Frizzled BRET sensors based on bioorthogonal labeling of unnatural amino acids reveal WNT-induced dynamics of the cysteine-rich domain

Frizzleds (FZD1-10) comprise a class of G protein-coupled receptors containing an extracellular cysteine-rich domain (CRD) that binds lipoglycoproteins of the Wingless/Int-1 family (WNTs). Despite the prominent role of the WNT/FZD system in health and disease, our understanding of how WNT binding to the FZD CRD is translated into receptor activation and transmembrane signaling remains limited. Current hypotheses dispute the roles for conformational dynamics and the involvement of the linker domain connecting the CRD with the seven-helical transmembrane core of FZD. To clarify the mechanism of WNT binding to FZD and to elucidate how WNT/FZD complexes achieve signaling pathway specificity, we devised conformational FZD-CRD biosensors based on bioluminescence-resonance-energy-transfer (BRET). Using FZD engineered with N-terminal nanoluciferase and fluorescently-labeled unnatural amino acids in the linker domain and extracellular loop 3, we show that WNT-3A and WNT-5A induce similar CRD conformational rearrangements despite promoting distinct downstream signaling pathways, and that CRD dynamics are not required for WNT/β-catenin signaling. Thus, the novel FZD-CRD biosensors we report provide insights into the stepwise binding, activation and signaling processes in FZDs. The sensor design is broadly applicable to explore fundamental events in signal transduction mediated by other membrane receptors.

Download Full-text

Faculty Opinions recommendation of Cellular incorporation of unnatural amino acids and bioorthogonal labeling of proteins.

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

10.3410/f.725901313.793522612 ◽

2016 ◽

Author(s):

Morten Meldal ◽

Sanne Schoffelen

Keyword(s):

Amino Acids ◽

Unnatural Amino Acids ◽

Bioorthogonal Labeling ◽

Cellular Incorporation

Download Full-text

The Linker Domain of SNAP25 Acts as a Flexible Molecular Spacer to Ensure Efficient S-Acylation

10.1101/2020.01.21.914333 ◽

2020 ◽

Author(s):

Christine Salaun ◽

Jennifer Greaves ◽

Nicholas C.O. Tomkinson ◽

Luke H. Chamberlain

Keyword(s):

Amino Acids ◽

Binding Motif ◽

Linear Motif ◽

Linker Region ◽

Rich Domain ◽

Enzyme Substrate ◽

Efficient Coupling ◽

Affinity Interaction ◽

Linker Domain ◽

Cysteine Rich Domain

ABSTRACTS-Acylation of the SNARE protein SNAP25 is mediated by a subset of Golgi zDHHC enzymes, in particular zDHHC17. The ankyrin repeat (ANK) domain of this enzyme interacts with a short linear motif known as the zDHHC ANK binding motif (zDABM) in SNAP25 (112-VVASQP-117), which is downstream of the S-acylated cysteine-rich domain (85-CGLCVCPC-92). In this study, we have investigated the importance of the flexible linker (amino acids 93-111; referred to as the “mini-linker” region) that separates the zDABM and S-acylated cysteines. Shortening the mini-linker had no effect of zDHHC17 interaction but blocked S-acylation. Insertion of additional flexible glycine-serine repeats had no effect on S-acylation, whereas extended and rigid alanine-proline repeats perturbed this process. Indeed, a SNAP25 mutant in which the mini-linker region was substituted with a flexible glycine-serine linker of the same length underwent efficient S-acylation. Furthermore, this mutant displayed the same intracellular localisation as wild-type SNAP25, showing that the sequence of the mini-linker is not important in this context. By using the results of previous peptide array experiments, we generated a SNAP25 mutant predicted to have a higher affinity zDABM, and this mutant showed enhanced interaction with zDHHC17 in cells. Interestingly, this mutant was S-acylated with reduced efficiency, implying that a lower affinity interaction of the SNAP25 zDABM with zDHHC17 is optimal for S-acylation efficiency. Overall, the results of this study show that amino acids 93-111 in SNAP25 act as a flexible molecular spacer to ensure efficient coupling of enzyme-substrate interaction and S-acylation.

Download Full-text

Differential Utilization of CD134 as a Functional Receptor by Diverse Strains of Feline Immunodeficiency Virus

Journal of Virology ◽

10.1128/jvi.80.7.3386-3394.2006 ◽

2006 ◽

Vol 80 (7) ◽

pp. 3386-3394 ◽

Cited By ~ 35

Author(s):

Brian J. Willett ◽

Elizabeth L. McMonagle ◽

Susan Ridha ◽

Margaret J. Hosie

Keyword(s):

Amino Acids ◽

Disease Progression ◽

Feline Immunodeficiency Virus ◽

Cell Tropism ◽

Receptor Function ◽

Rich Domain ◽

Immunodeficiency Virus ◽

Primary Determinant ◽

Functional Receptor ◽

Cysteine Rich Domain

ABSTRACT The feline homologue of CD134 (fCD134) is the primary binding receptor for feline immunodeficiency virus (FIV), targeting the virus preferentially to activated CD4+ helper T cells. However, with disease progression, the cell tropism of FIV broadens such that B cells and monocytes/macrophages become significant reservoirs of proviral DNA, suggesting that receptor utilization may alter with disease progression. We examined the receptor utilization of diverse strains of FIV and found that all strains tested utilized CD134 as the primary receptor. Using chimeric feline × human CD134 receptors, the primary determinant of receptor function was mapped to the first cysteine-rich domain (CRD1) of fCD134. For the PPR and B2542 strains, the replacement of CDR1 of fCD134 (amino acids 1 to 64) with human CD134 (hCD134) alone was sufficient to confer nearly optimal receptor function. However, evidence of differential utilization of CD134 was revealed, since strains GL8, CPGammer (CPG41), TM2, 0827, and NCSU1 required determinants in the region spanning amino acids 65 to 85, indicating that these strains may require a more stringent interaction for infection to proceed.

Download Full-text

Cellular Incorporation of Unnatural Amino Acids and Bioorthogonal Labeling of Proteins

Chemical Reviews ◽

10.1021/cr400355w ◽

2014 ◽

Vol 114 (9) ◽

pp. 4764-4806 ◽

Cited By ~ 577

Author(s):

Kathrin Lang ◽

Jason W. Chin

Keyword(s):

Amino Acids ◽

Unnatural Amino Acids ◽

Bioorthogonal Labeling ◽

Cellular Incorporation

Download Full-text

Zinc co-ordination by the DHHC cysteine-rich domain of the palmitoyltransferase Swf1

Biochemical Journal ◽

10.1042/bj20121693 ◽

2013 ◽

Vol 454 (3) ◽

pp. 427-435 ◽

Cited By ~ 19

Author(s):

Ayelén González Montoro ◽

Rodrigo Quiroga ◽

Javier Valdez Taubas

Keyword(s):

Amino Acids ◽

Tertiary Structure ◽

Random Mutagenesis ◽

Binding Pocket ◽

Essential Amino Acids ◽

Zinc Binding ◽

Loss Of Function ◽

Post Translational Modification ◽

Rich Domain ◽

Cysteine Rich Domain

S-acylation, commonly known as palmitoylation, is a widespread post-translational modification of proteins that consists of the thioesterification of one or more cysteine residues with fatty acids. This modification is catalysed by a family of PATs (palmitoyltransferases), characterized by the presence of a 50-residue long DHHC-CRD (Asp-His-His-Cys cysteine-rich domain). To gain knowledge on the structure–function relationships of these proteins, we carried out a random-mutagenesis assay designed to uncover essential amino acids in Swf1, the yeast PAT responsible for the palmitoylation of SNARE (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor) proteins. We identified 21 novel loss-of-function mutations, which are mostly localized within the DHHC-CRD. Modelling of the tertiary structure of the Swf1 DHHC domain suggests that it could fold as a zinc-finger domain, co-ordinating two zinc atoms in a CCHC arrangement. All residues predicted to be involved in the co-ordination of zinc were found to be essential for Swf1 function in the screen. Moreover, these mutations result in unstable proteins, in agreement with a structural role for these zinc fingers. The conservation of amino acids predicted to form each zinc-binding pocket suggests a shared function, as the selective pressure to maintain them is lost upon mutation of one of them. A Swf1 orthologue that lacks one of the zinc-binding pockets is able to complement a yeast swf1∆ strain, possibly because a similar fold can be stabilized by hydrogen bonds instead of zinc co-ordination. Finally, we show directly that recombinant Swf1 DHHC-CRD is able to bind zinc. Sequence analyses of DHHC domains allowed us to present models of the zinc-binding properties for all PATs.

Download Full-text