Solution NMR Structure of the SH3 Domain of Human Caskin1 Validates the Lack of a Typical Peptide Binding Groove and Supports a Role in Lipid Mediator Binding

SH3 domains constitute an important class of protein modules involved in a variety of cellular functions. They participate in protein-protein interactions via their canonical ligand binding interfaces composed of several evolutionarily conserved aromatic residues forming binding grooves for typical (PxxP) and atypical (PxxxPR, RxxK, RKxxY) binding motifs. The calcium/calmodulin-dependent serine protein kinase (CASK)-interacting protein 1, or Caskin1, a multidomain scaffold protein regulating the cortical actin filaments, is enriched in neural synapses in mammals. Based on its known interaction partners and knock-out animal studies, Caskin1 may play various roles in neural function and it is thought to participate in several pathological processes of the brain. Caskin1 has a single, atypical SH3 domain in which key aromatic residues are missing from the canonical binding groove. No protein interacting partner for this SH3 domain has been identified yet. Nevertheless, we have recently demonstrated the specific binding of this SH3 domain to the signaling lipid mediator lysophospatidic acid (LPA) in vitro. Here we report the solution NMR structure of the human Caskin1 SH3 domain and analyze its structural features in comparison with other SH3 domains exemplifying different strategies in target selectivity. The key differences revealed by our structural study show that the canonical binding groove found in typical SH3 domains accommodating proline-rich motifs is missing in Caskin1 SH3, most likely excluding a bona fide protein target for the domain. The LPA binding site is distinct from the altered protein binding groove. We conclude that the SH3 domain of Caskin1 might mediate the association of Caskin1 with membrane surfaces with locally elevated LPA content.

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A conserved proline-rich region of the Saccharomyces cerevisiae cyclase-associated protein binds SH3 domains and modulates cytoskeletal localization.

Molecular and Cellular Biology ◽

10.1128/mcb.16.2.548 ◽

1996 ◽

Vol 16 (2) ◽

pp. 548-556 ◽

Cited By ~ 90

Author(s):

N L Freeman ◽

T Lila ◽

K A Mintzer ◽

Z Chen ◽

A J Pahk ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Adenylyl Cyclase ◽

Sh3 Domain ◽

Regulatory Function ◽

Nucleotide Exchange ◽

Cortical Actin ◽

Rich Region ◽

Sh3 Domains ◽

Proline Rich Region

Saccharomyces cerevisiae cyclase-associated protein (CAP or Srv2p) is multifunctional. The N-terminal third of CAP binds to adenylyl cyclase and has been implicated in adenylyl cyclase activation in vivo. The widely conserved C-terminal domain of CAP binds to monomeric actin and serves an important cytoskeletal regulatory function in vivo. In addition, all CAP homologs contain a centrally located proline-rich region which has no previously identified function. Recently, SH3 (Src homology 3) domains were shown to bind to proline-rich regions of proteins. Here we report that the proline-rich region of CAP is recognized by the SH3 domains of several proteins, including the yeast actin-associated protein Abp1p. Immunolocalization experiments demonstrate that CAP colocalizes with cortical actin-containing structures in vivo and that a region of CAP containing the SH3 domain binding site is required for this localization. We also demonstrate that the SH3 domain of yeast Abp1p and that of the yeast RAS protein guanine nucleotide exchange factor Cdc25p complex with adenylyl cyclase in vitro. Interestingly, the binding of the Cdc25p SH3 domain is not mediated by CAP and therefore may involve direct binding to adenylyl cyclase or to an unidentified protein which complexes with adenylyl cyclase. We also found that CAP homologous from Schizosaccharomyces pombe and humans bind SH3 domains. The human protein binds most strongly to the SH3 domain from the abl proto-oncogene. These observations identify CAP as an SH3 domain-binding protein and suggest that CAP mediates interactions between SH3 domain proteins and monomeric actin.

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Evidence for physical and functional interactions among two Saccharomyces cerevisiae SH3 domain proteins, an adenylyl cyclase-associated protein and the actin cytoskeleton.

Molecular Biology of the Cell ◽

10.1091/mbc.8.2.367 ◽

1997 ◽

Vol 8 (2) ◽

pp. 367-385 ◽

Cited By ~ 115

Author(s):

T Lila ◽

D G Drubin

Keyword(s):

Saccharomyces Cerevisiae ◽

Actin Cytoskeleton ◽

Adenylyl Cyclase ◽

Sh3 Domain ◽

Actin Binding ◽

Cortical Actin ◽

Sh3 Domains ◽

Functional Interactions ◽

Independent Functions

In a variety of organisms, a number of proteins associated with the cortical actin cytoskeleton contain SH3 domains, suggesting that these domains may provide the physical basis for functional interactions among structural and regulatory proteins in the actin cytoskeleton. We present evidence that SH3 domains mediate at least two independent functions of the Saccharomyces cerevisiae actin-binding protein Abp1p in vivo. Abp1p contains a single SH3 domain that has recently been shown to bind in vitro to the adenylyl cyclase-associated protein Srv2p. Immunofluorescence analysis of Srv2p subcellular localization in strains carrying mutations in either ABP1 or SRV2 reveals that the Abp1p SH3 domain mediates the normal association of Srv2p with the cortical actin cytoskeleton. We also show that a site in Abp1p itself is specifically bound by the SH3 domain of the actin-associated protein Rvs167p. Genetic analysis provides evidence that Abp1p and Rvs167p have functions that are closely interrelated. Abp1 null mutations, like rvs167 mutations, result in defects in sporulation and reduced viability under certain suboptimal growth conditions. In addition, mutations in ABP1 and RVS167 yield similar profiles of genetic "synthetic lethal" interactions when combined with mutations in genes encoding other cytoskeletal components. Mutations which specifically disrupt the SH3 domain-mediated interaction between Abp1p and Srv2p, however, show none of the shared phenotypes of abp1 and rvs167 mutations. We conclude that the Abp1p SH3 domain mediates the association of Srv2p with the cortical actin cytoskeleton, and that Abp1p performs a distinct function that is likely to involve binding by the Rvs167p SH3 domain. Overall, work presented here illustrates how SH3 domains can integrate the activities of multiple actin cytoskeleton proteins in response to varying environmental conditions.

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Solution NMR structure and ligand identification of human Gas7 SH3 domain reveal a typical SH3 fold but a non-canonical ligand-binding mode

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2019.07.004 ◽

2019 ◽

Vol 516 (4) ◽

pp. 1190-1195

Author(s):

Yao Nie ◽

Jiang Zhu ◽

Theresa A. Ramelot ◽

Michael A. Kennedy ◽

Maili Liu ◽

...

Keyword(s):

Ligand Binding ◽

Binding Mode ◽

Sh3 Domain ◽

Nmr Structure ◽

Solution Nmr ◽

Ligand Identification

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Solution NMR structure of the SH3 domain of human nephrocystin and analysis of a mutation-causing juvenile nephronophthisis

Proteins Structure Function and Bioinformatics ◽

10.1002/prot.20344 ◽

2005 ◽

Vol 59 (2) ◽

pp. 347-355 ◽

Cited By ~ 6

Author(s):

Albane le Maire ◽

Thomas Weber ◽

Sophie Saunier ◽

Isabelle Broutin ◽

Corinne Antignac ◽

...

Keyword(s):

Sh3 Domain ◽

Nmr Structure ◽

Solution Nmr ◽

Juvenile Nephronophthisis

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Faculty Opinions recommendation of RNA helical packing in solution: NMR structure of a 30 kDa GAAA tetraloop-receptor complex.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1028364.337169 ◽

2005 ◽

Author(s):

Eric Westhof

Keyword(s):

Nmr Structure ◽

Solution Nmr ◽

Receptor Complex ◽

Helical Packing

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Expression and Solution NMR Study of Multi-site Phosphomimetic Mutant BCL-2 Protein

Protein and Peptide Letters ◽

10.2174/0929866526666190327121225 ◽

2019 ◽

Vol 26 (6) ◽

pp. 449-457

Author(s):

Ting Song ◽

Keke Cao ◽

Yu dan Fan ◽

Zhichao Zhang ◽

Zong W. Guo ◽

...

Keyword(s):

Structural Change ◽

Structural Biology ◽

Quantum Coherence ◽

Structural Changes ◽

Solution Nmr ◽

Flexible Loop ◽

Loop Region ◽

Loop Domain ◽

Nmr Study ◽

Binding Groove

Background: The significance of multi-site phosphorylation of BCL-2 protein in the flexible loop domain remains controversial, in part due to the lack of structural biology studies of phosphorylated BCL-2. Objective: The purpose of the study is to explore the phosphorylation induced structural changes of BCL-2 protein. Methods: We constructed a phosphomietic mutant BCL-2(62-206) (t69e, s70e and s87e) (EEEBCL- 2-EK (62-206)), in which the BH4 domain and the part of loop region was truncated (residues 2-61) to enable a backbone resonance assignment. The phosphorylation-induced structural change was visualized by overlapping a well dispersed 15N-1H heteronuclear single quantum coherence (HSQC) NMR spectroscopy between EEE-BCL-2-EK (62-206) and BCL-2. Results: The EEE-BCL-2-EK (62-206) protein reproduced the biochemical and cellular activity of the native phosphorylated BCL-2 (pBCL-2), which was distinct from non-phosphorylated BCL-2 (npBCL-2) protein. Some residues in BH3 binding groove occurred chemical shift in the EEEBCL- 2-EK (62-206) spectrum, indicating that the phosphorylation in the loop region induces a structural change of active site. Conclusion: The phosphorylation of BCL-2 induced structural change in BH3 binding groove.

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Solution NMR structure of titin N2A region Ig domain I83 and its interaction with metal ions

Journal of Molecular Biology ◽

10.1016/j.jmb.2021.166977 ◽

2021 ◽

pp. 166977

Author(s):

Colleen Kelly ◽

Nicola Pace ◽

Matthew Gage ◽

Mark Pfuhl

Keyword(s):

Metal Ions ◽

Nmr Structure ◽

Solution Nmr ◽

Ig Domain

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Solution NMR Structure of the Ca2+-bound N-terminal Domain of CaBP7

Journal of Biological Chemistry ◽

10.1074/jbc.m112.402289 ◽

2012 ◽

Vol 287 (45) ◽

pp. 38231-38243 ◽

Cited By ~ 6

Author(s):

Hannah V. McCue ◽

Pryank Patel ◽

Andrew P. Herbert ◽

Lu-Yun Lian ◽

Robert D. Burgoyne ◽

...

Keyword(s):

Nmr Structure ◽

Solution Nmr ◽

Terminal Domain

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NMR structure of the C-terminal domain of TonB protein from Pseudomonas aeruginosa

PeerJ ◽

10.7717/peerj.5412 ◽

2018 ◽

Vol 6 ◽

pp. e5412 ◽

Cited By ~ 4

Author(s):

Jesper S. Oeemig ◽

O.H. Samuli Ollila ◽

Hideo Iwaï

Keyword(s):

Pseudomonas Aeruginosa ◽

Structural Changes ◽

Nmr Structure ◽

Dynamics Simulation ◽

Solution Nmr ◽

Gram Negative Bacteria ◽

E Coli ◽

Terminal Domain ◽

Tonb Protein ◽

Monomeric Structure

The TonB protein plays an essential role in the energy transduction system to drive active transport across the outer membrane (OM) using the proton-motive force of the cytoplasmic membrane of Gram-negative bacteria. The C-terminal domain (CTD) of TonB protein is known to interact with the conserved TonB box motif of TonB-dependent OM transporters, which likely induces structural changes in the OM transporters. Several distinct conformations of differently dissected CTDs of Escherichia coli TonB have been previously reported. Here we determined the solution NMR structure of a 96-residue fragment of Pseudomonas aeruginosa TonB (PaTonB-96). The structure shows a monomeric structure with the flexible C-terminal region (residues 338–342), different from the NMR structure of E. coli TonB (EcTonB-137). The extended and flexible C-terminal residues are confirmed by 15N relaxation analysis and molecular dynamics simulation. We created models for the PaTonB-96/TonB box interaction and propose that the internal fluctuations of PaTonB-96 makes it more accessible for the interactions with the TonB box and possibly plays a role in disrupting the plug domain of the TonB-dependent OM transporters.

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