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, 388 (4), 682-90

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

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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

Evgeny A Fadeev et al. J Mol Biol.

Abstract

The integrase protein (Int) from bacteriophage lambda is the archetypal 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 Escherichia 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-type site sequence by determining the solution structure of its amino-terminal domain (Int(N), residues Met1 to Leu64) in complex with its P'2 DNA binding site. Previous studies have shown that Int(N) adopts a rare monomeric DNA binding fold that consists of a three-stranded antiparallel beta-sheet that is packed against a carboxy-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-type site. The solution structure presented here reveals how Int(N) specifically recognizes the arm-type site sequence. A novel feature of the new solution structure is the use of an 11-residue tail that is located at the amino terminus. DNA binding induces the folding of a 3(10) 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" amino terminus is required for recombination.

Figures

Figure 1
Figure 1. NMR data of the IntN-DNA complex
(a) Representative intermolecular NOE data that was used to determine the structure of the IntN-DNA complex. Selected panels from NOESY data are shown for amino acids that are in close proximity to the DNA. The amino acids and corresponding hydrogen atoms are indicated on the left side of each panel. Relevant intermolecular NOE crosspeaks to DNA protons are labeled and indicated by an arrow where resonance overlap exists. The top seven panels are taken from a 3D 13C-edited NOESY spectrum of the complex dissolved in deuterated buffer. The bottom two panels are taken from a 3D 15N-edited NOESY spectrum of the complex dissolved in protonated buffer. (b) A comparison of the 15N{1H} heteronuclear NOE data of the DNA-free and -bound forms of IntN showing that its amino-terminal tail becomes ordered upon interacting with DNA. Top and bottom panels correspond to IntN in its DNA free and bound states, respectively. The N-terminal domain of Int (IntN, residues Met 1 to Leu 64) labeled with 13C and 15N was produced as described previously . DNA corresponding to the P’2 arm-site (5′- G CAG TCA AAA T C - 3′// 3′- C GTC AGT TTT A G -5′, binding site underlined) was purchased from Biosource and purified by gel electrophoresis. Initially, a 0.1 mM sample of the IntN:DNA complex was formed in the presence of high salt (50 mM HEPES (pH 7), 500 mM NaCl, and 2 mM deuterated DTT). The complex was then concentrated using a centricon (Amersham) and dialyzed into low salt NMR buffer. Two samples were prepared: (a) 0.85 mM IntN:DNA complex dissolved in water containing 7% D2O, 25 mM HEPES (pH 7), 15 mM NaCl, 2 mM deuterated DTT, and 0.01% NaN3 and (b) 0.9 mM IntN:DNA complex dissolved in 100% D2O, 26 mM deuterated TRIS (pH 7), 15 mM NaCl, 5.3 mM deuterated DTT, 0.01% NaN3. NMR spectra were recorded at 37 °C using Bruker Avance-500, -600, and -800 MHz NMR spectrometers. The 1H, 13C, and 15N protein backbone and side chain resonance assignments were obtained using conventional methods and have been described previously . The 12C attached 1H resonances of the DNA were assigned by analyzing a 2D F1,F2 13C filtered NOESY spectrum . NOE cross-peaks were recorded using 3D 13C- and 15N- edited NOESY spectra recorded with mixing times of 100 ms . Intermolecular NOE’s were obtained from 2D F1 13C filtered NOESY, and 15N- and 13C-edited 3D NOESY experiments . NMR data were processed and analyzed using the programs NMRPipe and PIPP , respectively. The software package MARS was used to automatically assign the backbone resonances of the protein .
Figure 2
Figure 2. NMR solution structure of the IntN-DNA complex
(a) A cross-eyed stereo view showing the ensemble of 20 lowest energy structures of the IntN-DNA complex. The protein (amino acids 1 to 55) and DNA backbone (nucleotides Cyt2 to Ade10, and Thy15 to Gua23) are shown in blue and red, respectively. (b) Ribbon drawing of the lowest energy structure of the complex. The strands in the beta-sheet and the helices are labeled. The view in the left image is identical to that shown in panel a. The amino-terminal portion of the protein that becomes ordered upon binding DNA is colored green. The solution structure of the IntN:DNA complex was determined in two stages. The structure of IntN in the complex was determined using the ATNOS/CANDID software package, which identifies NOE distance restraints by automatically assigning the NOESY NMR data ; . Input spectra for the calculations included: a 3D 13C- edited NOESY spectrum recorded using the sample dissolved in 100% D2O and a 15N- edited NOESY spectrum of the sample dissolved in water. Chemical shift assignments for residues Met1-Asp11 were excluded from the ATNOS/CANDID calculations because long range NOE signals from this part of the protein were sparse and the software tended to miss-assign these signals. After seven rounds of calculations CANDID yielded a converged bundle of conformers representing the structure of IntN. In a separate set of calculations the program NIH-XPLOR was used to calculate the structure of the bound DNA molecule. Distance restraints for the DNA were obtained by manually assigning 2D F1,F2 13C filtered NOESY spectra of the complex. In addition, the structures were refined using dihedral angle restraints obtained from the program TALOS and loose DNA dihedral angle restraints for the DNA. The latter, maintained the DNA molecule in a B-form conformation and facilitated convergence, but otherwise did not alter the structure of the complex. NIH-XPLOR was then used to calculate structures of the complex . The structure was calculated using the previously determined structure of the DNA molecule and the protein in its unfolded state. The initial docking calculations made use of full-set of distance restraints for the DNA and protein, as well as a limited number of intermolecular NOE’s to orientate the protein and the duplex. The resultant structure was then refined in an iterative manner by manually inspecting the NMR data. During the refinement the program QUEEN was used to sort NOE restraints by decreasing information content . The fifty most significant restraints were checked manually and this process (restraint sorting by QUEEN and manual restraint checking) was repeated until all of the most significant restraints were correct. At the final stages of refinement intra-protein hydrogen bonds in regions of regular secondary structure were identified by inspecting NOE data for characteristic patterns. In addition to standard energy terms to maintain appropriate covalent geometry and to account for distance and dihedral angle data, a mean force potential was employed to improve the structure of the DNA molecule ,. The final calculations produced 200 structures, 84 of which completely satisfied the experimental data. The program MOLMOL was used to make figures for this paper.
Figure 3
Figure 3. Mechanism of DNA binding
(a) Expanded view of the major groove interface. Beta-sheet strands B1 (Leu16-Ile18), B2 (Tyr24-Arg27) and B3 (Glu34-Gly38) insert into the major groove. The side chains of Arg19 and Glu34 contact the Cyt6-Gua19 base pair and simultaneously form a salt-bridge. The carboxyl group of Glu34 interacts with the N4 atom of Cyt6 and the guanidine group of Arg19 donates a hydrogen-bond to the O6 atom of Gua19. The side chains of Asn20 and Lys33 contact phosphate groups of Glu19 and Ade7, respectively. The side chain of Asn21 is juxtaposed with the Gua4-Cyt21 base step in the major groove, stabilizing the binding interface. (b) Expanded view of the minor groove interface and role of the amino-terminus in DNA binding. The side chain of Met1, the backbone residues Met1-Gly2 and the side chain of Arg3 are deeply inserted into the minor groove. Gly2 contacts the Ade9-Ade10 base-step and side chain of Arg3 contacts the Thy17-Thy18 base step. Arginine residues 3, 4, 5, 9 and 10 and the N-terminal amino group are favorably positioned for electrostatic interactions with the phosphodiester backbone adjacent to the minor groove interface and the 310 helix. (c) Schematic summarizing the protein-DNA contacts in the structure of the IntN-DNA complex. Phosphodiester linkages are shown as circles, those that are contacting by IntN highlighted in blue. Bases shaded blue and green are contacted by the protein from the major- and minor-groove, respectively. A hydrogen bond is considered to be present when potential donor and acceptor atoms are separated by less than 3 Å. Salt-bridge interactions occur when appropriately charged groups are separated by less than 4.5 Å. Interactions shown in the figure occur in >40% of the structures within the ensemble.
Figure 4
Figure 4. Comparison of three-stranded beta-sheet DNA binding proteins
The image compares the structures of the DNA complexes of the following proteins: IntN from bacteriophage lambda (left), the ethylene responsive factor domain 1 from Arabidopsis thaliana (AtERF1) and the DNA binding domain from the integrase protein encoded by the Tn916 transposon (IntTn916) ; . Only the backbone of the DNA is shown. Each protein is colored blue, with the exception of the amino-terminal tail of IntN that becomes ordered upon DNA binding, which is colored green. None of the proteins share significant primary sequence homology with one another.

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