Pseudo-merohedral twinning and noncrystallographic symmetry in orthorhombic crystals of SIVmac239 Nef core domain bound to different-length TCRzeta fragments

Abstract

HIV/SIV Nef mediates many cellular processes through interactions with various cytoplasmic and membrane-associated host proteins, including the signalling zeta subunit of the T-cell receptor (TCRzeta). Here, the crystallization strategy, methods and refinement procedures used to solve the structures of the core domain of the SIVmac239 isolate of Nef (Nef(core)) in complex with two different TCRzeta fragments are described. The structure of SIVmac239 Nef(core) bound to the longer TCRzeta polypeptide (Leu51-Asp93) was determined to 3.7 A resolution (R(work) = 28.7%) in the tetragonal space group P4(3)2(1)2. The structure of SIVmac239 Nef(core) in complex with the shorter TCRzeta polypeptide (Ala63-Arg80) was determined to 2.05 A resolution (R(work) = 17.0%), but only after the detection of nearly perfect pseudo-merohedral crystal twinning and proper assignment of the orthorhombic space group P2(1)2(1)2(1). The reduction in crystal space-group symmetry induced by the truncated TCRzeta polypeptide appears to be caused by the rearrangement of crystal-contact hydrogen-bonding networks and the substitution of crystallographic symmetry operations by similar noncrystallographic symmetry (NCS) operations. The combination of NCS rotations that were nearly parallel to the twin operation (k, h, -l) and a and b unit-cell parameters that were nearly identical predisposed the P2(1)2(1)2(1) crystal form to pseudo-merohedral twinning.

Figure 1

TCRζ polypeptide crystallization screen. The polypeptide sequences are shown with residue position numbers assigned on the left. The boxed region contains the sequence of the first of the two reported SIV Nef interaction domains (Schaefer et al., 2000 ▶). The sequence of ITAM 1 is colored red.

Figure 2

Crystallization and diffraction. (a) Crystals of the SIVmac239 Nef_core–TCRζ_DP1 and SIVmac239 Nef_core–TCRζ_A63–R80 complexes. Both crystals grew to 750 × 150 × 150 µm at 277 K. (b) Diffraction patterns of crystals of the SIVmac239 Nef_core–TCRζ polypeptide complexes collected on beamline X29 at the National Light Synchrotron Light Source, Brookhaven National Laboratory. (c) Enlarged view of the diffraction patterns. The diffraction-pattern spot profiles are singular, with no evidence of split spots.

Figure 3

Detection of twinning and estimation of the twin fraction α. Top row, cumulative intensity difference plot of the intensity difference of local pairs of intensities that are not twin-related |L| {L = [I(h ₁) − I(h ₂)]/[I(h ₁) + I(h ₂)]} against the cumulative probability distribution N(L) of the parameter L (Padilla & Yeates, 2003 ▶). The expected plots for untwinned and twinned acentric data (red) and the calculated plots for the SIVmac239 Nef_core–TCRζ polypeptide data (blue) are shown. Middle row, estimation of the twin fraction α by Britton plot analysis (Britton, 1972 ▶). The percentage of negative intensities after detwinning is plotted as a function of the assumed value of α. Overestimation of the twin factor α results in an increase in the percentage of negative intensities. The estimated value of α is extrapolated from the linear fit (dashed line). Bottom row, estimation of the twin fraction α using the H plot (Yeates, 1988 ▶). The cumulative fractional intensity difference of acentric twin-related intensities H {H = |I(h ₁) − I(h ₂)|/[I(h ₁) + I(h ₂)]} is plotted against H. The initial slope (dashed line) of the distribution is a measure of α. The expected slopes for the indicated twin fractions 0.0–0.4 are shown (dotted lines).

Figure 4

Twinning in an orthorhombic P2₁2₁2₁ crystal. (a) A P4₃2₁2 space-group unit cell with one molecule (arrow) per ASU (eight per unit cell) is shown with axes a, b and c labeled. (b) A P2₁2₁2₁ space-group unit cell with one molecule per ASU (four per unit cell) is shown (left) with its twin unit cell (right) related by the twin operator (y, x, −z). (c) A P2₁2₁2₁ space-group unit cell with two molecules per ASU (four per unit cell) is shown (left) with its twin unit cell (right) related by the twin operator (y, x, −z). The ASU is comprised of one blue and one black arrow related by noncrystallographic symmetry.

Figure 5

Molecular-replacement solutions. Five molecular-replacement solutions (A–E) are shown with their translation-function Z (TFZ) scores denoted. The relationships and rotation matrices relating the molecular-replacement solutions are shown.

Figure 6

2F _o − F _c OMIT electron-density maps of the TCRζ polypeptide. 2F _o − F _c OMIT electron-density maps contoured at 1σ calculated from the detwinned P2₁2₁2₁ data of the SIVmac239 Nef_core–TCRζ_A63–R80 crystal (a) and the P4₃2₁2 data of the SIVmac239 Nef_core–TCRζ_DP1 crystal (b) are shown for the region encompassing the TCRζ polypeptide.

Figure 7

Crystal packing of the P4₃2₁2 and P2₁2₁2₁ crystal forms. (a) The crystal symmetry organization of the P4₃2₁2 crystal form (left) and the P2₁2₁2₁ crystal form (right) is shown viewed down the fourfold symmetry axis and the corresponding twofold symmetry axis for the two SIVmac239 Nef_core–TCRζ polypeptide complexes. In (b) the crystal packing along the c axis is shown for both crystal forms. SIVmac239 Nef_core and TCRζ are colored cyan and yellow (left) and magenta and green (right), respectively.

Figure 8

SIVmac239 Nef_core dimer interface in the P4₃2₁2 and P2₁2₁2₁ crystal forms. (a) Overlay of the two molecules in the asymmetric unit of the P2₁2₁2₁ crystal (magenta/green) and two symmetry-related molecules [(x, y, z), (y, x, −z)] in the P4₃2₁2 crystal (cyan/yellow). The SIVmac239 Nef_core is colored magenta (P2₁2₁2₁ crystal) or cyan (P4₃2₁2 crystal) and the TCRζ polypeptide is colored green (P2₁2₁2₁ crystal) or yellow (P4₃2₁2 crystal). The structures of the lower SIVmac239 Nef_core–TCRζ polypeptide complexes were aligned by least-squares methods. The relative 10° counterclockwise rotation of the top P2₁2₁2₁ crystal SIVmac239 Nef_core–TCRζ polypeptide complex is depicted. (b, c) Detailed view of the SIVmac239 Nef_core dimer interface in the P2₁2₁2₁ (magenta) and P4₃2₁2 (blue) crystals. The aligned lower SIVmac239 Nef_core–TCRζ polypeptide complex is colored grey and the side chains of residues involved in the interface are shown as stick models. (b) P2₁2₁2₁ crystal form (cyan) is highlighted. (c) P4₃2₁2 crystal form (magenta) is highlighted.

Figure 9

Variation in the crystal contact hydrogen-bond network. Overlay of the crystal-packing interface between two asymmetric units of the P2₁2₁2₁ crystal lattice (SIVmac239 Nef is shown in magenta and TCRζ is shown in green) and two symmetry-related molecules (y, x, −z) and (1/2 + y, 1/2 − x, 1/4 + z) of the P4₃2₁2 crystal lattice (SIVmac239 Nef is shown in cyan and TCRζ is shown in yellow). Alignment was performed by least-squares methods using one SIVmac239 Nef–TCRζ polypeptide complex [at the bottom in (a)]. Hydrogen bonds present in the crystal lattices are represented by dashed lines and are colored green and yellow for the P2₁2₁2₁ and P4₃2₁2 crystal forms, respectively.