From genetic to structural characterization of a new class of RNA-binding domain within the SacY/BglG family of antiterminator proteins.

SacY is the prototype of a family of regulatory proteins able to prevent transcription termination. It interacts with a 29 nucleotide RNA sequence able to fold into a stem-loop structure and partially overlapping with a terminator sequence located in the 5' leader mRNA region of the gene it controls. We show here that the N-terminal fragment of SacY, SacY(1-55), and the corresponding fragments of other members of the family have antiterminator activities with efficiency and specificity identical to those of the full-length proteins. In vitro, this activity correlates with the specific affinity of SacY(1-55) for its RNA target. UV melting experiments demonstrate that SacY(1-55) binding stabilizes the RNA target structure. The NMR solution structure of SacY(1-55) is very similar to that obtained in the crystal (van Tilbeurgh et al., 1997): the peptide is folded as a symmetrical dimer without any structural homology with other RNA-binding domains yet characterized. According to a preliminary NMR analysis of the SacY(1-55)-RNA complex, the protein dimer is not disrupted upon RNA binding and several residues implicated in RNA recognition are located at the edge of the dimer interface. This suggests a new mode of protein-RNA interaction.

Crystal structure of a new RNA-binding domain from the antiterminator protein SacY of Bacillus subtilis.

SacY belongs to a family of, at present, seven bacterial transcriptional antiterminators. The RNA-binding and antitermination capacity of SacY resides in the 55 amino acids at the N-terminal [SacY(1-55)]. The crystal structure at 2 A resolution shows that SacY(1-55) forms a dimer in the crystal, in accordance with the NMR solution structure. The structure of the monomer is a four-stranded beta-sheet with a simple beta1beta2beta3beta4 topology. One side of the sheet is covered by a long surface loop and the other side forms the dimer interface. The dimer is stabilized by the orthogonal stacking of the two beta-sheets. The crystal structure is in excellent agreement with the NMR solution structure (r.m.s. distance for C alpha coordinates is 1.3 A). The structure of SacY(1-55) reveals a new RNA-binding motif.

Crystallization of the RNA-binding domain of the transcriptional antiterminator protein SacY from Bacillus subtilis.

SacY is the antiterminator protein involved in the induction by sucrose of the expression of the levansucrase gene (sacB) of Bacillus subtilis. In the presence of sucrose, SacY is activated and prevents premature termination of transcription by binding to a RNA-antiterminator (RAT) sequence partially overlapping with the terminator sequence. SacY consists of a RNA-binding N-terminal domain, SacY(1-55), and a regulatory domain, SacY(56-280), sensitive to the sucrose concentration. SacY(1-55) is in itself capable of binding to the RAT sequence and preventing termination independently of the sucrose concentration. In this paper we describe the overexpression, the purification, and the crystallization of SacY(1-55). We obtained six different crystal forms, some of them diffracting to high resolution (> 1.5 A). Self rotation function calculations indicated the presence of a dimer in the asymmetric unit, which is in agreement with a proposed oligomeric state in solution as observed by high-resolution NMR measurements. The crystallization of some site-directed cysteine mutants opens the way of solving the structure by multiple isomorphous replacement.

Structural basis of ligand discrimination by two related RNA aptamers resolved by NMR spectroscopy.

In a previous study, an RNA aptamer for the specific recognition of arginine was evolved from a parent sequence that bound citrulline specifically. The two RNAs differ at only 3 positions out of 44. The solution structures of the two aptamers complexed to their cognate amino acids have now been determined by two-dimensional nuclear magnetic resonance spectroscopy. Both aptamers contain two asymmetrical internal loops that are not well ordered in the free RNA but that fold into a compact structure upon ligand binding. Those nucleotides common to both RNAs include a conserved cluster of purine residues, three of which form an uneven plane containing a G:G pair, and two other residues nearly perpendicular to that surface. Two of the three variant nucleotides are stacked on the cluster of purines and form a triple contact to the amino acid side chain, whereas the edge of the third variant nucleotide is capping the binding pocket.

Structural probing and damage selection of citrulline- and arginine-specific RNA aptamers identify base positions required for binding.

In a recent study, an RNA aptamer for the specific recognition of the amino acid L-arginine was evolved from an in vitro selected L-citrulline binding parent sequence [M. Famulok (1994) J. Am. Chem. Soc. 116, 1698-1706]. We have now carried out a structural analysis of these aptamers by using chemical modification experiments. Footprinting experiments and a damage selection approach were performed to identify those positions protected from modification in the presence of the amino acids and modifications that interfere with the binding of the ligand. It is shown that of the two bulged regions present in both aptamers one can be modified without loss of binding activity whereas in the other bulge nearly every position is shown to be involved in the recognition of the ligands. This might be indicative for non-canonical base pairing to occur within the non-Watson-Crick paired regions which might be stabilized by the complexed amino acid. Binding to the cognate amino acid significantly enhances the conformational stability of the RNA. We also tested the sensitivity of both aptamers towards lead (II) ion induced cleavage and identified a hypersensitive cleavage site within the invariant bulged region. Lead cleavage is inhibited by the complexed amino acid, indicating a conformational change of the aptamer upon ligand binding. NMR titration data obtained with both aptamers and their cognate ligands confirm the proposed conformational changes and indicate the formation of a 1:1 complex of RNA:amino acid.

Hydration and solution structure of nucleic acids.

Of particular interest among the revelations from recent new DNA structures is the finding that both strands of the repeated DNA sequences found in telomeres and centromeres may adopt alternative conformations. High-definition NMR studies yielded information on the residence time of the water molecules interacting with nucleic acids. A better knowledge of the residence time of the hydration water may be useful in assessing its contribution to nucleic acid structure.

Aromatic-aromatic interactions in the zinc finger motif. Analysis of the two-dimensional nuclear magnetic resonance structure of a mutant domain.

The folding and stability of globular proteins are determined by a variety of chemical mechanisms, including hydrogen bonds, salt bridges and the hydrophobic effect. Of particular interest are weakly polar interactions involving aromatic rings, which are proposed to regulate the geometry of closely packed protein interiors. Such interactions reflect the electrostatic contribution of pi-electrons and, unlike van der Waals' interactions and the hydrophobic effect, may, in principle, introduce a directional force in a protein's hydrophobic core. Although the weakly polar hypothesis is supported by a statistical analysis of protein structures, the general importance of such contributions to protein folding and stability is unclear. Here, we show the presence of alternative aromatic-aromatic interactions in the two-dimensional nuclear magnetic resonance structure of a mutant Zn finger. Changes in aromatic packing lead in turn to local and non-local differences between the structures of a wild-type and mutant domain. The results provide insight into the evolution of Zn finger sequences and have implications for understanding how geometric relationships may be chemically encoded in a simple sequence template.

Structure and dynamics of des-pentapeptide-insulin in solution: the molten-globule hypothesis.

Structures of insulin in different crystal forms exhibit significant local and nonlocal differences, including correlated displacement of elements of secondary structure. Here we describe the solution structure and dynamics of a monomeric insulin analogue, des-pentapeptide-(B26-B30)-insulin (DPI), as determined by two-dimensional NMR spectroscopy and distance geometry/restrained molecular dynamics (DG/RMD). Although the solution structure of DPI exhibits a general similarity to its crystal structure, individual DG/RMD structures in the NMR ensemble differ by rigid-body displacements of alpha-helices that span the range of different crystal forms. These results suggest that DPI exists as a partially folded state formed by coalescence of distinct alpha-helix-associated microdomains. The physical reality of this model is investigated by comparison of the observed two-dimensional nuclear Overhauser enhancement (NOE) spectroscopy (NOESY) spectrum with that predicted from crystal and DG/RMD structures. The observed NOESY spectrum contains fewer tertiary contacts than predicted by any single simulation, but it matches their shared features; such "ensemble correspondence" is likely to reflect the effect of protein dynamics on observed NOE intensities. We propose (i) that the folded state of DPI is analogous to that of a compact protein-folding intermediate rather than a conventional native state and (ii) that the molten state is the biologically active species. This proposal (the molten-globule hypothesis) leads to testable thermodynamic predictions and has general implications for protein design.

Receptor binding redefined by a structural switch in a mutant human insulin.

Crystal structures of insulin have been determined in various distinct forms, the relevance of which to receptor recognition has long been the subject of speculation. Recently the crystal structure of an inactive insulin analogue has been determined and, surprisingly, found to have a conformation identical to native insulin. On this basis Dodson and colleagues have suggested that the known insulin crystal structures reflect an inactive conformation, and that a change in conformation is required for activity--specifically, the carboxy terminal residues of the B-chain are proposed to separate from the amino terminal residues of the A-chain. Here we report the solution structure of an active insulin mutant, determined by two-dimensional NMR, which supports this hypothesis. In the mutant, the carboxy terminal beta-turn and beta-strand of the B-chain are destabilized and do not pack across the rest of the molecule. We suggest that analogous detachment of the carboxy terminal region of the B-chain occurs in native insulin on binding to its receptor. Our finding that partial unfolding of the B-chain exposes an alternative protein surface rationalizes the receptor-binding properties of a series of anomalous insulin analogues, including a mutant insulin associated with diabetes mellitus in man.

Architectural rules of the zinc-finger motif: comparative two-dimensional NMR studies of native and "aromatic-swap" domains define a "weakly polar switch".

The Zn-finger motif, encoding a globular minidomain with characteristic structure, provides a striking example of a sequence template for protein folding. Insight into architectural rules relating the amino acid sequence of a protein to its structure and stability may be obtained by comparative study of analogues. As our first step toward defining such rules for the Zn finger, we have recently described the design of an "aromatic-swap" analogue based on the ZFY two-finger repeat: a conserved alternation in sequence pattern observed among odd- and even-numbered domains in a family of sex-related vertebrate transcription factors. Consensus and "swapped" aromatic residues, introduced as revertants of less stable "aromaticless" analogues, were observed to provide equivalent contributions to the thermodynamic stability of the Zn finger. Here we describe and compare the solution structures of a wild-type domain and an aromatic-swap analogue, as determined by two-dimensional NMR and distance-geometry/restrained molecular dynamics calculations. The wild-type and aromatic-swap analogue each contain an N-terminal beta-sheet and a C-terminal alpha-helix (beta beta alpha motif), as observed in other systems, and exhibit a highly ordered hydrophobic core in which the native or swapped aromatic ring is closely packed. Remarkably, however, the two structures are stabilized by alternative aromatic-aromatic interactions, which in turn alter the respective DNA-binding surfaces. Our results suggest that native and swapped Zn-finger sequences encode a "weakly polar switch" between thermodynamically equivalent but functionally distinct architectures for DNA recognition.

Alternating zinc fingers in the human male-associated protein ZFY: HX3H and HX4H motifs encode a local structural switch.

The two-finger repeat in the human male-associated protein ZFY provides a model for comparative 2D-NMR studies of classical and variant Zn fingers. This repeat is defined in part by an alternation in spacing between consensus (HX3H) and variant (HX4H) histidine spacings. To investigate the effects of a "switch" between alternative histidine spacings, we have designed an HX3H analogue of a representative HX4H domain of known structure [ZFY-6; Kochoyan, M., Havel, T., Nguyen, D. T., Dahl, C. E., Keutmann, H. T., & Weiss, M. A. (1991) Biochemistry 30, 3371-3386]. The HX3H analogue (designated ZFY-switch) forms a tetrahedral Co2+ complex whose thermodynamic stability is similar to that of the parent peptide. 2D-NMR studies demonstrate that ZFY-switch and ZFY-6, although similar in overall structure, exhibit significant local changes near the site of deletion. Whereas the HX4H site in the native finger forms a nonstandard loop, the HX3H site in ZFY-switch folds as a 3(10) extension of the C-terminal alpha-helix, as observed in the NMR solution structure of a consensus HX3H domain [Lee, M. S., Gippert, G. P., Soman, K. V., Case, D. A., & Wright, P. E. (1989) Science 245, 635-637] and in the crystal structure of a representative Zn finger-DNA complex [Pavletich, N. P., & Pabo, C. O. (1991) Science 252, 809-817]. We propose that variant histidine spacings (HX3H and HX4H) encode a local switch between alternative surface architectures with implications for models of protein-DNA recognition.

Alternating zinc fingers in the human male associated protein ZFY: refinement of the NMR structure of an even finger by selective deuterium labeling and implications for DNA recognition.

ZFY, a male-associated Zn-finger protein encoded by the human Y chromosome, exhibits a distinctive two-finger repeat: whereas odd-numbered domains fit a general consensus, even-numbered domains exhibit systematic differences. Do these odd and even sequences encode structurally distinct surfaces for DNA recognition? As a first step toward answering this question, we have recently described the sequential 1H NMR assignment of a representative nonconsensus Zn finger (designated ZFY-6T) based on 2D NMR studies of a 30-residue peptide [Kochoyan, M., Havel, T.F., Nguyen, D.T., Dahl, C.E., Keutmann, H. T., & Weiss, M.A. (1991) Biochemistry 30, 3371-3386]. Initial structural modeling by distance geometry/simulated annealing (DG/SA) demonstrated that this peptide retained the N-terminal beta-hairpin and C-terminal alpha-helix (beta beta alpha motif) observed in consensus Zn fingers. However, the precision of this initial structure was limited by resonance overlap, which led to ambiguities in the assignment of key NOEs in the hydrophobic core. In this paper these ambiguities are resolved by selective deuterium labeling, enabling a refined structure to be calculated by DG/SA and restrained molecular dynamics. These calculations provide a detailed view of the hydrophobic core and protein surface, which are analyzed in reference to previously characterized Zn fingers. Variant (even) and consensus (odd) aromatic residues Y10 and F12, shown in an "aromatic swap" analogue to provide equivalent contributions to the hydrophobic core [Weiss, M.A., & Keutmann, H.T. (1990) Biochemistry 29, 9808-9813], nevertheless exhibit striking differences in packing interactions: Y10--but not F12--contributes to a contiguous region of the protein surface defined by putative specificity-determining residues. Alternating surface architectures may have implications for the mechanism of DNA recognition by the ZFY two-finger repeat.

Alternating zinc fingers in the human male associated protein ZFY: 2D NMR structure of an even finger and implications for "jumping-linker" DNA recognition.

ZFY, a sex-related Zn-finger protein encoded by the human Y chromosome, is distinguished from the general class of Zn-finger proteins by the presence of a two-finger repeat. Whereas odd-numbered domains and linkers fit a general consensus, even-numbered domains and linkers exhibit systematic differences. Because this alternation may have fundamental implications for the mechanism of protein-DNA recognition, we have undertaken biochemical and structural studies of fragments of ZFY. We describe here the solution structure of a representative nonconsensus (even-numbered) Zn finger based on 2D NMR studies of a 30-residue peptide. Structural modeling by distance geometry and simulated annealing (DG/SA) demonstrates that this peptide folds as a miniglobular domain containing a C-terminal beta--hairpin and N-terminal alpha-helix (beta beta alpha motif). These features are similar to (but not identical with) those previously described in consensus-type Zn fingers (derived from ADR1 and Xfin); the similarities suggest that even and odd ZFY domains bind DNA by a common mechanism. A model of the protein-DNA complex (designated the "jumping-linker" model) is presented and discussed in terms of the ZFY two-finger repeat. In this model every other linker is proposed to cross the minor groove by means of a putative finger/linker submotif HX4HX3-hydrophobic residue-X3. Analogous use of a hydrophobic residue in a linker that spans the minor groove has recently been described in crystallographic and 3D NMR studies of homeodomain-DNA complexes. The proposed model of ZFY is supported in part by the hydroxyl radical footprint of the TFIIIA-DNA complex [Churchill, M.E.A., Tullius, T.D., & Klug, A. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 5528-5532].

Processes of base-pair opening and proton exchange in Z-DNA.

Using proton magnetic resonance, we have investigated imino and amino proton exchange in the Z form of the four oligomers d(Cbr8GCGCbr8G), d(CGm5CGCG), d(CG)6, and d(CG)12. In the latter two oligomers, all five exchangeable protons have been assigned. We find that proton acceptors such as NH3 or the basic form of Tris enhance imino proton exchange. The base-pair lifetime can then be obtained by extrapolation of the exchange time to infinite concentration of proton acceptor. For d(CG)6 and d(CG)12, the values are ca. 3.5 ms at 80 degrees C and ca. 130 ms at 35 degrees C. The latter value is about 65 times longer than in the same oligomers in the B form. The activation energy of base-pair opening, 80 kJ/mol, is the same in the Z and the B forms of d(CG)12. At 5 degrees C, the base-pair lifetime is about 3 s, much smaller than the time constant of the Z to B transition, to which it is therefore unrelated. The base-pair dissociation constant at 35 degrees C, 0.5 X 10(-6), is 5 times smaller than for the same oligomers in the B form. In the absence of added catalyst, at pH 7, the exchange time of the imino proton is 30 min at 5 degrees C. That of both cytidine amino protons, assigned by NOE, is about 50 min. The longest proton exchange time, ca. 330 min, is assigned unambiguously to the guanosine amino protons. Thus assigned and interpreted in terms of exchange chemistry rather than structural kinetics, the exchange times do not support earlier models of Z-DNA internal motions.

Evidence from base-pair kinetics for two types of adenine tract structures in solution: their relation to DNA curvature.

We have measured the base-pair lifetimes in oligodeoxynucleotides containing tracts of A.T base pairs using imino proton magnetic resonance. When the tract contains more than four consecutive A.T base pairs, possibly including a 5'-AT step but not a 5'-TA step, anomalously long lifetimes are observed. For example, the lifetimes of the central A.T base pairs of the dodecamer 5'-d-CGCAAAAAAGCG are 122 and 91 ms at 15 degrees C whereas, in the same conditions, the lifetime of the central A.T pair of the decamer 5'-d-CGCGATCGCG is only 4 ms, a value similar to those measured in several other B-DNA oligoduplexes [Leroy et al. (1988) J. Mol. Biol. 200, 223-238]. This strongly suggests that, in tracts of four A.T pairs or more, a conformation distinct from standard B-DNA is formed cooperatively. All sequences known to generate curved DNA exhibit anomalously long base-pair lifetimes. This is the first local and physical property shown to correlate with DNA curvature. Our observations suggest that the structure responsible for the long lifetimes is involved in the curvature of DNA.

Study of structure, base-pair opening kinetics and proton exchange mechanism of the d-(AATTGCAATT) self-complementary oligodeoxynucleotide in solution.

Using proton magnetic resonance, we have investigated the structure and the base-pair opening kinetics of the d-(AATTGCAATT) self-complementary duplex. All the non-exchangeable (except H5',5") and most exchangeable proton resonances have been assigned. The structure belongs to the B family. Imino proton exchange, measured by line broadening, longitudinal relaxation and magnetization transfer from water, is catalyzed by proton acceptors. The base-pair lifetimes, obtained by extrapolation of the exchange times to infinite concentration of ammonia are 2 and 3 milliseconds for internal A.Ts and 18 ms for G.C at 15 degrees C. In the absence of added catalysts, the imino proton of the first A.T base pair exchanges faster than that of the unpaired thymidine of the duplex formed by the sequence d-(AATTGCAATTT). This gives strong evidence for intrinsic exchange catalysis. The exchange of adenine amino protons from the closed state has been observed. Hence amino proton exchange is ill-suited for the investigation of base-pair opening kinetics.

Characterization of base-pair opening in deoxynucleotide duplexes using catalyzed exchange of the imino proton.

Using nuclear magnetic resonance line broadening, longitudinal relaxation and magnetization transfer from water, we have measured the imino proton exchange times in the duplex form of the 10-mer d-CGCGATCGCG and in seven other deoxy-duplexes, as a function of the concentration of exchange catalysts, principally ammonia. All exchange times are catalyst dependent. Base-pair lifetimes are obtained by extrapolation to infinite concentration of ammonia. Lifetimes of internal base-pairs are in the range of milliseconds at 35 degrees C and ten times more at 0 degrees C. Lifetimes of neighboring pairs are different, hence base-pairs open one at a time. Lifetimes of d(G.C) are about three times longer than those of d(A.T). The nature of neighbors usually has little effect, but lifetime anomalies that may be related to sequence and/or structure have been observed. In contrast, there is no anomaly in the A.T base-pair lifetimes of d-CGCGA[TA]5TCGCG, a model duplex of poly[d(A-T)].poly[d(A-T)]. The d(A.T) lifetimes are comparable to those of r(A.U) that we reported previously. End effects on base-pair lifetimes are limited to two base-pairs. The low efficiency of exchange catalysts is ascribed to the small dissociation constant of the deoxy base-pairs, and helps to explain why exchange catalysis had been overlooked in the past. This resulted in a hundredfold overestimation of base-pair lifetimes. Cytosine amino proteins have been studied in the duplex of d-CGm5CGCG. Exchange from the closed base-pair is indicated. Hence, the use of an amino exchange rate to evaluate the base-pair dissociation constant would result in erroneous, overestimated values. Catalyzed imino proton exchange is at this time the safest and most powerful, if not the only probe of base-pair kinetics. We propose that the single base-pair opening event characterized here may be the only mode of base-pair disruption, at temperatures well below the melting transition.

Proton exchange and base-pair lifetimes in a deoxy-duplex containing a purine-pyrimidine step and in the duplex of inverse sequence.

Using proton relaxation and magnetization transfer from water we have measured the imino proton exchange kinetics in two dodecadeoxynucleotide duplexes. One is formed by the self-complementary sequence 5'-d(C-C-T-T-T-C-G-A-A-A-G-G), the other by the inverse sequence. The imino proton exchange rates are found to depend on the concentration of ammonia or imidazole, acting as basic catalysts of proton exchange. Extrapolation of exchange times to infinite catalyst concentration yields the base-pair lifetimes, for instance 40 milliseconds for the central G.C base-pair of the 5'-d(C-C-T-T-T-C-G-A-A-A-G-G) duplex and four milliseconds for its A.T neighbour, at 15 degrees C. These results differ markedly from those reported by other laboratories for similar deoxy compounds. An explanation of the discrepancy has been proposed recently. Differences between base-pair lifetimes indicate that opening is not co-operative. From the catalyst efficiency relative to exchange from isolated nucleosides, we estimate the dissociation constant of each base-pair, e.g. 0.3 x 10(-6) and 1.5 x 10(-5) at 15 degrees C, for the same G.C and A.T base-pairs. The lifetime and dissociation constant of corresponding base-pairs of the two duplexes are similar, except for the central G.C base-pair. This correlates with differences in the solution structures reported by others. We have completed the assignments of the imino protons and of the six cytosine amino protons of the 5'-d(G-G-A-A-A-G-C-T-T-T-C-C) 12-mer. A new base-pair numbering scheme is proposed.

A single mode of DNA base-pair opening drives imino proton exchange.

The opening of base pairs of double-stranded DNA is an important process, being a prerequisite for replication and transcription and possibly a factor in the recognition, flexibility and structure of DNA. The kinetics of base-pair opening have, however, been controversial. Base-pair opening can be studied by following the exchange of protons from imino groups with water, a process that seems only to occur from open base pairs. We have recently demonstrated catalysis by proton acceptors of imino proton exchange in nucleic acids. This has enabled us to determine the base-pair lifetimes, which are in the region of 10 ms at room temperature. In earlier reports it had been considered that proton exchange is limited by the rate of base-pair opening, which had led to estimates of base-pair lifetimes that were larger by one or two orders of magnitude. There are also important discrepancies between recent and early estimates of the base-pair dissociation constant. Earlier estimates of base-pair lifetimes correspond in fact to the time required for proton exchange in the absence of added catalyst (AAC exchange). This could be a distinct mode of base-pair opening with a very long open lifetime, different from the mode revealed by the effect of catalyst. The evidence reported here suggests on the contrary that there is only a single mode of here suggests on the contrary that there is only a single mode of base-pair opening and that proton exchange in the absence of added catalyst is in fact catalysed by a proton acceptor intrinsic to the nucleic acid, most probably the other base of the open pair.