Identification of the ferredoxin interaction sites on ferredoxin-dependent glutamate synthase from Synechocystis sp. PCC 6803.

Based on in silico docking methods, five amino acids in glutamate synthase (Gln-467, His-1144, Asn-1147, Arg-1162, and Trp-676) likely constitute key binding residues in the interface of a glutamate synthase:ferredoxin complex. Although all interfacial mutants studied showed the ability to form a complex under low ionic strength, these docking mutations showed significantly less ferredoxin-dependent activities, while still retaining enzymatic activity. Furthermore, isothermal titration calorimetry showed a possible 1:2 molar ratio between the wild-type glutamate synthase and ferredoxin. However, each of our interfacial mutants showed only a 1:1 complex with ferredoxin, suggesting that the mutations directly affect the glutamate synthase:ferredoxin heterodimer interface.

Nucleotide dependence of the dimerization of ATP binding cassette nucleotide binding domains.

ATP-binding cassette proteins are ubiquitously present throughout all known genomes. Their basic functional unit possesses two transmembrane domains and two nucleotide-binding domains. The nucleotide-binding domains are responsible for ATP binding and hydrolysis, and their 3-dimensional structure is conserved across ATP-binding cassette proteins. Binding of ATP produces nucleotide-binding domain dimerization, a step necessary for hydrolysis. However, the possibility that nucleotide-binding domains bind and/or hydrolyze nucleotide triphosphates different from ATP has not been explored in detail. Here, we studied that possibility using M. jannaschii MJ0796, a prototypical ATP-binding cassette nucleotide-binding domain. We found that nucleotide-binding domain dimerization occurs as a result of binding to the natural nucleotide triphosphates ATP, GTP, CTP and UTP, and also to the analog ATP-γ-S. All the natural nucleotide triphosphates are hydrolyzed at similar rates, whereas ATP-γ-S is not hydrolyzed. We also found that the non-hydrolyzable ATP analog AMP-PNP, frequently assumed to produce the nucleotide-bound conformation, failed to elicit nucleotide-binding domain dimerization. Our results raise the possibility that not all the nucleotide binding sites of nucleotide-binding domains are occupied by ATP under physiological conditions, and that ATP is not always the nucleotide hydrolyzed to dissociate the nucleotide-binding domain dimers.

Nanodysferlins support membrane repair and binding to TRIM72/MG53 but do not localize to t-tubules or stabilize Ca2+ signaling.

Mutations in the DYSF gene, encoding the protein dysferlin, lead to several forms of muscular dystrophy. In healthy skeletal muscle, dysferlin concentrates in the transverse tubules and is involved in repairing the sarcolemma and stabilizing Ca2+ signaling after membrane disruption. The DYSF gene encodes 7-8 C2 domains, several Fer and Dysf domains, and a C-terminal transmembrane sequence. Because its coding sequence is too large to package in adeno-associated virus, the full-length sequence is not amenable to current gene delivery methods. Thus, we have examined smaller versions of dysferlin, termed "nanodysferlins," designed to eliminate several C2 domains, specifically C2 domains D, E, and F; B, D, and E; and B, D, E, and F. We also generated a variant by replacing eight amino acids in C2G in the nanodysferlin missing domains D through F. We electroporated dysferlin-null A/J mouse myofibers with Venus fusion constructs of these variants, or as untagged nanodysferlins together with GFP, to mark transfected fibers We found that, although these nanodysferlins failed to concentrate in transverse tubules, three of them supported membrane repair after laser wounding while all four bound the membrane repair protein, TRIM72/MG53, similar to WT dysferlin. By contrast, they failed to suppress Ca2+ waves after myofibers were injured by mild hypoosmotic shock. Our results suggest that the internal C2 domains of dysferlin are required for normal t-tubule localization and Ca2+ signaling and that membrane repair does not require these C2 domains.

Kinetics of the association/dissociation cycle of an ATP-binding cassette nucleotide-binding domain.

Most ATP binding cassette (ABC) proteins are pumps that transport substrates across biological membranes using the energy of ATP hydrolysis. Functional ABC proteins have two nucleotide-binding domains (NBDs) that bind and hydrolyze ATP, but the molecular mechanism of nucleotide hydrolysis is unresolved. This is due in part to the limited kinetic information on NBD association and dissociation. Here, we show dimerization of a catalytically active NBD and follow in real time the association and dissociation of NBDs from the changes in fluorescence emission of a tryptophan strategically located at the center of the dimer interface. Spectroscopic and structural studies demonstrated that the tryptophan can be used as dimerization probe, and we showed that under hydrolysis conditions (millimolar MgATP), not only the dimer dissociation rate increases, but also the dimerization rate. Neither dimer formation or dissociation are clearly favored, and the end result is a dynamic equilibrium where the concentrations of monomer and dimer are very similar. We proposed that based on their variable rates of hydrolysis, the rate-limiting step of the hydrolysis cycle may differ among full-length ABC proteins.

Whole-genome sequencing identifies missense mutation in GRM6 as the likely cause of congenital stationary night blindness in a Tennessee Walking Horse.

BACKGROUND: The only known genetic cause of congenital stationary night blindness (CSNB) in horses is a 1378 bp insertion in TRPM1. However, an affected Tennessee Walking Horse was found to have no copies of this variant. OBJECTIVES: To identify the genetic cause for CSNB in an affected Tennessee Walking Horse. STUDY DESIGN: Case report detailing a whole-genome sequencing (WGS) approach to identify a causal variant. METHODS: A complete ophthalmic exam, including an electroretinogram (ERG), was performed on suspected CSNB-affected horse. WGS data were generated from the case and compared with data from seven other breeds (n = 29). One hundred candidate genes were evaluated for coding variants homozygous in the case and absent in all other horses. Protein modelling was used to assess the functional effects of the identified variant. A random cohort of 90 unrelated Tennessee Walking Horses and 273 horses from additional breeds were screened to estimate allele frequency of the GRM6 variant. RESULTS: ERG results were consistent with CSNB. WGS analysis identified a missense mutation in metabotropic glutamate receptor 6 (GRM6) (c.533C>T p.Thr178Met). This single nucleotide polymorphism (SNP) is predicted to be deleterious and protein modelling supports impaired binding of the neurotransmitter glutamate. This variant was not detected in 273 horses from three additional breeds. The estimated allele frequency in Tennessee Walking Horses is 10%. MAIN LIMITATIONS: Limited phenotype information for controls and no additional cases with which to replicate this finding. CONCLUSIONS: We identified a likely causal recessive missense variant in GRM6. Based on protein modelling, this variant alters GRM6 binding, and thus signalling from the retinal rod cell to the ON-bipolar cell, impairing vision in low light conditions. Given the 10% population allele frequency, it is likely that additional affected horses exist in this breed and further work is needed to identify and examine these animals.

Enzymatic cleavage of myoferlin releases a dual C2-domain module linked to ERK signalling.

Myoferlin and dysferlin are closely related members of the ferlin family of Ca2+-regulated vesicle fusion proteins. Dysferlin is proposed to play a role in Ca2+-triggered vesicle fusion during membrane repair. Myoferlin regulates endocytosis, recycling of growth factor receptors and adhesion proteins, and is linked to the metastatic potential of cancer cells. Our previous studies establish that dysferlin is cleaved by calpains during membrane injury, with the cleavage motif encoded by alternately-spliced exon 40a. Herein we describe the cleavage of myoferlin, yielding a membrane-associated dual C2 domain 'mini-myoferlin'. Myoferlin bears two enzymatic cleavage sites: a canonical cleavage site encoded by exon 38 within the C2DE domain; and a second cleavage site in the linker adjacent to C2DE, encoded by alternately-spliced exon 38a, homologous to dysferlin exon 40a. Both myoferlin cleavage sites, when introduced into dysferlin, can functionally substitute for exon 40a to confer Ca2+-triggered calpain cleavage in response to membrane injury. However, enzymatic cleavage of myoferlin is complex, showing both constitutive or Ca2+-enhanced cleavage in different cell lines, that is not solely dependent on calpains-1 or -2. The functional impact of myoferlin cleavage was explored through signalling protein phospho-protein arrays revealing specific activation of ERK1/2 by ectopic expression of cleavable myoferlin, but not an uncleavable isoform. In summary, we molecularly define two enzymatic cleavage sites within myoferlin and demonstrate 'mini-myoferlin' can be detected in human breast cancer tumour samples and cell lines. These data further illustrate that enzymatic cleavage of ferlins is an evolutionarily preserved mechanism to release functionally specialized mini-modules.

Crystallization and preliminary X-ray diffraction of the ZO-binding domain of human occludin.

Occludin is a tight-junction protein controlling the integrity of endothelial and epithelial cell layers. It forms complexes with the cytoplasmic proteins ZO-1, ZO-2 and ZO-3. The ZO-binding domain in the C-terminal cytoplasmic region of human occludin has previously been isolated and identified. This domain, as expressed in a bacterial system or isolated from native cellular occludin, maintains its ability to bind ZO-1 and ZO-2. The crystallization conditions of the human ZO-binding domain are reported here. The crystals diffract to 2.3 A resolution and were shown to belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 33.3, b = 35.4, c = 107.3 A.

Utility of Synechocystis sp. PCC 6803 glutaredoxin A as a platform to study high-resolution mutagenesis of proteins.

Glutaredoxin from the cyanobacterium Synechocystis sp. PCC 6803 is a small protein, containing only 88 amino acids, that participates in a large number of redox reactions, serving both as an electron donor for enzyme-catalyzed reductions and as a regulator of diverse metabolic pathways. The crystal structures of glutaredoxins from several species have been solved, including the glutaredoxin A isoform from the cyanobacterium Synechocystis sp. PCC 6803. We have utilized the small size of Synechocystis glutaredoxin A and its propensity to form protein crystals that diffract to high resolution to explore a long-standing question in biochemistry; i.e., what are the effects of mutations on protein structure and function? Taking advantage of these properties, we have initiated a long-term educational project that would examine the structural and biochemical changes in glutaredoxin as a function of single-point mutational replacements. Here, we report some of the mutational effects that we have observed to date.

A pyrene maleimide with a flexible linker for sampling of longer inter-thiol distances by excimer formation.

Pyrene-containing compounds are commonly used in a number of fluorescence-based applications because they can form excited-state dimers (excimers) by stacking interaction between excited-state and ground-state monomers. Their usefulness arises from the facts that excimer formation requires close proximity between the pyrenes and that the excimer emission spectrum is very different from that of the monomers. One of many applications is to assess proximity between specific sites of macromolecules labeled with pyrenes. This has been done using pyrene maleimide, a reagent that reacts with reduced thiols of cysteines, but its use for structural studies of proteins has been rather limited. This is because the introduction of two cysteines at sufficiently close distance from each other to obtain excimer fluorescence upon labeling with pyrene maleimide requires detailed knowledge of the protein structure or extensive site-directed mutagenesis trials. We synthesized and tested a new compound with a 4-carbon methylene linker placed between the maleimide and the pyrene (pyrene-4-maleimide), with the aim of increasing the sampling distance for excimer formation and making the use of excimer fluorescence simpler and more widespread. We tested the new compound on thiol-modified oligonucleotides and showed that it can detect proximity between thiols beyond the reach of pyrene maleimide. Based on its spectroscopic and chemical properties, we suggest that pyrene-4-maleimide is an excellent probe to assess proximities between cysteines in proteins and thiols in other macromolecules, as well as to follow conformational changes.