NHP6A is a chromatin‐associated protein from Saccharomyces cerevisiae belonging to the HMG1/2 family of non‐specific DNA binding proteins. NHP6A has only one HMG DNA binding domain and forms relatively stable complexes with DNA. We have determined the solution structure of NHP6A and constructed an NMR‐based model structure of the DNA complex. The free NHP6A folds into an L‐shaped three α‐helix structure, and contains an unstructured 17 amino acid basic tail N‐terminal to the HMG box. Intermolecular NOEs assigned between NHP6A and a 15 bp 13C, 15N‐labeled DNA duplex containing the SRY recognition sequence have positioned the NHP6A HMG domain onto the minor groove of the DNA at a site that is shifted by 1 bp and in reverse orientation from that found in the SRY–DNA complex. In the model structure of the NHP6A–DNA complex, the N‐terminal basic tail is wrapped around the major groove in a manner mimicking the C‐terminal tail of LEF1. The DNA in the complex is severely distorted and contains two adjacent kinks where side chains of methionine and phenylalanine that are important for bending are inserted. The NHP6A–DNA model structure provides insight into how this class of architectural DNA binding proteins may select preferential binding sites.
Non‐histone protein 6A (NHP6A) is one of a number of HMG box proteins found in Saccharomyces cerevisiae. The HMG box is a conserved domain of ∼80 amino acids which mediates DNA binding of many proteins. Proteins which contain the HMG domain are divided into two subfamilies based upon differences in amino acid sequence and specificity of DNA binding (Grosschedl et al., 1994; Bustin and Reeves, 1996). The first class are generally transcription factors that bind to DNA in a sequence‐specific fashion, contain one HMG box and are only expressed in a few cell types. Examples include the human sex‐determining factor SRY (Gubbay et al., 1990; Sinclair et al., 1990), the lymphoid enhancer binding factor LEF1 (Travis et al., 1991) and the T‐cell factor TCF‐1 (van de Wetering et al., 1991).
The second class of HMG box proteins is more abundant, often contain two or more tandem HMG boxes, and bind DNA with little or no sequence specificity. This second class of HMG proteins, typified by vertebrate HMG1 and HMG2, which contain two HMG boxes (Wen et al., 1989), are present at a level of one molecule per two or three nucleosomes and thus are a major constituent of eukaryotic chromatin (Kuehl et al., 1984). Although their biological functions are just beginning to be revealed, they have been shown to participate in reactions as diverse as DNA recombination, repair, activation and repression of transcription as well as nucleosome assembly and disassembly (Paull et al., 1993, 1996; Ge and Roeder, 1994; Shykind et al., 1995; Nightingale et al., 1996; Ura et al., 1996; van Gent et al., 1997). The HMG1/2 proteins strongly distort DNA upon binding and can stabilize bent and supercoiled DNA. This DNA architectural role in facilitating the formation of higher order nucleoprotein complexes is believed to be a critical component of their activity in many of these reactions. In addition, direct interactions with other proteins may be important in some cases (Zwilling et al., 1995; Zappavigna et al., 1996; Jayaraman et al., 1998).
The S.cerevisiae HMG box protein NHP6A is present in the nucleus at levels similar to HMG1/2 but contains only one HMG box, which is 45% identical to rat HMG1 Box B and 80% identical to the HMG box of yeast NHP6B (Kolodrubetz and Burgum, 1990). NHP6B is a functional homologue of NHP6A which is present in lower amounts. In common with other non‐sequence‐specific HMG box proteins, NHP6A binds linear DNA with little sequence specificity, induces a large bend when it binds, and displays higher affinity for distorted DNA structures such as microcircular and cisplatinated DNA (Paull and Johnson, 1995; Yen et al., 1998). NHP6A forms a more stable complex with DNA than does mammalian HMG1/2. NHP6A/B both contain a highly basic amino acid region that precedes the HMG box, and the 16 amino acid N‐terminal basic segment of NHP6A has been shown to be essential for high affinity binding and the formation of monomeric DNA complexes (Yen et al., 1998). In vivo, neither NHP6A nor NHP6B is essential since Δnhp6A and Δnhp6B mutants grow normally, but the double mutant grows slowly at 30°C and is non‐viable at 38°C (Costigan et al., 1994). The Δnhp6A/B mutants display a variety of morphological changes and are defective in activated transcription of a subset of genes (Costigan et al., 1994; Paull et al., 1996). An NHP6A mutant lacking the N‐terminal segment is incapable of rescuing growth or co‐activating transcription in a Δnhp6A/B background, demonstrating the critical importance of this region for its biological activity (Yen et al., 1998).
To date, four structures of the HMG box domain, including three from the non‐sequence‐specific subfamily: HMG1 Box A (Hardman et al., 1995), HMG1 Box B (Read et al., 1993; Weir et al., 1993), HMG‐D (Jones et al., 1994) and one from the sequence‐specific subfamily, SOX4 (van Houte et al., 1995), have been determined in the absence of DNA by NMR. Each of these structures has the same general shape, an L‐shaped fold of three α‐helices. The NMR structures of two sequence‐specific HMG protein–DNA complexes, SRY and LEF1, bound to their cognate DNA recognition sequence, have also been determined (Love et al., 1995; Werner et al., 1995b). In these complexes, the minor groove of the DNA is bound to the concave face of the L‐shaped protein and is greatly distorted, generating an overall curvature of ∼75° and 90° for SRY and LEF1, respectively. The DNA is severely underwound, resulting in a widened and shallow minor groove and a highly compressed major groove. In conjunction with the helical underwinding, large positive roll angles are induced by numerous DNA–protein contacts which include a partial insertion of an amino acid side chain into the minor groove of the DNA. Extensive hydrophobic interactions as well as specific hydrogen bonds between the proteins and the DNA sequence mediate the specificity of this class of HMG1/2 proteins. Although these structures provide valuable insight into the interaction of the HMG domain with DNA, they do not fully account for how HMG1/2 proteins can interact with DNA in a non‐sequence‐specific manner.
In this study, we present the solution structure of the yeast HMG box protein NHP6A. The structure most closely resembles the fold of its Drosophila equivalent HMG‐D, including a kink in the middle of helix 3 (Jones et al., 1994). The complexes of NHP6A with two different 15 bp DNA duplexes containing the recognition sequence for LEF1 and SRY were also studied by NMR, with the latter complex being investigated most extensively. We present a model for the complex between NHP6A and the DNAsry based on the structure of the free protein, observed intermolecular interactions and analogy with the LEF1 protein–DNA complex (Love et al., 1995). A series of mutant NHP6A proteins was constructed based on the structure of the free protein and the model structure of the complex, and their DNA binding properties were investigated using gel mobility shift and ligation assays. Taken together, the data reveal both similarities and unexpected differences between the NHP6A–DNA complex and the structures of the sequence‐specific HMG box–DNA complexes, and provide insight into DNA target selection by the non‐specific HMG box proteins.
Solution structure of NHP6A
NHP6A is a 93 residue protein with the sequence given in Figure 1A. The amino acid sequence of the protein differs from the other HMG domain proteins in having a basic N‐terminal tail of ∼16 amino acids, which has been shown to be required for high affinity binding. At 37°C the free protein appears to be unfolded, based on the narrow chemical shift dispersion in the 1H‐15N TROSY spectra (Czisch and Boelens, 1998; Salzmann et al., 1998) compared with that observed at 20°C (Figure 2A and C). Therefore, the structure of NHP6A was solved at 20°C (10 mM NaPO4 pH 5.5, 100 mM NaCl) by multidimensional heteronuclear NMR spectroscopy. Nearly complete 1H, 15N and 13C resonance assignments for all residues except the N‐terminal tail (1–16) were obtained using standard resonance assignment procedures (see Materials and methods). Input restraints and structure statistics for the ensemble of 30 converged structures are given in Table I.
NHP6A (Figures 2 and 3A) is unstructured up to residue 17 (Asp17) and adopts the typical L‐shaped fold of an HMG domain protein (Read et al., 1993; Weir et al., 1993; Jones et al., 1994; Hardman et al., 1995; Love et al., 1995; van Houte et al., 1995; Werner et al., 1995b) from Pro18 to Leu92 (Figure 3). The three α‐helices comprise 63% of the protein sequence: helix 1 (Ala27–Glu42), helix 2 (Phe48–Lys60) and helix 3 (Thr63–Leu92) (Figure 3C). Helix 2 is straight, while both helices 1 and 3 are kinked in their centers, at Arg36 and Asp77, respectively. These kinks create bends of 40° for helix 1 and 30° for helix 3, respectively. The kink in helix 1 is also found in most other HMG domain proteins, while the kink in helix 3 is only present in NHP6A and HMG‐D, and plays an important role in DNA binding (see Discussion).
The L‐shaped fold is stabilized by three hydrophobic cores (Figure 3B). Helix 1 and helix 2 are positioned antiparallel to each other and form the short arm of the L‐shape. They interact via a hydrophobic core formed by amino acid side chains from helix 1, helix 2 and the loop between helix 1 and helix 2. Part of the N‐terminus (Pro18–Ala24) is extended and interacts with helix 3 to form the long arm of the L‐shape. These two regions of the protein are packed by hydrophobic interactions between the side chains from residues in the extended N‐terminus and in helix 3 (Figure 3B). The two arms of the L are oriented at an angle of ∼80° to each other via the third hydrophobic core located at the vertex of the L.
In addition to the 35 hydrogen bonds in the protein backbone maintaining the α‐helices, several salt bridges and one hydrogen bond contribute to the stability of the structure. There are five possible salt bridges: between Glu42 and Lys58 stabilizing the interaction between helix 1 and helix 2, between Glu57 and either Lys54 or Lys60 stabilizing helix 2, and between Lys67 and Glu71, Lys79 and Glu82, Arg80 and Glu84 stabilizing helix 3. Similar salt bridges are present in the structures of other HMG proteins. In addition, a hydrogen bond between the Tyr28 hydroxyl group and Gly52 carbonyl oxygen is likely to be present considering the distance of 2.6 ± 0.2 Å between the two oxygens over the ensemble of converged structures.
Formation of NHP6A–DNA complexes
In order to study the interaction of NHP6A with DNA and to compare it directly with the interaction of the sequence‐specific HMG domain proteins SRY and LEF1 (King and Weiss, 1993; Haqq et al., 1994; Love et al., 1995; Werner et al., 1995a, b), we prepared two complexes with different 15 bp DNA oligonucleotides. One sequence includes the recognition site for the SRY protein (DNAsry) (King and Weiss, 1993; Haqq et al., 1994) and the second sequence includes the recognition sequence for the LEF1 protein (DNAlef) (Love et al., 1995) (Figure 1B). Complex formation was easily monitored by observing the changes in the imino proton spectra of the DNA and the appearance of the NHϵ resonance from Trp59 upon addition of NHP6A. Titration of the DNA by adding NHP6A showed that the DNA in both complexes is in slow exchange on the NMR time scale (spectra not shown) under low salt conditions.
The two NHP6A protein–DNA complexes were very sensitive to salt concentration and temperature. We found that optimal spectra were obtained for the complexes in low salt (10 mM NaCl, 2 mM NaPO4 pH 5.5) and at 37°C. We note that NMR studies of the sequence‐specific complexes were also done at similar salt concentrations (using KCl) and temperature (Love et al., 1995; Werner et al., 1995b). The 1H‐15N TROSY spectra of the two complexes at 37°C are very similar to one another and to that of the free protein at 20°C, showing that NHP6A adopts essentially the same structure in the two complexes as in its free form at 20°C (Figure 2, NHP6A–DNAlef complex spectrum not shown). The most substantial differences are for the resonances of the N‐terminus, which are highly overlapped in the free protein (Figure 2A) and become more resolved and have broader line widths in the complex (Figure 2B).
It is interesting that a stable protein–DNA complex forms at 37°C, since the free protein is apparently largely unfolded at this temperature based on the NMR spectra (Figure 2C) and CD data (Yen et al., 1998). Titration of the DNA into the protein at 37°C also shows formation of the protein–DNA complex (not shown). Furthermore, the same binding affinity as measured by polyacrylamide gel shifts is obtained for protein–DNA complexes formed and run at 37°C as for those at 23°C (data not shown). Therefore, the folded structure of NHP6A is not only stabilized in the complex, it appears that the protein is actually folding on the DNA at 37°C.
Assignments of the protein and DNA in the NHP6A–DNAsry complex
The complex of NHP6A with DNAsry was chosen for more extensive NMR analysis. Despite the general line broadening due to the increase in molecular weight (19 kDa), almost complete resonance assignments of the protein (Figure 2B) and of the DNA in the complex were obtained. Protein assignments were facilitated by the generally small chemical shift differences (maximum of 0.5 p.p.m. for 1H, i.e. Ala20 HN and Phe48 Hα, 3 p.p.m. for 15N, i.e. Gln19, and 1.5 p.p.m. for 13C, i.e. Phe48 and Met29 Cα) that were observed between the resonances of the free and the bound form and by the presence of the same sequential connectivities. Based on these results, it appears that the protein in the complex adopts almost the same structure in its free and bound form, with the exception of the N‐terminal extension (Lys7–Asp17) which becomes ordered in the complex.
For the extensive NMR study of the NHP6A–DNA complex, uniformly 13C,15N‐labeled DNAsry was also enzymatically synthesized (Masse et al., 1998), and protein–DNA complexes with both strands as well as each of the individual labeled strands were prepared and studied. The use of individually labeled strands along with the labeled duplex greatly facilitated the assignments of the DNA in the complex, and more importantly, the assignment of intermolecular NOEs (Masse et al., 1999). The pattern of sequential NOE connectivities and cross‐peak intensities seen for the DNAsry is similar to that reported for the sequence‐specific SRY–DNA complex and consistent with a bent and underwound DNA structure in the complex (Werner et al., 1995a).
Chemical shift mapping and line broadening of the protein–DNA interface
An analysis of the chemical shift differences (backbone and side chain) between the free and the bound forms of the protein leads to a first ‘footprint’ of the protein surface in contact with the DNA. The regions of the protein showing the largest chemical shift difference between the free and the bound forms of the protein are the N‐terminal region (Asp17–Ser26), helix 1 (Ala27–Gln33, Arg36 and Arg40), the whole helix 2, and a few residues of helix 3 (Lys78, Tyr81 and Lys85) (Figure 4A). In addition to the chemical shift changes created by the new chemical environment, the resonances of some residues at the interface show a large line broadening in the complex, probably because of dynamics at the protein–DNA interface. Severe line broadening is observed in particular for the side chain resonances of Thr11, Thr12, Tyr28, Met29, Asn33 (not shown), Thr47, Phe48 (Figure 4B) and the amide resonances of Gly49 and 52 (Figure 1A). A change in chemical shifts and increase in the line width is also observed for part of the N‐terminal region (Lys8–Asp17) in the complex, e.g. the amides of Thr11 and Thr12 and the Hϵ of Arg5, 10 and 12 (Figure 2A and B). These chemical shift and line width changes are consistent with the N‐terminal tail becoming ordered in the complex, probably by interacting with the DNA.
Similarly, chemical shift changes and severe line broadening are observed in some regions of the DNA in the complex (not shown). Resonance broadening is particularly clear for most of the thymine imino protons and most adenine H2s of the DNA in complex. These data map the protein binding site on the DNA to the 11 bp G4–A14/T17–C27, centered at the T9–G10/C21–A22 dinucleotide step (Figure 1B).
Intermolecular NOEs in the protein–DNA complex
Despite the severe line broadening observed for many resonances at the protein–DNA interface, a large number of intermolecular NOEs were observed in 3D double half filtered HMQC‐NOESY spectra (Lee et al., 1994) taken with 13C,15N‐labeled protein and unlabeled DNA as well as with 13C,15N‐labeled DNA (Figure 5). The use of labeled DNA proved essential for confirming the presence of weak cross‐peaks between protein and DNA as well as for obtaining the few unambiguous NOE assignments at the protein–DNA interface (Masse et al., 1999). Eight NOEs between Leu25 and the DNA were unambiguously assigned as follows: Leu25 CδH3s to the H1′ (weak), H3′ (weak), H4′, H5′ and H5″ of T9 and G10 (Figure 5A and Masse et al., 1999). Because of the severe spectral overlap of arginine and lysine protein side chains and the DNA H4′, H5′ and H5″, the majority of the intermolecular NOEs could only be assigned qualitatively. For example, Asn33, Tyr28, Phe48, Trp59 and several arginine and lysine side‐chains resonances (Hγ and Hδ of Arg, Hγ, Hδ and Hϵ of Lys) give NOEs to the unresolved deoxyribose H4′, H5′ and H5″, indicating contact to the DNA backbone from the minor groove of the DNA (Figure 5B). NOE correlations are observed from lysine and arginine side chains to T8 and T9 methyls (Figure 5C) and the deoxyribose H3′ region, indicating that interactions in the major groove are also taking place.
Modeling the NHP6A–DNA complex
Since only a small number of intermolecular NOEs could be assigned unambiguously, we calculated an NMR‐based model structure of the protein–DNA complex rather than a high resolution structure. Our model of the NHP6A–DNA complex is based on the free NHP6A structure, the previously published structure of the sequence‐specific LEF1–DNA complex (Love et al., 1995), the few assigned intermolecular NOEs and consistency with the partially assigned intermolecular NOEs in the NHP6A–DNAsry complex (Material and methods). We used the LEF1–DNA complex (Love et al., 1995) as the initial template for the model because: (i) the LEF1 protein is bound to a 15 bp DNA with contacts to both its major and minor grooves which is also the case for our NHP6A–DNAsry complex; (ii) the protein backbones of NHP6A (residue 25–75) and of LEF1 (residues 6–56) are the most similar of the complexes (r.m.s.d. 1.6 Å; Table I); and (iii) initial docking of the NHP6A structure on the LEF1–DNA template gave a much better fit than docking of the NHP6A structure onto the less bent SRY–DNA template. The free NHP6A structure was used to model the bound protein since the NMR spectroscopic evidence indicated that only the unstructured N‐terminus changes significantly upon DNA binding. The eight unambiguously assigned intermolecular NOEs between Leu25 and the DNA made it possible to localize the protein precisely on the DNAsry sequence. In the LEF1–DNA complex the residue equivalent to Leu25 (Leu6 in LEF1) contacts the sugar rings of two consecutive adenine residues (A23 and A24) (Love et al., 1995). In the NHP6A complex, Leu25 contacts the sugar rings of T9 and G10 (Figure 5A and Masse et al., 1999). Since the initial modeling resulted in Leu25 of NHP6A being positioned between A23 and A24 on the LEF1 DNA, we converted those nucleotides to T9 and G10 respectively, and then changed the rest of the nucleotides accordingly to correspond to the DNAsry sequence.
The model structure was refined using a series of restrained molecular dynamics steps with added constraints from the free protein structure and between the side chains and the phosphodiester backbone as described in the Materials and methods. After the model structure was calculated, the NMR spectra were examined for predicted NOE cross‐peaks based on short interproton distances between NHP6A and the DNA. Based on this analysis, numerous ambiguous intermolecular NOEs could be assigned. For example, Trp59 H2 and Tyr28 Hδ are <5 Å away from A22 H4′, H5′ and H5″ and Tyr28 Hϵ is less than 5 Å away from C21 H4′, H5′ and H5″, consistent with some of the intermolecular NOES shown in Figure 5B. Based on the model, the intermolecular NOEs from T8 and T9 methyl to Lys and Arg side chains (Figure 5C) could be assigned to Arg10 and Arg13 respectively. The observed intermolecular NOEs from Lys and Arg side chains (Hβ, Hγ, Hδ and Hϵ) to the DNA H4′, H5′ and H5″ are also explained by the close contacts between several Lys (22, 53 and 78) and Arg (5 and 23) side chains and the deoxyriboses in the model structure (Figure 6A).
The model structure is also consistent with other NMR results found in the spectra of the protein and DNA in the complex. The only significantly different chemical shifts in the complex in comparison with the free states were found for most of the residues located at the protein–DNA interface, as expected considering the change of chemical environment. The side chain resonances of Tyr28, Met29 and Phe48 show the largest chemical shift changes in the complex (Figure 4B), and these residues are intimately associated with DNA bases. In addition, some resonances from protein side chains found in the model to contact the DNA are severely broadened (see Thr11, Thr12, Tyr28, Met29, Thr47 and Phe48 in Figure 4B) possibly because of local motion at the protein–DNA interface. Chemical shift changes for the DNA resonances are also consistent with the location of amino acid side chains near DNA base pairs in the model structure. For example, there are significant chemical shift changes at the G10–T11 step and only minimal chemical shift changes at the T11–T12 step, consistent with partial insertion of the Phe48 between G10 and T11, as discussed below.
Description of the NHP6A–DNAsry model structure
In the model structure of the complex, NHP6A is bound to the highly distorted and bent DNA through a large number of electrostatic interactions with the phosphate backbone along both the major and minor grooves combined with hydrophobic interactions along the DNA minor groove. Seventeen arginines and lysines plus the side chains of Asn33, Trp59, Gln75, Tyr81 and Tyr88 neutralize the negatively charged non‐bridging phosphate oxygens (Figure 6A and B). Helices 2 and 3 follow the DNA backbone of strand 2 (C16–C30), with nine amino acid side chains contacting the minor groove via the non‐bridging oxygen O1P of the DNA nucleotides 21–27. A similar set of interactions between five amino acid side chains and the DNA O1P of nucleotides 9–12 is seen in strand 1. The hydrophobic side chains exposed on the concave surface of the protein contact the DNA bases and sugar rings (Figures 6A and 7). Of particular significance are the side chains of Met29 and Phe48, which both protrude out into the DNA binding surface in the free protein structure and are found inserted between adjacent base pairs in the model of the complex (Figure 7). Met29 is inserted between T9 and G10 where a large kink is present. The aromatic ring of Phe48 is inserted almost perpendicular to the minor groove edge of G10●C21 and T11●A20 and appears to be responsible for a second large kink between these base pairs, which occurred during the refinement of the model structure (Figures 6A and 7). Only two side chains are in a position to form hydrogen bonds with bases within the minor groove. The Ser26 OH is close to the N3 of A22, and the Tyr28 OH is positioned such that a network of hydrogen bonds involving the N2 of G10 and the carbonyl oxygen of Gly52 is possible (Figure 7B). As elaborated in the discussion, these contacts may impart some specificity to the choice of binding site.
The N‐terminal tail of NHP6A is in the major groove (Figure 6C and D), with the eight arginines and lysines interacting with the O2P along the backbone of both strands. In addition, there are possible hydrophobic interactions from Arg10, Arg13 and Thr11 side chains to the DNA major groove side of T8 and T9.
Mutant NHP6A proteins with amino acid substitutions on the DNA binding surface
A series of mutations were introduced into NHP6A to assess the functional importance of the different amino acid contacts observed in the model structure. The lysines and arginines within the HMG core domain at residues 22, 23, 36, 40, 53, 60, 67, 78 and 85, as well as Asn33, Tyr81 and Tyr88, which are all in a position to contact the phosphate backbone (Figure 6A and B) were individually substituted with alanine. These mutants all displayed a 1.4‐ to 4‐fold reduction in DNA binding as measured by gel mobility shift assays using a 98 bp DNA fragment (Table II). While some of the effects are small, they are statistically significant and reproducible. Loss of two of these contacts, as illustrated by the double mutants R23A/R36A and K53A/K60A, resulted in a loss of formation of discrete DNA complexes and up to a 10‐fold reduction in binding affinity (Figure 8A and Table II). On the other hand, alanine substitutions of K54 and K58, whose side chains are directed away from DNA, have no detectable effect on binding. An aspartic acid substitution of Leu25, whose side chain is in close proximity with the T9 and G10 riboses within the minor groove as directly established by the intermolecular NOEs, resulted in a 4‐fold reduction in binding affinity.
Mutations of two aromatic amino acids, Phe48 and Tyr28, along with Met29 (Yen et al., 1998), which are located at the T9–G10–T11 region that is strongly deformed in the NHP6A–DNAsry model structure result in proteins that have altered DNA bending properties. As shown in Figure 8A, the electrophoretic mobilities of the complexes formed with F48A are significantly faster than those produced by the wild type or most of the other mutants. In addition, microcircle formation by F48A is impaired. Over 20 times more F48A than wild‐type protein is required to generate maximum yields of 98 bp DNA fragments into circles (Figure 8B), even though the binding affinity is only reduced ∼2‐fold (Figure 8A, Table II). Moreover, maximum yields of circles produced with F48A is only ∼40% of that formed by the wild‐type protein. Because the Phe48 ring is packed against the central hydrophobic core of NHP6A, we were concerned that the alanine substitution may have affected the structure of the protein. CD analysis of the free protein, however, revealed no significant deviation from wild type (Figure 8C).
Y28A behaves similarly to F48A with respect to the faster mobilities of DNA bound complexes and inefficient microcircle formation (data not shown). In this case, the CD profile showed a demonstrable reduction in α‐helical content, suggesting that the structure of Y28A is partially disrupted at 25°C (Figure 8C). The 4‐fold decrease in DNA binding affinity by Y28A may therefore be a consequence, at least in part, of a folding defect. However, the faster migration of Y28A–DNA complexes and the poor microcircle formation even at high protein to DNA ratios suggest that loss of this side chain directly or indirectly alters DNA structure within the complex. In contrast to Y28A, the A74D mutant gave normal migrating DNA complexes upon gel electrophoresis and was almost as efficient as wild type in microcircle formation at high protein concentrations, even though its CD profile indicated a highly unfolded protein (Figure 8C), and it displayed a 10‐fold reduction in binding affinity.
Comparison of the NHP6A protein structure and other HMG domain proteins
We have determined the solution structure of NHP6A by multidimensional NMR spectroscopy (Figure 3). NHP6A adopts the L‐shaped fold common to proteins of the HMG1/2 class. Among the HMG domain proteins whose structures have been determined, NHP6A is most similar to HMG‐D (Jones et al., 1994), HMG1B (Read et al., 1993; Weir et al., 1993) and LEF1 (Love et al., 1995) (Table I). These four proteins all have a bend in helix 1 and an ∼80° angle between the two arms of the L, differing slightly from SRY (Werner et al., 1995b), SOX4 (van Houte et al., 1995) and HMG1A (Hardman et al., 1995). Helix 3 from NHP6A is seven residues longer than helix 3 from LEF1. The increased length of helix 3 in all of the non‐sequence‐specific HMG proteins reflects the absence of a proline at the C‐terminal end of the helix. NHP6A, HMG1B and SRY each contain a kink at a proline near the N‐terminal end of helix 3. Of more significance, however, is a pronounced bend in the center of helix 3 in NHP6A, which is also present in HMG‐D but not in other HMG proteins. In NHP6A, this 30° kink is stabilized by a hydrophobic contact between Ala24 and Asp77 and a salt bridge (i, i + 3) between Lys79 and Glu82. In contrast, a hydrophobic interaction between a Pro (for Ala24) and a Leu (for Asp77) and a salt bridge (i, i + 4) (the equivalent residue of Lys79 is a Glu in HMG1B) stabilizes the straight helix in HMG1B. As illustrated in Figure 6B and discussed further below, the central kink in helix 3 of NHP6A plus additional curvature extending towards the C‐terminus is important in positioning this helix together with the extended N‐terminus along the minor groove of the DNA in the complex.
Chemical shift mapping of the protein and DNA surfaces was initially obtained after resonance assignments of the protein and DNA in the complex and free forms were completed. The protein ‘footprint’ (Figure 4A) shows that the concave face of the L‐shaped NHP6A interacts with the DNA, consistent with the partial chemical shift mapping previously reported for the non‐sequence‐specific HMG box HMG1A bound to DNA (Hardman et al., 1995). The DNA ‘footprint’ for both the SRY and LEF1 sequences show that the binding site for NHP6A extends over 11 bp (Figure 1B), which corresponds to T5–A14 within the DNAsry sequence (Figure 6A), and that the primary interface is along the minor groove of DNA. A similarly sized DNA site was deduced for HMG‐D based on changes in the NMR spectra of the DNA upon binding (Churchill et al., 1995). The use of isotopically labeled DNA in combination with isotopically labeled protein enabled a limited number of unambiguous NOEs between NHP6A and the DNAsry sequence to be assigned, which made it possible to precisely position the protein onto the DNA. The structure of the DNA in the LEF1 complex provided a remarkably good initial fit when docked to the NHP6A surface at the location defined by the intermolecular NOEs, although a few clashes were present between the DNA and amino acids within the extended N‐terminus. In contrast, the less bent DNA from the SRY complex did not adequately fit onto the NHP6A structure. The refined model structure of the NHP6A–DNA complex is completely consistent with all of the NMR as well as biochemical and mutational analysis generated to date.
DNA minor groove interactions by the HMG domain of NHP6A
As discussed above, the binding surface of NHP6A conforms well to the highly distorted DNA in the LEF1–DNA complex, with only small changes in the DNA structure resulting from the model structure calculations. The DNA has an overall curvature of ∼90°, which is slightly lower than the value estimated from microcircle ligation and binding studies (Yen et al., 1998). The DNA binding face of the HMG domain of NHP6A presents a hydrophobic surface which is associated with the interior of the wide and shallow minor groove and is flanked by a series of basic residues that are interacting with the DNA phosphodiester backbone (Figure 6B and D). While there is striking complementarity between the protein and DNA surfaces, there is little opportunity for hydrogen bonding to the bases in the NHP6A–DNAsry model, unlike the sequence‐specific LEF1− and SRY–DNA structures. One strand of the phosphodiester backbone between C21 and C27 actually resides within a cleft in the protein surface and is stabilized by sequential salt bridges or hydrogen bonds involving Lys53, Trp59, Lys60, Lys67, Gln75, Lys78, Tyr81, Lys85 and Tyr88. Elimination of any of these contacts by individual alanine replacement (with the exception of Trp59 and Gln75 which were not tested) leads to a modest reduction in binding affinity (Table II). The predicted contacts from Lys78, Tyr81, Lys85 and Tyr88 from the C‐terminal half of helix III to the DNA are unique to NHP6A, since the equivalent residues in SRY and LEF1 do not contact the DNA. The five sequential salt bridges or hydrogen bonds involving Lys22, Arg23, Asn33, Arg36 and Arg40 on the other phosphodiester strand between T8 and T12 may play a more important role since individual alanine substitutions at these positions have a more detrimental effect on binding. Loss of two of these contacts, represented by the double mutant R23A/R36A, reduces DNA binding 10‐fold.
The largely hydrophobic DNA binding surface on NHP6A extends from Arg23 to Thr47 (Figures 6A and 7A). Several of these residues are intimately associated with the DNA near the center of the binding site at T9–G10–T11 and are responsible for the two large kinks within this region (Figure 7). The side chain of Met29 is inserted between T9 and G10. Met29 corresponds to Ile68 in the SRY and Met10 in the LEF1 HMG domains, which also insert between bases and are responsible for large kinks. M29A is defective in forming 66 and 75 bp microcircles and NHP6A M29A–DNA complexes show a slightly greater mobility than wild type, consistent with a compromised ability to induce severe bends in DNA (Yen et al., 1998). M29A functions poorly as a transcriptional co‐activator and only partly rescues the slow growth phenotype of Δnhp6A/B mutants. The SRY mutant I68T binds and bends DNA very poorly, leading to a sex reversal phenotype in humans (Haqq et al., 1994; Peters et al., 1995).
The aromatic ring of Tyr28 is closely packed against the bases within the T9–G10 step and thus is likely to stabilize the kink. Consistent with this, Y28A is defective in DNA bending as measured by its poor ligation efficiency and faster electrophoretic migration of Y28A–DNA complexes as compared with wild type. The position of the aromatic ring of the Tyr28 is stabilized by a hydrogen bond between the side chain OH and the main chain O of Gly52. The hydroxyl of Tyr28 can also act as a hydrogen bond acceptor for the amino group of G10 (Figure 7), therefore selecting for a G at this position. The only other potential hydrogen bond within this region involves Ser26 hydroxyl to A22 N3 (base paired to T9), which is not expected to impart sequence specificity since an equivalent hydrogen bond could form with any base in this position. Both a tyrosine and a serine at these locations are highly conserved among the non‐sequence‐specific HMG proteins, as opposed to the sequence‐specific class, which contains a Phe and an Asn, respectively (Figure 1A).
The aromatic side chain of Phe48 is partially stacked against A20 and abuts G10 in the NHP6A–DNAsry model structure (Figures 6A and 7) and thus causes a second severe kink in a manner that resembles insertions of the phenylalanines of TBP between T–A steps on either side of the TATA sequence (Kim et al., 1993a, b). A similarly located kink is not present in the SRY or LEF1 DNA complexes, which have polar amino acids in their analogous positions which are hydrogen bonded to bases. Loss of Phe48 results in only a 2‐fold reduction in binding affinity, but ligation and gel mobility experiments indicate that F48A–DNA complexes are less curved. The kink at the G10–T11 step induced by Phe48 in NHP6A may be a general property of the non‐sequence‐specific subclass of HMG proteins since these all tend to have an aromatic or branched chain amino acid at this position. A similar role for the equivalent amino acid in HMG‐D was proposed based on a molecular dynamics simulation of HMG‐D with the bent DNA of the TBP complex (Balaeff et al., 1998).
The basic N‐terminal tail of NHP6A binds in the major groove of the DNA
NHP6A differs from most HMG domain proteins in having a highly basic extension at its N‐terminus. We have previously shown that the N‐terminal basic tail is essential for the formation of stable NHP6A–DNA complexes (Yen et al., 1998). This region, which is unstructured in the free protein, contains two basic patches of amino acids beginning at residue 8, with the segment between Arg13 and Lys16 being most important for DNA binding. In the model of the NHP6A–DNAsry complex, the N‐terminal basic tail crosses the phosphodiester backbone and is inserted into the compressed major groove of the DNA (Figure 6C and D), thereby accounting for a series of intermolecular NOEs on the major groove side. The N‐terminal segment effectively wraps around the opposite side of the bound DNA from the expanded minor groove where the body of the protein is associated, and serves to clamp the protein onto the DNA. Every residue from Lys8 to Lys16 is positioned to contact the two phosphodiester strands in an alternating manner, thus complementing similar contacts from the minor groove side and stabilizing the bend towards the major groove. These contacts account for the critical importance of the basic extension at the N‐terminus in promoting the relatively strong DNA binding by NHP6A in comparison with many other non‐sequence‐specific HMG proteins.
The N‐terminal basic region of NHP6A in the model structurally and functionally mimics the C‐terminal basic region of LEF1 (Figure 9). The four basic residues closest to the HMG domain (Arg13–Lys16 for NHP6A and Lys78–Arg81 for LEF1) are the most critical for high affinity DNA binding (Yen et al., 1998). The basic tail of LEF1 is directed towards the major groove by a kink introduced at the C‐terminal end of helix III by Pro67. In NHP6A, a kink at Pro18 at the very beginning of the extended N‐terminus of the HMG domain directs the peptide chain into the major groove. A proline is present at the equivalent position in HMG1A and HMG1B and may play a similar role in orienting the N‐termini of these two HMG domains towards the DNA major groove. We have shown previously that an alanine substitution at Pro18 reduces DNA binding without affecting the structure of the HMG domain (Table II and Yen et al., 1998). The path of the basic N‐terminus of NHP6A is also indirectly mediated by the interaction of the extended N‐terminus (residues 18–25) with helix III. The curvature of helix III, and its interactions (Lys78, Tyr81, Lys85, Tyr88) with the phosphodiester backbone of strand 2 (Figure 6B) directs the basic N‐terminal residues starting with Lys16 in an orientation towards the major groove.
In a similar manner, the bend in helix 3 of HMG‐D could serve to bring its C‐terminal basic tail into the major groove (Jones et al., 1994). The short basic linker between the two HMG boxes of HMG1 is also likely interacting with the major groove, as evidenced by the cross‐link found between one of the basic residues (equivalent of Lys16 in NHP6A) and the DNA major groove of a cisplatinated DNA (Kane and Lippard, 1996). As also noted by others, interactions with a basic tail in the major groove either from the C‐terminus (in LEF1 and HMG‐D) or the N‐terminus (in NHP6A and HMG1B) appears to be a general feature that is indispensable for promoting a relatively stable DNA complex and for DNA bending toward the major groove (Love et al., 1995; Teo et al., 1995; Payet and Travers, 1997; Yen et al., 1998).
Cooperative folding of NHP6A and DNA upon complex formation at 37°C
A number of DNA binding proteins have been found to undergo significant conformational changes upon binding DNA, a phenomenon that has been referred to as an ‘induced fit’ model for DNA site recognition (Spolar and Record, 1994). Examples include the folding of otherwise disordered segments in BamHI, EcoRV, the N‐terminal arms of λ repressor and homeodomains, and the formation of the basic α‐helix in leucine zipper and helix–loop–helix proteins upon binding DNA (Jordan and Pabo, 1988; Luisi et al., 1991; Winkler et al., 1993; Ellenberger et al., 1994; Newman et al., 1995). The binding of NHP6A to DNA at 37°C is a particularly dramatic example of cooperative folding between a protein and its DNA binding site that would be expected to be coupled with a large favorable entropic change (Spolar and Record, 1994). NHP6A is largely unfolded at 37°C as measured by either NMR or CD (Yen et al., 1998), and the DNA must undergo a large structural change to conform to the NHP6A surface. Nevertheless, NHP6A readily forms DNA complexes that are stable to gel electrophoresis at 37°C, and the most optimal NMR spectra of the complex were obtained at this temperature. We imagine that at 37°C an unfolded NHP6A HMG domain initially associates with a DNA molecule through electrostatic forces and then a cooperative folding‐assembly occurs facilitated by hydrophobic interactions between the surfaces of the HMG domain and the DNA minor groove.
NHP6A binds at a different location than SRY within the same sequence
Previous studies have suggested that the sequence‐specific and non‐sequence‐specific HMG box proteins bind DNA in a similar manner (Churchill et al., 1995; Peters et al., 1995). We found, however, that NHP6A is positioned onto the DNA quite differently from the sequence‐specific HMG box proteins SRY and LEF1. The recognition sequences for SRY and LEF1 contain a common 5′‐TTG‐3′ motif (Figure 1B). In all three proteins, a series of hydrophobic residues at the convex corner of the L (Leu25, Met29, Tyr28, Trp59 for NHP6A), which have been described as forming a hydrophobic wedge in the case of SRY (Werner et al., 1995b), are localized at a dinucleotide step within the TTG motif. In the NHP6A–DNA complex, Leu25 contacts the deoxyriboses of T9 and G10, whereas the equivalent residues Met64 of SRY and Leu6 of LEF1 are located between the deoxyriboses of the two adenines on the opposite strand (Love et al., 1995; Werner et al., 1995b). Therefore, as illustrated in Figure 9 for the NHP6A and LEF1 complexes, NHP6A binds in an opposite orientation and one dinucleotide step shifted on the DNA from the sequence‐specific complexes.
The difference in binding between the sequence‐specific and non‐sequence‐specific HMG box proteins may be biologically important because if the much more abundant HMG1/2 proteins were to bind in the same manner as SRY or LEF1, they might promote aberrant transcription of the target genes. By binding in reverse orientation, the non‐sequence‐specific proteins would probably not be able to activate the promoter and may even function to inhibit inappropriate expression.
Selection of DNA binding site by NHP6A and other non‐specific HMG proteins
At the macrosequence level, NHP6A binds to B‐DNA non‐specifically as illustrated by gel mobility shift, DNase I footprint and ligase‐mediated circularization assays (Paull and Johnson, 1995; Yen et al., 1998). However, NHP6A, like other non‐specifically binding HMG1/2 proteins, binds with increased affinity to pre‐bent DNA such as microcircles and cisplatinated DNA (Pil and Lippard, 1992; Pil et al., 1993; Payet and Travers, 1997; Yen et al., 1998). Therefore at the microsequence level, NHP6A probably selectively binds to a DNA segment that can most easily adopt to the distorted configuration present in the complex. Indeed, in the present case, a specific site was chosen within the 15 bp SRY and LEF1 sequences. The greatest DNA deformations in the model structure occur at the center of the binding site and include the large kinks at T9–G10 and G10–T11 mediated by insertion of the Met29 and Phe48 side chains into the respective base stacks. Consistent with this, using in vitro selection experiments for preferred HMG‐D binding sites in random DNA sequences, Churchill et al. (1995) found that T–G plus G–T were the most over‐represented dinucleotides present after several rounds of selection. In addition, a large number of structural studies have demonstrated that a pyrimidine–purine dinucleotide step, such as T–G, has a strong tendency for generating positive roll because of its stacking properties and is often found at positions of protein‐induced kinks in DNA (Dickerson, 1998; Dornberger et al., 1998). NHP6A–DNA complex formation therefore may initiate at a pre‐bent pyrimidine–purine step where the protruding methionine side chain could most easily penetrate. In the NHP6A–DNAsry model structure, the amino group of the G is well accommodated at the interface and may even form a hydrogen bond to Tyr28 (Figure 7). On the other hand, a C instead of a T at the pyrimidine–purine step would be disfavored because of a steric clash by the amino group of the complementary G. Thus, preferential sites on the DNA may be targeted by these proteins because of their pre‐existing conformation (pyrimidine–purine dinucleotides, cisplatinated DNA, microcircular DNA and four‐way junctions), their ability to support a small number of hydrogen bonds (selectivity for a T–G dinucleotide), and their ability to adapt to the concave binding surface of the protein.
Materials and methods
Construction of NHP6A mutants and protein purification
The NHP6A mutants were constructed by direct cloning of two‐step PCR products using mutant oligonucleotide primers (Landt et al., 1990). The mutant genes were transferred into pET11a and transformed into RJ1878 [BL21 (DE3) hupA::cm hupB::km] for protein overexpression (Paull et al., 1996). Each mutant gene in pET11a was sequenced in its entirety. Purification of the wild‐type and mutant proteins was performed as described in Yen et al. (1998). For 15N‐labeled wild‐type NHP6A, cells were grown in 15 l of minimal medium A (Miller, 1992) with 8 mM (15NH4)2SO4 (Isotec) and 0.2% glucose. Uniformly 15N,13C‐labeled samples were prepared from cells grown in 8 l of the above medium containing 0.125% [13C]glucose (Isotec). Each of the wild‐type and mutant NHP6A preparations were judged to be >99% pure by Coomassie Blue staining of overloaded SDS–polyacrylamide gels. Protein concentrations were determined by Coomassie Blue staining of SDS–polyacrylamide gels containing known concentrations of NHP6A that were originally established by direct amino acid analysis (Yen et al., 1998).
Analysis of NHP6A mutants
Electrophoretic mobility shift, DNA ligase‐mediated circularization and CD analyses were performed as described in Yen et al. (1998) with the exception that bromophenol blue was not included in the binding reactions for gel shift assays. This modification resulted in binding affinities being ∼10‐fold greater for wild‐type NHP6A than reported previously (Paull and Johnson, 1995; Yen et al., 1998). Affinities were calculated by plotting the log of the protein concentration versus the log of (b/1−b), where b is the fraction of bound DNA. When the value of log (b/1−b) is zero, 50% of the DNA is bound. Dissociation constants are an average of at least four independent experiments.
NMR sample preparation
The free protein was extensively dialyzed against 10 mM NaPO4 pH 5.5, 100 mM NaCl, and used at a concentration of 1–2 mM for the NMR. The 15 nucleotide unlabeled DNA single strands of DNAsry and DNAlef were chemically synthesized on an Applied Biosystems 392 DNA synthesizer using standard phosphoramidite chemistry. Formation of the duplex DNA was monitored by NMR, by titration of one strand into the other until a 1:1 duplex was formed. Uniformly 13C,15N‐labeled DNAsry was prepared enzymatically as previously described (Masse et al., 1998). Three DNA duplex samples were prepared: one with both strands 13C,15N‐labeled, one with only strand 1 labeled, and the third with only strand 2 labeled. The use of these ‘half‐labeled’ duplexes was essential to help in the resonance assignment of the free and bound DNA and to unambiguously assign intermolecular NOEs to one DNA strand or the other. Sample conditions for the unlabeled and labeled DNA duplexes were 50 mM NaCl, 10 mM NaPO4 pH 6.0 and 1–2 mM DNA duplex.
NHP6A–DNA complexes were formed by titration of the protein into the DNA. The titration was monitored by 1D NMR at 310 K by adding an increasing amount of protein to the DNA until a 1:1 ratio was reached as judged by the relative height of the Trp59 NϵH to the DNA imino or by the disappearance of some imino protons of the free DNA. The sample (1–2 mM) was then dialyzed overnight against a low salt buffer (10 mM NaCl, 2 mM NaPO4 pH 5.8).
NMR spectra were recorded on Bruker DRX 500, 600 and 750 MHz spectrometers. Spectra were processed with Bruker Xwinnmr and analyzed with the Felix 97 software. All the spectra of the free protein were recorded at 293 K with the exception of an 1H‐15N sensitivity‐enhanced TROSY (Czisch and Boelens, 1998; Salzmann et al., 1998) recorded at 310 K. For spectral assignment, 3D CBCACONH (Grzesiek and Bax, 1992a), CBCANH (Grzesiek and Bax, 1992b), HCCH‐TOCSY (Bax et al., 1990), TOCSY‐HMQC and 2D homonuclear TOCSY (Briand and Ernst, 1991) were recorded. For the structure determination, a series of 2D homonuclear NOESY spectra at different mixing times (30, 60, 90 and 150 ms) in H2O and D2O, three 3D at 150 ms mixing time (a 15N NOESY‐HMQC and a 13C HSQC‐NOESY in H2O and a 13C NOESY‐HMQC in D2O) were recorded at 600 MHz.
All the spectra of the free DNA and the protein–DNA complex were recorded at 310 K. For spectral assignments of the free DNA, 2D homonuclear TOCSY (50 ms mixing time) (Briand and Ernst, 1991) and NOESY (300 ms mixing time) were recorded on the unlabelled sample. 2D 15N HMQC, 2D 13C HSQC and 3D 13C NOESY‐HMQC (300 ms mixing time) (Cavanagh et al., 1996) and HCCH‐TOCSY (Bax et al., 1990) were recorded on the isotopically labeled sample. The same set of heteronuclear spectra previously taken on the free labeled protein was recorded on the protein–DNA complex where the protein was isotopically labeled, and similarly for the complex where the DNA was isotopically labeled. All the NOESY spectra (2D and 3D) were recorded with a 150 ms mixing time. In addition, 3D double half‐filtered HMQC‐NOESY (Lee et al., 1994) (150 ms mixing time) were recorded on the complexes with only one of the two components isotopically labeled at a time to observe intermolecular NOEs (Masse et al., 1999).
Protein assignment strategy
The free protein was assigned applying well established methods. The backbone amide, CαH, CβH were assigned using the 3D CBCACONH (Grzesiek and Bax, 1992a), CBCANH (Grzesiek and Bax, 1992b), 15N TOCSY‐HMQC and 15N and 13C HSQC‐NOESY recorded in H2O. The side‐chain assignments were completed by the analysis of the 3D HCCH‐TOCSY and NOESY‐HMQC recorded in D2O. The backbone and side–chain assignment is complete except for the N‐terminal region (Met1–Lys16) which is unstructured and whose resonances (with the exception of Thr11 and Thr12) overlap.
Resonance assignment of the protein in the complex was first obtained by comparison with the free protein since fairly small chemical shift changes (backbone and side chain) were observed. They were later confirmed by the sequential connectivities. Despite severe line‐broadening for several residues, the resonance assignment is complete from Glu17 to Ala93. The resonances of the basic N‐terminal residues (Met1–Lys16) were broader and more dispersed but could not be assigned because of spectral overlap of the many lysine (five) and arginine (three) side‐chain resonances which all have very similar chemical shifts.
DNA assignment strategy
The resonances of the two DNA duplexes DNAsry and DNAlef1 were assigned using well established methodology. The imino and adenine H2 resonances were assigned using the sequential connectivities observed in the 2D NOESY in H2O using a 11 echo water suppression scheme. They confirmed the published assignments of the imino resonances of the SRY DNA (King and Weiss, 1993). All the other non‐exchangeable proton resonances except the H5′, H5″ were assigned using the 2D homonuclear NOESY and TOCSY spectra on the unlabeled sample. Confirmation of the deoxyribose proton assignments as well as assignment of the H5′, H5″ together with the 13C assignments were made with the 3D HCCH‐TOCSY and NOESY‐HMQC spectra recorded on the isotopically labeled DNA.
Most of the DNA imino proton resonances in the complex could not be assigned due to broadening of several resonances, particularly the T iminos, and spectral overlap (not shown). Initial assignments of the non‐exchangeable base and deoxyribose H1′, H2′, H2″, and H3′ resonances of the DNA in the complex were obtained, as described above for the free DNA. These assignments were confirmed and extended to the H4′ and H5′, H5″ resonances from analysis of 3D 1H‐13C NOESY‐HMQC spectra. All of the non‐exchangeable resonances with the exception of the A22 and A23 H2, H4′, H5′, H5″ were assigned. These latter resonances showed more line broadening than the other DNA nucleotides in the complex.
Structure calculations for NHP6A
Inter‐proton distance constraints were obtained from 2D homonuclear NOESY spectra at different mixing time and 3D 15N and 13C separated NOESY spectra at 150 ms mixing time in H2O and D2O. The volume of the NOE cross‐peaks assigned in the 2D NOESY and the 15N NOESY‐HMQC spectra were integrated with the program spscan (Glaser and Wuthrich, http://www.mol.biol.ethz.ch/wuthrich/software/spscan/) and converted into distance constraints using the subroutine CALIBA within the program XEASY (Guntert et al., 1991). Because of spectral overlap in the 13C NOESY‐HMQC in D2O, the peaks were not volume integrated, so the distance constraints derived from the assigned NOEs of this spectrum were all given an upper limit of 5 or 7 Å (for the very weak correlation) plus a pseudo atom correction. This analysis resulted in 1393 relevant distance constraints (Table I). Sixty‐eight loose dihedral ψ (160° ± 100°) and ϕ (160° ± 80°) angle constraints were added based on the deviation of Cα shift from the random coil value and hydrogen bond constraints were added based on the observation of slowly exchanging amide protons when the protein was freshly put into D2O. Fifty structures were calculated in dihedral angle space using the program DYANA (Guntert et al., 1997). The 30 lowest energy structures form the ensemble of converged structures which are further analyzed and described (Table I).
Structure calculations for the model of the NHP6A–DNA complex
The calculations were done within X‐PLOR 3.1 (Brïnger, 1992). The first phase of the modeling started with the lowest energy structure of the pdb structure 2LEF of the LEF1–DNA complex. The bases of the DNA in that complex were changed to the DNAsry sequence by best fit of sugar atoms using standard nucleotides from fiber diffraction data (Biosym). This change was made such that the A23–A24 step in the LEF1 DNA was replaced by T9–G10 in DNAsry and the other bases accordingly. The coordinates of the sugar and backbone atoms were left unchanged. The terminal G1●C30 base pair in the DNAsry was added since it had no equivalent in the DNAlef sequence. The LEF1 protein was then replaced with the lowest NHP6A structure by best fit of backbone atoms for residues 25–75 of NHP6A with LEF1 residues 6–56 (the r.m.s. difference between these two structures is 1.6 Å). At this point some bad base pair geometry and a few clashes between the protein side chains and the DNA were corrected using the standard simulated annealing protocol in X‐PLOR, except that the backbone of the DNA and residues 25–75 of the protein were restrained to their original positions with the harmonic coordinate restraint function in X‐PLOR (Brünger, 1992). DNA hydrogen bonds were enforced with distance restraints, and good base pair geometry was obtained by adding planarity restraints for the base atoms in each base pair. In the next phase, the same calculation was run including distance constraints used in the free protein structure calculation and the eight unambiguous intermolecular distance constraints (upper limits 5 and 6 Å) from Leu25 Hδs to T9 and G10 sugar ring. Examination of the complex at this point showed that the N‐terminus of the protein was in the major groove of the DNA and that several arginine and lysine side chains appeared to be close enough to interact with a DNA phosphate oxygen. Taking advantage of the striking sequence similarity between the stretch R13K14K15K16 in NHP6A and the stretch R81K80K79K78 in LEF1, we modeled R13–K16 similarly to the LEF1–DNA complex (Love et al., 1995) by using a distance constraint of 3 Å between the amino or guanidinium protons and the contacted phosphate oxygen. K22, R23, R36, R40, K53, K60, K67, K78 and K85 were modeled with a similar constraint to the closest phosphate oxygen in the model structure. Finally, K8, K9 and R10 were modeled based on a proposed model of interaction of stretches of arginine in the major groove of DNA (Hud et al., 1994) using the same type of constraints. Calculations with these latter constraints resulted in the NMR‐based model presented here. No violations were found either in the intermolecular NOEs or the side chain to phosphate oxygen constraints, and no violations of more than 0.2 Å were found for the constraints coming from the free protein structure. Indeed, the protein in the complex deviates with an r.m.s.d. of 1.5 Å from the lowest energy structure of the free NHP6A (superimposition of the backbone atoms from residue 20 to 90). Finally, the model of the complex has excellent geometry and van der Waals contacts.
Coordinates for the 30 lowest energy structures of free NHP6A have been deposited in the Protein Data Bank (accession number 1cg7). Coordinates for the model structure of the NHP6A–DNAsry complex are available upon request from the authors.
We thank Rick Fahrner for help on the early part of this work and for DNA purification, Lenore Landis for initial characterization of several NHP6A mutants, Michael Haykinson for computer support and the members of our laboratories for useful discussion. This work was supported by NIH grants GM48123 to J.F. and GM38509 to R.C.J., an American Cancer Society Faculty Research Award to R.C.J., an NIH NIGMS training grant GM08042, the Medical Scientist Training Program and the Aesculapians Fund of the UCLA School of Medicine to Y.‐M.Y., and a European Molecular Biology Organization and Human Frontiers Sciences Program Organization postdoctoral fellowships to F.H.‐T.A.
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