Barry M. Willardson
BA, Brigham Young University (1984)
Ph.D., Purdue University (1990)
Postdoctoral Fellow, University of California, Los Alamos National Lab (1990-92)
Staff Scientist, University of California, Los Alamos National Lab (1992-96)
Mechanisms of Assembly of Signaling Complexes
Most cellular functions are performed by proteins associated together into complexes. In fact, many proteins cannot exist in the cell without their binding partners. These protein complexes often require the help of other proteins, called chaperones, to bring the complexes together. This is certainly the case for protein complexes involved in cell signaling processes. Our work has focused on the mechanism of assembly of two types of signaling complexes, the G protein heterotrimer and the mTOR kinase complexes. It is through the G protein complex and its associated receptors and effectors that cells detect hormones, neurotransmitters, chemokines and sensory signals such as odorants, taste molecules and even photons of light. G proteins regulate almost every aspect of cellular physiology and as a result more than a third of current therapeutic drugs target G protein signaling pathways. The two mTOR complexes, mTORC1 and mTORC2, are also high-value drug targets because of their role in orchestrating cell survival, growth and metabolism in response to growth hormones and nutrient levels.
Both G protein and mTOR complexes are assembled with the help of the cytosolic chaperonin CCT (also called TRiC), a large protein folding machine with a double-ring structure of eight different chaperonin subunits in each ring. The center of each ring creates a protein folding chamber in which nascent proteins with intricate folding trajectories bind and are assisted in the folding process. One such protein fold is the b-propeller, which commonly has seven b-sheets that form the blades of a propeller-like circular structure. b-propellers have a unique folding trajectory that requires the C-terminus to interact with the N-terminus to make the last b-sheet that closes the b-propeller. CCT is believed to facilitate this process. We have found that the b-propellers of the G protein b subunit (Gb) and the mLST8 and Raptor subunits of mTOR complexes are folded by CCT prior to their assembly into complexes.
The process of G protein heterotrimer assembly begins with the association of the G protein β subunit (Gβ) with the G protein γ subunit (Gγ) into the Gβγ dimer. Gβγ is an obligate dimer, meaning that neither subunit is stable in the cell without the other. As a result, Gβ and Gγ must be brought together by chaperones. At some point during or after translation, the nascent Gβ subunit binds CCT and is folded into its b-propeller structure. However, the b-propeller is not stable in the absence of the Gg subunit, and Gβ cannot associate with Gγ until it is released from CCT. This conundrum is resolved by the CCT co-chaperone, phosducin-like protein 1 (PhLP1). PhLP1 binds Gβ in the CCT folding cavity and initiates the release of Gβ from CCT. Once released, Gγ is able to bind Gβ in the PhLP1-Gβ complex and form the stable Gβγ dimer. The G protein α subunit then associates with Gβγ, forming the active Gαβγ heterotrimer and simultaneously releasing PhLP1. All four of the typical Gβ subunits are assembled with their 12 associated Gγ subunits by this same mechanism involving CCT and PhLP1.
The atypical Gβ5 subunit forms a dimer with regulators of G protein signaling (RGS) proteins of the RGS7 subfamily. These dimers have a different function than Gβγ dimers. They turn off G protein signaling in neurons by accelerating the rate of GTP hydrolysis on the Gα subunit. We have found that CCT and PhLP1 also assist in the assembly of these Gβ5-RGS complexes. In fact, the conditional deletion of the PhLP1 gene in the rod photoreceptor cells of mice results in the loss of the Gβ5-RGS9 dimer from these cells in addition to the loss of Gβγ dimers. Consequently, G protein-dependent responses to light by rod photoreceptors were diminished and their recovery was slow. These findings have confirmed the importance of PhLP1 in Gβγ and Gβ5-RGS dimer formation in vivo.
Fig. 1. Location of Gb and mLST8 in the CCT folding chamber. The 3D reconstructions of Gb-CCT (white) and mLST8-CCT (tan) reveal the different locations of the two b-propellers.
In the case of mLST8 and Raptor, both of their b-propeller domains are folded by CCT. They then release from CCT independently of PhLP1 to associate with mTOR. Cryo-EM structural studies of the Gb-CCT and mLST8-CCT complexes, done in collaboration with the lab of Jose M. Valpuesta at the Centro National de Biotecnologia in Madrid Spain, show that the b-propellers of both proteins have reached a near-native state while bound to CCT, but they associate with CCT very differently despite their structural similarity (Fig. 1). Gb binds the CCT apical domains at the top of the CCT folding chamber similar to actin, another CCT substrate, while mLST8 binds CCT at the bottom of the folding chamber between the CCT rings, which has not been previously seen with any CCT substrate. These positions explain the effects of PhLP1, which can interact with Gb at the top of the chamber and mediate its release, but it cannot access mLST8 between the rings. These structural studies provide the molecular details needed for structure-based therapeutic design to control the folding and thereby the function of these important CCT folding substrates.
Cuéllar, J., Ludlam, W. G., Tensmeyer, N. C., Aoba, T., Dhavale M., Bueno-Carrasco, T., Santiago, C. Plimpton, R. L., Makaju, A., Mann, M. Franklin 3 , S., Willardson, B. M. and Valpuesta, J. M. “Structural and functional analysis of the role of the chaperonin CCT in mTOR complex assembly” Nature Communications 10, 2865.
Lord, N. P., Plimpton, R. L., Sharkey, C. R., Suvorov, A., Lelito, J. P., Willardson, B. M., Bybee, S. M. (2016). A cure for the blues: opsin duplication and subfunctionalization for short-wavelength sensitivity in jewel beetles (Coleoptera: Buprestidae).. BMC evolutionary biology, 16(1), 107.
Xie, K., Masuho, I., Shih, C.-C., Cao, Y., Sasaki, K., Lai, C. W. J., Han, P.-L., Ueda, H., Dessauer, C. W., Ehrlich, M. E., Xu, B., Willardson, B. M., Martemyanov, K. A. (2015). Stable G protein-effector complexes in striatal neurons: mechanism of assembly and role in neurotransmitter signaling. eLife.
Plimpton, R. L.*, Cuéllar, J.*, Lai, C. W. J., Aoba, T., Makaju, A., Franklin, S., Mathis, A. D., Prince, J. T., Carrascosa, J. L., Valpuesta, J. M. and Willardson, B. M. (2015) “Structures of the Gβ‑CCT and PhLP1‑Gβ‑CCT Complexes Reveal a Molecular Mechanism for G protein β Subunit Folding and βγ Dimer Assembly”. Proc. Natl. Acad. Sci. U.S.A. 112, 2413-2418 (*Equal contribution of these two authors)
Tracy, C. M.*, Kolesnikov A. V.*, Blake D. R., Chen, C.-K., Baehr, W., Kefalov, V. J. and Willardson B. M. (2015) “Retinal cone photoreceptors require phosducin-like protein 1 for G protein complex assembly and signaling.” PLOS ONE 10, e0117129 (*Equal contribution of these two authors)
Tracy, C. M., Gray A. J., Cuellar, J., Shaw, T.S., Howlett, A.C., Taylor, R.M., Prince, J.T., Ahn, N.G., Valpuesta, J.M. and Willardson, B.M. (2014) “Programmed cell death protein 5 interacts with the chaperonin CCT to regulate β-tubulin folding.” J. Biol. Chem. 289, 4490-4502 (Selected as paper of the week)
Lai, C. W. J., Kolesnikov, A.V., Frederick, J.M., Blake, D.R., Li, J., Stewart, J., Chen, C.-K., Barrow, J.R., Baehr, W., Kefalov, V.J. and Willardson, B.M. (2013) “Phosducin-like protein 1 is essential for G protein assembly and signaling in retinal rod photoreceptors.” J. Neurosci. 33, 7941-7951 (Selected in Faculty of 1000)
Smrcka, A. V., Kichik, N., Tarrago, T., Burroughs, M., Park, M., Stern, H., Itoga, N. K. Willardson, B. M. and Giralt, E. (2010) “NMR Analysis of G Protein βγ Subunit Complexes Reveals a Dynamic Gα-Gβγ Subunit Interface and Multiple Protein Recognition Modes” Proc. Natl. Acad. Sci. U. S. A. 107, 639-644.
Howlett, A. C., Gray, A. J., Hunter, J. M. and Willardson, B. M. (2009) “Role of Molecular Chaperones in G protein β5/Regulator of G protein Signaling Dimer Assembly and G protein βγ Dimer Specificity” J. Biol. Chem. 284, 16386-16399.
Lukov, G. L., Baker, C. M., Ludtke, P. J., Hu, T., Carter, M. D., Hackett, R. A., Thulin, C. D. and Willardson, B. M. (2006) “Mechanism of Assembly of G Protein βγ subunits by Protein Kinase CK2-phosphorylated Phosducin-like Protein and the Cytosolic Chaperonin Complex” J. Biol. Chem. 281, 22261-22274.
Lukov, G. L., Hu, T., McLaughlin, J. N., Hamm, H. E. and Willardson, B. M. (2005) “Phosducin-like protein acts as a molecular chaperone for G protein βγ dimer assembly” EMBO J. 24, 1965-1975.
Martin-Benito, J., Bertrand, S., Hu, T., Ludtke, P., McLaughlin, J. N., Willardson, B. M., Carrascosa, J. L. and Valpuesta, J. M. (2004) “Structure of the complex between phosduin-like protein and the cytosolic chaperonin complex” Proc. Natl. Acad. Sci. 101, 17410-17415.
McLaughlin, J. N., Thulin, C. D., Hart, S. J., Resing, K. A., Ahn, N. G. and Willardson, B. M. (2002) “Regulatory Interaction of Phosducin-like Protein with the Cytosolic Chaperonin Complex” Proc. Natl. Acad. Sci. U.S.A. 99, 7962-7967.
Member, Cancer Research Center at Brigham Young University
Member, American Society for Biochemistry and Molecular Biology
Ad hoc reviewer, National Institutes of Health.
Front Row left to right:
- Madhura Dhuvale – Graduate student
- Christopher Tracy – Graduate student
- Molly Clemens – Undergraduate student
- Rebecca Plimpton – Graduate student
- Chun Wan “Jeffrey” Lai – Post-doctoral Fellow
Back row left to right:
- Dr. Willardson
- Devon Blake – Undergraduate student
- Tanner Shaw – Undergraduate student
- Takuma Aoba – Graduate student