A water channel in the core of the vitamin B(12) RNA aptamer

BACKGROUND: The 3.0 A crystal structure of the vitamin B(12) RNA aptamer revealed an unusual tertiary structure that is rich in novel RNA structural motifs. Important details of the interactions that stabilize noncanonical base pairing and the role of solvent in the structure were not apparent owing to the limited resolution. RESULTS: The structure of the vitamin B(12) RNA aptamer in complex with its ligand has been determined at 2.3 A resolution by X-ray crystallography. The crystallographic asymmetric unit contains five independent copies of the aptamer-vitamin B(12) complex, making it possible to accurately define well-conserved features. The core of the aptamer contains an unusual water-filled channel that is buried between the three strands of an RNA triplex. Well-ordered water molecules positioned within this channel form bridging hydrogen bonds and stabilize planar base triples that otherwise lack significant direct base-base contacts. The water channel terminates at the interface between the RNA and the bound ligand, leaving a pair of water molecules appropriately positioned to hydrogen bond with the highly polarized cyanide nitrogen of vitamin B(12). Analysis of the general solvation patterns for each nucleotide suggests that water molecules are not precisely positioned, as observed in previous RNA duplex structures, but instead might adjust in response to the varying local environment. Unusual intermolecular base pairing contributes to the formation of three different dimerization contacts that drive formation of the crystal lattice. CONCLUSIONS: The structure demonstrates the important role of water molecules and noncanonical base pairing in driving the formation of RNA tertiary structure and facilitating specific interactions of RNAs with other molecules.     

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.     

Influence of ground-state structure and Mg2+ binding on folding kinetics of the guanine-sensing riboswitch aptamer domain

Riboswitch RNAs fold into complex tertiary structures upon binding to their cognate ligand. Ligand recognition is accomplished by key residues in the binding pocket. In addition, it often crucially depends on the stability of peripheral structural elements. The ligand-bound complex of the guanine-sensing riboswitch from Bacillus subtilis, for example, is stabilized by extensive interactions between apical loop regions of the aptamer domain. Previously, we have shown that destabilization of this tertiary loop-loop interaction abrogates ligand binding of the G37A/C61U-mutant aptamer domain (Gsw(loop)) in the absence of Mg(2+). However, if Mg(2+) is available, ligand-binding capability is restored by a population shift of the ground-state RNA ensemble toward RNA conformations with pre-formed loop-loop interactions. Here, we characterize the striking influence of long-range tertiary structure on RNA folding kinetics and on ligand-bound complex structure, both by X-ray crystallography and time-resolved NMR. The X-ray structure of the ligand-bound complex reveals that the global architecture is almost identical to the wild-type aptamer domain. The population of ligand-binding competent conformations in the ground-state ensemble of Gsw(loop) is tunable through variation of the Mg(2+) concentration. We quantitatively describe the influence of distinct Mg(2+) concentrations on ligand-induced folding trajectories both by equilibrium and time-resolved NMR spectroscopy at single-residue resolution.     

A structural intermediate pre-organizes the add adenine riboswitch for ligand recognition

Riboswitches are RNA sequences that regulate gene expression by undergoing structural changes upon the specific binding of cellular metabolites. Crystal structures of purine-sensing riboswitches have revealed an intricate network of interactions surrounding the ligand in the bound complex. The mechanistic details about how the aptamer folding pathway is involved in the formation of the metabolite binding site have been previously shown to be highly important for the riboswitch regulatory activity. Here, a combination of single-molecule FRET and SHAPE assays have been used to characterize the folding pathway of the adenine riboswitch from Vibrio vulnificus. Experimental evidences suggest a folding process characterized by the presence of a structural intermediate involved in ligand recognition. This intermediate state acts as an open conformation to ensure ligand accessibility to the aptamer and folds into a structure nearly identical to the ligand-bound complex through a series of structural changes. This study demonstrates that the add riboswitch relies on the folding of a structural intermediate that pre-organizes the aptamer global structure and the ligand binding site to allow efficient metabolite sensing and riboswitch genetic regulation.     

Metal-ion binding and metal-ion induced folding of the adenine-sensing riboswitch aptamer domain

Divalent cations are important in the folding and stabilization of complex RNA structures. The adenine-sensing riboswitch controls the expression of mRNAs for proteins involved in purine metabolism by directly sensing intracellular adenine levels. Adenine binds with high affinity and specificity to the ligand binding or aptamer domain of the adenine-sensing riboswitch. The X-ray structure of this domain in complex with adenine revealed an intricate RNA-fold consisting of a three-helix junction stabilized by long-range base-pairing interactions and identified five binding sites for hexahydrated Mg²⁺-ions. Furthermore, a role for Mg²⁺-ions in the ligand-induced folding of this RNA was suggested. Here, we describe the interaction of divalent cations with the RNA–adenine complex in solution as studied by high-resolution NMR spectroscopy. Paramagnetic line broadening, chemical shift mapping and intermolecular nuclear Overhauser effects (NOEs) indicate the presence of at least three binding sites for divalent cations. Two of them are similar to those in the X-ray structure. The third site, which is important for the folding of this RNA, has not been observed previously. The ligand-free state of the RNA is conformationally heterogeneous and contains base-pairing patterns detrimental to ligand binding in the absence of Mg²⁺, but becomes partially pre-organized for ligand binding in the presence of Mg²⁺. Compared to the highly similar guanine-sensing riboswitch, the folding pathway for the adenine-sensing riboswitch aptamer domain is more complex and the influence of Mg²⁺ is more pronounced.     

Towards understanding the mechanisms of molecular recognition by computer simulations of ligand-protein interactions

The thermodynamic and kinetic aspects of molecular recognition for the methotrexate (MTX)-dihydrofolate reductase (DHFR) ligand-protein system are investigated by the binding energy landscape approach. The impact of 'hot' and 'cold' errors in ligand mutations on the thermodynamic stability of the native MTX-DHFR complex is analyzed, and relationships between the molecular recognition mechanism and the degree of ligand optimization are discussed. The nature and relative stability of intermediates and thermodynamic phases on the ligand-protein association pathway are studied, providing new insights into connections between protein folding and molecular recognition mechanisms, and cooperativity of ligand-protein binding. The results of kinetic docking simulations are rationalized based on the thermodynamic properties determined from equilibrium simulations and the shape of the underlying binding energy landscape. We show how evolutionary ligand selection for a receptor active site can produce well-optimized ligand-protein systems such as MTX-DHFR complex with the thermodynamically stable native structure and a direct transition mechanism of binding from unbound conformations to the unique native structure.     

A model for the study of ligand binding to the ribosomal RNA helix h44

Oligonucleotide models of ribosomal RNA domains are powerful tools to study the binding and molecular recognition of antibiotics that interfere with bacterial translation. Techniques such as selective chemical modification, fluorescence labeling and mutations are cumbersome for the whole ribosome but readily applicable to model RNAs, which are readily crystallized and often give rise to higher resolution crystal structures suitable for detailed analysis of ligand–RNA interactions. Here, we have investigated the HX RNA construct which contains two adjacent ligand binding regions of helix h44 in 16S ribosomal RNA. High-resolution crystal structure analysis confirmed that the HX RNA is a faithful structural model of the ribosomal target. Solution studies showed that HX RNA carrying a fluorescent 2-aminopurine modification provides a model system that can be used to monitor ligand binding to both the ribosomal decoding site and, through an indirect effect, the hygromycin B interaction region.     

Folding of the SAM-I riboswitch: A tale with a twist

Riboswitches are ligand-dependent RNA genetic regulators that control gene expression by altering their structures. The elucidation of riboswitch conformational changes before and after ligand recognition is crucial to understand how riboswitches can achieve high ligand binding affinity and discrimination against cellular analogs. The detailed characterization of riboswitch folding pathways suggest that they may use their intrinsic conformational dynamics to sample a large array of structures, some of which being nearly identical to ligand-bound molecules. Some of these structural conformers can be "captured" upon ligand binding, which is crucial for the outcome of gene regulation. Recent studies about the SAM-I riboswitch have revealed unexpected and previously unknown RNA folding mechanisms. For instance, the observed helical twist of the P1 stem upon ligand binding to the SAM-I aptamer adds a new element in the repertoire of RNA strategies for recognition of small metabolites. From an RNA folding perspective, these findings also strongly indicate that the SAM-I riboswitch could achieve ligand recognition by using an optimized combination of conformational capture and induced-fit approaches, a feature that may be shared by other RNA regulatory sequences.     

Molecular dissection of the intrinsic factor-vitamin B12 receptor, cubilin, discloses regions important for membrane association and ligand binding.

Cubilin, the receptor for intrinsic factor-vitamin B12, is a novel type of high molecular weight receptor consisting of a 27 CUB (complement components C1r/C1s, Uegf, and bone morphogenic protein-1) domain cluster preceded by 8 epidermal growth factor repeats and a short N-terminal sequence. In addition to binding the vitamin B12-carrier complex, cubilin also binds receptor-associated protein. To delineate the structures for membrane association and ligand binding we established a panel of stable transfected Chinese hamster ovary cells expressing overlapping segments of rat cubilin. Analysis of conditioned media and cell extracts of transfected cells revealed that the N-terminal cubilin region conveys membrane association. Helical plotting of this region demonstrated a conserved amphipathic helix pattern (Lys74-Glu109) as a candidate site for hydrophobic interactions. Ligand affinity chromatography and surface plasmon resonance analysis of the secreted cubilin fragments showed ligand binding in the CUB domain region. Further dissection of binding-active fragments localized the binding site for intrinsic factor-vitamin B12 to CUB domains 5-8 and a receptor-associated protein-binding site to CUB domains 13-14. In conclusion, the N-terminal cubilin region seems crucial for membrane association, whereas the CUB domain cluster harbors distinct sites for ligand binding.     

Key molecular contacts promote recognition of the BAFF receptor by TNF receptor-associated factor 3: implications for intracellular signaling regulation

B cell-activating factor belonging to the TNF family receptor (BAFF-R), a member of the TNFR superfamily, plays a role in autoimmunity after ligation with BAFF ligand (also called TALL-1, BLyS, THANK, or zTNF4). BAFF/BAFF-R interactions are critical for B cell regulation, and signaling from this ligand-receptor complex results in NF-kappaB activation. Most TNFRs transmit signals intracellularly by recruitment of adaptor proteins called TNFR-associated factors (TRAFs). However, BAFF-R binds only one TRAF adaptor, TRAF3, and this interaction negatively regulates activation of NF-kappaB. In this study, we report the crystal structure of a 24-residue fragment of the cytoplasmic portion of BAFF-R bound in complex with TRAF3. The recognition motif (162)PVPAT(166) in BAFF-R is accommodated in the same binding crevice on TRAF3 that binds two related TNFRs, CD40 and LTbetaR, but is presented in a completely different structural framework. This region of BAFF-R assumes an open conformation with two extended strands opposed at right angles that each make contacts with TRAF3. The recognition motif is located in the N-terminal arm and intermolecular contacts mediate TRAF recognition. In the C-terminal arm, key stabilizing contacts are made, including critical hydrogen bonds with Gln(379) in TRAF3 that define the molecular basis for selective binding of BAFF-R solely to this member of the TRAF family. A dynamic conformational adjustment of Tyr(377) in TRAF3 occurs forming a new intermolecular contact with BAFF-R that stabilizes the complex. The structure of the complex provides a molecular explanation for binding affinities and selective protein interactions in TNFR-TRAF interactions.     

Human initiation protein Orc4 prefers triple stranded DNA

In higher eukaryotes mechanism of DNA replication origin recognition and binding by origin recognition complex (ORC) is still unknown. Origin transfer studies have shown that origin sites are genetically determined, containing functionally interchangeable modules. One of such modules from the human lamin B2 origin of replication has the ability to adopt unorthodox structure partly composed of intramolecular triplex. Sequences involved in triplex formation coincide with ORC binding sites both in vitro and in vivo. To explore potential significance of unorthodox DNA structures in origin recognition by ORC, we tested DNA binding properties of human ORC subunit 4 (HsOrc4) which has independent DNA binding activity in vitro and similar binding characteristics as ORC holocomplex. Our results demonstrated that DNA binding activity of HsOrc4 depends on length and structure of DNA with triplex being the protein’s preferred binding target. Such feature could play part in origin selection through directing ORC to DNA sequence prone to adopt unorthodox structure.     

Crystallographic studies on B12 binding proteins in eukaryotes and prokaryotes

The X-ray crystal structures of several important vitamin B12 binding proteins that have been solved in recent years have enhanced our current understanding in the vitamin B12 field. These structurally diverse groups of B12 binding proteins perform various important biological activities, both by transporting B12 as well as catalyzing various biological reactions. An in-depth comparative analysis of these structures was carried out using PDB coordinates of a carefully chosen database of B12 binding proteins to correlate the overall folding of the molecule with phylogeny, the B12 interactions, and with their biological function. The structures of these proteins are discussed in the context of this comparative analysis.     

Molecular dynamics simulation study of the binding of purine bases to the aptamer domain of the guanine sensing riboswitch

Riboswitches are a novel class of genetic control elements that function through the direct interaction of small metabolite molecules with structured RNA elements. The ligand is bound with high specificity and affinity to its RNA target and induces conformational changes of the RNA''s secondary and tertiary structure upon binding. To elucidate the molecular basis of the remarkable ligand selectivity and affinity of one of these riboswitches, extensive all-atom molecular dynamics simulations in explicit solvent (≈1 μs total simulation length) of the aptamer domain of the guanine sensing riboswitch are performed. The conformational dynamics is studied when the system is bound to its cognate ligand guanine as well as bound to the non-cognate ligand adenine and in its free form. The simulations indicate that residue U51 in the aptamer domain functions as a general docking platform for purine bases, whereas the interactions between C74 and the ligand are crucial for ligand selectivity. These findings either suggest a two-step ligand recognition process, including a general purine binding step and a subsequent selection of the cognate ligand, or hint at different initial interactions of cognate and noncognate ligands with residues of the ligand binding pocket. To explore possible pathways of complex dissociation, various nonequilibrium simulations are performed which account for the first steps of ligand unbinding. The results delineate the minimal set of conformational changes needed for ligand release, suggest two possible pathways for the dissociation reaction, and underline the importance of long-range tertiary contacts for locking the ligand in the complex.     

Solution structure of an informationally complex high-affinity RNA aptamer to GTP

Higher-affinity RNA aptamers to GTP are more informationally complex than lower-affinity aptamers. Analog binding studies have shown that the additional information needed to improve affinity does not specify more interactions with the ligand. In light of those observations, we would like to understand the structural characteristics that enable complex aptamers to bind their ligands with higher affinity. Here we present the solution structure of the 41-nt Class I GTP aptamer (K(d) = 75 nM) as determined by NMR. The backbone of the aptamer forms a reverse-S that shapes the binding pocket. The ligand nucleobase stacks between purine platforms and makes hydrogen bonds with the edge of another base. Interestingly, the local modes of interaction for the Class I aptamer and an RNA aptamer that binds ATP with a K(d) of 6 microM are very much alike. The aptamers exhibit nearly identical levels of binding specificity and fraction of ligand sequestered from the solvent (81%-85%). However, the GTP aptamer is more informationally complex (approximately 45 vs. 35 bits) and has a larger recognition bulge (15 vs. 12 nucleotides) with many more stabilizing base-base interactions. Because the aptamers have similar modes of ligand binding, we conclude that the stabilizing structural elements in the Class I aptamer are responsible for much of the difference in K(d). These results are consistent with the hypothesis that increasing the number of intra-RNA interactions, rather than adding specific contacts to the ligand, is the simplest way to improve binding affinity.     

The 1.3 A crystal structure of a biotin-binding pseudoknot and the basis for RNA molecular recognition

A pseudoknot-containing aptamer isolated from a pool of random sequence molecules has been shown previously to represent an optimal RNA solution to the problem of binding biotin. The affinity of this RNA molecule is nonetheless orders of magnitude weaker than that of its highly evolved protein analogs, avidin and streptavidin. To understand the structural basis for biotin binding and to compare directly strategies for ligand recognition available to proteins and RNA molecules, we have determined the 1.3 A crystal structure of the aptamer complexed with its ligand. Biotin is bound at the interface between the pseudoknot's stacked helices in a pocket defined almost entirely by base-paired nucleotides. In comparison to the protein avidin, the aptamer packs more tightly around the biotin headgroup and makes fewer contacts with its fatty acid tail. Whereas biotin is deeply buried within the hydrophobic core in the avidin complex, the aptamer relies on a combination of hydrated magnesium ions and immobilized water molecules to surround its ligand. In addition to demonstrating fundamentally different approaches to molecular recognition by proteins and RNA, the structure provides general insight into the mechanisms by which RNA function is mediated by divalent metals.     

NMR resonance assignments for the tetramethylrhodamine binding RNA aptamer 3 in complex with the ligand 5-carboxy-tetramethylrhodamine

RNA aptamers are used in a wide range of biotechnological or biomedical applications. In many cases the high resolution structures of these aptamers in their ligand-complexes have revealed fundamental aspects of RNA folding and RNA small molecule interactions. Fluorescent RNA-ligand complexes in particular find applications as optical sensors or as endogenous fluorescent tags for RNA tracking in vivo. Structures of RNA aptamers and aptamer ligand complexes constitute the starting point for rational function directed optimization approaches. Here, we present the NMR resonance assignment of an RNA aptamer binding to the fluorescent ligand tetramethylrhodamine (TMR) in complex with the ligand 5-carboxy-tetramethylrhodamine (5-TAMRA) as a starting point for a high-resolution structure determination using NMR spectroscopy in solution.     

Molecular interactions investigated by multi-dimensional solid-state NMR

Solid-state NMR (ssNMR) offers insight into the formation of protein complexes and ligand binding for a large range of molecular sizes and binding affinities. Recent instrumental and methodological progress has enabled novel possibilities for using multi-dimensional ssNMR to study molecular 3D structures and interactions in noncrystalline systems. Two-dimensional ssNMR correlation experiments were applied to study ligand binding in globular and membrane proteins and have enabled the investigation of molecular interfaces in the context of protein folding and aggregation. In lipid bilayers, a versatile set of ssNMR experiments has been developed to study molecular structure, topology and complex formation in a functional environment.