NMR structure and regulated expression in APL cell of human SH3BGRL3

SH3 domain binding glutamic acid-rich protein like 3 (SH3BGRL3) is the new member of thioredoxin (TRX) super family, whose posttranslational modified form was identified as tumor necrosis factor alpha (TNF-alpha) inhibitory protein, TIP-B1. In this paper, we determined its solution structure by multi-dimensional nuclear magnetic resonance spectroscopy. The overall structure of human SH3BGRL3 conformed to a TRX-like fold. To understand its function in vivo, the upregulated expression in acute promyelocytic leukemia cell line NB4 at both mRNA and protein level was elucidated. Immunofluorescence and immunohistochemistry staining with monoclonal antibody against SH3BGRL3 demonstrated that it was a cytoplasmic protein in both NB4 cell and human tissues. These results, as a whole, indicate that SH3BGRL3 may function as a regulator in all-trans retinoic acid-induced pathway.     

The identification of a novel human homologue of the SH3 binding glutamic acid-rich (SH3BGR) gene establishes a new family of highly conserved small proteins related to Thioredoxin Superfamily

The SH3 binding glutamic acid-rich (SH3BGR) gene was cloned in an effort to identify genes located to human chromosome 21, within the congenital heart disease region, and expressed in the developing heart. After the identification of SH3BGR, two human homologous genes, SH3BGRL and SH3BGRL3, were identified and mapped to chromosome Xq13.3 and 1p34.3-35, respectively. SH3BGRL and SH3BGRL3 code for small proteins similar to the N-terminal region of the SH3BGR protein. SH3BGRL3 protein shows a significant similarity to Glutaredoxin 1 of Escherichia coli, and all the three proteins are predicted to belong to Thioredoxin-like protein Superfamily. Here we describe the identification and characterization of an additional human homologue of SH3BGR, named SH3BGRL2. The SH3BGRL2 gene maps to chromosome 6q13-15 and its messenger RNA has a large 3' untranslated region containing several AUUUA repeats. SH3BGRL2 codes for a protein of 107 amino acids, which, like SH3BGRL and SH3BGRL3 proteins, is highly homologous to the N-terminal region of the SH3BGR protein and appears to be related to Glutaredoxins and to PKC-interacting cousin of thioredoxin homology domain. We propose that the identification of SH3BGRL2 establishes a novel family of human genes, coding for highly conserved small proteins belonging to Thioredoxin-like protein Superfamily.     

A novel human homologue of the SH3BGR gene encodes a small protein similar to Glutaredoxin 1 of Escherichia coli.

Glutaredoxins (GRXs) are ubiquitous GSH-dependent oxidoreductases, which catalyze the reduction of protein-glutathionyl-mixed disulfides and are considered to play an important role in the enzymatic regulation of redox-sensitive proteins. In this paper, we describe the identification and characterization of a new human homologue of the SH3BGR gene, named SH3BGRL3 (SH3 domain binding glutamic acid-rich protein like 3). SH3BGRL3 is widely expressed and codes for a highly conserved small protein, which shows a significant similarity to Glutaredoxin 1 (GRX1) of Escherichia coli and is predicted to belong to the Thioredoxin Superfamily. However, the SH3BGRL3 protein lacks both the conserved cysteine residues, which characterize the enzymatic active site of GRX. This structural feature raises the possibility that SH3BGRL3 could function as an endogenous modulator of GRX biological activity. EGFP-SH3BGRL3 fusion protein expressed in COS-7 cells localizes both to the nucleus and to the cytoplasm. The SH3BGRL3 gene was mapped to chromosome 1p34.3-35.     

Molecular mapping of functionalities in the solution structure of reduced Grx4, a monothiol glutaredoxin from Escherichia coli

The ubiquitous glutaredoxin protein family is present in both prokaryotes and eukaryotes, and is closely related to the thioredoxins, which reduce their substrates using a dithiol mechanism as part of the cellular defense against oxidative stress. Recently identified monothiol glutaredoxins, which must use a different functional mechanism, appear to be essential in both Escherichia coli and yeast and are well conserved in higher order genomes. We have employed high resolution NMR to determine the three-dimensional solution structure of a monothiol glutaredoxin, the reduced E. coli Grx4. The Grx4 structure comprises a glutaredoxin-like alpha-beta fold, founded on a limited set of strictly conserved and structurally critical residues. A tight hydrophobic core, together with a stringent set of secondary structure elements, is thus likely to be present in all monothiol glutaredoxins. A set of exposed and conserved residues form a surface region, implied in glutathione binding from a known structure of E. coli Grx3. The absence of glutaredoxin activity in E. coli Grx4 can be understood based on small but significant differences in the glutathione binding region, and through the lack of a conserved second GSH binding site. MALDI experiments suggest that disulfide formation on glutathionylation is accompanied by significant structural changes, in contrast with dithiol thioredoxins and glutaredoxins, where differences between oxidized and reduced forms are subtle and local. Structural and functional implications are discussed with particular emphasis on identifying common monothiol glutaredoxin properties in substrate specificity and ligand binding events, linking the thioredoxin and glutaredoxin systems.     

Solution structure of N-terminal SH3 domain of Vav and the recognition site for Grb2 C-terminal SH3 domain

The three-dimensional structure of the N-terminal SH3 domain (residues 583–660) of murine Vav, which contains a tetra-proline sequence (Pro 607-Pro 610), was determined by NMR. The solution structure of the SH3 domain shows a typical SH3 fold, but it exists in two conformations due to cis-trans isomerization at the Gly614-Pro615 bond. The NMR structure of the P615G mutant, where Pro615 is replaced by glycine, reveals that the tetra-proline region is inserted into the RT-loop and binds to its own SH3 structure. The C-terminal SH3 domain of Grb2 specifically binds to the trans form of the N-terminal SH3 domain of Vav. The surface of Vav N-terminal SH3 which binds to Grb2 C-terminal SH3 was elucidated by chemical shift mapping experiments using NMR. The surface does not involve the tetra-proline region but involves the region comprising the n-src loop, the N-terminal and the C-terminal regions. This surface is located opposite to the tetra-proline containing region, consistent with that of our previous mutagenesis studies.     

Backbone dynamics of the human MIA protein studied by (15)N NMR relaxation: implications for extended interactions of SH3 domains

The melanoma inhibitory activity (MIA) protein is a clinically valuable marker in patients with malignant melanoma as enhanced values diagnose metastatic melanoma stages III and IV. Here, we report the backbone dynamics of human MIA studied by (15)N NMR relaxation experiments. The folded core of human MIA is found to be rigid, but several loops connecting beta-sheets, such as the RT-loop for example, display increased mobility on picosecond to nanosecond time scales. One of the most important dynamic features is the pronounced flexibility of the distal loop, comprising residues Asp 68 to Ala 75, where motions on time scales up to milliseconds occur. Further, significant exchange contributions are observed for residues of the canonical binding site of SH3 domains including the RT-loop, the n-Src loop, for the loop comprising residues 13 to 19, which we refer to as the"disulfide loop", in part for the distal loop, and the carboxyl terminus of human MIA. The functional importance of this dynamic behavior is discussed with respect to the biological activity of several point mutations of human MIA. The results of this study suggest that the MIA protein and the recently identified highly homologous fibrocyte-derived protein (FDP)/MIA-like (MIAL) constitute a new family of secreted proteins that adopt an SH3 domain-like fold in solution with expanded ligand interactions.     

Effect of pH and salt bridges on structural assembly: molecular structures of the monomer and intertwined dimer of the Eps8 SH3 domain

The SH3 domain of Eps8 was previously found to form an intertwined, domain-swapped dimer. We report here a monomeric structure of the EPS8 SH3 domain obtained from crystals grown at low pH, as well as an improved domain-swapped dimer structure at 1.8 A resolution. In the domain-swapped dimer the asymmetric unit contains two "hybrid-monomers." In the low pH form there are two independently folded SH3 molecules per asymmetric unit. The formation of intermolecular salt bridges is thought to be the reason for the formation of the dimer. On the basis of the monomer SH3 structure, it is argued that Eps8 SH3 should, in principle, bind to peptides containing a PxxP motif. Recently it was reported that Eps8 SH3 binds to a peptide with a PxxDY motif. Because the "SH3 fold" is conserved, alternate binding sites may be possible for the PxxDY motif to bind. The strand exchange or domain swap occurs at the n-src loops because the n-src loops are flexible. The thermal b-factors also indicate the flexible nature of n-src loops and a possible handle for domain swap initiation. Despite the loop swapping, the typical SH3 fold in both forms is conserved structurally. The interface of the acidic form of SH3 is stabilized by a tetragonal network of water molecules above hydrophobic residues. The intertwined dimer interface is stabilized by hydrophobic and aromatic stacking interactions in the core and by hydrophilic interactions on the surface.     

Structure-function analysis of yeast Grx5 monothiol glutaredoxin defines essential amino acids for the function of the protein

Grx5 defines a family of yeast monothiol glutaredoxins that also includes Grx3 and Grx4. All three proteins display significant sequence homology with proteins found from bacteria to humans. Grx5 is involved in iron/sulfur cluster assembly at the mitochondria, but the function of Grx3 and Grx4 is unknown. Three-dimensional modeling based on known dithiol glutaredoxin structures predicted a thioredoxin fold structure for Grx5. Positionally conserved amino acids in this glutaredoxin family were replaced in Grx5, and the effect on the biological function of the protein has been tested. For all changes studied, there was a correlation between the effects on several different phenotypes: sensitivity to oxidants, constitutive protein oxidation, ability for respiratory growth, auxotrophy for a number of amino acids, and iron accumulation. Cys(60) and Gly(61) are essential for Grx5 function, whereas other single or double substitutions in the same region had no phenotypic effects. Gly(115) and Gly(116) could be important for the formation of a glutathione cleft on the Grx5 surface, in contrast to adjacent Cys(117). Substitution of Phe(50) alters the beta-sheet in the thioredoxin fold structure and inhibits Grx5 function. None of the substitutions tested affect the structure at a significant enough level to reduce protein stability.     

Crystal structure of the SH3 domain in human Fyn; comparison of the three-dimensional structures of SH3 domains in tyrosine kinases and spectrin.

The Src-homology 3 (SH3) region is a protein domain consisting of approximately 60 residues. It occurs in a large number of eukaryotic proteins involved in signal transduction, cell polarization and membrane--cytoskeleton interactions. The function is unknown, but it is probably involved in specific protein--protein interactions. Here we report the crystal structure of the SH3 domain of Fyn (a Src family tyrosine kinase) at 1.9 A resolution. The crystals have two SH3 molecules per asymmetric unit. These two Fyn SH3 domains are not related by a local twofold axis. The crystal structures of spectrin and Fyn SH3 domains as well as the solution structure of the Src SH3 domain show that these all have the same basic fold. A protein domain which has the same topology as SH3 is present in the prokaryotic regulatory enzyme BirA. The comparison between the crystal structures of Fyn and spectrin SH3 domains shows that a conserved surface patch, consisting mainly of aromatic residues, is flanked by two hairpin-like loops (residues 94-104 and 114-118 in Fyn). These loops are different in tyrosine kinase and spectrin SH3 domains. They could modulate the binding properties of the aromatic surface.     

A lack of peptide binding and decreased thermostability suggests that the CASKIN2 scaffolding protein SH3 domain may be vestigial

Background

CASKIN2 is a neuronal signaling scaffolding protein comprised of multiple ankyrin repeats, two SAM domains, and one SH3 domain. The CASKIN2 SH3 domain for an NMR structural determination because its peptide-binding cleft appeared to deviate from the repertoire of aromatic enriched amino acids that typically bind polyproline-rich sequences.

Results

The structure demonstrated that two non-canonical basic amino acids (K290/R319) in the binding cleft were accommodated well in the SH3 fold. An K290Y/R319W double mutant restoring the typical aromatic amino acids found in the binding cleft resulted in a 20 °C relative increase in the thermal stability. Considering the reduced stability, we speculated that the CASKIN2 SH3 could be a nonfunctional remnant in this scaffolding protein.

Conclusions

While the NMR structure demonstrates that the CASKIN2 SH3 domain is folded, its cleft has suffered two substitutions that prevent it from binding typical polyproline ligands. This observation led us to additionally survey and describe other SH3 domains in the Protein Data Bank that may have similarly lost their ability to promote protein-protein interactions.

     

Functional characterisation of ganglioside-induced differentiation-associated protein 1 as a glutathione transferase

Mutations in the ganglioside-induced differentiation-associated protein 1 (GDAP1) gene have been linked with Charcot-Marie-Tooth (CMT) disease. This protein, and its paralogue GDAP1L1, appear to be structurally related to the cytosolic glutathione S-transferases (GST) including an N-terminal thioredoxin fold domain with conserved active site residues. The specific function, of GDAP1 remains unknown. To further characterise their structure and function we purified recombinant human GDAP1 and GDAP1L1 proteins using bacterial expression and immobilised metal affinity chromatography. Like other cytosolic GSTs, GDAP1 protein has a dimeric structure. Although the full-length proteins were largely insoluble, the deletion of a proposed C-terminal transmembrane domain allowed the preparation of soluble protein. The purified proteins were assayed for glutathione-dependent activity against a library of 'prototypic' GST substrates. No evidence of glutathione-dependent activity or an ability to bind glutathione immobilised on agarose was found.     

Plasmodium falciparum glutaredoxin-like proteins

Glutaredoxin-like proteins form a new subgroup of glutaredoxins with a serine replacing the second cysteine in the CxxC-motif of the active site. Yeast Grx5 is the only glutaredoxin-like protein studied biochemically so far. We identified and cloned three genes encoding glutaredoxin-like proteins from the malaria parasite Plasmodium falciparum (Pf Glp1, Pf Glp2, and Pf Glp3) containing a conserved cysteine in the CGFS-, CKFS-, and CKYS-motif, respectively. Here, we describe biochemical properties of Pf Glp1 and Pf Glp2. Cys 99, the only cysteine residue in Pf Glp1, has a pK(a) value as low as 5.5 and is able to mediate covalent homodimerization. Monomeric and dimeric Pf Glp1 react with GSSG and GSH, respectively. Pf Glp2 is monomeric and both of its cysteine residues can be glutathionylated. Molecular models reveal a thioredoxin fold for the putative C-terminal domain of Pf Glp1, Pf Glp2, and Pf Glp3, as well as conserved residues presumably required for glutathione binding. However, Pf Glp1 and Pf Glp2 neither possess activity in a classical glutaredoxin assay nor display activity as glutathione peroxidase or glutathione S-transferase. Mutation of Ser 102 in the CGFS-motif of Pf Glp1 to cysteine did not generate glutaredoxin activity either. We conclude that, despite their ability to react with glutathione, glutaredoxin-like proteins are a mechanistically and functionally heterogeneous group with only little similarities to canonical glutaredoxins.     

NMR structure of oxidized glutaredoxin 3 from Escherichia coli

A high precision NMR structure of oxidized glutaredoxin 3 [C65Y] from Escherichia coli has been determined. The conformation of the active site including the disulphide bridge is highly similar to those in glutaredoxins from pig liver and T4 phage. A comparison with the previously determined structure of glutaredoxin 3 [C14S, C65Y] in a complex with glutathione reveals conformational changes between the free and substrate-bound form which includes the sidechain of the conserved, active site tyrosine residue. In the oxidized form this tyrosine is solvent exposed, while it adopts a less exposed conformation, stabilized by hydrogen bonds, in the mixed disulfide with glutathione. The structures further suggest that the formation of a covalent linkage between glutathione and glutaredoxin 3 is necessary in order to induce these structural changes upon binding of the glutathione peptide. This could explain the observed low affinity of glutaredoxins for S-blocked glutathione analogues, in spite of the fact that glutaredoxins are highly specific reductants of glutathione mixed disulfides.     

The v-Src SH3 domain facilitates a cell adhesion-independent association with focal adhesion kinase

Integrins facilitate cell attachment to the extracellular matrix, and these interactions generate cell survival, proliferation, and motility signals. Integrin signals are relayed in part by focal adhesion kinase (FAK) activation and the formation of a transient signaling complex initiated by Src homology 2 (SH2)-dependent binding of Src family protein-tyrosine kinases to the FAK Tyr-397 autophosphorylation site. Here we show that in viral Src (v-Src)-transformed NIH3T3 fibroblasts, an adhesion-independent FAK-Src signaling complex occurs. Co-expression studies in human 293T cells showed that v-Src could associate with and phosphorylate a Phe-397 FAK mutant at Tyr-925 promoting Grb2 binding to FAK in suspended cells. In vitro, glutathione S-transferase fusion proteins of the v-Src SH3 but not c-Src SH3 domain bound to FAK in lysates of NIH3T3 fibroblasts. The v-Src SH3-binding sites were mapped to known proline-X-X-proline (PXXP) SH3-binding motifs in the FAK N- (residues 371-377) and C-terminal domains (residues 712-718 and 871-882) by in vitro pull-down assays, and these sites are composed of a PXXPXXPhi (where Phi is a hydrophobic residue) v-Src SH3 binding consensus. Sequence comparisons show that residues in the RT loop region of the c-Src and v-Src SH3 domains differ. Substitution of c-Src RT loop residues (Arg-97 and Thr-98) for those found in the v-Src SH3 domain (Trp-97 and Ile-98) enhanced the binding of distinct NIH3T3 cellular proteins to a glutathione S-transferase fusion protein of the c-Src (Trp-97 + Ile-98) SH3 domain. FAK was identified as a c-Src (Trp-97 + Ile-98) SH3 domain target in fibroblasts, and co-expression studies in 293T cells showed that full-length c-Src (Trp-97 + Ile-98) could associate in vivo with Phe-397 FAK in an SH2-independent manner. These studies establish a functional role for the v-Src SH3 domain in stabilizing an adhesion-independent signaling complex with FAK.     

Structural basis of the differential binding of the SH3 domains of Grb2 adaptor to the guanine nucleotide exchange factor Sos1

Grb2-Sos1 interaction, mediated by the canonical binding of N-terminal SH3 (nSH3) and C-terminal SH3 (cSH3) domains of Grb2 to a proline-rich sequence in Sos1, provides a key regulatory switch that relays signaling from activated receptor tyrosine kinases to downstream effector molecules such as Ras. Here, using isothermal titration calorimetry in combination with site-directed mutagenesis, we show that the nSH3 domain binds to a Sos1-derived peptide containing the proline-rich consensus motif PPVPPR with an affinity that is nearly threefold greater than that observed for the binding of cSH3 domain. We further demonstrate that such differential binding of nSH3 domain relative to the cSH3 domain is largely due to the requirement of a specific acidic residue in the RT loop of the β-barrel fold to engage in the formation of a salt bridge with the arginine residue in the consensus motif PPVPPR. While this role is fulfilled by an optimally positioned D15 in the nSH3 domain, the chemically distinct and structurally non-equivalent E171 substitutes in the case of the cSH3 domain. Additionally, our data suggest that salt tightly modulates the binding of both SH3 domains to Sos1 in a thermodynamically distinct manner. Our data further reveal that, while binding of both SH3 domains to Sos1 is under enthalpic control, the nSH3 binding suffers from entropic penalty in contrast to entropic gain accompanying the binding of cSH3, implying that the two domains employ differential thermodynamic mechanisms for Sos1 recognition. Our new findings are rationalized in the context of 3D structural models of SH3 domains in complex with the Sos1 peptide. Taken together, our study provides structural basis of the differential binding of SH3 domains of Grb2 to Sos1 and a detailed thermodynamic profile of this key protein-protein interaction pertinent to cellular signaling and cancer.     

Structural basis for the thioredoxin-like activity profile of the glutaredoxin-like NrdH-redoxin from Escherichia coli.

NrdH-redoxin is a representative of a class of small redox proteins that contain a conserved CXXC motif and are characterized by a glutaredoxin-like amino acid sequence and thioredoxin-like activity profile. The crystal structure of recombinant Escherichia coli NrdH-redoxin in the oxidized state has been determined at 1.7 A resolution by multiwavelength anomalous diffraction. NrdH-redoxin belongs to the thioredoxin superfamily and is structurally most similar to E. coli glutaredoxin 3 and phage T4 glutaredoxin. The angle between the C-terminal helix alpha3 and strand beta4, which differs between thioredoxin and glutaredoxin, has an intermediate value in NrdH-redoxin. The orientation of this helix is to a large extent determined by an extended hydrogen-bond network involving the highly conserved sequence motif (61)WSGFRP(D/E)(67), which is unique to this subclass of the thioredoxin superfamily. Residues that bind glutathione in glutaredoxins are in general not conserved in NrdH-redoxin, and no glutathione-binding cleft is present. Instead, NrdH-redoxin contains a wide hydrophobic pocket at the surface, similar to thioredoxin. Modeling studies suggest that NrdH-redoxin can interact with E. coli thioredoxin reductase at this pocket and also via a loop that is complementary to a crevice in the reductase in a similar manner as observed in the E. coli thioredoxin-thioredoxin reductase complex.     

The structure of SOCS3 reveals the basis of the extended SH2 domain function and identifies an unstructured insertion that regulates stability

SOCS3 is essential for regulating the extent, duration, and specificity of cellular responses to cytokines such as G-CSF and IL-6. Here we describe the solution structure of SOCS3, the first structure determined for any SOCS protein, in complex with a phosphotyrosine-containing peptide from the IL-6 receptor signaling subunit gp130. The structure of the complex shows that seven peptide residues form a predominantly hydrophobic binding motif. Regions outside the SOCS3 SH2 domain are important for ligand binding, in particular, a single 15 residue alpha helix immediately N-terminal to the SH2 domain makes direct contacts with the phosphotyrosine binding loop and, in part, determines its geometry. The SH2 domain itself is remarkable in that it contains a 35 residue unstructured PEST motif insertion that is not required for STAT inhibition. The PEST motif increases SOCS3 turnover and affects its degradation pathway, implying that it has an important regulatory role inside the cell.     

The purification, characterization, and primary structure of a small redox protein from Methanobacterium thermoautotrophicum, an archaebacterium.

A small redox-active protein has been purified to homogeneity from cell-free extracts of the strictly anaerobic thermophilic methanogen, Methanobacterium thermoautotrophicum (strain Marburg). The purification consisted of streptomycin sulfate and acid treatments and three chromatographic steps using Sephadex G-75, Mono Q HR 10/10, and Superose 12 HR 10/30 columns. When these procedures were carried out under strictly anaerobic conditions, approximately 3 mg of this protein could be isolated from 45 g of wet cell paste. Like the thioredoxins and glutaredoxins, it is a small acidic protein (pI = 4.2) consisting of 83 amino acids (M(r) = 9136). In the presence of dithiothreitol or dihydrolipoate, the protein serves as a hydrogen donor for the ribonucleotide reductase from Escherichia coli, and it catalyzes the reduction of insulin. However, it does not interact with the thioredoxin reductases from E. coli or Corynebacterium nephridii and does not function as a hydrogen donor for the ribonucleotide reductase of C. nephridii. The amino acid sequences determined by automated Edman degradation of the 14C-carboxymethylated protein and of peptides derived from trypsin and chymotrypsin digestions show a redox-active site -Cys-Pro-Tyr-Cys-, typical of the glutaredoxins. Its amino acid sequence shows moderate identity with the known glutaredoxins (E. coli, yeast, rabbit bone marrow, calf thymus, and pig liver) when the proteins are aligned at the active site. The secondary structure of the glutaredoxin-like protein predicted by the Chou-Fasman procedure shows that it is similar to the known glutaredoxins. However, surprisingly, the protein does not function as a glutathione-disulfide oxidoreductase in the presence of glutathione and glutathione reductase. This glutaredoxin-like protein may be a component of a ribonucleotide-reducing system distinct from the previously described systems utilizing thioredoxin or glutaredoxin.     

Characterization of Escherichia coli null mutants for glutaredoxin 2.

Three Escherichia coli glutaredoxins catalyze GSH-disulfide oxidoreductions, but the atypical 24-kDa glutaredoxin 2 (Grx2, grxB gene), in contrast to the 9-kDa glutaredoxin 1 (Grx1, grxA gene) and glutaredoxin 3 (Grx3, grxC gene), is not a hydrogen donor for ribonucleotide reductase. To improve the understanding of glutaredoxin function, a null mutant for grxB (grxB(-)) was constructed and combined with other mutations. Null mutants for grxB or all three glutaredoxin genes were viable in rich and minimal media with little changes in their growth properties. Expression of leaderless alkaline phosphatase showed that Grx1 and Grx2 (but not Grx3) contributed in the reduction of cytosolic protein disulfides. Moreover, Grx1 could catalyze disulfide formation in the oxidizing cytosol of combined null mutants for glutathione reductase and thioredoxin 1. grxB(-) cells were more sensitive to hydrogen peroxide and other oxidants and showed increased carbonylation of intracellular proteins, particularly in the stationary phase. Significant up-regulation of catalase activity was observed in null mutants for thioredoxin 1 and the three glutaredoxins, whereas up-regulation of glutaredoxin activity was observed in catalase-deficient strains with additional defects in the thioredoxin pathway. The expression of catalases is thus interconnected with the thioredoxin/glutaredoxin pathways in the antioxidant response.     

Crystal structure of the amphiphysin-2 SH3 domain and its role in the prevention of dynamin ring formation.

The amphiphysins are brain-enriched proteins, implicated in clathrin-mediated endocytosis, that interact with dynamin through their SH3 domains. To elucidate the nature of this interaction, we have solved the crystal structure of the amphiphysin-2 (Amph2) SH3 domain to 2.2 A. The structure possesses several notable features, including an extensive patch of negative electrostatic potential covering a large portion of its dynamin binding site. This patch accounts for the specific requirement of amphiphysin for two arginines in the proline-rich binding motif to which it binds on dynamin. We demonstrate that the interaction of dynamin with amphiphysin SH3 domains, unlike that with SH3 domains of Grb2 or spectrin, prevents dynamin self-assembly into rings. Deletion of a unique insert in the n-Src loop of Amph2 SH3, a loop adjacent to the dynamin binding site, significantly reduces this effect. Conversely, replacing the n-Src loop of the N-terminal SH3 domain of Grb2 with that of Amph2 causes it to favour dynamin ring disassembly. Transferrin uptake assays show that shortening the n-Src loop of Amph2 SH3 reduces the ability of this domain to inhibit endocytosis in vivo. Our data suggest that amphiphysin SH3 domains are important regulators of the multimerization cycle of dynamin in endocytosis.