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

2013 ◽  
Vol 454 (3) ◽  
pp. 427-435 ◽  
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.

scholarly journals Involvement of the Zinc-Binding Capacity of Sendai Virus V Protein in Viral Pathogenesis

2000 ◽  
Vol 74 (17) ◽  
pp. 7834-7841 ◽  
Author(s):  
Cheng Huang ◽  
Katsuhiro Kiyotani ◽  
Yutaka Fujii ◽  
Noriko Fukuhara ◽  
Atsushi Kato ◽  
...  
Keyword(s):  
Fusion Proteins ◽  
Sendai Virus ◽  
Binding Capacity ◽  
Viral Pathogenesis ◽  
Growth Patterns ◽  
Zinc Binding ◽  
Wild Type ◽  
V Protein ◽  
Rich Domain ◽  
Cysteine Rich Domain

ABSTRACT The V protein of Sendai virus (SeV) is nonessential to virus replication in cell culture but indispensable to viral pathogenicity in mice. The highly conserved cysteine-rich zinc finger-like domain in its carboxyl terminus is believed to be responsible for this viral pathogenicity. In the present study, we showed that the cysteine-rich domain of the SeV V protein could actually bind zinc by using glutathione-S-transferase fusion proteins. When the seven conserved cysteine residues at positions 337, 341, 353, 355, 358, 362, and 365 were replaced individually, the zinc-binding capacities of the mutant proteins were greatly impaired, ranging from 22 to 68% of that of the wild type. We then recovered two mutant SeVs from cDNA, which have V-C341S and V-C365R mutations and represent maximal and minimal zinc-binding capacities among the corresponding mutant fusion proteins, respectively. The mutant viruses showed viral protein synthesis and growth patterns similar to those of wild-type SeV in cultured cells. However, the mutant viruses were strongly attenuated in mice in a way similar to that of SeV VΔC, which has a truncated V protein lacking the cysteine-rich domain, by exhibiting earlier viral clearance from the mouse lung and less virulence to mice. We therefore conclude that the zinc-binding capacity of the V protein is involved in viral pathogenesis.

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

2021 ◽  
Author(s):  
Maria Kowalski-Jahn ◽  
Hannes Schihada ◽  
Ainoleena Turku ◽  
Thomas Huber ◽  
Thomas P. Sakmar ◽  
...  
Keyword(s):  
Amino Acids ◽  
Unnatural Amino Acids ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Receptor Activation ◽  
Membrane Receptors ◽  
Rich Domain ◽  
Linker Domain ◽  
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.

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

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.

scholarly journals Use of L- arginine in the treatment of comorbid pathology in neurological patients

2017 ◽  
pp. 4-8
Author(s):  
N. Svyrydova ◽  
N. Zhhilova
Keyword(s):  
Amino Acids ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Essential Amino Acids ◽  
The Body ◽  
Special Importance ◽  
Post Translational Modification ◽  
Regulatory Pathways ◽  
Transcriptional Regulatory ◽  
Neurological Patients

Amino acids are the structural chemical units that make up proteins. The special importance of amino acids is that some of them - precursors of neurotransmitters which are necessary for normal functioning of the brain. The absence one of  essential amino acid suspend the formation of protein. Correction of deficiency of essential amino acids and replacement carried out by different drugs. Recently, interest in preparations that contain arginine significantly increased. This interest caused by the importance of L- arginine in the body functioning, as L- Arginine is a precursor of nitric oxide (NO). Arginine (Valagyn) is a common post-translational modification of proteins tsytoplazmatycheskyh that has a unique transcriptional regulatory pathways. L-arginine showed a protective effect in ischemia / reperfusion injury, which can be widely used in various diseases of the cardio-sudinnoyi system and metabolic disorders

scholarly journals Solution Structure of Synthetic Penaeidin-4 with Structural and Functional Comparisons with Penaeidin-3

2005 ◽  
Vol 280 (16) ◽  
pp. 16009-16018 ◽  
Author(s):  
Brandon J. Cuthbertson ◽  
Yinshan Yang ◽  
Evelyne Bachère ◽  
Erika E. Büllesbach ◽  
Paul S. Gross ◽  
...  
Keyword(s):  
Antimicrobial Peptide ◽  
Solution Structure ◽  
Tertiary Structure ◽  
Structural Characteristics ◽  
Target Specificity ◽  
Primary Sequence ◽  
Specific Domain ◽  
Rich Domain ◽  
Isoform Diversity ◽  
Cysteine Rich Domain

Antimicrobial peptide structure has direct implications for the complexity of functions and mechanisms of action. The penaeidin antimicrobial peptide family from shrimp is divided into multiple class designations based on primary structure. The penaeidin classes are not only characterized by variability in primary sequence but also by variation in target specificity and effectiveness. Whereas class 4 exhibits low isoform diversity within species and is highly conserved between species, the primary sequence of penaeidin class 3 is less conserved between species and exhibits considerable isoform diversity within species. All penaeidins, regardless of class or species, are composed of two dramatically different domains: an unconstrained proline-rich domain and a disulfide bond-stabilized cysteine-rich domain. The proline-rich domain varies in length and is generally less conserved, whereas the spacing and specific residue content of the cysteine-rich domain is more conserved. The structure of the synthetic penaeidin class 4 (PEN4-1) fromLitopenaeus setiferuswas analyzed using several approaches, including chemical mapping of disulfide bonds, circular dichroism analysis of secondary structural characteristics, and complete characterization of the solution structure of the peptide by proton NMR.L. setiferusPEN4-1 was then compared with the previously characterized structure of penaeidin class 3 fromLitopenaeus vannamei. Moreover, the specificity of these antimicrobial peptides was examined through direct comparison of activity against a panel of microbes. The penaeidin classes differ in microbial target specificity, which correlates to variability in specific domain sequence. However, the tertiary structure of the cysteine-rich domain and indeed the overall structure of penaeidins are conserved across classes.

Mechanism and function of DHHC S-acyltransferases

2013 ◽  
Vol 41 (1) ◽  
pp. 29-34 ◽  
Author(s):  
Maurine E. Linder ◽  
Benjamin C. Jennings
Keyword(s):  
Fatty Acids ◽  
Protein S ◽  
Enzyme Mechanism ◽  
Long Chain Fatty Acids ◽  
Post Translational Modification ◽  
Rich Domain ◽  
Cytoplasmic Face ◽  
Protein Membrane ◽  
And Function ◽  
Cysteine Rich Domain

Protein S-palmitoylation is a reversible post-translational modification of proteins with fatty acids. In the last 5 years, improved proteomic methods have increased the number of proteins identified as substrates for palmitoylation from tens to hundreds. Palmitoylation regulates protein membrane interactions, activity, trafficking and stability and can be constitutive or regulated by signalling inputs. A family of PATs (protein acyltransferases) is responsible for modifying proteins with palmitate or other long-chain fatty acids on the cytoplasmic face of cellular membranes. PATs share a signature DHHC (Asp-His-His-Cys) cysteine-rich domain that is the catalytic centre of the enzyme. The biomedical importance of members of this family is underscored by their association with intellectual disability, Huntington's disease and cancer in humans, and raises the possibility of DHHC PATs as targets for therapeutic intervention. In the present paper, we discuss recent progress in understanding enzyme mechanism, regulation and substrate specificity.

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

2006 ◽  
Vol 80 (7) ◽  
pp. 3386-3394 ◽  
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.

scholarly journals Cryo-EM structure of the activated RET signaling complex reveals the importance of its cysteine-rich domain

2019 ◽  
Vol 5 (7) ◽  
pp. eaau4202 ◽  
Author(s):  
Janna M. Bigalke ◽  
Shintaro Aibara ◽  
Robert Roth ◽  
Göran Dahl ◽  
Euan Gordon ◽  
...  
Keyword(s):  
Calcium Binding ◽  
Neurological Diseases ◽  
Receptor Activation ◽  
Loss Of Function ◽  
Calcium Binding Site ◽  
Signaling Complex ◽  
Rich Domain ◽  
Extracellular Region ◽  
Domain 3 ◽  
Cysteine Rich Domain

Signaling through the receptor tyrosine kinase RET is essential during normal development. Both gain- and loss-of-function mutations are involved in a variety of diseases, yet the molecular details of receptor activation have remained elusive. We have reconstituted the complete extracellular region of the RET signaling complex together with Neurturin (NRTN) and GFRα2 and determined its structure at 5.7-Å resolution by cryo-EM. The proteins form an assembly through RET-GFRα2 and RET-NRTN interfaces. Two key interaction points required for RET extracellular domain binding were observed: (i) the calcium-binding site in RET that contacts GFRα2 domain 3 and (ii) the RET cysteine-rich domain interaction with NRTN. The structure highlights the importance of the RET cysteine-rich domain and allows proposition of a model to explain how complex formation leads to RET receptor dimerization and its activation. This provides a framework for targeting RET activity and for further exploration of mechanisms underlying neurological diseases.

scholarly journals An integrated approach unravels a crucial structural property for the function of the insect steroidogenic Halloween protein Noppera-bo

2019 ◽  
Author(s):  
Kotaro Koiwai ◽  
Kazue Inaba ◽  
Kana Morohashi ◽  
Sora Enya ◽  
Reina Arai ◽  
...  
Keyword(s):  
Structural Property ◽  
Tertiary Structure ◽  
Structural Characteristics ◽  
Integrated Approach ◽  
Binding Pocket ◽  
Loss Of Function ◽  
Ecdysteroid Biosynthesis ◽  
17Β Estradiol

AbstractEcdysteroids are the principal insect steroid hormones essential for insect development and physiology. In the last 18 years, several enzymes responsible for ecdysteroid biosynthesis, encoded by Halloween genes, have been identified and well characterized, both genetically and biochemically. However, none of these proteins have yet been characterized at the tertiary structure level. Here, we report an integrated in silico, in vitro, and in vivo analyses of the Halloween glutathione S-transferase (GST) protein, Noppera-bo (Nobo). We determine crystal structures of Drosophila melanogaster Nobo (DmNobo) complexed with glutathione and 17β-estradiol, a DmNobo inhibitor. 17β-estradiol almost fully occupied the putative ligand-binding pocket, and a prominent hydrogen bond formed between Asp113 of DmNobo and 17β-estradiol. Asp113 is essential for inhibiting DmNobo enzymatic activity by 17β-estradiol, as 17β-estradiol does not inhibit and physically interacts less with the Asp113Ala DmNobo point mutant. Asp113 is highly conserved among Nobo proteins, but not among other GSTs, implying that Asp113 is important for endogenous Nobo function. Indeed, a homozygous nobo allele possessing the Asp113Ala point mutation exhibits embryonic lethality with undifferentiated cuticle structure, a phenocopy of complete loss-of-function nobo homozygotes. These results suggest that the nobo family of GST proteins has acquired a unique amino acid residue, which seems to be essential for binding an endogenous sterol substrate to regulate ecdysteroid biosynthesis. This is the first study to reveal the structural characteristics of insect steroidogenic Halloween proteins. This study also provides basic insight into applied entomology for developing a new type of insecticides that specifically inhibit ecdysteroid biosynthesis.Significance StatementInsect molting and metamorphosis are drastic and dynamic biological processes and, therefore, have fascinated many scientists. Ecdysteroids represent one class of insect hormones that are indispensable for inducing molting and metamorphosis. It is well known that proteins responsible for catalyzing ecdysteroid biosynthesis reactions are encoded by “Halloween” genes, most of which have names of ghosts and phantoms. However, no studies have focused on the structural properties of these biosynthetic proteins. In this study, we addressed this unsolved issue and successfully unraveled a structural property that is crucial for the function of the fruit fly Halloween protein, Noppera-bo (a Japanese faceless ghost). This is the first study to reveal the structural characteristics of an insect steroidogenic Halloween protein.

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