production and purification procedure was developed, and large quantities of various forms of stable isotope-labeled Tctex-1 were obtained for NMR structural studies. The1H-15N HSQC spectrum of 15N-labeled Tctex-1 showed that the majority of the backbone resonances of the protein were extraordinary broad at various protein concentrations tested (0.1–1 mm). The severe line broadening is likely to be a combined effect of slow to intermediate time scale conformational exchange and nonspecific protein aggregation (for more detail, see below). An array of triple resonance experiments on15N,13C-labeled Tctex-1, aiming to obtain the backbone assignment of the protein, failed. Both 15N- and13C-separated three-dimensional NOESY experiments of Tctex-1 showed unusually low amounts of NOE cross-peaks. To circumvent sample aggregation and conformational exchange-induced T2 shortening, partial deuteration (23Grzesiek S. Anglister J. Ren H. Bax A. J. Am. Chem. Soc... 1993; 115: 4369-4370Google Scholar, 24Yamazaki T. Lee W. Arrowsmith C.H. Muhandiram D.R. Kay L.E. J. Am. Chem. Soc... 1994; 116: 11655-11666Google Scholar) of the protein (∼80%) together with TROSY techniques (17Pervushin K. Braun D. Fernandez C. Wuthrich K. J. Biomol. NMR.. 2000; 17: 195-202Google Scholar) were used for NMR characterization of Tctex-1 in solution. Using a combination of TROSY-based HNCA, HN(CO)CA, HN(CA)CB, and HN(COCA)CB triple resonance experiments on a2H,13C,15N-triple labeled Tctex-1 sample, we were able to obtain essentially complete backbone resonance assignment of the protein. A 15N-separated three-dimensional NOESY experiment recorded on a15N-labeled Tctex-1, and an HSQC-NOESY-HSQC experiment recorded on a 2H,15N-labeled protein sample were used to confirm the assignment obtained by the triple resonance experiments. Fig. 1 A shows the TROSY-HSQC spectrum of Tctex-1 with each backbone amide resonance labeled with amino acid residue name and number. The completion of the backbone chemical shift assignment allowed us to determine the secondary structure of Tctex-1 using a slightly modified chemical shift index approach (25Wishart D.S. Sykes B.D. Richards F.M. Biochemistry.. 1992; 31: 1647-1651Google Scholar). As both 13Cα and 13Cβ shifts are sensitive to secondary structure, and as they shift to opposite fields in a given secondary structure, a combined13Cα/13Cβ secondary shift presented by subtracting the 13Cβ secondary shift of a residue from its 13Cα secondary shift enhances secondary structure-induced chemical shift changes of the protein (Fig.1 B). Presentation of the secondary structure chemical shifts using combined 13Cα/13Cβ shifts has the additional advantage of canceling potential secondary shift errors resulted from chemical shift referencing. Based on the data in Fig.1 B, we conclude that Tctex-1 is composed of two long N-terminal α-helices (α1 and α2, with starting and ending residues labeled in Fig. 1 B) followed by four β-strands (β1 to β4). The secondary structure of Tctex-1 derived from the chemical shift data was supported by the backbone NOE patterns of the protein derived from the two three-dimensional 15N-NOESY experiments (HSQC-NOESY-HSQC and 15N-separate NOESY, data not shown). The chemical shift values of the residues (Asp-3 to Thr-10) N-terminal to α1 indicate that this fragment of Tctex-1 appears to assume a random coil-like structure. The random coil structure of the N-terminal fragment inferred from chemical shift data is also supported by the exceptionally narrow line widths of these residues and the lack of detectable long range backbone NOE of the region. The secondary structure of Tctex-1 shown in Fig. 1 B is remarkably similar to that of another dynein light chain, DLC8 (26Liang J. Jaffrey S.R. Guo W. Snyder S.H. Clardy J. Nat. Struct. Biol... 1999; 6: 735-740Google Scholar, 27Fan J.-S. Zhang Q. Tochio H. Li M. Zhang M. J. Mol. Biol... 2001; 306: 97-108Google Scholar), although the two light chains have very limited amino acid sequence homology. Next, we determined the overall topology of Tctex-1. To obtain maximal backbone amide resolution, we used an HSQC-NOESY-HSQC experiment recorded on 2H,15N-labeled Tctex-1 to detect backbone HN-HN NOEs. Large numbers of long range, inter-β-strand NOEs were used to determine the folding topology of the Tctex-1 β-strands (Fig. 2). The four β-strands of Tctex-1 form an antiparallel β-sheet structure with a β3-β4-β1-β2 arrangement (Fig. 2). Several unambiguous inter-α-helical HN-HN NOEs spanning the entire helices (e.g. backbone amide NOEs between Asp-15 and Thr-55, Ile-20 and Thr-50, and Gly-30 and Val-39) indicate that the two α-helices of Tctex-1 are antiparallel to each other. The detection of long range backbone amide NOEs between amino acid residues from the α-helices and β-strands (e.g.NOEs between Ala-28 and Cys-104, Asn-32 and Trp-97, Asn-45 and Cys-67, and Leu-54 and Asp-86) indicate that the two helices pack against the antiparallel β-sheet of the protein (see Fig. 6, inset). The overall topology of Tctex-1 is again very similar to that of DLC8 (26Liang J. Jaffrey S.R. Guo W. Snyder S.H. Clardy J. Nat. Struct. Biol... 1999; 6: 735-740Google Scholar, 27Fan J.-S. Zhang Q. Tochio H. Li M. Zhang M. J. Mol. Biol... 2001; 306: 97-108Google Scholar).Figure 6Interaction of Tctex-1 with a DIC peptide. The figure shows an overlay plot of TROSY-HSQC spectra of Tctex-1 at the starting (blue) and end (red) points of titration with an 11-residue peptide (LGRRLHKLGVS) corresponding to the N-terminal part of the Tctex-1 binding domain of DIC. The concentration of Tctex-1 (15N-, 80% 2H-labeled) is 0.4 mm, and the protein was dissolved in 50 mm Tris-d11, pH 7.0, 2 mm dithiothreitol-d10. The molar ratio of the DIC peptide to Tctex-1 at the final point of the titration is 2:1. The residues that show significant chemical shift changes upon the addition of the DIC peptide are marked with open boxes, and these amino acid residues are mapped to the topology structure of Tctex-1 shown as an inset in the figure. The dashed lines in the topology diagram of Tctex-1 are used to indicate the uncertainty of the connections of the β-strands due to potential domain swapping of the Tctex-1 dimer.View Large Image Figure ViewerDownload (PPT) Earlier biochemical experiments showed that Tctex-1 and DIC cofractionated from a KI-treated cytoplasmic dynein complex preparation, suggesting that Tctex-1 and DIC might interact directly with each other (2King S.M. Dillman I.I.I., J.F. Benashski S.E. Lye R.J. Patel-King R.S. Pfister K.K. J. Biol. Chem... 1996; 271: 32281-32287Google Scholar, 11King S.M. Barbarese E. Dillman I.I.I., J.F. Benashski S.E. Do K.T. Patel-King R.S. Pfister K.K. Biochemistry.. 1998; 37: 15033-15041Google Scholar). We used purified recombinant proteins to test direct interaction between Tctex-1 and DIC. A GST-fused fragment spanning residues 1–213 of DIC was found to interact robustly with Tctex-1 in the GSH-Sepharose pull-down assay (Fig.3 B, lane 3), indicating that Tctex-1 and DIC can indeed interact with each other directly. As expected, purified, recombinant full-length DIC was also able bind to Tctex-1 (data not shown). In contrast, the C-terminal part of DIC which contains the highly conserved Trp-Asp repeats failed to bind to Tctex-1 (data not shown). We then mapped the exact Tctex-1 binding site of DIC by creating a series of DIC truncation mutations (Fig. 3 A). A 19-residue fragment (residues 124–142) of DIC was identified as sufficient for binding to Tctex-1 (for the amino acid sequence of the peptide fragment, see Fig. 3 A). Given the overall structural similarities between Tctex-1 and DLC8, we suspected that Tctex-1 might also bind to a short peptide fragment, as does DLC8 (27Fan J.-S. Zhang Q. Tochio H. Li M. Zhang M. J. Mol. Biol... 2001; 306: 97-108Google Scholar). To localize the precise Tctex-1 binding site further, we deleted a positive charged, 5-residue fragment (RRLHK) located at the N-terminal end of the 19-residue Tctex-1 binding fragment of DIC (Fig. 4). Deletion of the 5-residue fragment completely abolished the interaction between DIC and Tctex-1, indicating that this 5-residue cassette plays an important role in supporting the Tctex-1·DIC complex formation. However, this 5-residue fragment alone is not sufficient for effective binding to Tctex-1 because a GST fusion peptide containing this 5-residue fragment plus a few amino acid residues at both ends was not able to pull down Tctex-1 (Fig. 4 B, lane 5). These data indicate that both the N- and C-terminal parts of the 19-residue DIC fragment are required for the Tctex-1·DIC complex formation. We next set out to identify the DIC binding region in Tctex-1. To simplify the experiment, we used a synthetic peptide corresponding to the full-length Tctex-1 binding domain of DIC to titrate with partially deuterated 15N-labeled Tctex-1. Unfortunately, the 1H-15N HSQC of Tctex-1 in the presence of this peptide became exceedingly broad and beyond interpretation, suggesting a severe aggregation of the protein-peptide complex (data not shown). Inspection of the amino acid sequences of a number of Tctex-1 binding regions in various targets revealed a short stretch of consensus sequence of R/KR/KXXR/K (Fig.5). Although an 11-residue DIC peptide containing the consensus sequence fused to GST was not able to pull down Tctex-1 (the assay requires high affinity binding between the two proteins; Fig. 3), we anticipated that NMR titration of Tctex-1 with the same 11-residue peptide would allow us to detect the possible interaction between the protein and peptide (even if the interaction were relatively weak). To test this possibility, we titrated15N-labeled Tctex-1 with the 11-residue synthetic peptide (LGRRLHKLGVS from residue 124 to 134 of DIC, Fig. 4). Significant chemical shift changes of a number of backbone amide cross-peaks of Tctex-1 were observed during the titration, indicating that the 11-residue DIC peptide can indeed bind to the protein. Fig.6 shows an overlay plot of the HSQC spectra of Tctex-1 at the start (blue) and end (red) points of the titration. The amino acid residues that display significant chemical shift changes are labeled, and these amino acid residues are likely to be those involved in the peptide binding. The inset of Fig. 6 shows the distribution of the amino acids that undergo significant peptide-induced chemical shift changes, and these residues are found mainly at the end of β2 (Cys-83, Phe-84, Trp-85, and Asp-86) and the β2/β3-loop (Thr-89, Asp-90, and Gly-91) of Tetex-1. In addition, several residues that are in close proximity to this region (e.g. Thr-55 and Lys-56 at the end of α1, and Phe-61 and Lys-62 at the start of β1) also experience some chemical shift changes. Based on the data in Fig. 6, we conclude that the β2-strand and the β2/β3-loop are the main DIC binding region in Tctex-1. To assess the potential roles of the conserved amino acids in the consensus R/KR/KXXR/K sequence of DIC in Tctex-1 binding, we performed a systematic mutational analysis. The data indicate that no single residue in the consensus sequence (RRLHK) plays a dominant role in supporting the Tctex-1·DIC complex formation (Fig. 7). The second arginine residue in the consensus sequence appears to make a slightly larger contribution to the complex formation (Fig. 7 B, lane 5). However, deletion of the whole consensus sequence resulted in complete disruption of the Tctex-1·DIC complex (Fig. 4), suggesting that these amino acid residues may function additively in binding to Tctex-1. The DIC truncation experiment showed that residues C-terminal to the consensus RRLHK sequence (i.e. LGVSKVTQVDFL) are also involved in Tctext-1 binding (Fig. 4). Consistent with this, mutation of Leu-131 to Asn greatly diminished the interaction between Tctex-1 and DIC (Fig. 8, lane 9). However, a combination of the consensus RRLHK sequence and Leu-131 is not sufficient for strong binding because the GST-fused peptide containing both the consensus sequence and Leu-131 was not able to form a stable complex with Tctex-1 (Fig. 5, lane 5). Further mutational analysis will be required to identify the structural features that are essential for Tctex-1 binding. The Tctex-1 binding site (residues 124–142, LGRRLHKLGVSKVTQVDFL) identified in this study and the DLC8 binding site (151–155, KETQT) in DIC are immediately next to each other in the amino acid sequence of DIC (Fig. 9, and Ref. 31Lo K.W.-H. Naisbitt S. Fan J.-S. Sheng M. Zhang M. J. Biol. Chem.. 2001; 276: 14059-14066Google Scholar). Additionally, the Tctex-1 binding site contains a KVTQV sequence that is similar to the KETQT DLC8 binding motif (31Lo K.W.-H. Naisbitt S. Fan J.-S. Sheng M. Zhang M. J. Biol. Chem.. 2001; 276: 14059-14066Google Scholar). Therefore, there is a possibility that the binding of Tctex-1 and DLC8 on DIC are mutually exclusive. To test this possibility, we performed a binding competition experiment as shown in Fig. 8. In this experiment, equal molar amounts of GST-DIC and Tctex-1 were mixed with increasing molar ratio amounts of DLC8 (Fig. 8). The GST-DIC·Tctex-1·DLC8 ternary complex was pelleted by GSH-Sepharose beads and subsequently analyzed by SDS-polyacrylamide gel electrophoresis. Data in Fig. 8clearly demonstrate that excess DLC8 does not displace Tctex-1 from DIC, indicating that Tctex-1 and DLC8 can bind simultaneously to DIC. In a reverse experiment, we found that excess Tctex-1 does not compete with DLC8 for binding to DIC, consistent with our conclusion that the Tctex-1 binding site and the DLC8 binding site on DIC are mutually independent (data not shown). As a part of our continuing effort to understand the structure and function of cytoplasmic DLCs, we performed a detailed structural analysis of Tctex-1 in this study. Using multidimensional TROSY-enhanced NMR spectroscopic techniques, we determined the secondary structure and the topology of Tctex-1 in solution. Tctex-1 shares a remarkably similar structure with DLC8 both at the secondary structure and folding topology levels (26Liang J. Jaffrey S.R. Guo W. Snyder S.H. Clardy J. Nat. Struct. Biol... 1999; 6: 735-740Google Scholar, 27Fan J.-S. Zhang Q. Tochio H. Li M. Zhang M. J. Mol. Biol... 2001; 306: 97-108Google Scholar), although the two proteins display very limited amino acid sequence homology. Analogous to DLC8, both gel filtration and chemical cross-linking studies showed that Tctex-1 can exist as a dimer in solution (data not shown). A full structural determination of Tctex-1 and/or its complex with a target peptide is required to answer how the Tctex-1 dimer is assembled in solution. Detailed comparisons of the structures of Tctex-1 and DLC8, both in apo- and target-bound forms, will help to elucidate how the two structurally similar light chains distinguish their respective targets. In DLC8, target peptides bind to the protein by augmenting the β-sheet via the β2-strand in an antiparallel fashion (26Liang J. Jaffrey S.R. Guo W. Snyder S.H. Clardy J. Nat. Struct. Biol... 1999; 6: 735-740Google Scholar, 27Fan J.-S. Zhang Q. Tochio H. Li M. Zhang M. J. Mol. Biol... 2001; 306: 97-108Google Scholar). Our NMR studies indicated that Tctex-1 binds a target peptide derived from DIC using a mechanism similar to that of DLC8 (i.e. the β2-strand and the β2/β3-loop are the major target binding regions for both proteins) (Fig. 6). It is possible that the short DIC peptide (LGRRLHKLGVS) used in the NMR titration experiment may also bind to Tctex-1 by pairing with the β2-strand of the protein. However, we also notice significant differences between Tctex-1 and DLC8 in their respective target bindings. For example, DLC8 is capable of binding to a KXTQT motif-containing peptide, which is as short as 9 residues, with high affinity and specificity (27Fan J.-S. Zhang Q. Tochio H. Li M. Zhang M. J. Mol. Biol... 2001; 306: 97-108Google Scholar, 31Lo K.W.-H. Naisbitt S. Fan J.-S. Sheng M. Zhang M. J. Biol. Chem.. 2001; 276: 14059-14066Google Scholar). In contrast, the Tctex-1-binding peptide is likely to be significantly longer. An 11-residue DIC peptide containing a consensus R/KR/KXXR/K sequence with a few amino acid residue extensions at both ends was not able to bind to Tctex-1 with high affinity (Fig. 5). To achieve high affinity binding, an additional ∼10 amino acids C-terminal to the RRLHK sequence of DIC are required. Given the length of the Tctex-1-binding peptide, the target binding site on Tctex-1 is likely to involve regions other than the β2-strand and the β2/β3-loop of the protein. Unfortunately, because of the poor behavior of Tctex-1 complexed with a synthetic peptide containing the complete Tctex-1 binding region of DIC in solution, we were not able to determine the exact regions of Tctex-1 which are involved in target binding. Although the amino acid residues downstream of the consensus R/KR/KXXR/K sequence are necessary for effective binding of Tctex-1 and its targets, sequence alignment analysis shows that no obvious homology can be observed in these regions (Fig. 6). The sequence downstream of the consensus R/KR/KXXR/K sequence in DIC is a mix of hydrophobic and hydrophilic residues (LGVSKVTQV). The same region in CD5 is composed mainly of hydrophilic residues; only one hydrophobic residue occurs (KFRQKKQRQ). It seems that Tctex-1 is capable of binding to a number of targets with diverse amino acid sequences, a phenomenon that was also observed for DLC8 (27Fan J.-S. Zhang Q. Tochio H. Li M. Zhang M. J. Mol. Biol... 2001; 306: 97-108Google Scholar). Mutational analysis indicated that polar/charged interactions between the positively charged consensus sequence R/KR/KXXR/K and negatively charged amino acids from Tctex-1 are likely to play important roles in binding specificity because deletion of all three positively charged residues of DIC abolished its binding to Tctex-1. Hydrophobic interactions between amino acid residues downstream of the consensus sequence and Tctex-1 are also expected to provide favorable binding energy for complex formation, as mutation of Leu-131 of DIC to Asn greatly reduced its affinity for Tctex-1 (Fig. 7). Because of alternative splicing of the two DIC genes, there at least five different isoforms of DIC in mammals (8Vaughan K.T. Vallee R.B. J. Cell Biol... 1995; 131: 1507-1516Google Scholar, 10Susalka S.J. Hancock W.O. Pfister K.K. Biochim. Biophys. Acta.. 2000; 1496: 76-88Google Scholar, 28Nurminsky D.I. Nurminskaya M.V. Benevolenskaya E.V. Shevelyov Y.Y. Hartl D.L. Gvozdev V.A. Mol. Cell. Biol... 1998; 18: 6816-6825Google Scholar, 29Crackower M.A. Sinasac D.S. Xia J. Motoyama J. Prochazka M. Rommens J.M. Scherer S.W. Tsui L.C. Genomics.. 1999; 55: 257-267Google Scholar). Fig. 9 shows a schematic diagram of the domain organization of DIC. The function of the C-terminal Trp-Asp repeats is still unknown. The high amino acid sequence homology of the Trp-Asp repeats domain of DICs from both cytoplasmic and axonemal DICs suggests that this domain is likely to be responsible for attaching DICs to the heavy chains of the motor complex. The N-terminal ∼120 residues (including the two alternative splicing sites and the Ser-rich region) of DIC was identified to interact with dynactin. The discovery of the interaction between DIC and dynactin suggested that DIC functions as the adaptor to link the motor complex to its vesicle-based cargoes (8Vaughan K.T. Vallee R.B. J. Cell Biol... 1995; 131: 1507-1516Google Scholar). Following the dynactin binding region of DIC are the Tctex-1 and DLC8 binding regions. Because both light chains are capable of binding a large number of functional unrelated proteins, it is likely that the light chains function as motor adaptors for transporting various specific cargoes. This hypothesis is supported by the observation that the two target binding sites of DLC8 (and also likely Tctex-1) can simultaneously bind to DIC and one of its target proteins. 2K. W.-H. Lo, and M. Zhang, unpublished results. Unlike kinesins and myosins that contain a large number of isoforms, cytoplasmic dynein contains two copies of identical heavy chains with motor activities. It is likely that a combination of various light chains and light intermediate chains as well as intermediate chains allows a single cytoplasmic dynein complex to move a vast number of molecular cargoes along microtubules. The dynactin binding region of DIC contains alternative splicing sites and potential protein phosphorylation sites within the Ser-rich region (Fig. 9). The dynein light chain binding domains in DIC are situated immediately C-terminal to the second alternative splicing region. This means that neither alternative splicing nor phosphorylation of DIC would have any effect on Tctex-1 and DLC8 binding on DIC. A competition binding assay of Tctex-1 and DLC8 on DIC showed that the bindings of Tctex-1 and DLC8 on DIC are mutually independent (Fig. 8), suggesting that both Tctex-1 and DLC8, together with their unique cargoes, can bind to DIC simultaneously. Cytoplasmic dynein contains another light chain called RP3, which shares 52% amino acid identity to Tctex-1. However, RP3 displays distinct target binding properties when compared with Tctex-1. In contrast to what was observed for Tctex-1, RP3 does not interact with rhodopsin (15Tai A.W. Chuang J.-Z. Bode C. Wolfrum U. Sung C.-H. Cell.. 1999; 97: 877-887Google Scholar) or Doc2 (12Nagano F. Orita S. Sasaki T. Naito A. Sakaguchi G. Maeda M. Watanabe T. Kominami E. Uchiyama Y. Takai Y. J. Biol. Chem... 1998; 273: 30065-30068Google Scholar). Tctex-1 and RP3 are regulated differentially in both a developmental and tissue-specific manner. Functionally distinct populations of cytoplasmic dynein may contain different Tctex-1 family light chains. Subcellular localization of specific light chains, including Tctex-1, may also contribute to the functional differences of dynein complexes (30Tai A.W. Chuang J.-Z. Sung C.-H. J. Biol. Chem... 1998; 273: 19639-19649Google Scholar). We do not know whether RP3 directly binds to DIC, or if it does, whether Tctex-1 and RP3 bind to DIC in a mutually exclusive manner. Further work is required to address these questions. We further note that although the amino acid sequence of the DLC8 and Tctex-1 binding region shown in Fig. 9 is highly conserved in cytoplasmic DIC, these sequences are not clearly identifiable in axonemal DICs, suggesting a possible difference in the assembly of the light chains between axonemal and cytoplasmic dyneins. We thank Prof. Yoshimi Takai for providing the Tctex-1 gene and Dr. Jim Hackett for careful reading of the manuscript. dynein intermediate chain(s) dynein light chain glutathioneS-transferase nuclear Overhauser effect spectroscopy heteronuclear single quantum correlation transverse relaxation-optimized NMR spectroscopy" @default.
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