Efficient expression, purification, and visualization by cryo-EM of unliganded near full-length HER3

Abstract

Biochemical analyses of membrane receptor kinases have been limited by challenges in obtaining sufficient homogeneous receptor samples for downstream structural and biophysical characterization. Here, we report a suite of methods for the efficient expression, purification, and visualization by cryo-electron microscopy (cryo-EM) of near full-length Human Epidermal Growth Factor Receptor 3 (HER3), a receptor tyrosine pseudokinase, in the unliganded state. Through transient mammalian cell expression, a two-step purification with detergent exchange into lauryl maltose neopentyl glycol (LMNG), and freezing devoid of background detergent micelle, we obtained ~6Å reconstructions of the ~60kDa fully-glycosylated unliganded extracellular domain of HER3 from just 30mL of suspension culture. The reconstructions reveal previously unappreciated extracellular domain dynamics and glycosylation sites.

Keywords: Cryo-electron microscopy; HER receptors; Human epidermal growth factor receptors; Membrane proteins; Receptor tyrosine kinases.

Figure 1.. Construct Design and Mammalian Cell Expression of Near Full-Length HER3.

(A) Construct map of N-terminal Flag tagged HER3 (Δ1022-1342) with a C-terminal 3C protease site and twin-strep tag. (B) Cartoon of construct tagging scheme of near Full-Length HER3 comprising the extracellular, transmembrane, intracellular juxtamembrane, and pseudokinase domains. A 1x Flag tag precedes the N-terminus and the C-terminus is followed by a 3C protease cleavable twin-Strep tag. (C) Cartoon of expression method.

Figure 2.. Purification of Near Full-Length HER3.

(A) Left, Flag eluate sample of near full-length HER3 in the absence of bosutinib (-bos) immunoprecipitated with two other bands (1 and 2). Right, the addition of bosutinib (+bos) during expression reduces the other two species. (B) Superimposed gel filtration chromatograms of near full-length HER3 on a Superose6 Increase 10/300 GL column in the presence and absence of bosutinib. Both samples were run in the presence of 0.5 mM DDM. (C) Superimposed gel filtration chromatograms of near full-length HER3 on a Superose6 Increase 10/300 GL column after exchange to 0.01% LMNG or 0.1% over Flag wash. Both LMNG samples were run on gel filtration in a buffer without detergent. The 0.5 mM DDM is the same as in (B). (D) Superimposed gel filtration chromatograms of near full-length HER3 on a Superose6 Increase 10/300 GL column after exchange to 0.002% MNA-C12 or 0.06% GDN over Flag wash.

Figure 3.. Negative Stain and Cryo-EM Freezing of Near Full-Length HER3.

(A) Top, representative NS-EM class averages of near full-length HER3 in the presence of 0.5 mM DDM. Middle, representative NS-EM class averages of near full-length HER3 in the presence of 0.1% LMNG. Bottom, representative NS-EM class averages of near full-length HER3 in the presence of 0.06% GDN. Density corresponding to putative extracellular domains I and III are colored in blue. (B) Surface cartoon of the HER3 extracellular domain crystal structure (PDB ID: 1M6B) viewed from the front and side. (C) Representative cryo-EM micrograph with particles more clearly visualized in the insets.

Figure 4.. Reconstructions of the HER3 extracellular domain reveal glycosylation sites and dynamic properties.

(A) Representative cryo-EM 2D class averages of near full-length HER3 exchanged into 0.1% LMNG. (B) Rosetta fit model of the HER3 extracellular domain (PDB ID 1M6B) into the resulting cryo-EM reconstruction with N-linked glycans labeled. Insets highlight densities corresponding to N469 and N522. (C) Left, two resulting volumes after subclassification are superimposed. (D) Rosetta fit models for each volume overlayed to show movement of domain III relative to domain I in the inset.