Modified T4 Lysozyme Fusion Proteins Facilitate G Protein-Coupled Receptor Crystallogenesis

Abstract

G protein-coupled receptors (GPCRs) mediate the majority of cellular responses to hormones and neurotransmitters. Most GPCR crystal structures have been obtained using a fusion protein strategy where the flexible third intracellular loop is replaced by T4 lysozyme (T4L). However, wild-type T4L may not be ideally suited for all GPCRs because of its size and the inherent flexibility between the N- and C-terminal subdomains. Here we report two modified T4L variants, designed to address flexibility and size, that can be used to optimize crystal quality or promote alternative packing interactions. These variants were tested on the M3 muscarinic receptor (M3). The original M3-T4L fusion protein produced twinned crystals that yielded a 3.4 Å structure from a 70 crystal data set. We replaced T4L with the modified T4L variants. Both T4L variants yielded M3 muscarinic receptor crystals with alternate lattices that were not twinned, including one that was solved at 2.8 Å resolution.

Figure 1. T4 lysozyme is a two-domain protein with a flexible hinge region

(A) T4L is composed of a small N-terminal helix (helix A) (red) which extends into a larger N-terminal lobe including helix B (blue). The N-terminal lobe couples via its larger helix C (purple) to the C-terminal domain (green), which forms the core of the protein. Together the N- and C-terminal domains form a binding groove in which hydrolysis is catalyzed by E11 and D20 (orange). The positions of the cysteines introduced to make dsT4L are shown as yellow spheres. (B) T4Ls of GPCR fusion proteins previously crystalized in our laboratory (PDB IDs; 3UON, 4DKL, 4EJ4, 3VW7, 4DAJ, 3SN6 and 2RH1) were superimposed by their C-terminal lobes illustrating the flexibility of the N-terminal lobes relative to the C-terminal lobes. (C) Schematic representation of the primary structure of wtT4L compared with the structure of the two recombinant versions of T4L, dsT4L and mT4L used in the current study. The two disulfide bridges introduced in dsT4L between position 3 and 97 and 21 and 142 are represented by orange bars. The glycine/serine linker (-GGSGG-) replacing the N-terminal domain in mT4L is shown in blue. See also figure S3.

Figure 2. Crystal packing interactions for M3-wtT4L, M3-dsT4L and M3-mT4L

(A and B) M3-wtT4L was previously crystallized in a P1 crystal in which the receptor (green) forms arrays of anti parallel dimers stacked in between layers of T4L (orange with the N-terminal domain shown in blue). The packing of T4L alternates in every second layer (panels C and D). (E and F) Packing interactions in M3-dsT4L are similar to M3 wtT4L with the receptor forming arrays of anti parallel dimers. But unlike M3 wtT4L, dsT4L has only one T4L packing arrangement (panel G). (H and I) In M3-mT4L receptor formed two oligomerization interfaces such that the receptor positions in a linear arrangement in the crystal. The first weaker interaction is mediated by helix 1 and 2. A second interface is formed by an antiparallel interaction between helix 4 and 5 on each receptor. The dimeric packing of mT4L is mediated by the C-terminal surface of T4L that is exposed upon removal of the N-terminal domain. (Figure J).

Figure 3. Features of the M3 mT4L and dsT4L structures

(A) Close up showing the formation of a disulfide bridge between dsT4L position C21 and C142 (top panel) and position C3 and C97 (bottom panel). The 2F_o-F_c electron density map around the disulfide bridges contoured at 1.0σ is shown in grey. Shown in green is a F_o-F_c omit map, refined with the disulfide bond omitted, contoured at 3.0σ. Maps are carved at 2.0 A around the disulfide atoms. (B, top panel) Side view of the dsT4L and wtT4L showing that the disulfide bridge between dsT4L position C21 and C142 stabilizes a more closed conformation of dsT4L (blue) compared with wtT4L (orange and light orange). (B, bottom panel) In contrast, the distance between positions C3 and C97 in dsT4L is nearly the same as in wtT4L. (C) Surfaces of M3 involved in packing interactions for M3- wtT4L, M3-dsT4L and M3-mT4L. Note that the right light green monomer in M3- mT4L is parallel to the center grey monomer. In M3-wtT4L and M3-dsT4L this interface is antiparallel.

Figure 4. Differences in T4L have only small effects on M3 structure

(A) Alignment of the M3-mT4L (green) (PDB 4U15), M3-dsT4L (blue) (PDB 4U15) and M3-wtT4L (orange) (PDB 4DAJ) structures showing that the M3 structures are highly similar. (B) Alignment of the binding pockets of M3-mT4L, M3-dsT4L and M3-wtT4L reveals nearly identical interactions with the antagonist tiotropium. The binding mode of methylscopolamine and a PEG molecule occupying the alloteric binding pocket are shown in figure S2.