Solution structure of the complex formed by the two N-terminal RNA-binding domains of nucleolin and a pre-rRNA target

Nucleolin is a 70 kDa multidomain protein involved in several steps of eukaryotic ribosome biogenesis. In vitro selection in combination with mutagenesis and structural analysis identified binding sites in pre-rRNA with the consensus (U/G)CCCG(A/G) in the context of a hairpin structure, the nucleolin recognition element (NRE). The central region of the protein contains four tandem RNA-binding domains (RBDs), of which the first two are responsible for the RNA-binding specificity and affinity for NREs. Here, we present the solution structure of the 28 kDa complex formed by the two N-terminal RNA-binding domains of nucleolin (RBD12) and a natural pre-rRNA target, b2NRE. The structure demonstrates that the sequence-specific recognition of the pre-rRNA NRE is achieved by intermolecular hydrogen bonds and stacking interactions involving mainly the beta-sheet surfaces of the two RBDs and the linker residues. A comparison with our previously determined NMR structure of RBD12 in complex with an in vitro selected RNA target, sNRE, shows that although the sequence-specific recognition of the loop consensus nucleotides is the same in the two complexes, they differ in several aspects. While the protein makes numerous specific contacts to the non-consensus nucleotides in the loop E motif (S-turn) in the upper part of the sNRE stem, nucleolin RBD12 contacts only consensus nucleotides in b2NRE. The absence of these upper stem contacts from the RBD12/b2NRE complex results in a much less stable complex, as demonstrated by kinetic analyses. The role of the loop E motif in high-affinity binding is supported by gel-shift analyses with a series of sNRE mutants. The less stable interaction of RBD12 with the natural RNA target is consistent with the proposed role of nucleolin as a chaperone that interacts transiently with pre-rRNA to prevent misfolding.     

Contributions of the RNA-binding and linker domains and RNA structure to the specificity and affinity of the nucleolin RBD12/NRE interaction

Nucleolin is a multidomain phosphoprotein involved in ribosome biogenesis. In vitro selection and binding studies with pre-rRNA fragments have shown that the first two RNA-binding domains (RBDs) in nucleolin (RBD12) recognize the consensus sequence (U/G)CCCG(A/G) in the context of a stem-loop structure (nucleolin-recognition element = NRE). Structural studies of nucleolin RBD12 in complex with an in vitro selected NRE (sNRE) and a natural pre-rRNA NRE (b2NRE) have revealed that sequence-specific binding of the consensus NRE is achieved in a similar manner in both complexes using residues in both RBDs as well as the linker connecting them. Using fluorescence anisotropy (FA) and nuclear magnetic resonance (NMR), we demonstrate the importance of the linker for NRE affinity by showing that only the individual RBDs with the linker attached retain the ability to specifically bind, albeit weakly, to sNRE and b2NRE. Binding of RBD1 and RBD2 to the NREs in trans is not detected even when one of the RBDs has the linker attached, which suggests that the linker also contributes to the affinity by tethering the two RBDs. To determine if binding of nucleolin RBD12 to natural NREs is dependent on a specific RNA stem-loop structure, as was the case for the sNRE, we conducted FA and NMR binding assays with nucleolin RBD12 and a single-stranded NRE. The results show that nucleolin RBD12 sequence-specifically binds a single-stranded NRE with an affinity similar to that for b2NRE, indicating that a stem-loop structure is not required for the nucleolin RBD12/pre-rRNA NRE interaction.     

Recognition of pre-formed and flexible elements of an RNA stem-loop by nucleolin

Nucleolin is an abundant nucleolar protein which is essential for ribosome biogenesis. The first two of its four tandem RNA-binding domains (RBD12) specifically recognize a stem-loop structure containing a conserved UCCCGA sequence in the loop called the nucleolin-recognition element (NRE). We have determined the structure of the consensus SELEX NRE (sNRE) by NMR spectroscopy. In both the free and bound RNA the top part of the stem forms a loop E (or S-turn) motif. In the absence of protein, the structure of the hairpin loop is not well defined due to conformational heterogeneity, and appears to be in equilibrium between two families of conformations. Titrations of RBD1, RBD2, and RBD12 with the sNRE show that specific binding requires RBD12. In complex with RBD12, the hairpin loop interacts specifically with the protein and adopts a well-defined structure which shares some of the features of the free form. The loop E motif also has specific interactions with the protein. Implications of these findings for the mechanism of recognition of RNA structures by modular proteins are discussed.     

Molecular basis of sequence-specific recognition of pre-ribosomal RNA by nucleolin

The structure of the 28 kDa complex of the first two RNA binding domains (RBDs) of nucleolin (RBD12) with an RNA stem-loop that includes the nucleolin recognition element UCCCGA in the loop was determined by NMR spectroscopy. The structure of nucleolin RBD12 with the nucleolin recognition element (NRE) reveals that the two RBDs bind on opposite sides of the RNA loop, forming a molecular clamp that brings the 5' and 3' ends of the recognition sequence close together and stabilizing the stem-loop. The specific interactions observed in the structure explain the sequence specificity for the NRE sequence. Binding studies of mutant proteins and analysis of conserved residues support the proposed interactions. The mode of interaction of the protein with the RNA and the location of the putative NRE sites suggest that nucleolin may function as an RNA chaperone to prevent improper folding of the nascent pre-rRNA.     

Solution structure of the two N-terminal RNA-binding domains of nucleolin and NMR study of the interaction with its RNA target

Nucleolin is an abundant 70 kDa nucleolar protein involved in many aspects of ribosomal RNA biogenesis. The central region of nucleolin contains four tandem consensus RNA-binding domains (RBD). The two most N-terminal domains (RBD12) bind with nanomolar affinity to an RNA stem-loop containing the consensus sequence UCCCGA in the loop. We have determined the solution structure of nucleolin RBD12 in its free form and have studied its interaction with a 22 nt RNA stem-loop using multidimensional NMR spectroscopy. The two RBDs adopt the expected beta alpha beta beta alpha beta fold, but the position of the beta 2 strand in both domains differs from what was predicted from sequence alignments. RBD1 and RBD2 are significantly different from each others and this is likely important in their sequence specific recognition of the RNA. RBD1 has a longer alpha-helix 1 and a shorter beta 2-beta 3 loop than RBD2, and differs from most other RBDs in these respects. The two RBDs are separated by a 12 amino acid flexible linker and do not interact with one another in the free protein. This linker becomes ordered when RBD12 binds to the RNA. Analysis of the observed NOEs between the protein and the RNA indicates that both RBDs interact with the RNA loop via their beta-sheet. Each domain binds residues on one side of the loop; specifically, RBD2 contacts the 5' side and RBD1 contacts the 3'.     

Two different combinations of RNA-binding domains determine the RNA binding specificity of nucleolin

Nucleolin is an abundant nucleolar protein involved in several steps of ribosome biogenesis. The protein is highly conserved through evolution and possesses four RNA-binding domains (RBD), which are likely to determine its RNA binding specificity. Previous studies have shown that nucleolin interacts with two different RNA targets. The first is a small stem-loop structure, the nucleolin recognition element (NRE), found all along the pre-ribosomal RNA. The second is a short single-stranded RNA sequence, the evolutionary conserved motif (ECM), located five nucleotides downstream of the first processing site in the pre-ribosomal RNA 5' external transcribed spacer. Biochemical, genetic, and structural studies have shown that the first two RBD of nucleolin are necessary and sufficient for the specific interaction of nucleolin with the NRE motif. In this work, we have studied the interaction of nucleolin with the ECM sequence. Deletion and mutational analyses showed that all four RBDs of hamster nucleolin were required for the interaction with the ECM sequence. This RNA binding specificity is conserved between hamster and Xenopus laevis, whereas the Xenopus protein does not interact with the NRE. Nucleolin is the first example of a protein that requires four RBDs for its interaction with an RNA target, demonstrating that a single protein can use different combinations of RBD to interact specifically with several RNA sequences.     

RNA recognition by the joint action of two nucleolin RNA-binding domains: genetic analysis and structural modeling.

The interaction of nucleolin with a short stem-loop structure (NRE) requires two contiguous RNA-binding domains (RBD 1+2). The structural basis for RNA recognition by these RBDs was studied using a genetic system in Escherichia coli. Within each of the two domains, we identified several mutations that severely impair interaction with the RNA target. Mutations that alter RNA-binding specificity were also isolated, suggesting the identity of specific contacts between RBD 1+2 amino acids and nucleotides within the NRE stem-loop. Our data indicate that both RBDs participate in a joint interaction with the NRE and that each domain uses a different surface to contact the RNA. The constraints provided by these genetic data and previous mutational studies have enabled us to propose a three-dimensional model of nucleolin RBD 1+2 bound to the NRE stem-loop.     

Structure of the RNA Binding Domain of a DEAD-Box Helicase Bound to Its Ribosomal RNA Target Reveals a Novel Mode of Recognition by an RNA Recognition Motif

DEAD-box RNA helicases of the bacterial DbpA subfamily are localized to their biological substrate when a carboxy-terminal RNA recognition motif domain binds tightly and specifically to a segment of 23S ribosomal RNA (rRNA) that includes hairpin 92 of the peptidyl transferase center. A complex between a fragment of 23S rRNA and the RNA binding domain (RBD) of the Bacillus subtilis DbpA protein YxiN was crystallized and its structure was determined to 2.9 Å resolution, revealing an RNA recognition mode that differs from those observed with other RNA recognition motifs. The RBD is bound between two RNA strands at a three-way junction. Multiple phosphates of the RNA backbone interact with an electropositive band generated by lysines of the RBD. Nucleotides of the single-stranded loop of hairpin 92 interact with the RBD, including the guanosine base of G2553, which forms three hydrogen bonds with the peptide backbone. A G2553U mutation reduces the RNA binding affinity by 2 orders of magnitude, confirming that G2553 is a sequence specificity determinant in RNA binding. Binding of the RBD to 23S rRNA in the late stages of ribosome subunit maturation would position the ATP-binding duplex destabilization fragment of the protein for interaction with rRNA in the peptidyl transferase cleft of the subunit, allowing it to “melt out” unstable secondary structures and allow proper folding.     

Contribution of the C-terminal tail of U1A RBD1 to RNA recognition and protein stability.

The N-terminal RNA binding domain (RBD1) of the human U1A protein interacts specifically with a short RNA hairpin containing the U1 snRNA stem/loop II sequence. Previous RNA binding studies have suggested that the C-terminal tail of RBD1 contributes to RNA recognition in addition to interactions on the beta-sheet surface of the protein. To evaluate the contributions of these C-terminal residues in RBD1 to RNA binding affinity and specificity, as well as to study the thermodynamic stability of RBDs, a number of RBD1 mutants with truncated tails, with single amino acid substitutions, and with both a truncation and an amino acid substitution, have been constructed. The thermodynamic stabilities of these mutants have been measured and compared by GdnHCI unfolding experiments. The RNA binding affinity and specificity of these mutant proteins have been assessed by measuring the binding of each protein to the wild-type RNA hairpin and to selected RNA mutants with nucleotide substitutions in the RNA loop. The results demonstrate first that, although the C-terminal tail of RBD1 makes significant contributions to RNA binding affinity, it is not required for RNA binding, and second, its contributions to binding specificity are mediated only through selected nucleotides in the RNA loop, for in the absence of the tail, the protein continues to use other nucleotides to discriminate among RNAs. In these truncated proteins, the secondary structure intrinsic to the C-terminal tail is absent, yet their affinity and discrimination for RNAs are not lost. Thus, a structured tail is not required for RNA recognition.     

Contribution of the tyrosines to the structure and function of the human U1A N-terminal RNA binding domain.

RNA binding domains (RBDs) are members of a large family of proteins that share minimal sequence conservation but adopt an alpha beta sandwich global fold. Defining the contributions of specific amino acids to RBD structure and RNA binding is critical to understanding the functions of these proteins. In these experiments with the human U1A N-terminal RNA binding domain (RBD1), the contributions from each of its four tyrosines to protein structure, stability, and RNA binding were measured. Each tyrosine was substituted with phenylalanine and one other selected residue, and the resulting proteins were characterized by chemical denaturation to measure their unfolding free energy, by binding free energies to the wild-type RNA hairpin, and by 19F NMR to probe for structural changes. Features of the protein identified in these experiments include a possible tyrosine/lysine contact in an alpha-helix, which may be an example of an energetically favorable aromatic/amino side chain interaction. One long loop of the protein, which shows unusual 15N backbone and tyrosine side-chain dynamics, is implicated in protein:protein association. The diverse interactions of the four tyrosine residues in the organization of RBD1 illustrate how each member of this family of proteins will have unique molecular details that contribute to function.     

Crosslinking of an iodo-uridine-RNA hairpin to a single site on the human U1A N-terminal RNA binding domain.

The N-terminal RNA binding domain (RBD) of the human U1A snRNP protein binds tightly and specifically to an RNA hairpin that contains a 10-nucleotide loop. The protein is one of a class of RNA binding proteins that adopts a beta alpha beta beta alpha beta global fold, which in turn forms a four-stranded antiparallel beta-sheet. This sheet forms the primary binding surface for the RNA, as shown by the crosslinking results described here, and in more detail by a recently described co-crystal of this RBD with an RNA hairpin (Oubridge C, et al., 1994, Nature 372:432-438). The RNA hairpin sequence used in the crosslinking experiments, containing 5-iodo-uridine, is a variant of the normal U1 snRNA sequence which is able to form a crosslink with the protein, in contrast to the wild-type sequence, which does not. This single uridine substitution in the 10-nucleotide loop is the site of cross-linking to one tyrosine (Tyr 13) in the beta 1 strand of the U1A N-terminal RBD. This same uridine is also crosslinked to a mutant Tyr 13 Phe RBD, at this Phe 13 substitution.     

Solution structure of a purine rich hexaloop hairpin belonging to PGY/MDR1 mRNA and targeted by antisense oligonucleotides

A preferential target of antisense oligonucleotides directed against human PGY/MDR1 mRNA is a hairpin containing a stem with a G*U wobble pair, capped by the purine-rich 5'r(GGGAUG)3' hexaloop. This hairpin is studied by multidimensional NMR and restrained molecular dynamics, with special emphasis on the conformation of south sugars and non-standard phosphate linkages evidenced in both the stem and the loop. The hairpin is found to be highly structured. The G*U wobble pair, a strong counterion binding site, displays structural particularities that are characteristic of this type of mismatch. The upper part of the stem undergoes distortions that optimize its interactions with the beginning of the loop. The loop adopts a new fold in which the single-stranded GGGA purine tract is structured in A-like conformation stacked in continuity of the stem and displays an extensive hydrogen bonding surface for recognition. The remarkable hairpin stability results from classical inter- and intra-strand interactions reinforced by numerous hydrogen bonds involving unusual backbone conformations and ribose 2'-hydroxyl groups. Overall, this work emphasizes numerous features that account for the well-ordered structure of the whole hairpin and highlights the loop properties that facilitate interaction with antisense oligonucleotides.     

The C-terminal domain of nucleolin accelerates nucleic acid annealing

We report that the abundant nucleolar protein nucleolin accelerates nucleic acid annealing. Nucleolin accelerates annealing of complementary oligonucleotides and of oligonucleotides that contain a limited number of mismatches. The annealing activity of nucleolin can be localized to a C-terminal region consisting of two RNA binding domains (RBD3 and RBD4) and the RGG(9) domain (RBD3-RBD4-RGG(9)). This same region mediates self-association of nucleolin. The RGG(9) domain of nucleolin, believed to mediate interactions between nucleolin and several ribosomal proteins, is neither sufficient for self-association, as determined by small-angle X-ray scattering, nor can it independently accelerate annealing. Acceleration of nucleic acid annealing by nucleolin is likely to depend on self-association of nucleolin molecules bound to nucleic acid.     

Defining the orientation of the human U1A RBD1 on its UTR by tethered-EDTA(Fe) cleavage.

The N-terminal RNA binding domain of the human U1A protein (RBD1) specifically binds an RNA hairpin of U1 snRNA as well as two internal loops in the 3' UTR of its own mRNA. Here, a single cysteine has been introduced into Loop 1 of RBD1, which is subsequently used to attach (EDTA-2-aminoethyl) 2-pyridyl disulfide-Fe3+ (EPD-Fe). This EDTA-Fe derivative is used to generate hydroxyl radicals to cleave the proximal RNA sugar-phosphate backbone in the RNA-RBD complexes. RBD1(K20C)-EPD-Fe cleaves the 5' strand of the RNA hairpin stem, centered four base pairs away from the base of the loop, and cleaves the UTR in two places, again centered on the 5' side of the fourth base pair from each internal loop. These data, extrapolated to the position of Lys 20 in RBD1, orient the two proteins bound to the UTR, and provide direct biochemical evidence for the proposed model of the RBD1:UTR complex.     

Modulation of Integrin Activation by an Entropic Spring in the β-Knee

We show that the length of a loop in the β-knee, between the first and second cysteines (C1-C2) in integrin EGF-like (I-EGF) domain 2, modulates integrin activation. Three independent sets of mutants, including swaps among different integrin β-subunits, show that C1-C2 loop lengths of 12 and longer favor the low affinity state and masking of ligand-induced binding site (LIBS) epitopes. Shortening length from 12 to 4 residues progressively increases ligand binding and LIBS epitope exposure. Compared with length, the loop sequence had a smaller effect, which was ascribable to stabilizing loop conformation, and not interactions with the α-subunit. The data together with structural calculations support the concept that the C1-C2 loop is an entropic spring and an emerging theme that disordered regions can regulate allostery. Diversity in the length of this loop may have evolved among integrin β-subunits to adjust the equilibrium between the bent and extended conformations at different set points.     

The solution structure of a d[C(TTCG)G] DNA hairpin and comparison to the unusually stable RNA analogue.

The solution structure of the DNA analogue of the unusually stable r[C(UUCG)G] RNA hairpin, 5'-d[GGA-C(TTCG)GTCC]-3', has been determined by NMR spectroscopy, and its structure has been compared to that of the RNA molecule. The RNA molecule is compact and rigid with a highly structured loop. However, the DNA molecule is much less structured. The DNA hairpin contains a B-form stem of four base pairs. The terminal base pair frays, and the 3'-terminal nucleotides, C11 and C12, are in equilibrium between 2'-endo and 3'-endo conformations. Unlike the RNA loop, the DNA loop contains no syn nucleotides, and there is no evidence for base-base or base-phosphate hydrogen bonding in the loop. The loop is flexible, and reveals no specific internucleotide interactions.     

Restraining the conformation of HIV-1 gp120 by removing a flexible loop

The trimeric HIV/SIV envelope glycoprotein, gp160, is cleaved to noncovalently associated fragments, gp120 and gp41. Binding of gp120 to viral receptors leads to large structural rearrangements in both fragments. The unliganded gp120 core has a disordered beta3-beta5 loop, which reconfigures upon CD4 binding into an ordered, extended strand. Molecular modeling suggests that residues in this loop may contact gp41. We show here that deletions in the beta3-beta5 loop of HIV-1 gp120 weaken the binding of CD4 and prevent formation of the epitope for monoclonal antibody (mAb) 17b (which recognizes the coreceptor site). Formation of an encounter complex with CD4 binding and interactions of gp120 with mAbs b12 and 2G12 are not affected by these deletions. Thus, deleting the beta3-beta5 loop blocks the gp120 conformational change and may offer a strategy for design of restrained immunogens. Moreover, mutations in the SIV beta3-beta5 loop lead to greater spontaneous dissociation of gp120 from cell-associated trimers. We suggest that the CD4-induced rearrangement of this loop releases structural constraints on gp41 and thus potentiates its fusion activity.