The structure of an RNA "kissing" hairpin complex of the HIV TAR hairpin loop and its complement

We have used nuclear magnetic resonance (NMR) to obtain the structure of an RNA "kissing" hairpin complex formed between the HIV-2 TAR hairpin loop and a hairpin with a complementary loop sequence. Kissing hairpins are important in natural antisense reactions; their complex is a specific target for protein binding. The complex has all six nucleotides of each loop paired to form a bent quasicontinuous helix of three coaxially stacked helices: two stems plus a loop-loop interaction helix. Experimental constraints derived from heteronuclear and homonuclear NMR data on 13C and 15N-labeled RNA led to a structure for the loop-loop helix with an average root-mean-square deviation of 0.83 (+/-0.10) A for 33 converged structures relative to the average structure. The loop-loop helix of the kissing complex is distorted compared to A-form RNA. Its major groove is blocked by the phosphodiester bonds that connect the first loop residue of each hairpin with its own stem, and it is flanked by two negatively charged phosphate clusters. The loop-loop helix has alternating helical twists between adjacent base-pairs. The base-pairs at the helix junctions are overwound and three base-pairs near the helix junctions adopt high propeller twists. All these changes reduce the distance needed for the bridging phosphodiester bonds connecting each stem and loop to cross the major groove of the loop-loop helix, and result in a deformed RNA helix with localized perturbations in the minor groove surface. The alternating helical twist pattern, plus other distortions in the loop-loop helix may be important for Rom protein recognition of the kissing hairpin complex.     

Functional analysis of peptide motif for RNA microhelix binding suggests new family of RNA-binding domains

RNA microhelices that recreate the acceptor stems of transfer RNAs are charged with specific amino acids. Here we identify a two-helix pair in alanyl-tRNA synthetase that is required for RNA microhelix binding. A single point mutation at an absolutely conserved residue in this motif selectively disrupts RNA binding without perturbation of the catalytic site. These results, and findings of similar motifs in the proximity of the active sites of other tRNA synthetases, suggest that two-helix pairs are widespread and provide a structural framework important for contacts with bound RNA substrates.     

Strong selective pressure to use G:U to mark an RNA acceptor stem for alanine

The identity of alanine tRNAs is dependent on a G:U base pair at the 3:70 position of the acceptor helix. This system of molecular recognition is widely distributed from bacteria to human-cell cytoplasm. In contrast, some mitochondrial alanine acceptor helices are markedly different and contain nucleotides known to block aminoacylation by a nonmitochondrial enzyme. Thus, acceptor helix recognition may differ in these systems and may not depend on G:U. Here we report an example of a Caenorhabditis elegans mitochondrial system where the G:U pair is preserved but where proximal nucleotides known to block charging by a nonmitochondrial enzyme are also present. We show that, as expected, the mitochondrial substrate is not charged by the bacterial enzyme. In contrast, the cloned mitochondrial enzyme charged both mitochondrial and bacterial microhelices. Strikingly, charging of each required the G:U pair. Thus, G:U recognition persists even with an acceptor helix context that inactivates nonmitochondrial systems. The results suggest strong selective pressure to use G:U in a variety of contexts to mark an acceptor stem for alanine. Separate experiments also demonstrate that, at least for the mitochondrial enzyme, helix instability or irregularity is not important for recognition of G:U.     

Antisense RNA control of plasmid R1 replication. The dominant product of the antisense rna-mrna binding is not a full RNA duplex

The replication frequency of plasmid R1 is controlled by an antisense RNA (CopA) that binds to its target site (CopT) in the leader region of repA mRNA and inhibits the synthesis of the replication initiator protein RepA. Previous studies on CopA-CopT pairing in vitro revealed the existence of a primary loop-loop interaction (kissing complex) that is subsequently converted to an almost irreversible duplex. However, the structure of more stable binding intermediates that lead to the formation of a complete duplex was speculative. Here, we investigated the interaction between CopA and CopT by using Pb(II)-induced cleavages. The kissing complex was studied using a truncated antisense RNA (CopI) that is unable to form a full duplex with CopT. Furthermore, RNase III, which is known to process the CopA-CopT complex in vivo, was used to detect the existence of a full duplex. Our data indicate that the formation of a full CopA-CopT duplex appears to be a very slow process in vitro. Unexpectedly, we found that the loop-loop interaction persists in the predominant CopA-CopT complex and is stabilized by intermolecular base pairing involving the 5'-proximal 30 nucleotides of CopA and the complementary region of CopT. This almost irreversible complex suffices to inhibit ribosome binding at the tap ribosome binding site and may be the inhibitory complex in vivo.     

Aggregation behaviour and Zn2+ binding properties of secretin

Dynamic light scattering measurements were carried out on secretin in aqueous solution (2 mM; pH 5.0). The results indicated that the molecule exists as a fairly compact hexamer under these solution conditions. Secondary structural properties of the secretin hexameric complex were evaluated using CD and NMR spectroscopy. Specifically, the spectral properties of secretin in water were examined as a function of peptide concentration. Results from the analyses indicated a 2-fold increase (17-32%) in alpha-helical content within the region Ser11-Arg21 as the peptide concentration was increased from 0.1 to 2 mM. Displacement of the alphaH proton chemical shifts relative to random coil values did not alter significantly with increasing peptide concentration. This observation confirmed that the length of the helical segment is independent of peptide concentration between 0.1 and 2 mM. The nature of the helix was furthermore determined as amphipathic, and thus the potential for a cooperative intermolecular association through the apolar helical face of individual monomers was indicated. These findings suggest that secretin aggregates into symmetric hexamers at millimolar concentrations and, furthermore, that the helical domain is stabilized through this intermolecular association. The potential for secretin to bind divalent cations, including Ca2+ and Zn2+, was also examined by CD1 and NMR spectroscopy. The results revealed that Zn2+ specifically coordinates to the His1 and Asp3 residues of each secretin monomer without disrupting the peptide's helical structure, whereas Ca2+ did not exhibit any interaction with the peptide hormone. It was concluded from these studies that secretin may be stored in a hexameric form within its secretory tissues and that zinc may play a role in the storage of secretin through a specific interaction with the N-terminal histidine and aspartic acid residues.     

Microhelix aminoacylation by a class I tRNA synthetase. Non-conserved base pairs required for specificity

Nucleotides in tRNAs that are conserved among isoacceptors are typically considered as candidates for tRNA synthetase recognition, with less importance attached to non-conserved nucleotides. Although the anticodon is an important contributor to the identity of methionine tRNAs, the class I methionine tRNA synthetase aminoacylates microhelices with high specificity. The microhelix substrates are comprised of as few as the 1st 4 base pairs of the acceptor stems of the elongator and initiator methionine tRNAs. For these two tRNAs, only the central 2:71 and 3:70 base pairs are common to the 1st 4 acceptor stem base pairs. We show here that, although the flanking 4:69 base pair is not conserved, a particular substitution at this position substantially reduces the gel electrophoresis-detected aminoacylation of an acceptor stem substrate that has the conserved 2:71 and 3:70 base pairs. Although the two methionine tRNAs have either U:A or G:C at position 4:69, substitution with C:G reduces charging of 9- or 4-base pair substrates that recreate part or all of the acceptor stem of a methionine tRNA. This effect is sufficient for methionine tRNA synthetase to discriminate between the closely related methionine and isoleucine tRNA acceptor stems. The ability to distinguish G:C and U:A from C:G is contrary to a simple scheme for recognition of atoms in the RNA minor groove.     

Non-canonical interactions in a kissing loop complex: the dimerization initiation site of HIV-1 genomic RNA

Retroviruses encapsidate two molecules of genomic RNA that are noncovalently linked close to their 5' ends in a region called the dimer linkage structure (DLS). The dimerization initiation site (DIS) of human immunodeficiency virus type 1 (HIV-1) constitutes the essential part of the DLS in vitro and is crucial for efficient HIV-1 replication in cell culture. We previously identified the DIS as a hairpin structure, located upstream of the major splice donor site, that contains in the loop a six-nucleotide self-complementary sequence preceded and followed by two and one purines, respectively. Two RNA monomers form a kissing loop complex via intermolecular interactions of the six nucleotide self-complementary sequence. Here, we introduced compensatory mutations in the self-complementary sequence and/or a mutation in the flanking purines. We determined the kinetics of dimerization, the thermal stabilities and the apparent equilibrium dissociation constants of wild-type and mutant dimers and used chemical probing to obtain structural information. Our results demonstrate the importance of the 5'-flanking purine and of the two central bases of the self-complementary sequence in the dimerization process. The experimental data are rationalized by triple interactions between these residues in the deep groove of the kissing helix and are incorporated into a three-dimensional model of the kissing loop dimer. In addition, chemical probing and molecular modeling favor the existence of a non-canonical interaction between the conserved adenine residues at the first and last positions in the DIS loop. Furthermore, we show that destabilization of the kissing loop complex at the DIS can be compensated by interactions involving sequences located downstream of the splice donor site of the HIV-1 genomic RNA.     

Solution structure of the tobramycin-RNA aptamer complex

We have solved the solution structure of the aminoglycoside antibiotic tobramycin complexed with a stem-loop RNA aptamer. The 14 base loop of the RNA aptamer 'zippers up' alongside the attached stem through alignment of four mismatches and one Watson-Crick pair on complex formation. The tobramycin inserts into the deep groove centered about the mismatch pairs and is partially encapsulated between its floor and a looped out guanine base that flaps over the bound antibiotic. Several potential intermolecular hydrogen bonds between the charged NH3 groups of tobramycin and acceptor atoms on base pair edges and backbone phosphates anchor the aminoglycoside antibiotic within its sequence/structure specific RNA binding pocket.     

Mutations in the conserved P loop perturb the conformation of two structural elements in the peptidyl transferase center of 23 S ribosomal RNA

Evidence is presented for the participation of the P loop (nucleotides G2250-C2254) of 23 S rRNA in establishing the tertiary structure of the peptidyl transferase center. Single base substitutions were introduced into the P loop, which participates in peptide bond formation through direct interaction with the CCA end of P site-bound tRNA. These mutations altered the pattern of reactivity of RNA to chemical probes in a structural subdomain encompassing the P loop and extending roughly from G2238 to A2433. Most of the effects on chemical modification in the P loop subdomain occurred near sites of tertiary interactions inferred from comparative sequence analysis, indicating that these mutations perturb the tertiary structure of this region of RNA. Changes in chemical modification were also seen in a subdomain composed of the 2530 loop (nucleotides G2529-A2534) and the A loop (nucleotides U2552-C2556), the latter a site of interaction with the CCA end of A site-bound tRNA. Mutations in the P loop induced effects on chemical modification that were commensurate with the severity of their characterized functional defects in peptide bond formation, tRNA binding and translational fidelity. These results indicate that, in addition to its direct role in peptide bond formation, the P loop contributes to the tertiary structure of the peptidyl transferase center and influences the conformation of both the acceptor and peptidyl tRNA binding sites.     

CD spectra of 5-methyl-2-thiouridine in tRNA-Met-f from an extreme thermophile

5-Methyl-2-thiouridine (S) in tRNA-Met-f from an extreme thermophile is located in the TpsiC region, replacing T, and has a positive CD band centered at 310 nm. Upon heating, the profiles of the change in this band were similar to the UV melting profiles of the change monitored at 260 nm. This strongly suggests a close relation between heat denaturation of the tRNA and the conformation of the S base. Oligonucleotides containing S showed negative CD bands at 320-330 nm, like the monomer S itself, but when the 3'-2/5 fragment containing S formed a complex with the complementary 5'-3/5 fragment, a positive CD band appeared at 310 nm. These results suggest that combination of the TpsiC loop containing S with the hU loop is necessary for the positive band of S at 310 nm. S may serve to strengthen the association of the TpsiC loop with the hU loop in tRNA of the thermophile.     

Molecular recognition in the FMN-RNA aptamer complex

We report on a combined NMR-molecular dynamics calculation approach that has solved the solution structure of the complex of flavin mononucleotide (FMN) bound to the conserved internal loop segment of a 35 nucleotide RNA aptamer identified through in vitro selection. The FMN-RNA aptamer complex exhibits exceptionally well-resolved NMR spectra that have been assigned following application of two, three and four-dimensional heteronuclear NMR techniques on samples containing uniformly 13C, 15N-labeled RNA aptamer in the complex. The assignments were aided by a new through-bond NMR technique for assignment of guanine imino and adenine amino protons in RNA loop segments. The conserved internal loop zippers up through the formation of base-pair mismatches and a base-triple on complex formation with the isoalloxazine ring of FMN intercalating into the helix between a G.G mismatch and a G.U.A base-triple. The recognition specificity is associated with hydrogen bonding of the uracil like edge of the isoalloxazine ring of FMN to the Hoogsteen edge of an adenine at the intercalation site. There is significant overlap between the intercalated isoalloxazine ring and its adjacent base-triple platform in the complex. The remaining conserved residues in the internal loop participate in two G.A mismatches in the complex. The zippered-up internal loop and flanking stem regions form a continuous helix with a regular sugar-phosphate backbone except at a non-conserved adenine, which loops out of the helix to facilitate base-triple formation. Our solution structure of the FMN-RNA aptamer complex is to our knowledge the first structure of an RNA aptamer complex and outlines folding principles that are common to other RNA internal and hairpin loops, and molecular recognition principles common to model self-replication systems in chemical biology.     

Bactericidal antibody recognition of meningococcal PorA by induced fit. Comparison of liganded and unliganded Fab structures

MN12H2 is a bactericidal antibody directed against outer membrane protein PorA epitope P1.16 of Neisseria meningitidis. Binding of MN12H2 to PorA at the meningococcal surface activates the classical complement pathway resulting in bacterial lysis. We have determined the crystal structure of the unliganded MN12H2 Fab fragment in two different crystal forms and compared it with the structure of the Fab in complex with a P1.16-derived peptide. The unliganded Fabs have elbow bend angles of 155 degrees and 159 degrees, whereas the liganded Fab has a more closed elbow bend of 143 degrees. Substantial differences in quaternary and tertiary structure of the antigen binding site are observed between the unliganded and liganded MN12H2 Fab structures that can be attributed to peptide binding. The variable light and heavy chain interface of the liganded Fab is twisted by a 5 degrees rotation along an axis approximately perpendicular to the plane of the interface. Hypervariable loops H1, H2, and framework loop FR-H3 follow this rotation. The hypervariable loop H3 undergoes conformational changes but remains closely linked to hypervariable loop L1. In contrast with the binding site expansion seen in other Fab-peptide structures, the MN12H2 binding site is narrowed upon peptide binding due to the formation of a "false floor" mediated by arginine residue 101 of the light chain. These results indicate that PorA epitope P1.16 of N. meningitidis is recognized by the complement-activating antibody MN12H2 through induced fit, allowing the formation of a highly complementary immune complex.     

Induced fit of a peptide loop of methionyl-tRNA formyltransferase triggered by the initiator tRNA substrate

A 16-aa insertion loop present in eubacterial methionyl-tRNA formyltransferases (MTF) is critical for specific recognition of the initiator tRNA in Escherichia coli. We have studied the interactions between this region of the E. coli enzyme and initiator methionyl-tRNA (Met-tRNA) by using two complementary protection experiments: protection of MTF against proteolytic cleavage by tRNA and protection of tRNA against nucleolytic cleavage by MTF. The insertion loop in MTF is uniquely sensitive to cleavage by trypsin. We show that the substrate initiator Met-tRNA protects MTF against trypsin cleavage, whereas a formylation-defective mutant initiator Met-tRNA, which binds to MTF with approximately the same affinity, does not. Also, mutants of MTF within the insertion loop (which are defective in formylation) are not protected by the initiator Met-tRNA. Thus, a functional enzyme-substrate complex is necessary for protection of MTF against trypsin cleavage. Along with other data, these results strongly suggest that a segment of the insertion loop, which is exposed and unstructured in MTF, undergoes an induced fit in the functional MTF.Met-tRNA complex but not in the nonfunctional one. Footprinting experiments show that MTF specifically protects the acceptor stem and the 3'-end region of the initiator Met-tRNA against cleavage by double and single strand-specific nucleases. This protection also depends on formation of a functional MTF.Met-tRNA complex. Thus, the insertion loop interacts mostly with the acceptor stem of the initiator Met-tRNA, which contains the critical determinants for formylation.     

Protein engineering of chimeric Serpins: an investigation into effects of the serpin scaffold and reactive centre loop length

The exposed Serpin reactive centre loop controls the specificity of the serpin proteinase interaction. Mutations within this region have been used to generate novel potentially therapeutic inhibitors. In this study we examine the effect of the serpin scaffold and reactive centre loop length upon the generation of such inhibitors. The reactive centre loop regions, P7-P3', of alpha1-antitrypsin and alpha1-antichymotrypsin were replaced by the corresponding residues of the viral serpin, Serp1, to form AT/Serp1 and ACT/Serp1, respectively. AT/Serp1 formed SDS stable complexes with a range of proteinases with association rate constants for plasmin, tissue plasminogen activator, urokinase, thrombin and factor Xa of approximately 10(4) M(-1)s(-1) and a stoichiometry of inhibition of approximately 1 for all of them. ACT/Serp1, however, formed SDS-stable complexes with only plasmin and thrombin with association rate constant 100-fold slower than AT/Serp1 and an increased stoichiometry of inhibition. The reactive centre loop of ACT/Serp1 is four amino acid residues longer than AT/Serp1. These four additional residues (VETR) were inserted into AT/Serp1 to form AT/Serp1(VETR). AT/Serp1(VETR) formed SDS stable complexes with plasmin, thrombin and tissue plasminogen activator similar to AT/Serp1, however, the association rate constants were 10-fold slower than those observed with AT/Serp1, while the stoichiometry of inhibition remained around 1. These results suggest that the additional reactive centre loop residues effect the rate of initial complex formation by placing the reactive centre loop in a non-ideal conformation. This study demonstrates that both reactive centre loop length and serpin scaffold are important in defining the inhibitory characteristics of a serpin.     

Alpha-helix nucleation by a calcium-binding peptide loop

A 12-residue peptide AcDKDGDGYISAAENH2 analogous to the third calcium-binding loop of calmodulin strongly coordinates lanthanide ions (K = 10(5) M-1). When metal saturated, the peptide adopts a very rigid structure, the same as in the native protein, with three last residues AAE fixed in the alpha-helical conformation. Therefore, the peptide provides an ideal helix nucleation site for peptide segments attached to its C terminus. NMR and CD investigations of peptide AcDKDGDGYISAAEAAAQNH2 presented in this paper show that residues A13-Q16 form an alpha-helix of very high stability when the La3+ ion is bound to the D1-E12 loop. In fact, the lowest estimates of the helix content in this segment give values of at least 80% at 1 degreesC and 70% at 25 degreesC. This finding is not compatible with existing helix-coil transition theories and helix propagation parameters, s, reported in the literature. We conclude, therefore, that the initial steps of helix propagation are characterized by much larger s values, whereas helix nucleation is even more unfavorable than is believed. In light of our findings, thermodynamics of the nascent alpha-helices is discussed. The problem of CD spectra of very short alpha-helices is also addressed.     

Interaction between photoactivated rhodopsin and the C-terminal peptide of transducin alpha-subunit studied by FTIR spectroscopy

Structural changes in the complex formed between photolyzed bovine rhodopsin and a synthetic 11-mer peptide corresponding to the C-terminal region of the transducin alpha-subunit (Gtalpha) were analyzed by means of Fourier transform infrared spectroscopy. A complex with a protonated Schiff base appears at the beginning, accompanying the formation of an alpha-helix. This complex evolves into another which abolishes the original structure but retains the protonated Schiff base. This complex exhibits the same spectral shape as that of the final stable complex with an unprotonated Schiff base. The Fourier transform infrared spectrum for the formation of this final complex was compared to that with transducin [Nishimura, S., Sasaki, J., Kandori, H., Matsuda, T., Fukada, Y., and Maeda, A. (1996) Biochemistry 35, 13267-13271]. A large part of the frequency shifts of the peptide carbonyl vibrations which form upon complex formation with transducin but are absent with the synthetic 11-mer peptide must be structural changes in other sites, such as the nucleotide binding site in Gtalpha. The peptide, like transducin, shows the perturbation of a carboxylic acid in an extremely apolar environment. Some of the changes in the peptide backbone remain in the complex formed with the peptide. These are due to sites where rhodopsin interacts with the C-terminal region of Gtalpha. Specifically, the labeling of the peptide amide corresponding to Leu349 of transducin by 15N reveals weakening of the hydrogen bond of the peptide N-H of Leu349 and/or distortion of a peptide bond between Gly348 and Leu349 upon complex formation.     

Structure-activity relationships of the Kvbeta1 inactivation domain and its putative receptor probed using peptide analogs of voltage-gated potassium channel alpha- and beta-subunits

Certain beta-subunits exert profound effects on the kinetics of voltage-gated (Kv) potassium channel inactivation through an interaction between the amino-terminal "inactivation domain" of the beta-subunit and a "receptor" located at or near the cytoplasmic mouth of the channel pore. Here we used a bacterial random peptide library to examine the structural requirements for this interaction. To identify peptides that bind Kv1.1 we screened the library against a synthetic peptide corresponding to the predicted S4-S5 cytoplasmic loop of the Kv1.1 alpha-subunit (residues 313-328). Among the highest affinity interactors were peptides with significant homology to the amino terminus of Kvbeta1. We performed a second screen using a peptide from the amino terminus of Kvbeta1 (residues 2-31) as "bait" and identified peptide sequences with significant homology to the S4-S5 loop of Kv1.1. A series of synthetic peptides containing mutations of the wild-type Kvbeta1 and Kv1.1 sequences were examined for their ability to inhibit Kvbeta1/Kv1.1 binding. Amino acids Arg20 and Leu21 in Kvbeta1 and residues Arg324 and Leu328 in Kv1.1 were found to be important for the interaction. Taken together, these data provide support for the contention that the S4-S5 loop of the Kv1.1 alpha subunit is the likely acceptor for the Kvbeta1 inactivation domain and provide information about residues that may underlie the protein-protein interactions responsible for beta-subunit mediated Kv channel inactivation.