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NMR analysis of G-protein betagamma subunit complexes reveals a dynamic G(alpha)-Gbetagamma subunit interface and multiple protein recognition modes.

ABSTRACT: G-protein betagamma (Gbetagamma) subunits interact with a wide range of molecular partners including: G(alpha) 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 Gbetagamma, a method for site-specific (15)N-labeling of Gbeta-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 Gbetagamma 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 GbetaW99 indole was very intense, suggesting a high level of mobility on the protein surface and molecular dynamics simulations indicate that GbetaW99 is highly mobile on the nanosecond timescale in comparison with other Gbeta tryptophans. Binding of peptides and phosducin dramatically altered the mobility of GbetaW99 and GbetaW332 in the binding site and the chemical shifts at sites distant from the direct binding surface in distinct ways. In contrast, binding of G(alpha)(i1)-GDP to Gbetagamma 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 Gbetagamma subunits explore a range of conformations that can be exploited during molecular recognition by diverse binding partners.

SUBMITTER: Smrcka AV

PROVIDER: S-EPMC2818914 | biostudies-literature | 2010 Jan

REPOSITORIES: biostudies-literature

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NMR analysis of G-protein betagamma subunit complexes reveals a dynamic G(alpha)-Gbetagamma subunit interface and multiple protein recognition modes.

Smrcka Alan V AV Kichik Nessim N Tarragó Teresa T Burroughs Michael M Park Min-Sun MS Itoga Nathan K NK Stern Harry A HA Willardson Barry M BM Giralt Ernest E

Proceedings of the National Academy of Sciences of the United States of America 20091216 2

G-protein betagamma (Gbetagamma) subunits interact with a wide range of molecular partners including: G(alpha) 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 Gbetagamma, a method for site-specific (15)N-labeling of Gbeta-Trp res ...[more]

PMID: 20018744

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Project description:Proteins involved in interactions throughout the course of evolution tend to co-evolve and compensatory changes may occur in interacting proteins to maintain or refine such interactions. However, certain residue pair alterations may prove to be detrimental for functional interactions. Hence, determining co-evolutionary pairings that could be structurally or functionally relevant for maintaining the conservation of an inter-protein interaction is important. Inter-protein co-evolution analysis in several complexes utilizing multiple existing methodologies suggested that co-evolutionary pairings can occur in spatially proximal and distant regions in inter-protein interactions. Subsequently, the Co-Var (Correlated Variation) method based on mutual information and Bhattacharyya coefficient was developed, validated, and found to perform relatively better than CAPS and EV-complex. Interestingly, while applying the Co-Var measure and EV-complex program on a set of protein-protein interaction complexes, co-evolutionary pairings were obtained in interface and non-interface regions in protein complexes. The Co-Var approach involves determining high degree co-evolutionary pairings that include multiple co-evolutionary connections between particular co-evolved residue positions in one protein with multiple residue positions in the binding partner. Detailed analyses of high degree co-evolutionary pairings in protein-protein complexes involved in cancer metastasis suggested that most of the residue positions forming such co-evolutionary connections mainly occurred within functional domains of constituent proteins and substitution mutations were also common among these positions. The physiological relevance of these predictions suggested that Co-Var can predict residues that could be crucial for preserving functional protein-protein interactions. Finally, Co-Var web server (http://www.hpppi.iicb.res.in/ishi/covar/index.html) that implements this methodology identifies co-evolutionary pairings in intra and inter-protein interactions.

Dataset Information

NMR analysis of G-protein betagamma subunit complexes reveals a dynamic G(alpha)-Gbetagamma subunit interface and multiple protein recognition modes.

Publications

NMR analysis of G-protein betagamma subunit complexes reveals a dynamic G(alpha)-Gbetagamma subunit interface and multiple protein recognition modes.

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