PDBsum entry 2wcc

Protein/DNA

PDB id: 2wcc

Contents

Protein chain

64 a.a. *

DNA/RNA

* Residue conservation analysis

PDB id:

2wcc

Name:

Protein/DNA

Title:

Phage lambda intdbd1-64 complex with p prime 2 DNA

Structure:

DNA (5'-d( Dcp Dgp Dap Dgp Dtp Dcp Dap Dap Dap Dap Dtp Dc)-3'). Chain: 1. Engineered: yes. Other_details: intn(1-64) complex with 12 mer double stranded p'2 DNA. DNA (5'-d( Dgp Dap Dtp Dtp Dtp Dtp Dgp Dap Dcp Dtp Dgp Dc)-3'). Chain: 2.

Source:

Synthetic: yes. Enterobacteria phage lambda. Phage lambda. Organism_taxid: 10710. Expressed in: escherichia coli. Expression_system_taxid: 469008.

NMR struc:

20 models

Authors:

E.A.Fadeev,M.D.Sam,R.T.Clubb

Key ref:

E.A.Fadeev et al. (2009). 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. J Mol Biol, 388, 682-690. PubMed id: 19324050 DOI: 10.1016/j.jmb.2009.03.041

Date:

11-Mar-09 Release date: 07-Apr-09

Protein chain

P03700  (VINT_LAMBD) -  Integrase from Escherichia phage lambda

Seq: 356 a.a.

Struc: 64 a.a.

DNA/RNA chains

G-C-A-G-T-C-A-A-A-A-T-C 12 bases

G-A-T-T-T-T-G-A-C-T-G-C 12 bases

Enzyme reactions

• Enzyme class 1: E.C.2.7.7.- - ?????
• Enzyme class 2: E.C.3.1.-.-

Note, where more than one E.C. class is given (as above), each may correspond to a different protein domain or, in the case of polyprotein precursors, to a different mature protein.

DOI no: 10.1016/j.jmb.2009.03.041

J Mol Biol 388:682-690 (2009)

PubMed id: 19324050

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.

E.A.Fadeev, M.D.Sam, R.T.Clubb.

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.

Selected figure(s)

Figure 2.
Fig. 2. NMR solution structure of the Int^N–DNA complex. (a) A cross-eyed stereo view showing the ensemble of 20 lowest-energy structures of the Int^N–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 (a). The amino-terminal portion of the protein that becomes ordered upon binding DNA is colored green. The solution structure of the Int^N–DNA complex was determined in two stages. The structure of Int^N in the complex was determined using the ATNOS/CANDID software package, which identifies NOE distance restraints by automatically assigning the NOESY NMR data.^[24]^ and ^[25] Input spectra for the calculations included a 3D ^13C-edited NOESY spectrum recorded using the sample dissolved in 100% D[2]O 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 misassign these signals. After seven rounds of calculations, CANDID yielded a converged bundle of conformers representing the structure of Int^N. 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.^26 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 a full set of distance restraints for the DNA and protein, as well as a limited number of intermolecular NOEs 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.^27 The 50 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, intraprotein 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.^[17]^ and ^[28] The final calculations produced 200 structures, 84 of which completely satisfied the experimental data.

Figure 3.
Fig. 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 the side chain of Arg3 contacts the Thy17–Thy18 base step. Arginine residues 3, 4, 5, 9, and 10 and the amino-terminal amino group are favorably positioned for electrostatic interactions with the phosphodiester backbone adjacent to the minor groove interface and the 3[10] helix. (c) Schematic summarizing the protein–DNA contacts in the structure of the Int^N–DNA complex. Phosphodiester linkages are shown as circles; those that are contacting by Int^N are 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.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2009, 388, 682-690) copyright 2009.

Figures were selected by an automated process.