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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
J Mol Biol. Author manuscript; available in PMC 2010 May 15.
Published in final edited form as:
PMCID: PMC2739574

NMR Structure of the amino-terminal domain of the lambda integrase protein in complex with DNA: immobilization of a flexible tail facilitates beta-sheet recognition of the major groove


The integrase protein (Int) from bacteriophage lambda is the archetype member of the tyrosine recombinase family, a large group of enzymes that rearrange DNA in all domains of life. Int catalyzes the insertion and excision of the viral genome into and out of the E. coli chromosome. Recombination transpires within higher-order nucleoprotein complexes that form when its amino-terminal domain binds to arm-type DNA sequences that are located distal to the site of strand exchange. Arm-site binding by Int is essential for catalysis, as it promotes Int mediated bridge structures that stabilize the recombination machinery. We have elucidated how Int is able to sequence specifically recognize the arm-sites by determining the solution structure of its amino-terminal domain (IntN, residues Met1 to Leu64) in complex with its P’2 DNA binding site. Previous studies have shown that IntN adopts a rare monomeric DNA binding fold that consists of a three-stranded anti-parallel beta-sheet that is packed against a C-terminal alpha helix. A low resolution crystal structure of the full-length protein also revealed that the sheet is inserted into the major groove of the arm-site. The solution structure presented here reveals how IntN specifically recognizes the arm-site sequence. A novel feature of the new solution structure is the use of an eleven residue tail that is located at the amino terminus. DNA binding induces the folding of a 310 helix in the tail that projects the amino-terminus of the protein deep into the minor groove for stabilizing DNA contacts. This finding reveals the structural basis for the observation that the ‘unstructured’ N-terminus is required for recombination.


Bacteriophage lambda employs a tightly regulated set of site-specific DNA recombination reactions that integrate and excise its genome into and out of the E. coli chromosome (reviewed in 1). Phage integration occurs when the attachment site (attP) on the phage and a specific site on the bacterial chromosome (attB) recombine to generate the hybrid recombination sites attL and attR at the phage-host DNA junction. Under the appropriate inducing signals, the attR and attL sites recombine regenerating an excised circular lambda chromosome and a reconstituted attB site on the bacterial chromosome. Although each attachment site contains identical 15 base pair ‘core-type’ sequences that are recombined by the phage-encoded integrase (Int) 2; 3, the directionality of recombination is controlled by protein binding to distally positioned sites within the P and P’ arms (the location of these sites is shown in Fig. S1). The latter interactions promote the assembly of distinct higher-order nucleoprotein complexes called ‘intasomes’, in which the excisive and integrative recombination reactions transpire. Structurally distinct intasomes excise and integrate the phage, and differ in the specific arm-type sites that are occupied by Int, which accessory factors are present, and the specific phage attachment sites that are involved in recombination. Integrative recombination requires Int 4 and the E. coli-encoded integration host factor (IHF) 5; 6, while excisive recombination requires the phage-encoded excise (Xis) protein in addition to Int and IHF 7. The host-encoded factor for inversion stimulation (Fis) is also required for efficient excision in vivo and at limiting concentrations of Xis in vitro 8; 9.

The integrase protein (Int) is the archetypal member of the tyrosine recombinase family, a large group of enzymes that rearrange DNA in archaebacteria, eubacteria, and yeast 1; 10. It belongs to an important subgroup of these enzymes called heterobivalent recombinases, which mobilize bacteriophages and transposons. Heterobivalent recombinases are distinguished by their ability to bind to two distinct DNA sites and by the high degree of control they exert over the directionality of recombination. In the case of Int, its C-terminal catalytic and core domains bind to the core-type sites that bracket the site of strand exchange, while its N-terminal domain interacts with arm-type DNA sequences located 50 to 150 base-pairs from the site of recombination 11. Simultaneous occupancy of the core- and arm-type is essential for recombination because it creates Int-mediated bridges that stabilize the excisive and integrative intasomes 12. Arm-site binding by N-terminal domain also biases the reaction towards product formation and allosterically regulates the activity of the catalytic domain 13; 14.

A NMR structure of the N-terminal domain solved in the absence of DNA revealed that it adopts a three-stranded beta-sheet DNA binding fold 15. In addition, a crystal structure of full-length Int bound to DNA has shown that the sheet in the N-terminal domain interacts with the major groove 16. However, the crystal structure was determined at a resolution of only 4.4 Å and thus yielded limited insight into the origins of nucleotide sequence specific binding. Most importantly, the crystal structure failed to reveal how the amino-terminal tail of Int participates in recombination. The tail precedes the N-terminal domain and is essential for recombination and DNA binding, but clear electron density for its amino acids was missing in the crystal structure 15; 16. In this communication, we report the NMR structure of the N-terminal domain of the lambda integrase protein in complex with DNA. The structure reveals the role of the essential amino-terminal tail in arm-site binding and recombination.

NMR structure determination of the IntN-DNA complex

NMR was used to determine the structure of the N-terminal domain of Int (IntN, residues Met 1 to Leu 64) in complex with a 15-base pair oligonucleotide corresponding to the P’2 arm-type site within the phage (5′- G CAG TCA AAA T C -3′// 3′- C GTC AGT TTT A G -5′, binding site underlined). The NMR spectra of the 14.8 kDa IntN-DNA complex were of good quality enabling the identification of 29 intermolecular NOEs (Fig. 1A). Representative NMR spectra and a summary of the chemical shift, NOE and coupling constant data is also provided as supplemental material (Figs. S2-S4). The solution structure of the complex was calculated using simulated annealing methods from a total of 1,163 experimental derived restraints (1,023 NOE distance, 120 dihedral angle and 20 coupling constant restraints). The coordinate precision of the ensemble of 20 conformers to the average structure is 0.60 ± 0.11 Å for the IntN and DNA backbone atoms (residues Met1 to Leu55 of IntN and base pairs Cyt2 to Ade10) (Fig. 2A). All of the structures exhibit good covalent geometry and are compatible with the NMR data (Table 1). The coordinates and experimental data have been deposited at the protein data bank (accession code 2WCC).

Figure 1
NMR data of the IntN-DNA complex
Figure 2
NMR solution structure of the IntN-DNA complex
Table 1
Structural statistics for the solution structure of IntN-DNA complex

Sequence specific recognition of the arm-site

Int recognizes the P’2 arm-site by inserting a three-stranded beta-sheet into the major groove, and by wrapping its amino-terminal tail around the duplex (Fig. 2B). These interactions bury ~2,220 Å2 of solvent accessible surface area, but only modestly bend the duplex by ~35°. Similar to previously reported structures, the N-terminal domain consists of a three-stranded anti-parallel beta-sheet (strands B1 (Leu16-Ile18), B2 (Tyr24-Arg27) and B3 (Glu34-Gly38)) that is packed against a C-terminal α-helix (Arg41-Leu55) 15; 16. However, in the structure of the IntN-DNA complex this core domain is supplemented by an ordered amino-terminal extension that has not been observed previously (colored green in Fig. 2B). This appendage immediately precedes the beta-sheet and consists of a 310 helix (Glu8 to Arg10) that rests against the phosphodiester backbone of the duplex, and an amino-terminal tail that is inserted deep into the minor groove. The presence of the 310 helix and other secondary structural elements is substantiated by characteristic NOE patterns in the NOESY data (Fig. S2).

Sequence specific binding is achieved by major groove contacts from amino acids located in the beta-sheet. The side chain atoms in the ensemble are reasonably well defined by the NMR data and have a coordinate precision of 1.07 ± 0.10 Å. Within the major groove the side chains of Arg19 (strand B1) and Glu34 (strand B3) project from opposing sides of the sheet to form a salt-bridge (Fig. 3A). This positions the carboxyl group of Glu34 for interactions with the N4 group of Cyt6 and enables the guanidine group of Arg19 to donate a hydrogen-bond to the O6 atom of Gua19. This interface is stabilized by the side chains of Asn20 and Lys33, which interact with the phosphate groups of Gua19 and Ade7, respectively. All arm-type binding sites contain a conserved Cyt6-Gua19 base pair suggesting that these interactions are required for nucleotide sequence specific binding. A second base specific contact originates from Asn21, whose side chain amide is appropriately positioned to donate and accept hydrogen bonds from the Gua6 O6 atom and the N4 amine group of Cyt21, respectively. Contacts at this interface may explain why Int binds to sites P1 and P1’ approximately 2-3 times more tightly than to sites P2, P’2 and P’3 (Fig. S5) 17. These higher affinity sites contain a guanine base on the 3′ side of this contact interface that likely participate in complementary hydrogen-bonding interactions to the amide group of Asn21. In contrast, all of the weaker binding sites contain an adenine at this position whose N8 amine group presumably precludes these favorable interactions. The structure of the complex also explains why modification of the side chain of Cys25 with the sulfhydryl-directed reagent N-ethylmaleimide (NEM) disrupts Int’s capacity to bind arm-type DNA and, consequently the ability to carry out recombination 18. The thiol side chain of Cys25 is positioned adjacent to the Arg19-Glu34 salt bridge and its modification likely creates a steric clash with the O4 atom of Thy5 that disrupts binding. A list of the intermolecular protein-DNA contacts present in the complex is summarized in Fig. 3C.

Figure 3
Mechanism of DNA binding

The essential amino-terminal tail of integrase is immobilized in the minor groove

Alteration of the first five amino acids of Int disrupts recombination and DNA binding 15. In the NMR structure of IntN solved the absence of DNA these residues are part of an eleven amino acid disordered tail that precedes strand B1 15. Electron density for these residues is also absent in all previously reported crystal structures of full-length Int 16. Our results indicate that the tail undergoes a disordered-ordered transition upon binding the duplex as result of extensive hydrogen bonding to the minor groove. This is evident by analyzing the 15N{1H} heteronuclear NOE values of the backbone amide groups of the protein in its DNA-free and - bound states (Fig. 1B). In the apo-form, residues preceding strand B1 are unfolded and dynamic as evidenced by small magnitude or negative NOE values. However, upon complex formation the entire N-terminus becomes ordered, as its residues exhibit large NOE values similar to amino acids in the remainder of the domain. Interestingly, other parts of the Int protein also undergo DNA induced folding as recent studies have shown that the core domain is poorly structured in its free form, but folds upon binding to DNA 19.

Inspection of the NMR structure reveals that DNA binding induces the folding of residues Glu8-Arg10 into a 310 helix as a result of stabilizing contacts from the side chains of Ser6 and Arg9 to the phosphates of Thy18 and Thy17, respectively (Fig. 3B). Residues Met1-Gly2-Arg3 preceding the 310 helix then extend into the minor groove enabling the backbone amide of Gly2 to hydrogen bond to the N3 group of Ade10 and the side chain of Met1 to form non-polar interactions with the Ade10-Thy11 base step (Fig. 3B). The tail is further positioned by contacts from by the side chain of Arg3, which is poised to interact with the phosphate of Gua19. Previous studies have investigated the function of residues Arg3, Arg4 and Arg5 in DNA binding and recombination 15. Deleting a single arginine amino acid within this tripeptide has little impact, while protein mutants that eliminate two or more of the arginines are defective in binding and recombination. These results are compatible with the structure, since it is likely that the polypeptide segment preceding the 310 helix can accommodate the removal of a single amino acid without altering its position within the minor groove, while larger deletions would necessitate major changes in the positioning of the Met1-Gly2-Arg3 tripeptide. Because the single amino acid deletion mutant also contains a Gly2Lys substitution, this data suggests that Gly2 can be replaced with lysine without affecting DNA binding. This is compatible with the structure of the complex, since the side chain hydrogen of Gly2 projects into the solvent and is therefore presumably tolerant to mutation. In contrast to the amino-terminal tail, DNA binding causes only modest structural changes in the remainder of the IntN domain as the backbone atoms of residues Leu12 to Leu55 can be superimposed with a rmsd of 0.55 Å when the NMR structures of the DNA-free and -bound forms of IntN are compared 15. Interestingly, in the 4.4 Å crystal structure of full-length Int bound to DNA electron density for residues Met1 to His7 is missing, and residues Glu8 to Arg10 project away from the duplex instead of forming the DNA contacting 310-helix observed in the NMR structure of the complex. The origin of this difference remains unknown, but could be caused by crystal packing effects.

Comparison to other three-stranded beta-sheet DNA binding domains

The three-stranded beta-sheet DNA binding domain present in IntN is very rare and only two other proteins containing this fold have been visualized in complex with DNA: the DNA binding domain from the integrase protein encoded by the Tn916 transposon (IntTn916) 20; 21 and the ethylene responsive factor domain 1 from Arabidopsis thaliana (AtERF1) 22. These proteins share no significant sequence homology with one another, but adopt similar three-dimensional structures that engage the major groove through a three-stranded beta-sheet (Fig. 4). In each protein, the anti-parallel beta-sheet is formed by residues that are contiguous in the primary sequence (B1-B2-B3 topology of secondary structural elements). The sheet is also similarly positioned next to the duplex. In this orientation strand B3 runs from its amino to carboxyl terminus, while the adjacent phosphodiester backbone of the DNA moves from its 3′ to 5′ end. An opposite polarity exists on the other side of the sheet-DNA interface. Here, strand B1 is placed in an amino to carboxyl orientation, while the proximal DNA strand runs from 5′ to 3′. This orientation maximizes the protein-DNA contact surface because the inherent right-handed twist in the beta strands causes the B1-B2 portion of the sheet to form a concave surface that is complementary to the curvature of the duplex 23; 24. In the IntN-DNA complex this enables Arg19 in strand B1, as well as the side chains of Asn20 and Asn21 to make major groove contacts. A comparison of the structures reveals that the sheet in the IntN domain is smaller than the sheets present in the AtERF1 and the IntTn916 proteins because its B2 and B3 strands are shorter. As a result, IntN only projects seven residues from its beta-sheet towards the duplex as compared to nine in the AtERF1 and the IntTn916 proteins. IntN presumably compensates for the reduced size of its sheet-DNA interface by forming additional stabilizing contacts from its amino-terminal tail that are absent in the AtERF1- and the IntTn916-DNA complexes (in IntTn916 and AtERF1 several amino acids precede the DNA binding domain, but they share no significant sequence homology with IntN and do not interact with the minor groove). A similar strategy to increase the size of the DNA contact interface, and thus affinity, is used by helical binding motifs 25. The IntN-DNA NMR structure forms the foundation for future studies to elucidate how the Xis and Fis proteins control the directionality of recombination. Recent studies have shown that these key accessory factors regulate recombination by cooperatively assembling with Int onto attR to form a Int-Xis-Xis-Xis-Fis:DNA complex 26; 27; 28. Moreover, Xis cooperatively binds DNA with Int by interacting with the IntN domain 29; 30; 31. The structure of the IntN-DNA complex serves as a foundation for structural studies of these larger multi protein-DNA complexes that comprise the intasome that mediates phage excision 12; 32.

Figure 4
Comparison of three-stranded beta-sheet DNA binding proteins

Supplementary Material



We thank Dr. Scott Robson for useful discussions and Dr. Robert Peterson for his assistance with NMR experiments. This work was supported by grant from the U.S. Department of Energy (DE-FC-03-87ER60615).


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Accession Numbers: Coordinates and structure factors have been deposited in the Protein Data Bank with accession number 2WCC.


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