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. 2013;9(10):e1003672.
doi: 10.1371/journal.ppat.1003672. Epub 2013 Oct 17.

Molecular basis for oligomeric-DNA binding and episome maintenance by KSHV LANA

John F Domsic  1 , Horng-Shen Chen, Fang Lu, Ronen Marmorstein, Paul M Lieberman
Affiliations

Molecular basis for oligomeric-DNA binding and episome maintenance by KSHV LANA

John F Domsic et al. PLoS Pathog. 2013.

Abstract

LANA is the KSHV-encoded terminal repeat binding protein essential for viral replication and episome maintenance during latency. We have determined the X-ray crystal structure of LANA C-terminal DNA binding domain (LANADBD) to reveal its capacity to form a decameric ring with an exterior DNA binding surface. The dimeric core is structurally similar to EBV EBNA1 with an N-terminal arm that regulates DNA binding and is required for replication function. The oligomeric interface between LANA dimers is dispensable for single site DNA binding, but is required for cooperative DNA binding, replication function, and episome maintenance. We also identify a basic patch opposite of the DNA binding surface that is responsible for the interaction with BRD proteins and contributes to episome maintenance function. The structural features of LANADBD suggest a novel mechanism of episome maintenance through DNA-binding induced oligomeric assembly.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Crystal structure of LANADBD.
(A) The structure of the LANADBD dimer is shown in orange. (B) This fold is conserved in the functional homolog EBNA1 (PDB 1vhi; . (C) In the crystal structure, five LANADBD dimers interact to form a decameric ring. Each dimer is highlighted in a different color. (D) A zoom-in of the boxed area of (C) highlights the residues involved in the formation of the tetramer interface. It is composed of Phe1037, Phe1041 and Met1117, each shown as sticks.
Figure 2
Figure 2. Oligomeric interface affects DNA binding and cooperativity.
(A) Dissociation constants for mutants of LANADBD as determined by fluorescence polarization using an LBS1 oligomer. (B) Representative isotherms of the FP assays for wild-type (cooperative), F1037A/F1041A (non-cooperative), 1021–1153 (increased cooperativity), and M1117A (negative cooperativity) to illustrate the fit of the data using a Hill coefficient. The error bars represent the standard deviation of three independent replicates. Fits not shown in this panel are available in Figure S3.
Figure 3
Figure 3. The tetramer interface is required for cooperative DNA binding.
(A) DNA binding induces oligomerization of LANADBD. In the absence of crosslinker (EGS), wild-type and F1037A/F1041A migrate as monomers (lanes 1 and 2). Addition of increasing concentrations of EGS produces mostly dimers and tetramers of wild-type (lanes 3–5). Addition of LBS1 DNA induces oligomerization (each successive band is the addition of one monomer) in wild-type (lanes 6–9). The oligomer interface mutant F1037A/F1041A has a greatly reduced propensity to form higher molecular weight oligomers (lanes 9–11). (B) Agarose gel EMSA of full-length FLAG-tagged LANA wild-type or mutant proteins (indicated above each lane) binding to DNA probes for LBS1 (lanes 1–9), or LBS1/2 (lanes 10–18). (C) Western blot of affinity purified FLAG-LANA proteins used for EMSA in panel B.
Figure 4
Figure 4. Plasmid replication activity is dependent on DNA binding activity and the oligomerization interface.
(A) Quantification of DNA replication assays of wild-type or mutant LANA (as indicated) after transient transfection with p8xTR in 293T cells. Activity is relative to wild-type LANA and error bars represent the standard deviation for three independent replicates. (B–G) Representative Southern blot replication assay showing BglII linearization (B and E) or BglII+DpnI (C and E) digestion of p8xTR. Arrows indicate full-length DpnI resistant replicated plasmid used for quantification in panel A. Smaller DNA fragments represent unreplicated input or incomplete replication products of p8xTR plasmid. (D and G) Western blot for detection of LANA proteins used in replication assays.
Figure 5
Figure 5. Mutations that affect DNA binding and oligomerization are deleterious to plasmid maintenance activity.
(A) Southern blot analysis of p8xTR plasmids at 7 days post-transfection in BJAB cell lines expressing wild-type or mutant LANA, as indicated above. The same number of cells were loaded in each lane. (B) Western blot of LANA protein expression levels in BJAB cells used for plasmid maintenance assays shown in panel A probed for LANA or cellular Actin. (C) Quantification of plasmid maintenance assays normalized to wild-type LANA (lane 1). Error bars represent the standard deviation for three independent replicates.
Figure 6
Figure 6. LANADBD presents unique surface features.
(A) The N-terminal arm of the domain lies across the DNA binding face. (B) Superimposition with the structure of EBNA1 bound to DNA (PDB 1b3t; [59]) shows that, in this position, the NTA of LANADBD would occlude DNA from binding. (C) An electrostatic surface potential rendering (red = acidic, blue = basic and white = neutral) highlights the coverage of this basic patch opposite the DNA binding face on LANADBD. This view is rotated 90° about the x-axis in (A), as indicated. (D) In EBNA1, the same patch is generally acidic, with a slightly lower overall charge density.
Figure 7
Figure 7. The basic patch of LANADBD interacts with the ET domain of BRD2 and BRD4.
(A) Wild-type LANA (lane 2) is able to co-immunoprecipitate BRD4 and mutations in the N-terminal arm (lanes 11–15), the tetrameric interface (lanes 3–5 and 7), and the interior portion of the basic patch (lanes 9–10) do not affect this interaction. However, mutation of Lys1138, Lys1140, and Lys1141 results in decreased levels of BRD4 interaction (lanes 6 and 8). IP and input are shown for BRD4 (top panels) and LANA (lower panels). (B–E) LANADBD is able to interact with BRD2 (B) and BRD4 (C) ET domains. Ni-NTA resin was loaded with LANA or His-tagged BRD ET domain (input, I) and the flow-through (F) was collected. Untagged LANA was then added and the unbound (U) fraction was collected. Complex formation is indicated by the presence of BRD (dark arrows) and LANA (open arrows) in the elution fraction (E). EBNA1DBD was not able to interact with either BRD2 or BRD4 ET domains (D, E).
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