NMR analysis of G-protein βγ subunit complexes reveals a dynamic Gα-Gβγ subunit interface and multiple protein recognition modes

Abstract

G-protein βγ (Gβγ) subunits interact with a wide range of molecular partners including: Gα subunits, effectors, peptides, and small molecule inhibitors. The molecular mechanisms underlying the ability to accommodate this wide range of structurally distinct binding partners are not well understood. To uncover the role of protein flexibility and alterations in protein conformation in molecular recognition by Gβγ, a method for site-specific 15 N-labeling of Gβ-Trp residue backbone and indole amines in insect cells was developed. Transverse Relaxation Optimized Spectroscopy-Heteronuclear Single-Quantum Coherence Nuclear Magnetic Resonance (TROSY-HSQC NMR) analysis of 15 N-Trp Gβγ identified well-dispersed signals for the individual Trp residue side chain and amide positions. Surprisingly, a wide range of signal intensities was observed in the spectrum, likely representing a range of backbone and side chain mobilities. The signal for GβW99 indole was very intense, suggesting a high level of mobility on the protein surface and molecular dynamics simulations indicate that GβW99 is highly mobile on the nanosecond timescale in comparison with other Gβ tryptophans. Binding of peptides and phosducin dramatically altered the mobility of GβW99 and GβW332 in the binding site and the chemical shifts at sites distant from the direct binding surface in distinct ways. In contrast, binding of Gα i1 -GDP to Gβγ had relatively little effect on the spectrum and, most surprisingly, did not significantly alter Trp mobility at the subunit interface. This suggests the inactive heterotrimer in solution adopts a conformation with an open subunit interface a large percentage of the time. Overall, these data show that Gβγ subunits explore a range of conformations that can be exploited during molecular recognition by diverse binding partners.Beta gamma subunits | clam shell | Hot Spots | Molecular Recognition | subunit interactions

Intramolecular allosteric interaction in the phospholipase C-δ1 pleckstrin homology domain

“…S7E). Although the reason for the high intensity of the Lys-140 signal is unclear, a similar phenomenon was discussed in the NMR study of the G-protein βγ subunit, and it was predicted that such strong NMR signals would result from unusually high mobility [39]. As the N-and C-terminal regions were disordered in the crystal structure of the PLC-δ1 PH domain (dashed lines in Fig.…”

Understanding Molecular Recognition by G protein βγ Subunits on the Path to Pharmacological Targeting

“…An NMR method was developed for monitoring G␤␥ conformational alterations and dynamics (Smrcka et al, 2010). In part because of protein size limitations in NMR, a specific labeling protocol was adopted in which all of the Trp positions in G␤␥ were labeled with 15 N at both indole and amide positions.…”

Direct-reversible binding of small molecules to G protein βγ subunits

“…One possibility is that the compounds bind to a single conformation explored by the Gβγ binding surface. It is widely thought that Gβγ does not undergo major conformational changes but recent NMR data suggests that the Gβγ hot spot is highly dynamic [37]. If the binding surface is exploring multiple conformations it could be that a specific minor conformation is required for small molecule binding.…”