Termini restraining of small membrane proteins enables structure determination at near-atomic resolution

Abstract

Small membrane proteins are difficult targets for structural characterization. Here, we stabilize their folding by restraining their amino and carboxyl termini with associable protein entities, exemplified by the two halves of a superfolder GFP. The termini-restrained proteins are functional and show improved stability during overexpression and purification. The reassembled GFP provides a versatile scaffold for membrane protein crystallization, enables diffraction to atomic resolution, and facilitates crystal identification, phase determination, and density modification. This strategy gives rise to 14 new structures of five vertebrate proteins from distinct functional families, bringing a substantial expansion to the structural database of small membrane proteins. Moreover, a high-resolution structure of bacterial DsbB reveals that this thiol oxidoreductase is activated through a catalytic triad, similar to cysteine proteases. Overall, termini restraining proves exceptionally effective for stabilization and structure determination of small membrane proteins.

Fig. 1. Termini restraining of small membrane proteins facilitates their entire structure determination process.

(Left) Difficulties with traditional structural biology approaches (blue box). (I) Overexpression of membrane proteins increases their tendency to unfold and aggregate in cells. The unfolding involves large relative motions between TMs (dashed arrow). (II) Protein purification in detergents is often disruptive to the native folded state of membrane proteins. (III) Small membrane proteins in detergent micelles often contain either small exposed regions or large flexible loops, both of which increase the difficulty of making crystal contact. These proteins are also too small for structural determination by cryo-EM. (Right) Termini-restraining strategy (orange box). (I′) Two associable protein entities (coupler; green) are fused to the flexible N and C termini of a membrane protein (blue), providing a loose restraint to stabilize its folded state during protein overexpression in cells (I′) and protein purification in detergents (II′). (III′) Introducing the coupler protein also provides a large surface for crystal packing and facilitates structure determination.

Fig. 2. Termini restraining maintains the functions of small membrane proteins and enhances their stability during overexpression and purification.

(A) Cellular activities of termini-restrained (TR) and unrestrained constructs of VKOR and VKORL in response to warfarin inhibition. (B) Activities of purified VKOR and VKORL constructs at the same protein concentration. (C) DsbB-catalyzed disulfide bond formation. Left: Transformations restore the motility (measured by colony size) of ∆dsbB E. coli. EV, empty vector. Right: Disulfide-mediated inactivation (white colonies) of MalF-fused β-galactosidase. (D) Cldn4-mediated cell junction. Fluorescence images are of live human embryonic kidney (HEK) 293 cells transfected with Cldn4 constructs. (E) Fe²⁺ uptake activity of purified Mfrn1 in liposomes (50). Cal, calcimycin. See Materials and Methods for explanation of assays in (A) to (E). (F) Fluorescence images of live Pichia cells overexpressing ER membrane proteins with C-GFP tag or sfGFP restraining. The merged images combine GFP fluorescence with ER marker. Red arrows indicate potential protein accumulation in the ER and/or vacuoles. DIC, differential interference contrast. (G) Comparison of extractable folded membrane protein fraction with or without the termini restraining. The immunoblots are shown for target proteins extracted from whole cells with SDS, SDS/urea, or DDM. (H) FSEC-based thermostability assay of purified proteins showing change of Tm (ΔTm) with termini restraining. The unrestrained (U) and sfGFP-restrained constructs both carry a C-terminal mCherry tag for fluorescence detection.

Fig. 3. The sfGFP coupler enables crystallization of small membrane proteins.

VKORL and SPCS1 are shown as examples here; other proteins are shown in fig. S2. (A) Identification of initial crystallization hits (left) of sfGFP-restrained VKORL through fluorescence imaging. The optimized crystals (right) are shown for comparison. (B) Electrostatic surfaces of VKORL and SPCS1 showing their different shapes and surface charges (blue, positive charge; red, negative charge). (C) sfGFP serves as a crystal packing scaffold that is highly adaptable to accommodate various small membrane proteins. Each crystal is packed with alternative layers (dashed lines) of sfGFP (green) and small membrane protein (SMP) molecules. The TMs (numbered) are shown in blue, and extramembrane regions are shown in red. Different membrane protein molecules in the crystals are indicated (A and B). (D) Zoom-in view of versatile crystal packing interactions formed among sfGFP molecules. The large polar surface of sfGFP can accommodate a multitude of crystal contacts (purple surface). The β strands in sfGFP are numbered to illustrate location of the contacts.

Fig. 4. Restraining-based methods for crystal diffraction and phase improvements.

(A) Shortening the restrained termini improves the crystal diffraction of VKORL. Diffraction patterns are shown for full-length (left) and termini-shortened (right) VKORL restrained by sfGFP. Arrows indicate visible diffraction spots near the diffraction limit. (B) Change of crystal packing interactions. The coloring and annotations are the same as in Fig. 3C. (C) Change of relative orientation between the membrane protein and sfGFP after termini shortening. The dashed lines indicate disordered termini. (D) Improvement of electron density maps through cross-crystal averaging with the coupler protein. The cross-crystal averaging is performed between the sfGFP (2B3P) crystal (averaging mask shown in orange) and the sfGFP-restrained VKORL crystal. In addition, the twofold NCS (molecules A and B) is used. (E) Electron density maps of the VKORL region before and after cross-crystal averaging of the low-resolution data. Arrows indicate regions with improved electron density. (F) Superimposition of the low-resolution (4.3 Å; blue) and high-resolution models (2.4 Å; red) showing that the cross-crystal averaged map allows building of a relatively reliable model at low resolution. (G) Electron density maps of the high-resolution VKORL structure after solvent flattening. All the maps are contoured at 1σ.

Fig. 5. High-resolution crystal structures of small membrane proteins provide new mechanistic insights into their functions.

(A) New membrane protein structures (resolution in parenthesis) determined by the termini-restraining strategy. The membrane boundary and flexible ends of membrane proteins fused to the sfGFP are indicated. The double arrows indicate the distance (Cα-Cα atoms) between the ends of N- and C-terminal TMs. (B) Side view of the restrained DsbB Cys¹⁰⁴Ser structure [same colors as in (A)] superimposed with an independently published crystal structure (PDB code 2ZUQ; blue). PL, periplasmic loop; H, helix. The Fab bound to DsbB in 2ZUQ is omitted for clarity. (C) Top view of the restrained DsbB structure, superimposed with an NMR structure (2K74; green) that captures a similar electron-transfer state (fig. S7). (D) Structural comparison of the catalytic triad in E. coli DsbB and hepatitis A virus 3C protease (1CQQ), a representative cysteine protease (45, 46). (E) Similarity of catalytic triads in DsbB and cysteine proteases. Histidine deprotonation of the catalytic cysteine (top) generates the reactive thiolate (bottom). The thiolate in DsbB forms a charge-transfer complex with the quinone (UQ), and the thiolate in cysteine protease attacks the scissile bond carbonyl carbon in a peptide substrate.