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

Walter M Kim,Lawrence J Stern,Alexander B Sigalov

doi:10.1107/s090744490904880x

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.

Highlights

Protein crystallization occurs under supersaturating conditions where protein molecules organize by either noncrystallographic or crystallographic symmetry operations into repeating unit cells that pack to form a crystal lattice
In order to determine the structure of the Nef–T-cell receptor (TCR) complex, mixtures of the structured core domain of SIVmac239 Nef (Asp95–Ser235) with various polypeptides spanning the putative binding regions of TCR (Schaefer et al, 2000) were screened for crystal formation
Initial crystallization experiments were aimed towards crystallizing the complex of Nef conserved core domain (Nefcore) (SIVmac239, HIV-1 ELI and NL4-3) with the full-length cytoplasmic domain of TCR (TCRcyt), but these were unsuccessful

Summary

Introduction

Protein crystallization occurs under supersaturating conditions where protein molecules organize by either noncrystallographic or crystallographic symmetry operations into repeating unit cells that pack to form a crystal lattice. Crystal twinning occurs when two or more crystal packings intersperse in one larger aggregate crystal. When the lattices of each crystal packing in the aggregate crystal do not overlap in three dimensions, the crystal exhibits epitaxial, or nonmerohedral, twinning, which can be detected by the presence of split reflections in the crystal’s X-ray diffraction pattern. When the lattice axes of the individual crystals are parallel the crystal is considered to be merohedrally twinned and the X-ray diffraction pattern will not provide any visual cues of crystal twinning. Merohedrally twinned crystals exist predominately as hemihedrally twinned crystals (Yeates, 1997) which contain two distinct twin domains that are related to each other by a doi:10.1107/S090744490904880X 163 research papers twin-law operation. The diffraction pattern of a hemihedrally twinned crystal is the superimposition of two unique diffraction patterns, one from each twin domain, where each reflection intensity is the weighted sum of two twin-related intensities (Grainger, 1969), Iobsðh1Þ 1⁄4 ð1 À ÞIðh1Þ þ Iðh2Þ ð1aÞ

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