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• W2058746483 abstract "Poly(C)-binding proteins (PCBPs) are important regulatory proteins that contain three KH (hnRNP K homology) domains. Binding poly(C) D/RNA sequences via KH domains is essential for multiple PCBP functions. To reveal the basis for PCBP-D/RNA interactions and function, we determined the structure of a construct containing the first two domains (KH1-KH2) of human PCBP2 by NMR. KH1 and KH2 form an intramolecular pseudodimer. The large hydrophobic dimerization surface of each KH domain is on the side opposite the D/RNA binding interface. Chemical shift mapping indicates both domains bind poly(C) DNA motifs without disrupting the KH1-KH2 interaction. Spectral comparison of KH1-KH2, KH3, and full-length PCBP2 constructs suggests that the KH1-KH2 pseudodimer forms, but KH3 does not interact with other parts of the protein. From NMR studies and modeling, we propose possible modes of cooperative binding tandem poly(C) motifs by the KH domains. D/RNA binding may induce pseudodimer dissociation or stabilize dissociated KH1 and KH2, making protein interaction surfaces available to PCBP-binding partners. This conformational change may represent a regulatory mechanism linking D/RNA binding to PCBP functions. Poly(C)-binding proteins (PCBPs) are important regulatory proteins that contain three KH (hnRNP K homology) domains. Binding poly(C) D/RNA sequences via KH domains is essential for multiple PCBP functions. To reveal the basis for PCBP-D/RNA interactions and function, we determined the structure of a construct containing the first two domains (KH1-KH2) of human PCBP2 by NMR. KH1 and KH2 form an intramolecular pseudodimer. The large hydrophobic dimerization surface of each KH domain is on the side opposite the D/RNA binding interface. Chemical shift mapping indicates both domains bind poly(C) DNA motifs without disrupting the KH1-KH2 interaction. Spectral comparison of KH1-KH2, KH3, and full-length PCBP2 constructs suggests that the KH1-KH2 pseudodimer forms, but KH3 does not interact with other parts of the protein. From NMR studies and modeling, we propose possible modes of cooperative binding tandem poly(C) motifs by the KH domains. D/RNA binding may induce pseudodimer dissociation or stabilize dissociated KH1 and KH2, making protein interaction surfaces available to PCBP-binding partners. This conformational change may represent a regulatory mechanism linking D/RNA binding to PCBP functions. Poly(C)-binding proteins (PCBPs) 2The abbreviations used are: PCBP, poly(C)-binding proteins; KH, K homology; UTR, untranslated region; PDB, Protein Data Bank; R.M.S.D., root mean square deviation. 2The abbreviations used are: PCBP, poly(C)-binding proteins; KH, K homology; UTR, untranslated region; PDB, Protein Data Bank; R.M.S.D., root mean square deviation. are KH (hnRNP-K-homology) domain-containing proteins that specifically recognize poly(C) D/RNA sequences (1Makeyev A.V. Liebhaber S.A. RNA. 2002; 8: 265-278Crossref PubMed Scopus (338) Google Scholar, 2Gamarnik A.V. Andino R. J. Virol. 2000; 74: 2219-2226Crossref PubMed Scopus (183) Google Scholar). There are five PCBPs in mammalian cells: PCBP1-4 and hnRNP K. Each PCBP contains three KH domains: two consecutive domains at the N terminus and a third domain at the C terminus; an intervening sequence of variable length is present between the second and third domains (Fig. 1A). PCBPs regulate gene expression at various levels, including transcription, mRNA processing, mRNA stabilization, and translation, among others. For example, specific binding of hnRNP K and PCBP1 to the single-stranded pyrimidine-rich promoter sequence of the human c-myc gene and mu-opioid receptor (MOR) gene, respectively, activates transcription (3Tomonaga T. Levens D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5830-5835Crossref PubMed Scopus (161) Google Scholar, 4Malik A.K. Flock K.E. Godavarthi C.L. Loh H.H. Ko J.L. Brain Res. 2006; 1112: 33-45Crossref PubMed Scopus (13) Google Scholar). Binding of PCBP1 or PCBP2 to cellular mRNAs harboring tandem poly(C) motifs within the 3′-UTRs stabilize these mRNAs, including α-globin, β-globin, collagen-α1, tyrosine hydroxylase, erythropoietin, rennin, and hTERT mRNAs (5Weiss I. Liebhaber S. Mol. Cell. Biol. 1995; 15: 2457-2465Crossref PubMed Google Scholar, 6Chkheidze A.N. Lyakhov D.L. Makeyev A.V. Morales J. Kong J. Liebhaber S.A. Mol. Cell. Biol. 1999; 19: 4572-4581Crossref PubMed Google Scholar, 7Yu J. Russell J.E. Mol. Cell. Biol. 2001; 21: 5879-5888Crossref PubMed Scopus (71) Google Scholar, 8Stefanovic B. Hellerbrand C. Holcik M. Briendl M. Aliebhaber S. Brenner D. Mol. Cell. Biol. 1997; 17: 5201-5209Crossref PubMed Google Scholar, 9Lindquist J.N. Kauschke S.G. Stefanovic B. Burchardt E.R. Brenner D.A. Nucleic Acids Res. 2000; 28: 4306-4316Crossref PubMed Google Scholar, 10Paulding W.R. Czyzyk-Krzeska M.F. J. Biol. Chem. 1999; 274: 2532-2538Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 11Czyzyk-Krzeska M.F. Bendixen A.C. Blood. 1999; 93: 2111-2120Crossref PubMed Google Scholar, 12Persson P.B. Skalweit A. Mrowka R. Thiele B. Am. J. Physiol. Regul Integr Comp Physiol. 2003; 285: R491-R497Crossref PubMed Scopus (26) Google Scholar, 13Emerald B.S. Chen Y. Zhu T. Zhu Z. Lee K.-O. Gluckman P.D. Lobie P.E. J. Biol. Chem. 2007; 282: 680-690Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). In the case of α-globin mRNA, it was established that the stoichiometry of the RNA-protein complex (the α-complex) is 1:1, and a minimum RNA sequence of 20-nt (5′-CCCAACGGGCCCUCCUCCCC-3′) is necessary and sufficient for forming the complex (14Waggoner S.A. Liebhaber S.A. Exp. Biol. Med. 2003; 228: 387-395Crossref PubMed Scopus (96) Google Scholar). Interaction of two PCBPs, hnRNP K, and PCBP1/2, with a multiply tandem C-rich sequence (differentiation control element, DICE.) within the 3′-UTR of 15-lipoxygenase (LOX) mRNA leads to translational silencing of the mRNA in erythroid precursor cells (15Ostareck-Lederer A. Ostareck D.H. Standart N. Thiele B.J. EMBO J. 1994; 13: 1476-1481Crossref PubMed Scopus (222) Google Scholar, 16Ostareck D.H. Ostareck-Lederer A. Wilm M. Thiele B.J. Mann M. Hentze M.W. Cell. 1997; 89: 597-606Abstract Full Text Full Text PDF PubMed Scopus (423) Google Scholar, 17Ostareck D.H. Ostareck-Lederer A. Shatsky I.N. Hentze M.W. Cell. 2001; 104: 281-290Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar). DICE contains 10 gapless C-rich repeats. The sequence for one repeat is 5′-CCCCACCCUCUUCCCCAAG-3′. A minimum of two repeats is required for efficient translational suppression. PCBPs can also activate translation of cellular mRNAs. For example, binding of PCBP1 to an 18-nt C-rich sequence (5′-CUCCAUUCCCACUCCCU-3′) within the 5′-UTR of folate receptor mRNA up-regulates its translation (18Xiao X. Tang Y.S. Mackins J.Y. Sun X.L. Jayaram H.N. Hansen D.K. Antony A.C. J. Biol. Chem. 2001; 276: 41510-41517Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Binding of PCBP1/2 to the acute box cis-element in human heavy ferritin mRNA 5′-UTR also enhances translation (19Thomson A.M. Cahill C.M. Cho H.-H. Kassachau K.D. Epis M.R. Bridges K.R. Leedman P.J. Rogers J.T. J. Biol. Chem. 2005; 280: 30032-30045Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Besides cellular mRNAs, PCBPs also participate in regulating critical viral RNA functions. Binding of PCBP1/2 to two cisacting C-rich sequence-containing RNA elements within the 5′-UTR of poliovirus mRNA (also the genomic RNA) is critical for regulation of cap-independent translation and replication of the viral RNA (20Gamarnik A.V. Andino R. RNA. 1997; 3: 882-892PubMed Google Scholar, 21Gamarnik A.V. Andino R. Gene Dev. 1998; 12: 2293-2304Crossref PubMed Scopus (400) Google Scholar, 22Parsley T.B. Towner J.S. Blyn L.B. Ehrenfeld E. Semler B.L. RNA. 1997; 3: 1124-1134PubMed Google Scholar, 23Blyn L.B. Swiderek K.M. Richards O. Stahl D.C. Semler B.L. Ehrenfeld E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11115-11120Crossref PubMed Scopus (168) Google Scholar, 24Blyn L.B. Towner J.S. Semler B.L. Ehrenfeld E. J. Virol. 1997; 71: 6243-6246Crossref PubMed Google Scholar). The mechanistic details are not well understood for any of the PCBP functions. What emerges as a common feature is the binding of PCBPs to C-rich sequence motifs (often present in tandem) of the target D/RNAs. The molecular basis of PCBP KH domains-D/RNA interactions has been revealed by a number of crystal structures of individual KH1 or KH3 domain from PCBPs in complex with C-rich D/RNA sequences (25Backe P.H. Messias A.C. Ravelli R.B. Sattler M. Cusack S. Structure (Camb). 2005; 13: 1055-1067Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 26Du Z. Lee J.K. Tjhen R. Li S. Pan H. Stroud R.M. James T.L. J. Biol. Chem. 2005; 280: 38823-38830Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 27Fenn S. Du Z. Lee J.K. Tjhen R. Stroud R.M. James T.L. Nucleic Acids Res. 2007; 35: 2651-2660Crossref PubMed Scopus (28) Google Scholar, 28Du Z. Lee J.K. Fenn S. Tjhen R. Stroud R.M. James T.L. RNA. 2007; 13: 1043-1051Crossref PubMed Scopus (35) Google Scholar). However, there are no structures with KH2. Little is known about the events subsequent to any KH domain-D/RNA interaction. Pertinent to this point, there are no structures of PCBP constructs containing more than one KH domain. While studies suggest that protein-protein interactions occur and are vital for function, e.g. see Ref. 28Du Z. Lee J.K. Fenn S. Tjhen R. Stroud R.M. James T.L. RNA. 2007; 13: 1043-1051Crossref PubMed Scopus (35) Google Scholar), knowledge about how PCBPs engage in protein-protein interaction is also very limited. While a protein-D/RNA interaction seems essential for initiating the sequence of events, protein-protein interactions most likely are responsible for connectivity to various functions in most cases. It is therefore important to explore the missing link between protein-D/RNA and protein-protein interactions. To address these critical issues regarding the mechanisms of PCBP functions, we have determined the solution structure of the N terminus-half of human PCBP2 containing the KH1 and KH2 domains (corresponding to residues 11-169 of the full-length protein, referred to as PCBP2 KH1-KH2 herein). The sequence of the construct, as well as homologous sequences from other PCBPs, is shown in Fig. 1B. We have also investigated D/RNA binding properties of the KH1-KH2 construct by chemical shift mapping. Spectral comparison of the KH1-KH2, KH3, and full-length protein constructs provides insights into how the three KH domains might be arranged in the full-length protein. Based on results of our studies and available biochemical data, we propose possible regulatory mechanisms for PCBP functions that involve the interplay of D/RNA binding and conformational rearrangement of the KH domains. Sample Preparation—Three protein constructs of human PCBP2 were prepared containing: KH1-KH2 (residues 11-169 of the full-length sequence), KH3 (residues 285-359), and the “full-length” (residues 11-359). The proteins were expressed with N-terminal His tag (MKH6K; all but the last K could be removed by TAGzyme from Qiagen). The proteins were overexpressed in the BL21(DE3) strain of Escherichia coli (Stratagene). The preparation of properly folded PCBP2 KH1-KH2 proteins involved refolding from denatured proteins obtained from inclusion bodies. Methods for refolding and isotope labeling of the KH1-KH2 construct have been described previously (28Du Z. Lee J.K. Fenn S. Tjhen R. Stroud R.M. James T.L. RNA. 2007; 13: 1043-1051Crossref PubMed Scopus (35) Google Scholar). For most samples, the final NMR buffer contains 50 mm deuterated sodium acetate (pH 5.4), 2 mm dithiothreitol, 0.1 m EDTA, in either 90% H2O/10% D2O or 100% D2O. Preparation of the KH3 construct has also been described previously (27Fenn S. Du Z. Lee J.K. Tjhen R. Stroud R.M. James T.L. Nucleic Acids Res. 2007; 35: 2651-2660Crossref PubMed Scopus (28) Google Scholar). For the full-length construct, protein expression was induced with 0.1 mm isopropyl-β-d-thiogalactopyranoside at A600 = 0.6-0.8 at 10 °C for 48 h before harvest. The His-tagged proteins present in the supernatant were purified using Ni-nitrilotriacetic acid resin. The purified proteins were concentrated and buffer-exchanged by centrifugation. DNA oligonucleotides were purchased from Integrated DNA Technology. Samples of the KH1-KH2-DNA complexes were prepared by titrating a solution of DNA into a solution of the KH1-KH2 protein (molar ratio 2:1) at low concentration (∼10 μm), and subsequently concentrating to ∼0.5 mm. It was found that mixing the DNA with protein and subsequent concentration of the complex led to some precipitation, presumably due to nonspecific aggregation. For this reason, the accurate DNA/protein molar ration for the NMR sample could not be determined. NMR Spectroscopy and Structure Refinement—Data for structure determination of KH1-KH2 were collected on three isotopically labeled samples: uniformly 13C/15N, 13C/15N with 60% random fractional deuteration, and 13C/15N/2H/(Ile/Leu/Val)-methyl-protonated KH1-KH2. For most of the NMR experiments, the MKH6K-tag was not removed. All NMR experiments were performed on a Varian Inova spectrometer, operating at 600 MHz for protons, equipped with a cryoprobe. Spectra were processed with NMRPipe/NMRDraw (29Delaglio F. Grzesiek S. Vuister G.W. Zhu G. Pfeifer J. Bax A. J. Biomolec. NMR. 1995; 6: 277-293Crossref PubMed Scopus (11279) Google Scholar) and analyzed with SPARKY (30Goddard T.D. Kneller D.G. SPARKY. 3.0 Ed. University of California, San Francisco1998Google Scholar). All NMR experiments were carried out at 25 °C. 1H, 15N, and 13C resonance assignments were achieved using standard double and triple resonance experiments. Interproton distance restraints were derived from a number of 15N or 13C-separated NOESY three-dimensional experiments. NOE-derived inter-proton distance restraints were classified into four categories with a lower bound of 1.8 Å and an upper bound of 2.7, 3.5, 5.0, and 6.0 Å corresponding to strong, medium, weak, and very weak NOEs, respectively. Torsion angle restraints were derived from TALOS (31Cornilescu G. Delaglio F. Bax A. J. Biomol. NMR. 1999; 13: 289-302Crossref PubMed Scopus (2727) Google Scholar). Generic hydrogen bond distance restraints were utilized for regions of regular secondary structure that were based on characteristic NOE patterns and chemical shifts characteristic of the secondary structure. Structure refinement was carried out by simulated annealing in torsion angle space using established procedures implemented in the program XPLOR-NIH (32Schwieters C.D. Kuszewski J.J. Tjandra N. Clore G.M. J. Magn. Res. 2003; 160: 66-74Crossref Scopus (1831) Google Scholar). NMR restraint violations and structure quality were analyzed via the programs AQUA and PROCHECK_NMR (33Laskowski R.A. Rullmann J.A.C. MacArthur M.W. Kaptein R. Thornton J.M. J. Biomolec. NMR. 1996; 8: 477-486Crossref PubMed Scopus (4283) Google Scholar). The final ensemble of structures contains the twelve lowest energy conformers, from 50 randomized initial structures; all calculated structures converge to the same fold. For the measurements of the longitudinal relaxation time (T1) and transverse relaxation time (T2) of the backbone amide 15N nuclei, a series of 15N-HSQC spectra with varied relaxation delays were used (10, 60, 110, 280, 440, 610, 880, 1320 ms for T1; 10, 30, 50, 70, 90, 110, 130, 150 ms for T2). The relaxation experiments were performed on a 15N-labeled sample at 298 K. Relaxation parameters (reported in Fig. 2) were calculated by fitting peak heights measured with the program SPARKY (30Goddard T.D. Kneller D.G. SPARKY. 3.0 Ed. University of California, San Francisco1998Google Scholar). Structure figures were generated using PyMol (DeLano, W. L. The PyMOL Molecular Graphics System (2002). PCBP2 KH1-KH2 Exists as a Monomer in Solution—The PCBP2 KH1-KH2 protein (with a removable N-terminal His tag) was expressed in inclusion bodies. Properly folded proteins suitable for structural study were obtained by refolding. To investigate whether refolded KH1-KH2 is in monomeric or multimeric states, we employed the following method. Two KH1-KH2 samples, one unlabeled and the other 15N-labeled, were prepared by refolding. For the 15N-labeled sample, the His tag was removed. Equal amounts of unlabeled (with His tag) and 15N-labeled (without His tag) samples were mixed and denatured in 8 m urea. The denatured protein mixtures were then refolded. The refolded proteins were recovered from the refolding solution by Ni-NTA resin. If the refolded proteins were in multimeric states, the sample recovered by the resin would contain both the His-tagged unlabeled and tag-removed 15N-labeled proteins. SDS-PAGE electrophoresis and NMR experiments showed that only the His-tagged unlabeled protein was present in the recovered sample, indicating that no intermolecular protein-protein interaction took place during refolding: the refolded KH1-KH2 is in a monomeric state. Results from gel filtration chromatography also suggested that KH1-KH2 exists as a monomer (data not shown). Structure and Dynamics of PCBP2 KH1-KH2—Nearly complete 1H/13C/15N chemical shifts assignments for PCBP2 KH1-KH2 were obtained and deposited in the BioMagResBank (BMRB accession number 15049). The structure was determined using distance restraints derived from NOE measurements, torsion angle restraints derived from TALOS, as well as generic hydrogen bond restraints for regular secondary structure elements. Structure calculation statistics are summarized in Table 1. The structure ensemble and two views of the lowest energy structure as a representative are shown in Fig. 1C.TABLE 1NMR and structure determination statistics for PCBP2 KH1-KH2NOE-derived distance restraints1319Intra-residue297Sequential495Medium range (i+2 to i+5)317Long range (>i+5)210Total hydrogen bond restraintsaIncludes two restraints per hydrogen bond.170Total torsion angle restraints256Deviations from idealized covalent geometry Bonds (Å)0.0019 ± 0.0008 Angles (°)0.3376 ± 0.0081 Impropers (°)0.2228 ± 0.0115Backbone R.M.S.D. from mean structure (Å)bAveraged over the 12 accepted structures. Secondary structure region0.85 All but the linker1.18Ramachandran plot analysiscChecked by PROCHECK_NMR (33). Residues in most favored regions (%)83.2 Residues in additional allowed regions (%)13.1 Residues in generously allowed regions (%)2.4 Residues in disallowed regions (%)1.3a Includes two restraints per hydrogen bond.b Averaged over the 12 accepted structures.c Checked by PROCHECK_NMR (33Laskowski R.A. Rullmann J.A.C. MacArthur M.W. Kaptein R. Thornton J.M. J. Biomolec. NMR. 1996; 8: 477-486Crossref PubMed Scopus (4283) Google Scholar). Open table in a new tab In PCBP2 KH1-KH2, both the KH1 and KH2 domains adopt the classical type-I KH fold, which consists of three α-helices and three β-strands arranged in the order β1-α1-α2-β2-β3-α3. The conserved GXXG invariable loop (30GKKG and 114GKGG for KH1 and KH2, respectively) is located between α1 and α2; the variable loop (Ser50-Pro55 and Ala134-Thr142 for KH1 and KH2, respectively) is between β2 and β3 (Fig. 1, B and C). The three β-strands form an antiparallel β-sheet, with a spatial order of β1-β3-β2; the three α-helices are packed against one side of the β-sheet. Regions of the secondary structures and GXXG loops are well defined. But the variable loops within the two KH domains and the linker between the two KH domains are not. These loop regions of the molecule most likely are relatively flexible (Fig. 1C). Our previous work on crystal structures of the PCBP2 KH1 domain in complex with D/RNAs (26Du Z. Lee J.K. Tjhen R. Li S. Pan H. Stroud R.M. James T.L. J. Biol. Chem. 2005; 280: 38823-38830Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 28Du Z. Lee J.K. Fenn S. Tjhen R. Stroud R.M. James T.L. RNA. 2007; 13: 1043-1051Crossref PubMed Scopus (35) Google Scholar) revealed that the KH1 domain could engage in KH1-KH1 homodimerization. The protein interaction interface is a large and continuous hydrophobic surface comprised of residues from the β1-strand and α3-helix. It is clear from the present structure that the KH2 domain also has a hydrophobic surface comparable to that of the KH1 domain (Fig. 3A, left and middle). The two hydrophobic surfaces of the KH1 and KH2 domains interact with each other, resulting in an intramolecular KH1-KH2 association that buries 1528 Å2 of molecular surface from the two KH domains. The buried surface area is significantly larger than the estimated minimal area of 1200 Å2 required for a stable protein-protein complex (34Chothia C. Janin J. Nature. 1975; 256: 705-708Crossref PubMed Scopus (842) Google Scholar, 35Jones S. Thornton J.M. Prog. Biophys. Mol. Biol. 1995; 63: 31-59Crossref PubMed Scopus (490) Google Scholar). The KH1 and KH2 domains are arranged in a head-to-toe manner. The protein-protein interaction is defined by the anti-parallel positioning of the longest α-helix (α3) and β-strand (β1) in the protein domains (Fig. 3B). A number of hydrophobic residues from these structural elements are involved in hydrophobic protein-protein interaction (Fig. 3, A and B). KH1-KH2 association also brings the two three-stranded antiparallel β-sheets of the KH domains together in such a way that a six-stranded antiparallel β-sheet is formed (Fig. 1C, right). Amideamide NOEs between Arg17 and Val103, Leu19, and Arg101 were observed (28Du Z. Lee J.K. Fenn S. Tjhen R. Stroud R.M. James T.L. RNA. 2007; 13: 1043-1051Crossref PubMed Scopus (35) Google Scholar), characteristic of antiparallel β-strands. While hydrophobic forces are clearly the driving impetus for KH1 interaction with KH2, other factors, such as shape complementarities, hydrogen bonds, and electrostatic interactions may also play a role. There are two generic hydrogen bonds between backbone carbonyl and amide groups of the Arg17/Val103 and Arg101/Leu19 pairs of the interdomain antiparallel β-sheet. The guanidino groups of Arg17/Arg101, and the ϵ-amino group of Lys160 may mediate electrostatic interactions between the KH1 and KH2 domains (Fig. 3, A and B). Substantial interactions between the KH1 and KH2 domains show that both domains harbor a genuine protein interaction interface. Structures of type-I KH domain-D/RNA complexes (25Backe P.H. Messias A.C. Ravelli R.B. Sattler M. Cusack S. Structure (Camb). 2005; 13: 1055-1067Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 27Fenn S. Du Z. Lee J.K. Tjhen R. Stroud R.M. James T.L. Nucleic Acids Res. 2007; 35: 2651-2660Crossref PubMed Scopus (28) Google Scholar, 28Du Z. Lee J.K. Fenn S. Tjhen R. Stroud R.M. James T.L. RNA. 2007; 13: 1043-1051Crossref PubMed Scopus (35) Google Scholar, 36Lewis H.A. Musunuru K. Jensen K.B. Edo C. Chen H. Darnell R.B. Burley S.K. Cell. 2000; 100: 323-332Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar, 37Liu Z.H. Luyten I. Bottomley M.J. Messias A.C. Houngninou-Molango S. Sprangers R. Zanier K. Kramer A. Sattler M. Science. 2001; 294: 1098-1102Crossref PubMed Scopus (184) Google Scholar, 38Braddock D.T. Louis J.M. Baber J.L. Levens D. Clore G.M. Nature. 2002; 415: 1051-1056Crossref PubMed Scopus (140) Google Scholar, 39Braddock D.T. Baber J.L. Levens D. Clore G.M. EMBO J. 2002; 21: 3476-3485Crossref PubMed Scopus (110) Google Scholar, 40Du Z. Yu J. Chen Y. Andino R. James T.L. J. Biol. Chem. 2004; 279: 48126-48134Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar) show that type-I KH domains recognize their D/RNA targets using a common binding interface, which is mainly defined by helices α1, α2, and strand β2. This D/RNA binding interface is located on the molecular surface opposite the protein interaction interface. Interaction of the KH1 and KH2 domains results in placement of the two D/RNA binding interfaces of the KH1 and KH2 domains on opposite molecular surfaces in the structure of PCBP2 KH1-KH2, far from each other (Fig. 4A). Modeling of the bound D/RNA can readily be achieved by comparison with the complexes entailing a single KH domain. The 14-residue long linker (Ser84 to Pro97) between the KH1 and KH2 domains does not assume a regular secondary structure (Fig. 1C). This region of the molecule is the most ill-defined due to lack of constraints. However, chemical shifts for all of the linker residues were assigned. Indeed, residues from the linker region in general yielded stronger resonance signals than residues from the KH domains, indicating that the linker region probably experiences a higher degree of internal mobility. To characterize the backbone dynamics of the PCBP2 KH1-KH2 construct, we measured the 15N longitudinal and transverse relaxation times (T1 and T2) of the protein construct. The experimental T1 and T2 values for 131 residues are shown in Fig. 2. The construct contains 158 native residues including 6 prolines. T1 and T2 values for 21 other residues could not be determined due to either absence of cross-peak or spectral overlap. It is apparent that the measured T1 and T2 values indicate a rather non-uniform dynamic behavior of the protein construct. Excluding the terminal residues, there are four stretches of amino acids that show lower-than-average T1 values, corresponding to the variable loop of KH1, the linker between KH1 and KH2, the GXXG and variable loops of KH2, respectively (Fig. 2, top). Most residues from the two variable loops and the linker also show higher-than-average T2 values (Fig. 2, bottom). These data clearly indicate that the two variable loops within the KH domains and the linker region between the KH domains experience higher backbone mobility than other parts of the molecule. Residues in the linker region exhibit some of the lowest values of T1 and highest values of T2. A stretch of 10 amino acids (Ser86 to Arg95) within the linker has an average T2 value of 176 ms, doubling the average T2 value for the entire protein construct (88 ms). Overall, the relaxation data show that the linker region between the two KH domains is the most flexible part of the molecule. Within the two KH domains, the variable loop in each of the domains experiences increased internal molecular motion. Residues from the other secondary structural elements of the two KH domains in general show higher T1 and lower T2 values than the averages, indicating that these secondary structural elements are tightly packed in the individual KH domains, with reduced molecular mobility. Comparison with the Two Previous Structures of Tandem KH Domains—There are two published structures of tandem type-I KH domains: the KH3-KH4 domains of the FUSE-binding protein (FBP) in complex with single-stranded DNA (38Braddock D.T. Louis J.M. Baber J.L. Levens D. Clore G.M. Nature. 2002; 415: 1051-1056Crossref PubMed Scopus (140) Google Scholar) and the KH1-KH2 domains of fragile X mental retardation protein (FMRP) (42Valverde R. Pozdnyakova I. Kajander T. Venkatraman J. Regan L. Structure. 2007; 15: 1090-1098Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). In the crystal structure of FMRP KH1-KH2, the linker between the two domains has only one residue (Glu) separating the last α-helix of KH1 and the first β-strand of KH2 (Fig. 4B). Compared with most other published structures of type-I KH domains (25Backe P.H. Messias A.C. Ravelli R.B. Sattler M. Cusack S. 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