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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.

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.

SUBMITTER: Fadeev EA

PROVIDER: S-EPMC2739574 | biostudies-literature | 2009 May

REPOSITORIES: biostudies-literature

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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.

Fadeev Evgeny A EA Sam My D MD Clubb Robert T RT

Journal of molecular biology 20090324 4

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 ...[more]

PMID: 19324050

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Project description:BackgroundThe 18 residue tail abutting the SH3 fold that comprises the heart of the C-terminal domain is the only part of HIV-1 integrase yet to be visualized by structural biology. To ascertain the role of the tail region in integrase function and HIV-1 replication, a set of deletion mutants that successively lacked three amino acids was constructed and analyzed in a variety of biochemical and virus infection assays. HIV-1/2 chimers, which harbored the analogous 23-mer HIV-2 tail in place of the HIV-1 sequence, were also studied. Because integrase mutations can affect steps in the replication cycle other than integration, defective mutant viruses were tested for integrase protein content and reverse transcription in addition to integration. The F185K core domain mutation, which increases integrase protein solubility, was furthermore analyzed in a subset of mutants.ResultsPurified proteins were assessed for in vitro levels of 3' processing and DNA strand transfer activities whereas HIV-1 infectivity was measured using luciferase reporter viruses. Deletions lacking up to 9 amino acids (1-285, 1-282, and 1-279) displayed near wild-type activities in vitro and during infection. Further deletion yielded two viruses, HIV-1(1-276) and HIV-1(1-273), that displayed approximately two and 5-fold infectivity defects, respectively, due to reduced integrase function. Deletion mutant HIV-1(1-270) and the HIV-1/2 chimera were non-infectious and displayed approximately 3 to 4-fold reverse transcription in addition to severe integration defects. Removal of four additional residues, which encompassed the C-terminal beta strand of the SH3 fold, further compromised integrase incorporation into virions and reverse transcription.ConclusionHIV-1(1-270), HIV-1(1-266), and the HIV-1/2 chimera were typed as class II mutant viruses due to their pleiotropic replication defects. We speculate that residues 271-273 might play a role in mediating the known integrase-reverse transcriptase interaction, as their removal unveiled a reverse transcription defect. The F185K mutation reduced the in vitro activities of 1-279 and 1-276 integrases by about 25%. Mutant proteins 1-279/F185K and 1-276/F185K are therefore highlighted as potential structural biology candidates, whereas further deleted tail variants (1-273/F185K or 1-270/F185K) are less desirable due to marginal or undetectable levels of integrase function.

Project description:Phosphoprotein enriched in astrocytes, 15 kDa (PEA-15) exerts its regulatory roles on several critical cellular pathways through protein-protein interactions depending on its phosphorylation states. It can either inhibit the extracellular signal-regulated kinase (ERK) activities when it is dephosphorylated or block the assembly of death-inducing signaling complex (DISC) and the subsequent activation of apoptotic initiator, caspase-8, when it is phosphorylated. Due to the important roles of PEA-15 in regulating these pathways that lead to opposite cellular outcomes (cell proliferation vs. cell death), we proposed a phosphostasis (phosphorylation homeostasis) model, in which the phosphorylation states of the protein are vigorously controlled and regulated to maintain a delicate balance. The phosphostasis gives rise to the protective cellular functions of PEA-15 to preserve optimum cellular conditions. In this article, using advanced multidimensional nuclear magnetic resonance (NMR) techniques combined with a novel chemical shift (CS)-Rosetta algorithm for de novo protein structural determination, we report a novel conformation of PEA-15 death-effector domain (DED) upon interacting with ERK2. This new conformation is modulated by the irregularly structured C-terminal tail when it first recognizes and binds to ERK2 at the d-peptide recruitment site (DRS) in an allosteric manner, and is facilitated by the rearrangement of the surface electrostatic and hydrogen-bonding interactions on the DED. In this ERK2-bound conformation, three of the six helices (?2, ?3, and ?4) comprising the DED reorient substantially in comparison to the free-form structure, exposing key residues on the other three helices that directly interact with ERK2 at the DEF-docking site (docking site for ERK, FxF) and the activation loop. Additionally, we provide evidence that the phosphorylation of the C-terminal tail leads to a distinct conformation of DED, allowing efficient interactions with Fas-associated death domain (FADD) protein at the DISC. Our results substantiate the allosteric regulatory roles of the C-terminal tail in modulating DED conformation and facilitating protein-protein interactions of PEA-15.

Dataset Information

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.

Publications

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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