Allosteric regulators selectively prevent Ca2+-feedback of CaV and NaV channels

Calmodulin (CaM) serves as a pervasive regulatory subunit of CaV1, CaV2, and NaV1 channels, exploiting a functionally conserved carboxy-tail element to afford dynamic Ca2+-feedback of cellular excitability in neurons and cardiomyocytes. Yet this modularity counters functional adaptability, as global changes in ambient CaM indiscriminately alter its targets. Here, we demonstrate that two structurally unrelated proteins, SH3 and cysteine-rich domain (stac) and fibroblast growth factor homologous factors (fhf) selectively diminish Ca2+/CaM-regulation of CaV1 and NaV1 families, respectively. The two proteins operate on allosteric sites within upstream portions of respective channel carboxy-tails, distinct from the CaM-binding interface. Generalizing this mechanism, insertion of a short RxxK binding motif into CaV1.3 carboxy-tail confers synthetic switching of CaM regulation by Mona SH3 domain. Overall, our findings identify a general class of auxiliary proteins that modify Ca2+/CaM signaling to individual targets allowing spatial and temporal orchestration of feedback, and outline strategies for engineering Ca2+/CaM signaling to individual targets.

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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.

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Sprouty proteins: modified modulators, matchmakers or missing links?

Journal of Endocrinology ◽

10.1677/joe-09-0110 ◽

2009 ◽

Vol 203 (2) ◽

pp. 191-202 ◽

Cited By ~ 86

Author(s):

G R Guy ◽

R A Jackson ◽

P Yusoff ◽

S Y Chow

Keyword(s):

Protein Phosphatase ◽

Binding Motif ◽

Main Question ◽

Interaction Domain ◽

E3 Ligases ◽

Rich Domain ◽

N Terminus ◽

Phosphatase 2A ◽

Resultant Increase ◽

Cysteine Rich Domain

Sprouty proteins are involved in organogenesis, particularly during the branching of endothelial tubes, and existing evidence suggests that Sprouty's point of action lies downstream of receptor signaling to inhibit the activation of the central Ras/Erk pathway. How Sprouty proteins accomplish their inhibitory action and whether they interact with other signaling pathways are significant questions. Sprouty proteins are devoid of any recognizable protein interaction domain, and clues as to how they function have been mainly derived from screening for interacting partners. Conserved across all the Sprouty proteins are three sequences: a Cbl-tyrosine kinase-binding (TKB) binding motif centered on an obligatorily phosphorylated tyrosine (Y55 in Sprouty2), a serine-rich motif (SRM) and a cysteine-rich domain (CRD). With the exception of a handful of proteins that bind to the N-terminus, most of the binding to Sprouty occurs via the CRD, predominantly by serine/threonine kinases that target sites within the SRM on Sprouty. Some of the resultant increase in phosphorylation is opposed by activated protein phosphatase 2A that binds to the N-terminal Cbl-TKB binding motif. Significantly, two ubiquitin E3 ligases also bind to the N-terminus of Sprouty: c-Cbl binds with high affinity to the TKB binding motif and SIAH2 binds constitutively to a different site; both proteins are able to direct the ubiquitination of Sprouty proteins and its destruction. The collective evidence points to Sprouty proteins as being substantially covalently-modified to control its location, stability, association, and destruction. With such stringent control of the Sproutys, the main question is what key proteins does this facilitator bring together?

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The Evolution of the Scavenger Receptor Cysteine-Rich Domain of the Class A Scavenger Receptors

Frontiers in Immunology ◽

10.3389/fimmu.2015.00342 ◽

2015 ◽

Vol 6 ◽

Cited By ~ 10

Author(s):

Nicholas V. L. Yap ◽

Fiona J. Whelan ◽

Dawn M. E. Bowdish ◽

G. Brian Golding

Keyword(s):

Scavenger Receptor ◽

Scavenger Receptors ◽

Class A ◽

Rich Domain ◽

Cysteine Rich Domain

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The Combined Human Genotype of Truncating TTN and RBM20 Mutations Is Associated with Severe and Early Onset of Dilated Cardiomyopathy

Genes ◽

10.3390/genes12060883 ◽

2021 ◽

Vol 12 (6) ◽

pp. 883

Author(s):

Anna Gaertner ◽

Julia Bloebaum ◽

Andreas Brodehl ◽

Baerbel Klauke ◽

Katharina Sielemann ◽

...

Keyword(s):

Heart Failure ◽

Dilated Cardiomyopathy ◽

Early Onset ◽

Target Genes ◽

Frameshift Mutation ◽

Rna Binding ◽

Splicing Factor ◽

Binding Motif ◽

Rich Domain ◽

Generation Sequencing

A major cause of heart failure is cardiomyopathies, with dilated cardiomyopathy (DCM) as the most common form. Over 40 genes are linked to DCM, among them TTN and RBM20. Next Generation Sequencing in clinical DCM cohorts revealed truncating variants in TTN (TTNtv), accounting for up to 25% of familial DCM cases. Mutations in the cardiac splicing factor RNA binding motif protein 20 (RBM20) are also known to be associated with severe cardiomyopathies. TTN is one of the major RBM20 splicing targets. Most of the pathogenic RBM20 mutations are localized in the highly conserved arginine serine rich domain (RS), leading to a cytoplasmic mislocalization of mutant RBM20. Here, we present a patient with an early onset DCM carrying a combination of (likely) pathogenic TTN and RBM20 mutations. We show that the splicing of RBM20 target genes is affected in the mutation carrier. Furthermore, we reveal RBM20 haploinsufficiency presumably caused by the frameshift mutation in RBM20.

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Studies on the structure and binding properties of the cysteine-rich domain of rat low affinity nerve growth factor receptor (p75NGFR).

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)42451-9 ◽

1992 ◽

Vol 267 (12) ◽

pp. 8352-8359

Author(s):

A.N. Baldwin ◽

C.M. Bitler ◽

A.A. Welcher ◽

E.M. Shooter

Keyword(s):

Nerve Growth Factor ◽

Growth Factor ◽

Growth Factor Receptor ◽

Nerve Growth Factor Receptor ◽

Binding Properties ◽

Nerve Growth ◽

Rich Domain ◽

Cysteine Rich Domain

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The unusual structure of the PiggyMac cysteine-rich domain reveals zinc finger diversity in PiggyBac-related transposases

Mobile DNA ◽

10.1186/s13100-021-00240-4 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Marc Guérineau ◽

Luiza Bessa ◽

Séverine Moriau ◽

Ewen Lescop ◽

François Bontems ◽

...

Keyword(s):

Zinc Finger ◽

Dna Cleavage ◽

Trichoplusia Ni ◽

Catalytic Domain ◽

Structural Diversity ◽

Paramecium Tetraurelia ◽

Lysine Methylation ◽

Unusual Structure ◽

Rich Domain ◽

Cysteine Rich Domain

Abstract Background Transposons are mobile genetic elements that colonize genomes and drive their plasticity in all organisms. DNA transposon-encoded transposases bind to the ends of their cognate transposons and catalyze their movement. In some cases, exaptation of transposon genes has allowed novel cellular functions to emerge. The PiggyMac (Pgm) endonuclease of the ciliate Paramecium tetraurelia is a domesticated transposase from the PiggyBac family. It carries a core catalytic domain typical of PiggyBac-related transposases and a short cysteine-rich domain (CRD), flanked by N- and C-terminal extensions. During sexual processes Pgm catalyzes programmed genome rearrangements (PGR) that eliminate ~ 30% of germline DNA from the somatic genome at each generation. How Pgm recognizes its DNA cleavage sites in chromatin is unclear and the structure-function relationships of its different domains have remained elusive. Results We provide insight into Pgm structure by determining the fold adopted by its CRD, an essential domain required for PGR. Using Nuclear Magnetic Resonance, we show that the Pgm CRD binds two Zn2+ ions and forms an unusual binuclear cross-brace zinc finger, with a circularly permutated treble-clef fold flanked by two flexible arms. The Pgm CRD structure clearly differs from that of several other PiggyBac-related transposases, among which is the well-studied PB transposase from Trichoplusia ni. Instead, the arrangement of cysteines and histidines in the primary sequence of the Pgm CRD resembles that of active transposases from piggyBac-like elements found in other species and of human PiggyBac-derived domesticated transposases. We show that, unlike the PB CRD, the Pgm CRD does not bind DNA. Instead, it interacts weakly with the N-terminus of histone H3, whatever its lysine methylation state. Conclusions The present study points to the structural diversity of the CRD among transposases from the PiggyBac family and their domesticated derivatives, and highlights the diverse interactions this domain may establish with chromatin, from sequence-specific DNA binding to contacts with histone tails. Our data suggest that the Pgm CRD fold, whose unusual arrangement of cysteines and histidines is found in all PiggyBac-related domesticated transposases from Paramecium and Tetrahymena, was already present in the ancestral active transposase that gave rise to ciliate domesticated proteins.

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