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Originally published In Press as doi:10.1074/jbc.M508183200 on September 25, 2005

J. Biol. Chem., Vol. 280, Issue 46, 38823-38830, November 18, 2005
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Crystal Structure of the First KH Domain of Human Poly(C)-binding Protein-2 in Complex with a C-rich Strand of Human Telomeric DNA at 1.7 Å*

Zhihua Du{ddagger}, John K. Lee§, Richard Tjhen§, Shang Li§, Hu Pan§, Robert M. Stroud§, and Thomas L. James{ddagger}1

From the Departments of {ddagger}Pharmaceutical Chemistry, and §Biochemistry and Biophysics, University of California, San Francisco, California 94143-2280

Received for publication, July 27, 2005 , and in revised form, September 14, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Recognition of poly(C) DNA and RNA sequences in mammalian cells is achieved by a subfamily of the KH (hnRNP K homology) domain-containing proteins known as poly(C)-binding proteins (PCBPs). To reveal the molecular basis of poly(C) sequence recognition, we have determined the crystal structure, at 1.7-Å resolution, of PCBP2 KH1 in complex with a 7-nucleotide DNA sequence (5'-AACCCTA-3') corresponding to one repeat of the human C-rich strand telomeric DNA. The protein-DNA interaction is mediated by the combination of several stabilizing forces including hydrogen bonding, electrostatic interactions, van der Waals contacts, and shape complementarities. Specific recognition of the three cytosine residues is realized by a dense network of hydrogen bonds involving the side chains of two conserved lysines and one glutamic acid. The co-crystal structure also reveals a protein-protein dimerization interface of PCBP2 KH1 located on the opposite side of the protein from the DNA binding groove. Numerous stabilizing protein-protein interactions, including hydrophobic contacts, stacking of aromatic side chains, and a large number of hydrogen bonds, indicate that the protein-protein interaction interface is most likely genuine. Interaction of PCBP2 KH1 with the C-rich strand of human telomeric DNA suggests that PCBPs may participate in mechanisms involved in the regulation of telomere/telomerase functions.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
K-homology domain (KH2 domain, originally identified in hnRNP-K) is one of the most frequently occurring and conserved nucleic acid-binding protein motifs. So far, a large number of KH domain-containing proteins have been identified in a wide variety of species ranging from bacteria to human. These KH domain proteins assume a wide spectrum of biological functions, including transcriptional and translational controls, mRNA stabilization, and mRNA splicing, among others. Different KH domains possess quite different nucleic acid binding specificities. Unveiling how different KH domains interact specifically with their nucleic acid targets and how these interactions contribute to the complex regulatory processes is of central importance to a better understanding of how KH domain proteins function.

One of the most distinctive nucleic acid binding specificities achieved by the KH domains is manifested by a subfamily of KH domain-containing proteins known as poly(C)-binding proteins (PCBPs). As implied by the family name, PCBPs recognize poly(C) RNA or DNA sequences with high affinity and specificity (for reviews, see Refs. 1 and 2). To date, five evolutionarily related PCBPs have been identified in mammalian cells: PCBP1-4 (also known as {alpha}CP1-4; PCBP1 and -2 are also known as hnRNP E1 and E2) and hnRNP K. Each PCBP contains three KH domains: two consecutive KH domains at the N terminus and a third KH domain at the C terminus with an intervening sequence of variable length between the second and third KH domains (Fig. 1A). In general, corresponding KH domains share a higher degree of homology than KH domains within each protein (Fig. 1B). No other discernable nucleic acid binding motif is present in PCBPs; the KH domains are responsible for the ability of the PCBPs to interact with poly(C) sequences.

The established examples of functional roles carried out by PCBPs indicate that these proteins are key mediators in a number of important cellular processes (1). Binding of PCBP1 or PCBP2 to target RNA sequences harboring tandem poly(C) stretches within the 3'-UTRs of a number of cellular mRNAs confers unusual stability to these mRNAs, including {alpha}-globin (3, 4), collagen-{alpha}1 (5, 6), tyrosine hydroxylase (7), and erythropoietin (8) mRNAs. In the case of {alpha}-globin mRNA, it was established that the stoichiometry of the RNA-protein complex is 1:1; and a minimum RNA sequence of 20 nt (5'-CCCAACGGGCCCUCCUCCCC-3') was able to form the complex (9). Interaction of two PCBPs, hnRNP K and PCBP1 or -2, with a C-rich sequence within the 3'-UTR of some mRNAs can also result in translational silencing, as seen in 15-lipoxygenase (LOX) mRNA (10-12). A recent study identified 160 mRNA species that associate in vivo with PCBP2 from a human hematopoietic cell line (13), suggesting that the contribution of PCBPs in post-transcriptional regulation of cellular genes may be far more profound than currently known.

Besides cellular RNAs, PCBPs also participate in regulating critical viral RNA functions. Binding of PCBP1 or -2 to two cis-acting C-rich sequence containing RNA elements within the 5'-UTR of Poliovirus mRNA (which is also the genomic RNA) is critical for regulation of cap-independent translation and replication of the viral RNA (14-18).

Biological functions of PCBPs are further diversified by their ability to interact specifically with not only RNA but also DNA sequences. Specific binding of hnRNP K to the single-stranded C-rich sequence in the promoter of the human c-myc gene activates transcription (19). It was also shown that hnRNP K and PCBP1 could recognize the C-rich strand of human telomeric DNA with high affinity in vitro (20, 21); whether such an interaction is functionally significant is a subject for further biochemical/biological investigations.

It should be noted that although PCBP functions are diverse, they are nearly all dependent on the ability of the KH domains to recognize single-stranded C-rich RNA or DNA sequences with high specificity and affinity. To reveal the molecular basis of KH domain-poly(C) DNA/RNA interaction, we have previously used NMR to determine the solution structure of the first KH domain (KH1) from human PCBP2, and characterize its interaction with various DNA/RNA molecules (22). In this study, we report the 1.7-Å resolution crystal structure of the PCBP2 KH1 domain in complex with a seven-nucleotide single-stranded DNA sequence (5'-AACCCTA-3') corresponding to one repeat of the C-rich strand of human telomeric DNA (htDNA). The structure shows that PCBP2 KH1 makes substantial contacts with four nucleotides (5'-ACCC-3', the core recognition sequence). Interestingly, each of the three cystosines is specifically recognized by a network of strong hydrogen bonds to the Watson-Crick positions involving the side chain functional group of a particular amino acid. Another outstanding feature of the PCBP KH1 domain is the presence of a protein-protein dimerization interface located on the other side of KH1 domain from the DNA binding interface. A large number of protein-protein interactions, including hydrophobic contacts, stacking of aromatic side chains, and numerous hydrogen bonds, stabilize the dimerization interface, a feature not observed in any other KH domain structure previously characterized. Insights into mechanisms of PCBP functions, in the context of comparison with other available KH domain-nucleic acids complex structures, are discussed.



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  		FIGURE 1.
A, schematic diagram of the domain structure of human PCBP2. Similar domain structures are observed in other members of the PCBP family. B, sequence alignment of the KH domains from the known PCBP proteins PCBP1-4 and hnRNP-K. As a comparison, the sequence of the KH3 domain from NOVA2, which is not a member of the PCBP family, is also included in the alignment. The crystal structure of NOVA2 KH3 in complex with an RNA was the only crystal structure available for a KH domain-nucleic acid complex prior to this study. Alignments were carried out using the program ClustalX. The sequence shown for PCBP2-KH1 corresponds to residues 11-82 in the full-length protein. Secondary structures were based on the crystal structure. Residues involved in hydrogen bonds to the DNA bases are labeled as: S, side chain base hydrogen bond; B, backbone base hydrogen bond, including water-mediated interactions.

 

   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Sample Preparation and Crystallization—N-terminal His-tagged PCBP2 KH1 was overexpressed in BL21(DE3) strain of Escherichia coli (Stratagene). For Se-Met-labeled protein, the bacteria were grown in M9 minimal medium until they reached an A600 of 0.6-0.8, whereupon leucine, isoleucine, lysine, phenylalanine, threonine, and valine were added to the culture to inhibit methionine biosynthesis. After 15 min, L-seleno-methionine (50 mg/liter) was added, followed by isopropyl-{beta}-D-thiogalactopyranoside to a final concentration of 0.4 mM to induce expression of the Se-Met-labeled protein. After purification by Ni-nitrilotriacetic acid resin (Qiagen), the His tag was removed by the TAG-zyme system from Qiagen. Crystals of the PCBP2 KH1-DNA (5'-AACCCTA-3') complex were obtained by hanging drop vapor diffusion against 25% polyethylene glycol 8000, 100 mM sodium acetate, 100 mM sodium cacodylate, pH 6.1, at 22 °C. The protein concentration was about 250 µM with a 1:1.2 protein:DNA ratio. Orthorhombic crystals grew to useful size within 1 day with diffraction to 1.7 Å. The crystals are in space group P21212 (a = 65.60 Å, b = 115.18 Å, c = 45.53 Å), with four protein-DNA complexes in one asymmetric unit.

Data Collection, Structure Determination, and Refinement—A single SAD data set was collected at the peak wavelength of the selenium K absorption edge from a single frozen selenomethionine-containing crystal using Beamline 8.3.1 of the Advanced Light Source (ALS) at Berkeley National Laboratory. Diffraction intensities were integrated and reduced by using the program DENZO and were scaled by using SCALEPACK (23) (TABLE ONE). All twelve selenium atoms from the four protein molecules in an asymmetric unit were located by CNS (24). An interpretable electron density map was obtained after solvent flattening. The model was built by MOLOC (25) and refined in CNS to an R factor of 21.5% (Rfree = 23.8%). The final model includes all of the protein residues 11-82 (the PCBP2 numbering is used), four DNA molecules (two molecules have all of the seven nucleotides built; the other two molecules have five nucleotides built), and 222 water molecules. Analysis of the geometry shows that all parameters are well within expected values at this resolution (TABLE ONE). Structure figures were generated using PyMol.3


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  		TABLE ONE
Crystallographic refinement statistics

 


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Overall Structure of the PCBP2 KH1-htDNA Complex—There are four PCBP2 KH1-htDNA complexes in one asymmetric unit, labeled as complex A, B, C, and D in Fig. 2; the two complexes A and B form a dimer; the two complexes C and D form another dimer. Electron density is clearly present for every protein residue in all four complexes. All seven DNA residues were built in complexes A and C; five DNA residues were built for complexes B and D, but the two terminal residues were not built because of a lack of electron density. Structures of the four complexes are otherwise very similar (non-crystallographic symmetry averaging was not applied; average pair-wise RMSD is ~0.22 Å), including details in molecular recognition.



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  		FIGURE 2.
Structures of the PCBP2 KH1-htDNA complexes in the asymmetric unit. There are four complexes in the asymmetric unit, colored red, blue, green, and cyan for complex A, B, C, and D, respectively.

 
The overall structure of the complex (using complex A as a representative) is depicted in Fig. 3, A and B. The DNA-bound form of PCBP2 KH1 is very similar to the NMR structure of the free protein we previously determined (22). The structure consists of three {alpha}-helices and three {beta}-strands arranged in the order {beta}1-{alpha}1-{alpha}2-{beta}2-{beta}3-{alpha}3. The evolutionarily conserved invariable Gly30-Lys31-Lys32-Gly33 loop is located between {alpha}1 and {alpha}2; the variable loop (Ser50-Pro55) is between {beta}2 and {beta}3. The three {beta}-strands form an antiparallel {beta}-sheet, with a spatial order of {beta}1-{beta}3-{beta}2; the three {alpha}-helices are packed against one side of the {beta}-sheet. Hydrophobic interactions seem to play an important role in the packing of the structural elements to form a compact global fold; the residues in the core are exclusively hydrophobic.



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  		FIGURE 3.
A, overall structure of the PCBP2 KH1-htDNA complex. The KH domain is rendered by ribbon representation in cyan. The htDNA is shown using a sticks representation in magenta. Secondary structure elements of the KH domain are labeled. The side chains of R40, E51, and R57, which are involved in hydrogen bonds (depicted as yellow dashed lines) to the bases of the central poly(C) residues, are shown as sticks in blue. B, comparison of free (NMR, in red) and DNA-bound (x-ray, in cyan) structures of the PCBP KH1 domain. The bound DNA is also shown as sticks in magenta.

 
The C-rich strand of human telomeric DNA binds to PCBP2 KH1 in a groove defined by the juxtaposition of two {alpha}-helices ({alpha}1 and {alpha}2), two {beta}-strands ({beta}2 and {beta}3; only one residue from {beta}3, Arg57, participates in direct contact with the DNA), and two connecting loops (the GKKG loop and the variable loop). This binding groove is consistent with what we previously determined by NMR chemical shift perturbations (22); note that chemical shifts are reported in the Supplementary Materials in Ref. 22. The limited length of the groove only allows the accommodation of four DNA residues (Ade-2 to Cyt-5) as the core recognition motif. Other flanking DNA residues are not involved in direct contact with the protein (Fig. 3A).

Specific Recognition of the Core Sequence—Recognition of the core motif, Ade-2 to Cyt-5, is achieved by the combination of several forces including hydrogen bonding, electrostatic interactions, van der Waals contacts, and shape complementarities. The human telomeric DNA binds in a groove running from the C-terminal region to the N-terminal region of PCBP2 KH1 domain (Fig. 3A). The overall orientation of the DNA backbone is similar to that previously observed in other DNA/RNA-KH domain complexes (26-30), with the 5'- and 3'-ends interacting with the C- and N-terminal regions of the KH domain, respectively.



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  		FIGURE 4.
DNA recognition. A, surface representation of PCBP KH1 illustrating contributions of hydrogen bonds to DNA phosphate groups, electrostatic interaction, van der Waals contacts, and shape complementarities to DNA binding. Positively charged, negatively charged, uncharged hydrophilic, and hydrophobic residues are colored in blue, red, yellow, and green, respectively; glycines are in white. The DNA backbone is stabilized by two hydrogen bonds (depicted as yellow dashed lines; one of the hydrogen bonds is mediated by a bound water, depicted as a red sphere) and electrostatic interactions with the positively charged residues located on the left ridge of the DNA binding groove. Van der Waals contacts with the hydrophobic residues forming the floor of the binding groove (V25, G26, I29, V36, I47, and I49) also provide an important stabilizing force for DNA recognition. B, 2Fo - Fc electron density map contoured at 1{sigma} showing recognition of the three cytosines by the side chain functional groups of R57, R40, and E51, respectively. C, stereoview for the recognition of the three cytosine bases. The dense network of hydrogen bonds (yellow dashed lines) responsible for specific recognition is shown. Four water molecules (depicted as red spheres) are integral parts of this network. The DNA is represented as magenta sticks. The protein is represented as a ribbon in pale cyan, with sticks shown for those residues whose side chains (in blue) or backbones (in cyan) are involved in hydrogen bonds. To illustrate the binding environment of cytosine-5 in the crystal lattice, a symmetry-related adenosine (labeled as sym_Ade-7 in gold) is shown.

 
Two phosphate groups of the DNA participate in intermolecular hydrogen bonds (Fig. 4A). O1P of Cyt-3 accepts a hydrogen bond from the backbone amide of Lys32 within the conserved GKKG loop. This hydrogen bond formation explains the large NMR chemical shift of the Lys32 amide proton (downfield, 0.86 ppm) we previously observed (22). A water molecule forms a bridge between the phosphate group of Thy-6 and the backbone carbonyl of Ile47.

The DNA backbone roughly runs along the left ridge of the binding groove, where four positively charged residues (Lys31 and Lys32 from the invariable GKKG loop, Lys37 and Arg40 from helix {alpha}2) make close contacts with the phosphate groups of Cyt-3, Cyt-4, Cyt-5, and Thy-6 (Fig. 4A). Conservation of these positively charged residues suggests that electrostatic interactions may play a role in interaction with the DNA backbone.

The ribose and base moieties of the core recognition sequence fit nicely inside the binding groove (Fig. 4A). For Ade-2, no specific interactions are observed, but van der Waals contacts are mainly provided by Gly26, Ser27, and Lys31. Base-stacking with Ade-1 is observed. Binding at this position does not seem to be sequence-specific. For the three cytosines, most of their Watson-Crick functional groups of the bases point to the right, forming specific hydrogen bonds with protein residues from the {beta}-strands ({beta}2 and {beta}3) and variable loop. The size of the groove may dictate the preference for pyrimidine bases at these positions. There are extensive hydrophobic contacts between the riboses/hydrophobic faces of the cytosine bases and the aliphatic side chains that dominate the floor of the binding groove (Fig. 4A). For Cyt-3, van der Waals contacts are provided by Val25, Gly26, and Ile29; for Cyt-4, the hydrophobic environment is created by Ile29, Val36, and Ile49. For Cyt-5, Ile47 is involved. These conserved hydrophobic residues are presumably important for the function of the KH domain. A single mutation of an isoleucine to an asparagine in the second KH domain of FMR1 (corresponding to Val36 in PCBP2 KH1) is known to cause a particularly severe presentation of the mental retardation syndrome (31). In our hand, mutation of Val36 to an asparagine caused PCBP2 KH1 domain to aggregate in the inclusion body; we were not able to refold the protein despite substantial efforts. It seems as though the conserved hydrophobic residues are somehow important for proper folding and/or solubility of PCBP2 KH1 domain.

Specific recognition of the poly(C) sequence is achieved by an extensive network of hydrogen bonding interactions (Fig. 4, B and C). For Cyt-3, the side chain of Arg57 provides two hydrogen bond donors to the O2 and N3 acceptors of Cyt-3. The side chain conformation of Arg57 is rather extended in order to reach Cyt-3 from strand {beta}3; this extended conformation is further stabilized by two hydrogen bonds to the backbone carbonyl oxygen of Ser50. Hydrogen bonds involving the N4 amino group of Cyt-3 entail an intrastrand hydrogen bond to the O1P phosphate group of Ade-2 and a water bridge to the backbone carbonyl oxygen atoms of Gly22 and Cys54. This network of hydrogen bonds provides a molecular mimicry of a guanine to form Watson-Crick-like interactions with Cyt-3, therefore defining the specificity for a cytosine at this position of the DNA sequence.

For Cyt-4, the N4 amino group of Cyt-4 forms two hydrogen bonds: one with the backbone carbonyl oxygen of Ile49, the other with a water which bridges to the amide group of Gly52. The N3 group of Cyt-4 forms a hydrogen bond with another water, which bridges to the amide of Ile49 and the O2 group of Cyt-5. Involvement of the amide group of Ile49 in a hydrogen bond explains the big downfield NMR chemical shift change (1.31 ppm) we previously observed for Ile49 amide proton upon complex formation with the htDNA (22). The O2 group of Cyt-4 forms hydrogen bonds with the side chain of Arg40 from helix {alpha}2. The extended conformation of the Arg40 side chain is stabilized by hydrogen bonds to the backbone carbonyl of Ile47 from strand {beta}2. Intrastrand base stacking with Cyt-5 also contributes to defining the binding environment for Cyt-4.

For Cyt-5, the side chain of Glu51 from the variable loop forms two hydrogen bonds to the N4 and N3 groups. The conformation of the Glu51 side chain is further stabilized by a water bridge to the side chain carbonyl oxygen of Asn48. The base of Cyt-5 engages in stacking interactions with the base of Cyt-4 on one face and the base of an adenosine from a symmetry-related DNA strand (labeled as sym_Ade-7 in Fig. 4C) from an adjacent complex in the crystal lattice on the other face.



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  		FIGURE 5.
Dimerization of the KH domain. A, view of the PCBP2 KH1 homodimer. The two monomers are colored green and cyan in the ribbon representation; residues located at the dimerization interface are shown as sticks. The bound DNAs are shown in magenta. The DNA binding groove and the dimerization interface are located on opposite sites of the molecular surface. Four generic (involving only backbone amide and carbonyl groups) intermolecular hydrogen bonds at the dimerization interface are depicted as yellow dashed lines; two of these are bridged by water, shown as red spheres. A number of non-generic hydrogen bonds (involving side chain functional groups) also present at the dimerization interface (not shown, see text for details). B, surface representation of a monomer showing the large hydrophobic area (green) of the dimerization interface. The view is ~180° from that of Fig. 4A; the coloring scheme is the same. C, close-up view of the PCBP2 KH1 dimerization interface.

 
Dimerization of the PCBP2-KH1 Domain—Numerous interactions stabilize the formation of the PCBP2 KH1 homodimer (Fig. 5, A, B, and C). The two protein domains in the dimer are arranged in a head-to-toe manner, with the dimerization interface located on the opposite side of the protein from the DNA binding groove. The dimerization interface is defined by the anti-parallel positioning of the longest {alpha}-helix ({alpha}3) and {beta}-strand ({beta}1) in the protein domain. The molecular interactions mediating the dimerization of the KH domains are truly remarkable in that they are the kinds of interactions normally encountered in the folding of a compact, integral protein motif. There is a hydrophobic interior core defined by the hydrophobic side chains of Leu14, Ile16, Leu18 from the two {beta}-strands ({beta}1s), and Ile68, Phe72, Ile76, Leu79 from the {alpha}-helices ({alpha}3s). Stacking of the two Phe72 aromatic rings is also observed. Dimerization orients the two three-stranded antiparallel {beta}-sheets of the monomers in such a way that a six-stranded antiparallel {beta}-sheet is formed. Four generic (backbones only) hydrogen bonds stabilize the antiparallel arrangement of the two {beta}1-strands. These include: two from Leu19 amide to Arg17 carbonyl oxygen, and two water bridges from Arg17 amide to Leu19 carbonyl oxygen. There are also quite a number of non-generic (involving side chain functional groups) intermolecular hydrogen bonds formed at the dimerization interface, including two from the side chain of Arg17 in molecule A to the side chain of Glu56 in molecule B, one from the side chain of Arg17 in molecule B to the side chain of Glu56 in molecule A, one from Val12 backbone in molecule A to the side chain of Glu80 in molecule B, one from the side chain hydroxyl of Thr65 in molecule B to Glu80 side chain in molecule A, and one water bridge from the amide of Thr65 in molecule A to the side chain of Glu80 in molecule B.

Formation of the dimer buries 1188 Å2 of solvent-accessible surface area in each monomer. The molecular surface forming the dimerization interface is rather hydrophobic in nature (Fig. 5B). Such a big area of hydrophobic surface should provide a significant driving force for formation of the protein-protein interface. Whether the dimerization of the PCBP2 KH1 domain depicted in Fig. 5, A and C is biologically significant is not clear at the present time. However, the details about the driving forces for dimer formation as revealed by our crystal structure strongly suggest that the dimer should be very stable. KH domain dimers were also observed in the crystal structures of free and RNA-bound forms of NOVA2 KH3 (32), and DNA-bound form of hnRNP K KH3 (32). Only the dimerization interface of free NOVA2 KH3 is similar to that of PCBP2 KH1 in terms of the involvement of both strand {beta}1 and helix {alpha}3 in an antiparallel arrangement. Other interfaces do not possess such a full set of "native-like" stabilizing interactions as we observe in the PCBP2 KH1 interface. The same PCBP2 KH1 dimer is also present in the crystal structures of PCBP2 KH1 in complex with a 12-nt DNA and RNA with different crystal packings.4 Given the remarkable features and reoccurrence of the PCBP2 KH1 dimer, it is tempting to speculate that some KH domains may have a natural propensity to dimerize (self-association, forming heterodimers with other KH domains, or interacting with other proteins) through the {beta}1/{alpha}3 interface; depending on the properties of the residues present at the interface. For such KH domains, they cannot only play a role in interaction with nucleic acids, but also in protein-protein interactions. Moreover, because there is no overlap between the nucleic acids and protein interaction, binding to nucleic acids and protein partners can happen simultaneously, which may be functionally important in certain scenarios.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The DNA-bound form crystal structure of the PCBP2 KH1 is very similar to the unliganded structure we previously determined by NMR (22). The most noticeable structural change upon DNA binding is seen in the variable loop region. In the free form structure, the variable loop is pointing outward, resulting in a more open binding groove; in the DNA-bound form, the variable loop wraps back toward the groove to achieve close contacts with the DNA (Fig. 3B). In several other KH domain-nucleic acid interactions (26-30), the nucleic acid binding groove is also largely preformed. This appears to be a common property among KH domains.

Prior to this study, there were two KH domain-nucleic acid co-crystal structures available in the literature: the 2.4-Å structure of NOVA2 KH3 in complex with a SELEX RNA stem loop (28), and the 1.8-Å structure of hnRNP K KH3 in complex with a 6-nt DNA (30). There are also several complex structures determined by NMR: splicing factor 1 (SF1) KH with single-stranded RNA (29), FUSE-binding protein (FBP) KH3 and KH4 with ssDNA (27), and hnRNP K KH3 with ssDNA (26). A previous analysis of these structures (30) revealed that the NMR structures of the KH domains of hnRNP K and FBP with ssDNA differed from other KH domain-nucleic acid complexes in that they employed weak methyl-mediated hydrogen bonds to achieve specific recognition of the Watson-Crick positions of the DNA bases, with different relative positioning of the DNA bases as a possible result. A comparison of the other structures led the authors to the following conclusions: each KH domain recognized a core motif of four nucleotides; only pyrimidines were found at the first and fourth positions; recognition of the second and third bases were in general realized by hydrogen bonds from the protein residues to the Watson-Crick edges of the bases. With the latest entry of our PCBP2 KH1-htDNA complex into the structural data base of KH domain-DNA/RNA interactions, we discuss how our structure reinforces some of the common features of the interactions, while also providing new structural insights. We mainly compare our structure with the crystal structures of NOVA2 KH3-RNA and hnRNP K KH3-DNA complexes.



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  		FIGURE 6.
Comparison of the PCBP2 KH1-htDNA and the NOVA2 KH3-RNA co-crystal structures. The sequences of PCBP2 KH1 and NOVA2 KH3 are ~23% identical and ~47% similar. A, structure of the PCBP2 KH1-htDNA complex. This rendering is virtually identical to that in Fig. 4C except that the viewing window includes the whole structure, and the residues defining the hydrophobic floor of the binding groove are shown as sticks (labeled in green). See Fig. 4C for a detailed annotation. B, crystal structure of the NOVA2 KH3-RNA complex (28). For clarity, only five residues out of the 20-nt crystallization stemloop RNA are shown. Coloring and annotation are comparable to the PCBP2 KH1-htDNA complex. Stick representations are shown for residues involved in hydrogen bonds with the bases of the RNA and defining the hydrophobic floor of the binding groove, colored in blue and green, respectively.

 
Although the sequence specificity for nucleic acid recognition of NOVA2 KH3 is different from that of PCBP2 KH1, comparison of the two co-crystal structures nonetheless reveals some very interesting similarities (Fig. 6, A and B). The overall structures of the two KH domains are similar, and a common binding groove is utilized for nucleic acid recognition. More remarkably, the four RNA/DNA residues of the core recognition sequence, 5'-UCAY-3' (RNA, Y is a pyrimidine) for NOVA2 KH3 and 5'-ACCC-3' (DNA) for PCBP2 KH1, adopt quite similar conformations; the bases and riboses of corresponding nucleic acid residues occupy virtually the same location, with mostly similar orientations.

The identity of the nucleotide at the first position of the core recognition motif differs for PCBP2 KH1 and NOVA2 KH3 (A versus U). In the PCBP2 KH1 complex, this position does not involve base-specific interactions. The base of the first nucleotide is positioned on top of helix {alpha}1; the backbone of the nucleic acid winds around the helix downward, placing the base of the second nucleotide close to the bottom of helix {alpha}1. The bases of the first and second nucleotides act like a pair of molecular tongs grasping the helix (Fig. 6, A and B). Two glycines in this helical segment are absolutely conserved (Gly26 and Gly30 in the PCBP2 KH1 numbering scheme. See Fig. 1B for sequence alignments). Other amino acids with larger side chains at these positions presumably would hinder binding of the nucleic acid.

Recognition of the second nucleotide in the core sequence (cytosine in both cases) is highly specific and very similar for the two complexes. The O2 and N3 positions of the cytosine form two hydrogen bonds to the guanidino group of a conserved arginine. One of the hydrogen bonds involving the N4 group is also identical: an intrastrand hydrogen bond to the backbone O1P of the preceding residue. Although the other hydrogen bond involving the N4 group is different, the protein residue involved occupies the corresponding position in both proteins (Gly22 in PCBP2 KH1 and Glu14 in NOVA2 KH3). Van der Waals contacts with the second position cytosine are provided by a set of conserved hydrophobic residues (Val25, Gly26, and Ile29 in PCBP2 KH1; Val17, Gly18, and Leu in NOVA2 KH3). At the third position, the residues differ (Ade for NOVA2 KH3 and Cyt for PCBP2 KH1). As a result, the hydrogen bonds responsible for specific recognition of the third position residue are different. However, the amino groups (N6 in Ade and N4 in Cyt) have the same hydrogen-bonding partner: the backbone carbonyl oxygen of a conserved isoleucine (Ile49 in PCBP2 KH1 and Ile41 in NOVA2 KH3). The van der Waals interactions for the third position residue are also very similar: a set of hydrophobic residues are conserved (Ile29, Val36, and Ile49 in PCBP2 KH1; Leu21, Leu28, and Ile41 in NOVA2 KH3), and the third and fourth position bases are stacked in both complexes. Recognition of the residue at the fourth position of the core motif is less specific for NOVA2 KH3. However, in both complexes, the fourth residue base is stabilized by stacking with bases on each side (Fig. 6, A and B), and van der Waals contacts are provided by a conserved isoleucine (Ile47 and Ile39 in PCBP2 KH1 and NOVA2 KH3, respectively).

PCBP2 and hnRNP K both belong to the PCBP family, with similar sequence specificity for poly(C) DNA/RNA sequences. Comparing the sequences of PCBP2 KH1 and hnRNP K KH3, they are ~32% identical and ~55% similar. Although the coordinates of the hnRNP K KH3-DNA complex have not yet been deposited, a comparison can still be made based on the description of the structure (30). The core recognition tetranucleotides of PCBP2 KH1 and hnRNP K KH3 differ only in the first position. It is clear from our structure that the first core recognition position can also accommodate a purine; since there is no base-specific interaction involved, this position for PCBP2 KH1 should also permit other types of residues. The rest of the core sequence is identical, but the specific hydrogen bonds involved in recognition are not. Most noticeably, recognition of the cytosine at the fourth position in the hnRNP K KH3 complex involves only water-mediated hydrogen bonds, whereas in the PCBP2 KH1 complex the side chain of Glu51 directly forms two hydrogen bonds to the cytosine (Fig. 4B). Intriguingly, a glutamate residue is also present at the corresponding position in the hnRNP K KH3 sequence (Fig. 1B). We noticed that the variable loop of PCBP2 KH1 (within which Glu51 is located) is shorter than that of hnRNP K KH3. While most of the residues from the variable loop of PCBP2 KH1 actively participate in hydrogen bonding with the DNA bases (Fig. 1B, notice the labels above the PCBP2 KH1 sequence), the opposite is true for hnRNP K KH3; only one of the variable loop residues is involved in protein-DNA interactions in one complex of the crystallographic dimer. (None of the variable loop residues interacts with the DNA in the other complex of the dimer.) Correspondingly, the variable loop of PCBP2 KH1 wraps back toward the nucleic acid binding groove and becomes more ordered upon binding (Fig. 3B), whereas the variable loop of hnRNP K KH3 remains poorly ordered before and after binding (30).

Recognition of the cytosines at the second and third positions is very similar. The specific hydrogen bonds are mostly conserved, presumably dictated by sequence conservation. Three comparably positioned water molecules are involved in the network of hydrogen bonds in both complexes. Interestingly, although each water bridge bonds to the same position of the DNA bases, mostly different partners are found on the other side of the bridge. This may reflect a sequence-dependent optimization of the binding interactions.

The proteins of the PCBP family contain three copies of the KH nucleic acids binding domains (Fig. 1A), but how many of these domains are capable of binding poly(C) sequences? From the crystal structures, it is now clear that PCBP2 KH1 and hnRNP K KH3 can both do so as an isolated domain. Based on knowledge about the critical residues responsible for specific recognition gained by the crystal structures and sequence alignments (Fig. 1B), all KH1 domains (probably except hnRNP K KH1, which has an aspartic acid instead of glutamic acid at position 51) and KH3 domains should be able to bind poly(C) sequences in a way similar (if not identical) to PCBP2 KH1 and hnRNP K KH3, respectively. We have prepared an 15N-labeled sample of the PCBP2 KH3 domain (which has an Asp at the position corresponding to Glu51 in PCBP2 KH1) and observed significant chemical shift changes in the fingerprint amide resonances upon addition of the same 7-nt htDNA (Supplementary Materials, Ref. 22), indicating a specific binding event. Because of conservation of the two arginines at positions 40 and 57 (PCBP2 KH1 numbering), all KH2 domains should also be able to specifically recognize at least two cytosines at the second and third positions of the tetranucleotide core motif. It is likely that all three of the KH domains within each PCBP are capable of poly(C) binding. Interdomain interactions as well as protein-protein interactions could mediate binding of particular nucleic acids to the proteins of this three KH domain family.

In the PCBP1/2-{alpha}-globin mRNA complex, it was established that a minimum RNA construct containing three stretches of poly(C) sequences formed a 1:1 complex with PCBP2 or PCBP1 (9), consistent with each KH domain recognizing one stretch of the poly(C) sequence. This kind of nucleic acid-PCBP interaction would increase the sequence specificity and affinity of interaction on the one hand, and might help to constrain one or both of the interacting partners (the nucleic acid and the PCBP) in certain biologically significant conformations on the other hand. Of course, other ways of interaction exist. One established example is the interaction of PCBP1 or 2 with two C-rich sequence-harboring RNA structures within the 5'-UTR of poliovirus type-1. The KH1 domain is the major determinant for interaction with both RNAs. Therefore, although PCBP2 KH1 and KH3 (very likely also KH2) domains can all bind single-stranded DNA/RNA, they are clearly not functionally equivalent to one another in these cases. The crystal structures of the PCBP2 KH1 and hnRNP K KH3 complexes reveal some different structural features in binding to poly(C) sequences. These may correlate to distinguishable differences in binding affinity and specificity (regarding the identity of the first nucleotide in the core motif). It is also possible that some KH domains, such as PCBP2 KH1, have evolved some special features to cope with recognition of poly(C) sequences presented in more constrained conformations within highly structured RNAs.

Another important insight into PCBP function gained from our crystal structure is the revelation that PCBP2 KH1 domain has a protein-protein interaction interface (the dimerization interface), suggesting that given an appropriate composition of amino acids residues on the {alpha}3 and {beta}1 surface, some KH domains are capable of assuming non-excluding dual functional roles in nucleic acid and protein interactions. Although the nucleic acid binding interface and the protein interaction interface are located on opposite sites of the domain, a study on the Nova2 KH3 domain (33) suggested that the processes of nucleic acid binding and protein interaction might be correlated. Binding to one interface induced stiffening in other regions of the protein and therefore reduced the entropic cost of binding to the other interface.

Most of the published studies on PCBP2 have been directed to functions dependent on RNA binding events, with only a few reports (21, 22) suggesting its possible involvement in mechanisms associated with DNA recognition. Our co-crystal structure of the PCBP2 KH1-htDNA complex, in conjunction with our previous NMR study of the complex in solution (21, 22), proves that PCBP2 has the ability to bind to poly(C) DNA sequences specifically; poly(G), poly(A), poly(T), and poly(U) sequences did not yield chemical shift changes. This feature should enable PCBP2 to assume functional roles in mechanisms dependent on DNA binding, such as transcriptional regulation and telomere maintenance.

The tandem arrangement of poly(C) stretches on the human C-rich strand telomeric DNA is very similar to some of the known RNA targets for PCBP1 and PCBP2 (such as the 3'-UTR of {alpha}-globin mRNA and some other ultrastable mRNAs). It is fully possible that PCBP1 or -2 would bind to the C-rich strand of htDNA in a manner somewhat similar to the {alpha}-globin complex. Recent progress on telomere/telomerase biology has shown that the telomere/telomerase complex can exist in different stages (34). Whereas the C-rich strand may be present in a double-stranded form with the complementary G-rich strand, at certain stages the DNA telomere or telomerase RNA may have the C-rich htDNA or the RNA template in a single-stranded conformation. Such a scenario would permit the involvement of proteins of the PCBP family in regulation of telomere and telomerase activities through specific binding to the exposed C-rich strands. To this end, we have confirmed, through antibody pull-down experiments and mass spectroscopy, that PCBP1 is one of the nucleic acid-binding proteins present in the human telomere/telomerase complex.4 Further structural and biological studies are being carried out to increase our knowledge of the exact involvement of PCBPs in telomere/telomerase regulation.


   FOOTNOTES
 
The atomic coordinates and structure factors (code 2AXY) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

* This work was supported in part by National Institutes of Health Grants AI46967 (to T. L. J.) and GM51232 (to R. M. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Dept. of Pharmaceutical Chemistry, 600 16th St., Genentech Hall, University of California, San Francisco, CA 94143-2280. Tel.: 415-476-1916; Fax: 415-502-8298; E-mail: james{at}picasso.ucsf.edu.

2 The abbreviations used are: KH domain, hnRNP-K homology domain; PCBP, poly(C)-binding protein; KH1, the first KH domain of PCBP2; UTR, untranslated region; RMSD, root mean square deviation; nt, nucleotide; hnRNP, heterogeneous nuclear ribonucleoprotein; ssDNA, single-stranded DNA; htDNA, human telomeric DNA. Back

3 W. L. DeLano (2002) The PyMOL Molecular Graphics System on the World Wide Web, www.pymol.org. Back

4 Z. Du, J. K. Lee, R. Tjhen, L. Shang, R. M. Stroud, and T. L. James, unpublished results. Back


   ACKNOWLEDGMENTS
 
We thank Chris Waddling for managing the UCSF X-ray Crystallization Laboratory.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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