The macro domain as fusion tag for carrier-driven crystallization

Abstract

Obtaining well-ordered crystals remains a significant challenge in protein X-ray crystallography. Carrier-driven crystallization can facilitate crystal formation and structure solution of difficult target proteins. We obtained crystals of the small and highly flexible SPX domain from the yeast vacuolar transporter chaperone 4 (Vtc4) when fused to a C-terminal, non-cleavable macro tag derived from human histone macroH2A1.1. Initial crystals diffracted to 3.3 Å resolution. Reductive protein methylation of the fusion protein yielded a new crystal form diffracting to 2.1 Å. The structures were solved by molecular replacement, using isolated macro domain structures as search models. Our findings suggest that macro domain tags can be employed in recombinant protein expression in E. coli, and in carrier-driven crystallization.

Keywords: carrier-driven crystallization; crystallization tag; histone macroH2A; macro domain; recombinant protein expression.

Figure 1

The VTC complex harbors N‐terminal SPX domains. Structure based sequence alignment of VTC subunits ScVtc2 (S. cerevisiae Vtc2; UniProt Acc. P43585), ScVtc3 (S. cerevisiae Vtc3; Q02725) and ScVtc4 (S. cerevisiae Vtc4; P47075) and including a secondary structure assignment calculated with the program DSSP48 and based on PDB entry 5IIG.14 Invariant and conserved residues are highlighted in dark‐ and light‐purple, respectively. SPX^ScVtc 2 shows 62% and 32% sequence identity with SPX^ScVtc 3 and SPX^ScVtc 4, respectively. The two long core helices of the SPX domain are colored in light blue, surrounding helices are colored from yellow to red. The C‐terminal catalytic TTM domain (α‐helices in green, β‐strands in blue) is connected to the SPX domain via a variable linker (in yellow).

Figure 2

A stable SPX^ScVtc4‐macro fusion protein can be recombinantely expressed in E. coli. (A) Four SPX^ScVtc 4 constructs were assayed for crystallization, all of which contained a C‐terminal 6xHis tag and either no fusion protein, a macro tag (macro domain of the human histone macroH2A1.126, 27), trxA (E. coli thioredoxinA¹¹) or the catalytic TTM domain22, 23 of ScVtc4. (B) Overview of the constructed macro domain expression plasmid providing a macro tag (human macroH2A1.1 residues 181‐366) connected to the N‐terminal SPX domain via a Ala‐Gly‐Ser linker and followed by a non‐cleavable 6xHis tag. Target proteins can be inserted using the restriction enzymes XbaI/BamHI. (C) SDS‐PAGE analysis of purified SPX^ScVtc4−6xHis and SPX^ScVtc4‐macro‐6xHis fusion proteins expressed in E. coli.

Figure 3

The known macro domain structures allow for structure solution of SPX^ScVtc 4 by molecular replacement. (A) Structural superposition of different, previously described macro H2A1.1 macro domain structures indicates that N‐ and C‐termini are flexible. R.m.s.d. is between 0.7‐2.6 Å comparing 180 corresponding C_α atoms. (B) Table summary of molecular replacement calculations using the different known macroH2A1.1 structures. 1xRFZ: rotation function Z‐score for the 1^st molecule to be placed. 1xTFZ: translation function Z‐score for the 1^st molecule to be placed. 2xRFZ: rotation function Z‐score for the 2^nd molecule to be placed. 2xTFZ: translation function Z‐score for the 2^nd molecule. LLG: Log Likelihood Gain of the refined solution. (C) C_α traces of two SPX^ScVtc4‐macro molecules (SPX domain are shown in light‐blue and yellow, the C‐terminal macro domains in dark‐blue and orange, respectively) related by a pseudo 2‐fold axis in the asymmetric unit of the I2₁2₁2₁ crystal form. The central core helices of the SPX domains are highlighted (ribbon diagrams).

Figure 4

Reductively and non‐methylated SPX^ScVtc4‐macro protein adopts different conformations. (A) C_α trace of the SPX^ScVtc4‐macro tetramer in the asymmetric unit of the P2₁ crystal form. The C‐terminal helix of the SPX domain is highlighted in red. The C‐terminal helix of human SPX^HsXPR1 has a similar configuration, again contacting a symmetry‐related molecule (PDB‐ID 5IJH¹⁴). Comparison of (B) non‐methylated and (C) reductively methylated SPX^ScVtc4‐macro fusion proteins (in ribbon representation). The zoom‐in shows the connecting Ala‐Gly‐Ser linker in bonds representation (colored in yellow) and including a 2Fo‐Fc omit electron density map contoured at 1.0 σ (blue mesh). (D) Reductively methylated and (E) non‐methylated SPX^ScVtc4‐macro fusion proteins form different crystal lattices, involving SPX–SPX, macro–macro and SPX–macro interactions. Colors are as in (A) and Figure 3(C).