Conserved VP16-core region. (A) Schematic representation of VP16 homologs from five herpesviruses and delineation of the VP16-core protein used for crystallography. The VP16 homologs are derived from HSV-1 (strain 17; Campbell et al. 1984; Dalrymple et al. 1985), VZV (Davison and Scott 1986), GHV-1 (Yanagida et al. 1993), EHV-1 (Telford et al. 1992), and BHV-1 (Carpenter and Misra 1992). A conserved VP16-core region is shown in dark gray and the carboxy-terminal HSV-1 transcriptional activation domain (AD) is shown in light gray. The HSV-1 VP16 core (49–412) protein is shown at the bottom with the structured regions identified by x-ray diffraction shown in black. (B) Electrophoretic mobility retardation assay of GST–VP16 fusion proteins synthesized in E. coli. A labeled (OCTA⁺)TAATGARAT site from the HSV-1 ICP0 promoter (Stern et al. 1989) was used for electrophoretic mobility retardation as described (Huang and Herr 1996). (Lane 1) Free probe; (lanes 2–6), partially purified HeLa-cell Oct-1 and HCF (the wheat germ agglutinin fraction; Wilson et al. 1993); (lane 3) GST–VP16ΔC; (lane 4) VP16ΔC; (lane 5) GST–VP16_49–412 core; (lane 6) VP16_49–412 core. The positions of the free probe, Oct-1–DNA (Oct-1) complex, and VP16-induced complex (VIC) are indicated on the left.

Ribbon (Carson 1991) diagrams of the VP16-core protein, colored red (residues 49–145), green (146–247), light blue (248–323), and yellow (324–412). (A) Front view; (B) right-side view.

Views of defined elements in the VP16 structure. (A) Network of internal charged interactions centered on residue K343. (B) Loop L-BC (red) between helix αK (yellow) and the seat bottom (blue). (C) Solvent-exposed and invariant arginines 214 and 217 and the discontinuity point of helix αG. [Produced by MOLSCRIPT (Kraulis 1991)].

Molecular surface representation (Nicholls et al. 1991) of the electrostatic potential of the VP16 core. The surface charge distribution was computed at neutral pH and displayed at the level of the solvent-accessible surface, assuming a probe radius of 1.4 Å to represent a water molecule, and dielectric constants of 80 and 2 for the solvent and protein, respectively. The surface is colored: blue for positive (20 k_BT), red for negative (−20 k_BT), and white for neutral, where k_B is the Boltzmann’s constant and T is the temperature. Charged side chains are labeled. (A) Left-side view. (B) front view; (C) right-side view. A green cross indicates the location of a phosphate ion.

Sequence alignment of the conserved VP16-core region from HSV-1, VZV, GHV-1, EHV-1, and BHV-1 and alignment with structural elements of the VP16-core protein. The HSV-1 residue numbering is shown above the sequence alignment. Amino acids highlighted are either invariant (white against black) among the five proteins or similar (shaded) as defined by the following groupings: V, L, I, and M; F, Y, and W; K and R; E and D; Q and N; S and T; and A, G, and P. The color coding is as described in the text; α helices are labeled A–K (helix αG is subdivided into helices αG′ and αG′′ owing to a discontinuity in this helix); β strands are labeled 1–6; turns and loops (L) are referred to by their flanking secondary structures; broken lines indicate disordered regions. Polar interactions among conserved residues that are seen to exert their effect on the structure of VP16 as a whole include ion pairings (R64–D68, D97–R162, E160–H131, R164–E237, E165–K343, R214–E218, and R308–E313), polar side-chain– side-chain interactions (D87–S90, Y149–Q246, Y99–E165, S106–D111, Y168–H229, Y231–K343), polar side-chain–main-chain interactions (N85–H326, N85–A330, S106–L108, S186–Y182, Y215–F104, Y216–S106, R237–L302, R264–S90, N296–V307, R299–P305, R299–L269), and water-mediated polar interactions (S106-H₂O–S348, E160-H₂O–E237, Y182-H₂O–R214, Y335-H₂O–A330).

Position of insertion mutations (green) prepared by Ace et al. (1988) on a molecular surface representation of VP16 (Nicholls et al. 1991). (A) Rear view; (B) Left-side view.

The structured region of the conserved VP16 core is involved in DNA-sequence recognition. (A) Electrophoretic mobility retardation assay comparing the TAATGARAT-element recognition properties of HSV-1 VP16 (H-VP16) lacking the transcriptional activation domain (residues 1–412), full-length BHV-1 VP16 (B-VP16), and a recombinant VP16 protein (B/H-VP16) containing BHV-1 VP16 residues 1–356 and HSV-1 VP16 residues 353–412. All samples contained partially purified HeLa-cell Oct-1 and HCF (the wheat germ agglutinin fraction; Wilson et al. 1993). Odd-numbered lanes contained the TAATGARAT DNA probe H-TAAT_P; even-numbered lanes contained the TAATGARAT DNA probe B-TAAT_P described previously (Huang and Herr 1996). (Lanes 1,2) No VP16; (lanes 3,4) H-VP16; (lanes 5,6) B/H-VP16; (lanes 7,8) B-VP16. Only the Oct-1-DNA (Oct-1) and VP16-induced complexes (VIC) are shown. (B) Diagrams of the H-VP16, B-VP16, and recombinant B/H-VP16 proteins. The dark (H-VP16) and light (B-VP16) shaded segments represent the conserved VP16-core sequences from HSV-1 and BHV-1, respectively.

Model of VP16 bound to a TAATGARAT element with the Oct-1 POU domain. The presentation of VP16 is similar to that shown in Fig. 7B. The nucleotide sequence of the DNA in the model is shown at the bottom and originates from the HSV ICP0 gene promoter. In the graphic, the bases are color coded as follows: A (red), T (blue), G (yellow), and C (purple). The blue DNA strand represents the top strand; the red strand represents the bottom strand of the sequence shown below. The Oct-1 POU_S (POU-S) and POU_H (POU-H) domain structures and the Oct-1 DNA-binding site structure are from Klemm et al. (1994).