Structural and Molecular Basis of the Assembly of the TRPP2/PKD1 Complex

Yu, Yong; Ulbrich, Maximilian H.; Li, Minghui; Buraei, Zafir; Chen, Xing-Zhen; Ong, Albert; Tong, Liang; Isacoff, Ehud Y.; Yang, Jian

doi:10.1016/j.bpj.2009.12.1864

Cited by 52 publications

(115 citation statements)

References 18 publications

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“…TRPP2 and PKD1 associate directly through their C termini (25,26), forming a complex containing three TRPP2 and one PKD1 (27). This association involves coiled-coil domains in the C termini of the two proteins (25,27).…”

mentioning

confidence: 99%

“…This association involves coiled-coil domains in the C termini of the two proteins (25,27). The TRPP2 coiled-coil domain forms a homotrimer, both in solution (27)(28)(29) and in crystals (27).…”

mentioning

confidence: 99%

“…The TRPP2 coiled-coil domain forms a homotrimer, both in solution (27)(28)(29) and in crystals (27). Disruption of this trimer abolishes the assembly of heteromeric TRPP2/PKD1 complexes (27). Although a highresolution crystal structure of the TRPP2 coiled-coil trimer has been solved (27), how the PKD1 coiled-coil binds to this trimer is still unknown.…”

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confidence: 99%

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Structural model of the TRPP2/PKD1 C-terminal coiled-coil complex produced by a combined computational and experimental approach

Zhu

Ulbrich

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in TRPP2 and PKD1, which form an ion channel/receptor complex containing three TRPP2 and one PKD1. A TRPP2 C-terminal coiled-coil trimer, critical for the assembly of this complex, associates with a single PKD1 C-terminal coiled-coil. Many ADPKD pathogenic mutations result in the abolishment of the TRPP2/PKD1 coiled-coil complex. To gain molecular and functional insights into this heterotetrameric complex, we computationally constructed a structural model by using a two-step docking strategy, based on a known crystal structure of the TRPP2 coiled-coil trimer. The model shows that this tetrameric complex has a novel di-trimer configuration: An upstream trimer made of three TRPP2 helices and a downstream trimer made of two TRPP2 helices and one PKD1 helix. Mutagenesis and biochemical analysis identified critical TRPP2/ PKD1 interface contacts essential for the heteromeric coiled-coil complex. Mutation of these interface positions in the full-length proteins showed that these interactions were critical for the assembly of the full-length complex in cells. Our results provide a means to specifically weaken the TRPP2 and PKD1 association, thus facilitating future in vitro and in vivo studies on the functional importance of this association.

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Structural model of the TRPP2/PKD1 C-terminal coiled-coil complex produced by a combined computational and experimental approach

Zhu

Ulbrich

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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“…Although a functional PC2 trimeric channel has not been reported to present, recent studies are compatible with this hypothetical arrangement (9,22). These analyses, however, were performed with incomplete PC2ts.…”

Section: Discussionmentioning

confidence: 89%

“…Previous work has suggested that a coiled-coil subdomain identified in the PC2t (PC2t-CC) plays critical roles in PC2 oligomerization and in its physical interaction with PC1 to form the PC1-PC2 complex, a process that also involves a coiled-coil subdomain in PC1t (1). Another recent study, however, suggests that the PC2t-CC assembles as a homotrimer (9). A Ca þþ -binding EF-hand subdomain has also been predicted in PC2t and been proposed to function as a Ca þþ -sensing switch (2,10), whereas the PC2 channel activity has been shown to be regulated by calcium (4, 5, 11).…”

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Macromolecular assembly of polycystin-2 intracytosolic C-terminal domain

Ferreira

Oliveira

Germino

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Mutations in PKD2 are responsible for approximately 15% of the autosomal dominant polycystic kidney disease cases. This gene encodes polycystin-2, a calcium-permeable cation channel whose C-terminal intracytosolic tail (PC2t) plays an important role in its interaction with a number of different proteins. In the present study, we have comprehensively evaluated the macromolecular assembly of PC2t homooligomer using a series of biophysical and biochemical analyses. Our studies, based on a new delimitation of PC2t, have revealed that it is capable of assembling as a homotetramer independently of any other portion of the molecule. Our data support this tetrameric arrangement in the presence and absence of calcium. Molecular dynamics simulations performed with a modified all-atoms structure-based model supported the PC2t tetrameric assembly, as well as how different populations are disposed in solution. The simulations demonstrated, indeed, that the bestscored structures are the ones compatible with a fourfold oligomeric state. These findings clarify the structural properties of PC2t domain and strongly support a homotetramer assembly of PC2.

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Single‐molecule pull‐down (SiMPull) for new‐age biochemistry

Aggarwal

2014

BioEssays

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Macromolecular interactions play a central role in many biological processes. Protein-protein interactions have mostly been studied by co-immunoprecipitation, which cannot provide quantitative information on all possible molecular connections present in the complex. We will review a new approach that allows cellular proteins and biomolecular complexes to be studied in real-time at the single-molecule level. This technique is called single-molecule pull-down (SiMPull), because it integrates principles of conventional immunoprecipitation with the powerful single-molecule fluorescence microscopy. SiMPull is used to count how many of each protein is present in the physiological complexes found in cytosol and membranes. Concurrently, it serves as a single-molecule biochemical tool to perform functional studies on the pulled-down proteins. In this review, we will focus on the detailed methodology of SiMPull, its salient features and a wide range of biological applications in comparison with other biosensing tools.

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Structural and Molecular Basis of the Assembly of the TRPP2/PKD1 Complex

Cited by 52 publications

References 18 publications

Structural model of the TRPP2/PKD1 C-terminal coiled-coil complex produced by a combined computational and experimental approach

Structural model of the TRPP2/PKD1 C-terminal coiled-coil complex produced by a combined computational and experimental approach

Macromolecular assembly of polycystin-2 intracytosolic C-terminal domain

Single‐molecule pull‐down (SiMPull) for new‐age biochemistry

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