Structure determination protocol for transmembrane domain oligomers

Abstract

The transmembrane (TM) anchors of cell surface proteins had been one of the "blind spots" in structural biology because they are generally very hydrophobic and sometimes dynamics, and are thus difficult targets for structural characterization. A plethora of examples showed that these membrane anchors are not merely anchors but can multimerize specifically to activate signaling receptors on the cell surface or to stabilize the envelope proteins in viruses. Through a series of studies of the TM domains of immune receptors and viral membrane proteins, we have established a robust protocol for determining atomic resolution structures of TM oligomers by nuclear magnetic resonance (NMR) in bicelles that closely mimic a lipid bilayer. Here, we provide the details of the protocol consisting of five major sections: 1) general expression, purification, and bicelle reconstitution of hydrophobic TM and membrane-proximal domains; 2) determination of the oligomeric state of TM domains in bicelles; 3) detection of inter-molecular contacts; 4) structure determination; and 5) characterization of the protein TM partition. This protocol is broadly applicable to filling the structural gaps in membrane for many Type I/II membrane proteins.

Structural basis of transmembrane coupling of the HIV-1 envelope glycoprotein

“…Recording a 3D 15 N-edited NOESY-TROSY-HSQC (τ mix = 200 ms) on this sample allowed identification of NOEs exclusively between the 15 N-attached protons of one protomer and the aliphatic protons of the neighboring one. The identified NOEs were unambiguously confirmed by performing the 3D J CH -modulated NOE experiment 9,23 , in which two interleaved NOESY spectra were recorded with varying the J CH evolution (J CH = 0 ms and J CH = 8 ms) before the NOE mixing. Subtraction of the two spectra allowed selection of only the inter-chain NOEs.…”

“…The structure of the trimeric TMD-CT LLP2 in bicelles was determined using mainly two types of NMR-derived structural restraints. One is the inter-chain proton-proton distance, derived from nuclear Overhauser effects (NOEs) using previously established protocols 9,23 (see the Methods section and Supplementary Fig. 4).…”

“…The gp41 fragment TMD-CT LLP2 (residues 677-788) from a clade D HIV-1 isolate 92UG024.2 was synthesized by GenScript (Piscataway, NJ). The expression construct was created by fusing the TMD-CT LLP2 to the C-terminus of the His 9 -TrpLE expression sequence in pMM-LR6 vector with an added methionine in-between for subsequent cleavage during protein purification, as previously described 8,9,23 . Mutants and additional constructs (e.g., TMD-KS and CT LLP2 , Supplementary Tables 5 and 6) were generated by standard PCR protocols and confirmed by DNA sequencing.…”

“…9, lanes 1 and 2 of the gels, respectively). Therefore, their native oligomeric states were quantified using the non-denaturing method known as OG-label 23,27 .…”

“…NMR-based membrane partition analyses. The membrane partition of the TMD-CT LLP2 was determined using the paramagnetic probe titration (PPT) method 23,25 . As previously shown, DMPC/DHPC bicelle with sufficiently large q (≥0.5) allows direct use of measurable paramagnetic relaxation enhancement (PRE) to probe residue-specific immersion depth of the protein in the bilayer region of the bicelle.…”