Investigation of the multimerization region of the Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) protein K-bZIP: the proposed leucine zipper region encodes a multimerization domain with an unusual structure

Abstract

The K8 gene of Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) shares many functional similarities with the BZLF1 gene of Epstein-Barr virus. The protein products of K8 and BZLF1, K-bZIP (RAP, K8) and Zta (BZLF1, ZEBRA, Z) have both been proposed to be members of the bZIP family of transcription factors, forming multimers via a coiled-coil motif termed a leucine zipper. Substantial evidence supporting this model for Zta is published. Here, we demonstrate that the proposed leucine zipper region of K-bZIP (amino acids 182 to 218) is required for multimer formation but that it does not fold as a coiled coil.

FIG. 1.

Schematic diagram of Zta and K-bZIP. A. A schematic diagram of the known features of Zta is shown. The striped box represents the basic region, the stippled box the coiled coil, and the filled box the CT region. B. Analysis of the primary structure of K-bZIP (KbZip) and Zta using the bioinformatics program ClustalW revealed the indicated region of homology. Conserved amino acids are shown in on a light grey background, and conservative substitutions are shown on a dark grey background. The dots above the Zta sequence indicate those amino acids in the leucine zipper at “d” positions in the proposed coiled coil. The propensity of equivalent regions of Zta and K-bZIP to fold as coiled coils were investigated using the predictive program COILS and are shown as black bars below or above each sequence. The extent of the synthetic peptides used for biophysical analyses are indicated as boxes above or below each sequence. The position of the junction within the Z-K hybrid is indicated by an arrow.

FIG. 2.

K-bZIP domain swap proteins do not function as bZIPs. A. Vectors encoding hybrid proteins composed of the Zta transactivation and basic domains and the K-bZIP putative zipper domain were generated and are shown schematically here. The Zta basic region is shown in black. The region of K-bZIP from the proposed zipper to the carboxy terminus is shown as a grey box. B. Electrophoretic mobility shift assay reactions were undertaken with equal quantities of the indicated proteins and probes (as described in reference 15). Following electrophoresis, the locations of the probe and DNA complexes were visualized using phosphorimaging. C. Products from the indicated translation reactions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and quantitated by phosphorimaging. The migration of protein molecular weight markers (in kDa) is indicated on the left. D. Following the electrophoretic mobility shift assay analysis with equal amounts of protein, the specific binding of His-Zta and each hybrid protein to DNA is shown.

FIG. 3.

The formation of multimers of K-bZIP requires amino acids within the proposed leucine zipper region. A. [³⁵S]methionine-labeled His-K-bZIP, His-K-ΔZIP, and the Z-K hybrid series were generated in reticulocyte lysate. Products from the indicated translation reactions were analyzed by SDS-PAGE and quantitated by phosphorimaging. The migration of protein molecular weight markers (in kDa) is indicated on the left of each gel. B. In vitro association assays with GST and GST-K-bZIP agarose beads were undertaken with equal quantities of the indicated proteins. C. The products from the indicated translation reactions were analyzed by SDS-PAGE and quantitated by phosphorimaging.

FIG. 4.

The proposed leucine zipper region of K-bZIP does not fold as a coiled coil in vitro. A synthetic peptide corresponding to amino acids 182 to 218 of K-bZIP was synthesized and analyzed by circular dichroism spectroscopy as described in references and . A. The normalized spectrum of a 50 μM peptide solution at pH 3.7 is shown. The arrows indicate the two minima, which are characteristic of α-helical secondary structure. The observed minimum, 218 nm, is characteristic of a β-sheet. B. The FTIR-ATR spectrum of peptide is shown. The position of the amide I peak is indicated. C. The second derivative spectrum of the amide I peak revealed three distinct contributions at the indicated wavelengths. The characteristic structure responsible for generating a peak at that wavelength is shown.