Orientation and interactions of an essential tryptophan (Trp-38) in the capsid subunit of Pf3 filamentous virus

Abstract

The filamentous bacteriophage Pf3 consists of a covalently closed DNA single strand of 5833 nucleotides sheathed by approximately 2500 copies of a 44-residue capsid subunit. The capsid subunit contains a single tryptophan residue (Trp-38), which is located within the basic C-terminal sequence (-RWIKAQFF) and is essential for virion assembly in vivo. Polarized Raman microspectroscopy has been employed to determine the orientation of the Trp-38 side chain in the native virus structure. The polarized Raman measurements show that the plane of the indolyl ring is tilted by 17 degrees from the virion axis and that the indolyl pseudo-twofold axis is inclined at 46 degrees to the virion axis. Using the presently determined orientation of the indolyl ring and side-chain torsion angles, chi(1) (N-C(alpha)-C(beta)-C(gamma)) and chi(2,1) (C(alpha)-C(beta)-C(gamma)-C(delta1)), we propose a detailed molecular model for the local structure of Trp-38 in the Pf3 virion. The present Pf3 model is consistent with previously reported Raman, ultraviolet-resonance Raman and fluorescence results suggesting an unusual environment for Trp-38 in the virion assembly, probably involving an intrasubunit cation-pi interaction between the guanidinium moiety of Arg-37 and the indolyl moiety of Trp-38. Such a C-terminal Trp-38/Arg-37 interaction may be important for the stabilization of a subunit conformation that is required for binding to the single-stranded DNA genome during virion assembly.

FIGURE 1

(Top) Coordinate system for the uniaxially oriented Pf3 fiber (abc) in relation to the α-helical subunit and a tryptophan side chain. The drawing (not to scale) represents a small segment of the ∼700-nm virion and a few α-helical turns of one subunit. (Bottom) The Eulerian angles θ and χ define the orientation of the coordinate system (xyz) for a tryptophan Raman tensor with respect to the (abc) system. θ is the tilt angle (c–O–z) between the Raman tensor principal axis z and the fiber axis c. χ is the angle (y–O–N) formed by the Raman tensor principal axis y and the line of intersection between the plane (ab) normal to the fiber axis and the plane (xy) of the indolyl ring.

FIGURE 2

Raman spectra (600–1800 cm⁻¹, 514.5-nm excitation) of solutions of unlabeled Pf3 virus (middle trace) and labeled Pf3 virus containing deuteriotryptophan (Trp-d₅) at subunit residue 38 (top trace). The normalized difference spectrum is also shown (bottom trace). Samples were prepared at ∼100 mg/mL in 10 mM Tris at pH 7.8 and data were collected at 20°C.

FIGURE 3

Polarized Raman spectra (800–1700 cm⁻¹, 514.5-nm excitation) of an oriented Pf3 fiber in cc (top trace, I_cc) and bb (bottom trace, I_bb) polarizations.

FIGURE 4

Isointensity contours of I_cc/I_bb in (θ, χ)-space for the Trp-38 Raman markers of Pf3 at 1368 cm⁻¹ (W7″) and 1548 cm⁻¹ (W3). Contours were obtained by substituting the appropriate Raman tensors from Table 1 into Eq. 1. The observed Raman intensity ratios, [I_cc/I_bb]₁₃₆₈ = 7 ± 2 and [I_cc/I_bb]₁₅₄₈ = 1.3 ± 0.3, restrict the acceptable Eulerian angles to the circled area of (θ, χ)-space, i.e., (73 ± 4°, 44 ± 3°).

FIGURE 5

Stereo model of the refined structure of the Pf3 capsid subunit (Molscript software (Kraulis, 1991)). The top panel shows two neighboring subunits (indices 11 and 17 of the assembled capsid). The bottom panel shows residues 34–44 of the subunit C-terminus with labels indicating Arg-37 and Trp-38. In this energy-minimized model (CNS software (Brunger et al., 1998)), the guanidinium group of Arg-37 is close to the indolyl ring of Trp-38 (Nη2 to Cζ2 distance of ∼3.5 Å) implying a cation-π interaction similar to that suggested (Gallivan and Dougherty, 1999) for the tyrosine kinase domain of the insulin receptor (Hubbard et al., 1994).