A synergistic approach to protein crystallization: combination of a fixed-arm carrier with surface entropy reduction

Abstract

Protein crystallographers are often confronted with recalcitrant proteins not readily crystallizable, or which crystallize in problematic forms. A variety of techniques have been used to surmount such obstacles: crystallization using carrier proteins or antibody complexes, chemical modification, surface entropy reduction, proteolytic digestion, and additive screening. Here we present a synergistic approach for successful crystallization of proteins that do not form diffraction quality crystals using conventional methods. This approach combines favorable aspects of carrier-driven crystallization with surface entropy reduction. We have generated a series of maltose binding protein (MBP) fusion constructs containing different surface mutations designed to reduce surface entropy and encourage crystal lattice formation. The MBP advantageously increases protein expression and solubility, and provides a streamlined purification protocol. Using this technique, we have successfully solved the structures of three unrelated proteins that were previously unattainable. This crystallization technique represents a valuable rescue strategy for protein structure solution when conventional methods fail.

Figure 1

The tandem fixed-arm MBP/SER System. A: Schematic of the pMALX polylinker region. Sequences in yellow originated from the commercially available pMAL-c2X vector (New England Biolabs). Alanine substitutions in the C-terminal α-helix are highlighted in red with the nucleotide changes shown above or below in black. The sequence boxed in light blue shows the point of truncation of the MBP sequence after Asn367, deletion of the Factor Xa cleavage site, and insertion of NotI and NheI restriction sites. B: Ribbon diagram of the structure of MBP (PDB ID code 1HSJ38) showing the residues included in the crystallization cassettes. The C-terminal α-helix bearing three alanine substitutions is shown in purple. The bound maltose is drawn in stick (cyan). The positions of the residues that were substituted with alanine for the SER mutations are drawn in stick in orange. This figure was generated using MolScript.

Figure 2

Crystal-structure of MBP(wt)-2OST (PDB ID code 3F5F). The maltose bound to the MBP is drawn in stick in cyan. The PAP co-factor bound to the 2OST is drawn in stick in green. Ribbon diagrams were generated using PyMOL. A: Ribbon diagram of the crystal structure of the MBP(wt)-2OST fusion protein. The structure of the MBP is shown in gray. α-helices from the 2OST structure are shown in blue, β-strands are shown in red, and the random coil regions are shown in yellow. B: Ribbon diagram of the trimeric MBP(wt)-2OST. Molecule 1 is shown in green, Molecule 2 in blue, and Molecule 3 is shown in orange. 2OST molecules are drawn in ribbon and the MBP molecules are shown as surface renderings. C: Ribbon diagram of the MBP(wt)-2OST interaction surface. Secondary structural elements of the 2OST are shown in blue. The C-terminal α-helix of MBP is shown in gray, with the K359A and K362A mutations drawn in stick.

Figure 3

Crystal structure of the MBP(C)-RACK1A fusion protein (PDB ID code 3DM0). A: Ribbon diagram of the MBP(C)-RACK1A fusion protein. The bound maltose is drawn in stick (cyan). The structure of the MBP is shown in gray, relative to that of the RACK1A (multicolored). The β-strands of the RACK1A β-propeller domain are shown as ribbons, with each WD motif colored and labeled. The residues of the disordered loop (Lys277-Lys294) in WD6 (purple) are labeled. B,C, and D: Secondary structural elements of the MBP(C) protein are drawn as gray ribbons, with the SER mutations drawn in stick. The positions of symmetry-related MBP molecules (gray) and RACK1A (green, blue, yellow) are drawn in stick. B: Position of the D82A/K83A SER mutations in crystal lattice formation. C: Position of the K239A mutation in crystal lattice formation. D: Role of Asn173 in crystal lattice formation.

Figure 4

Crystal structure of the MBP(E)-Der p 7 fusion protein (PDB ID code 3H4Z). A: The ribbon diagram of Molecule A of MBP(E)-Der p 7. The structure of the MBP is shown in gray, with the bound maltose drawn in stick (cyan). α-helices of the Der p 7 structure are shown in green, β-strands in dark blue, and random coil regions in yellow. B: Intermolecular interactions comprising the asymmetric unit of the MBP(E)-Der p 7 crystals. Surface renderings of the MBP molecules (Molecule A in light green, Molecule B in light purple, Molecule C in light gold) illustrating the extensive MBP-MBP interactions within the asymmetric unit. Minimal interactions between the Der p 7 of Molecule B (dark purple) with the MBP of Molecule C (yellow). C: Position of the K239A mutation in the crystal lattice. The K239A mutations of Molecules A (green) and B (purple) lie along an intermolecular interaction surface.