Mapping of a Myosin-binding Domain and a Regulatory Phosphorylation Site in M-Protein, a Structural Protein of the Sarcomeric M Band

The myofibrils of cross-striated muscle fibers contain in their M bands cytoskeletal proteins whose main function seems to be the stabilization of the three-dimensional arrangement of thick filaments. We identified two immunoglobin domains (Mp2–Mp3) of M-protein as a site binding to the central region of light meromyosin. This binding is regulated in vitro by phosphorylation of a single serine residue (Ser76) in the immediately adjacent amino-terminal domain Mp1. M-protein phosphorylation by cAMP-dependent kinase A inhibits binding to myosin LMM. Transient transfection studies of cultured cells revealed that the myosin-binding site seems involved in the targeting of M-protein to its location in the myofibril. Using the same method, a second myofibril-binding site was uncovered in domains Mp9–Mp13. These results support the view that specific phosphorylation events could be also important for the control of sarcomeric M band formation and remodeling.

The Role of the Membrane-spanning Domain Sequence in Glycoprotein-mediated Membrane Fusion

The role of glycoprotein membrane-spanning domains in the process of membrane fusion is poorly understood. It has been demonstrated that replacing all or part of the membrane-spanning domain of a viral fusion protein with sequences that encode signals for glycosylphosphatidylinositol linkage attachment abrogates membrane fusion activity. It has been suggested, however, that the actual amino acid sequence of the membrane-spanning domain is not critical for the activity of viral fusion proteins. We have examined the function of Moloney murine leukemia virus envelope proteins with substitutions in the membrane-spanning domain. Envelope proteins bearing substitutions for proline 617 are processed and incorporated into virus particles normally and bind to the viral receptor. However, they possess greatly reduced or undetectable capacities for the promotion of membrane fusion and infectious virus particle formation. Our results imply a direct role for the residues in the membrane-spanning domain of the murine leukemia virus envelope protein in membrane fusion and its regulation. They also support the thesis that membrane-spanning domains possess a sequence-dependent function in other protein-mediated membrane fusion events.

Complex Formation with Focal Adhesion Kinase: A Mechanism to Regulate Activity and Subcellular Localization of Src Kinases

Tyrosine phosphorylation of focal adhesion kinase (FAK) creates a high-affinity binding site for the src homology 2 domain of the Src family of tyrosine kinases. Assembly of a complex between FAK and Src kinases may serve to regulate the subcellular localization and the enzymatic activity of members of the Src family of kinases. We show that simultaneous overexpression of FAK and pp60c-src or p59fyn results in the enhancement of the tyrosine phosphorylation of a limited number of cellular substrates, including paxillin. Under these conditions, tyrosine phosphorylation of paxillin is largely cell adhesion dependent. FAK mutants defective for Src binding or focal adhesion targeting fail to cooperate with pp60c-src or p59fyn to induce paxillin phosphorylation, whereas catalytically defective FAK mutants can direct paxillin phosphorylation. The negative regulatory site of pp60c-src is hypophosphorylated when in complex with FAK, and coexpression with FAK leads to a redistribution of pp60c-src from a diffuse cellular location to focal adhesions. A FAK mutant defective for Src binding does not effectively induce the translocation of pp60c-src to focal adhesions. These results suggest that association with FAK can alter the localization of Src kinases and that FAK functions to direct phosphorylation of cellular substrates by recruitment of Src kinases.

Recombinant immunotoxins specific for a mutant epidermal growth factor receptor: Targeting with a single chain antibody variable domain isolated by phage display

EGFRvIII is a mutant epidermal growth factor receptor found in glioblastoma, and in carcinoma of the breast, ovary, and lung. The mutant receptor has a deletion in its extracellular domain that results in the formation of a new, tumor-specific extracellular sequence. Mice were immunized with a synthetic peptide corresponding to this sequence and purified EGFRvIII. A single chain antibody variable domain (scFv) phage display library of 8 × 106 members was made from the spleen of one immunized mouse. A scFv specific for EGFRvIII was isolated from this library by panning with successively decreasing amounts of synthetic peptide. This was used to make an immunotoxin by fusing the scFv DNA sequence to sequences coding for domains II and III of Pseudomonas exotoxin A. Purified immunotoxin had a Kd of 22 nM for peptide and a Kd of 11 nM for cell-surface EGFRvIII. The immunotoxin was very cytotoxic to cells expressing EGFRvIII, with an IC50 of 1 ng/ml (16 pM) on mouse fibroblasts transfected with EGFRvIII and an IC50 of 7–10 ng/ml (110–160 pM) on transfected glioblastoma cells. There was no cytotoxic activity at 1000 ng/ml on the untransfected parent glioblastoma cell line. The immunotoxin was completely stable upon incubation at 37°C for 24 h in human serum. The combination of good affinity, cytotoxicity and stability make this immunotoxin a candidate for further preclinical evaluation.

Substitutions in the aspartate transcarbamoylase domain of hamster CAD disrupt oligomeric structure

Aspartate transcarbamoylase (ATCase; EC 2.1.3.2) is one of three enzymatic domains of CAD, a protein whose native structure is usually a hexamer of identical subunits. Alanine substitutions for the ATCase residues Asp-90 and Arg-269 were generated in a bicistronic vector that encodes a 6-histidine-tagged hamster CAD. Stably transfected mammalian cells expressing high levels of CAD were easily isolated and CAD purification was simplified over previous procedures. The substitutions reduce the ATCase Vmax of the altered CADs by 11-fold and 46-fold, respectively, as well as affect the enzyme's affinity for aspartate. At 25 mM Mg2+, these substitutions cause the oligomeric CAD to dissociate into monomers. Under the same dissociating conditions, incubating the altered CAD with the ATCase substrate carbamoyl phosphate or the bisubstrate analogue N-phosphonacetyl-l-aspartate unexpectedly leads to the reformation of hexamers. Incubation with the other ATCase substrate, aspartate, has no effect. These results demonstrate that the ATCase domain is central to hexamer formation in CAD and suggest that the ATCase reaction mechanism is ordered in the same manner as the Escherichia coli ATCase. Finally, the data indicate that the binding of carbamoyl phosphate induces conformational changes that enhance the interaction of CAD subunits.

The crayfish plasma clotting protein: A vitellogenin-related protein responsible for clot formation in crustacean blood

Coagulation in crayfish blood is based on the transglutaminase-mediated crosslinking of a specific plasma clotting protein. Here we report the cloning of the subunit of this clotting protein from a crayfish hepatopancreas cDNA library. The ORF encodes a protein of 1,721 amino acids, including a signal peptide of 15 amino acids. Sequence analysis reveals that the clotting protein is homologous to vitellogenins, which are proteins found in vitellogenic females of egg-laying animals. The clotting protein and vitellogenins are all lipoproteins and share a limited sequence similarity to certain other lipoproteins (e.g., mammalian apolipoprotein B and microsomal triglyceride transfer protein) and contain a stretch with similarity to the D domain of mammalian von Willebrand factor. The crayfish clotting protein is present in both sexes, unlike the female-specific vitellogenins. Electron microscopy was used to visualize individual clotting protein molecules and to study the transglutaminase-mediated clotting reaction. In the presence of an endogenous transglutaminase, the purified clotting protein molecules rapidly assemble into long, flexible chains that occasionally branch.

Identification of a nuclear domain with deacetylase activity

Here, we describe the identification and characterization of a nuclear body (matrix-associated deacetylase body) whose formation and integrity depend on deacetylase activity. Typically, there are 20–40 0.5-μM bodies per nucleus, although the size and number can vary substantially. The structure appears to contain both class I and the recently described class II histone deacetylases (HDAC)5 and 7 along with the nuclear receptor corepressors SMRT (silencing mediator for retinoid and thyroid receptor) and N-CoR (nuclear receptor corepressor). Addition of the deacetylase inhibitors trichostatin A and sodium butyrate completely disrupt these nuclear bodies, providing a demonstration that the integrity of a nuclear body is enzyme dependent. We demonstrate that HDAC5 and 7 can associate with at least 12 distinct proteins, including several members of the NuRD and Sin3A repression complexes, and appear to define a new but related complex.

Elucidating the mechanism of familial amyloidosis– Finnish type: NMR studies of human gelsolin domain 2

Familial amyloidosis–Finnish type (FAF) results from a single mutation at residue 187 (D187N or D187Y) within domain 2 of the actin-regulating protein gelsolin. The mutation somehow allows a masked cleavage site to be exposed, leading to the first step in the formation of an amyloidogenic fragment. We have performed NMR experiments investigating structural and dynamic changes between wild-type (WT) and D187N gelsolin domain 2 (D2). On mutation, no significant structural or dynamic changes occur at or near the cleavage site. Areas in conformational exchange are observed between β-strand 4 and α-helix 1 and within the loop region following β-strand 5. Chemical shift differences are noted along the face of α-helix 1 that packs onto the β-sheet, suggesting an altered conformation. Conformational changes within these areas can have an effect on actin binding and may explain why D187N gelsolin is inactive. {1H-15N} nuclear Overhauser effect and chemical shift data suggest that the C-terminal tail of D187N gelsolin D2 is less structured than WT by up to six residues. In the crystal structure of equine gelsolin, the C-terminal tail of D2 lies across a large cleft between domains 1 and 2 where the masked cleavage site sits. We propose that the D187N mutation destabilizes the C-terminal tail of D2 resulting in a more exposed cleavage site leading to the first proteolysis step in the formation of the amyloidogenic fragment.

A scrapie-like unfolding intermediate of the prion protein domain PrP(121–231) induced by acidic pH

The infectious agent of transmissible spongiform encephalopathies is believed to consist of an oligomeric isoform, PrPSc, of the monomeric cellular prion protein, PrPC. The conversion of PrPC to PrPSc is characterized by a decrease in α-helical structure, an increase in β-sheet content, and the formation of PrPSc amyloid. Whereas the N-terminal part of PrPC comprising residues 23–120 is flexibly disordered, its C-terminal part, PrP(121–231), forms a globular domain with three α-helices and a small β-sheet. Because the segment of residues 90–231 is protease-resistant in PrPSc, it is most likely structured in the PrPSc form. The conformational change of the segment containing residues 90–120 thus constitutes the minimal structural difference between PrPC and a PrPSc monomer. To test whether PrP(121–231) is also capable to undergo conformational transitions, we analyzed its urea-dependent unfolding transitions at neutral and acidic pH. We identified an equilibrium unfolding intermediate of PrP(121–231) that is exclusively populated at acidic pH and shows spectral characteristics of a β-sheet protein. The intermediate is in rapid equilibrium with native PrP(121–231), significantly populated in the absence of urea at pH 4.0, and may have important implications for the presumed formation of PrPSc during endocytosis.

Design of three-dimensional domain-swapped dimers and fibrous oligomers

Three-dimensional (3D) domain-swapped proteins are intermolecularly folded analogs of monomeric proteins; both are stabilized by the identical interactions, but the individual domains interact intramolecularly in monomeric proteins, whereas they form intermolecular interactions in 3D domain-swapped structures. The structures and conditions of formation of several domain-swapped dimers and trimers are known, but the formation of higher order 3D domain-swapped oligomers has been less thoroughly studied. Here we contrast the structural consequences of domain swapping from two designed three-helix bundles: one with an up-down-up topology, and the other with an up-down-down topology. The up-down-up topology gives rise to a domain-swapped dimer whose structure has been determined to 1.5 Å resolution by x-ray crystallography. In contrast, the domain-swapped protein with an up-down-down topology forms fibrils as shown by electron microscopy and dynamic light scattering. This demonstrates that design principles can predict the oligomeric state of 3D domain-swapped molecules, which should aid in the design of domain-swapped proteins and biomaterials.

Structure of a flavin-binding plant photoreceptor domain: Insights into light-mediated signal transduction

Phototropin, a major blue-light receptor for phototropism in seed plants, exhibits blue-light-dependent autophosphorylation and contains two light, oxygen, or voltage (LOV) domains and a serine/threonine kinase domain. The LOV domains share homology with the PER-ARNT-SIM (PAS) superfamily, a diverse group of sensor proteins. Each LOV domain noncovalently binds a single FMN molecule and exhibits reversible photochemistry in vitro when expressed separately or in tandem. We have determined the crystal structure of the LOV2 domain from the phototropin segment of the chimeric fern photoreceptor phy3 to 2.7-Å resolution. The structure constitutes an FMN-binding fold that reveals how the flavin cofactor is embedded in the protein. The single LOV2 cysteine residue is located 4.2 Å from flavin atom C(4a), consistent with a model in which absorption of blue light induces formation of a covalent cysteinyl-C(4a) adduct. Residues that interact with FMN in the phototropin segment of the chimeric fern photoreceptor (phy3) LOV2 are conserved in LOV domains from phototropin of other plant species and from three proteins involved in the regulation of circadian rhythms in Arabidopsis and Neurospora. This conservation suggests that these domains exhibit the same overall fold and share a common mechanism for flavin binding and light-induced signaling.

The wing in yeast heat shock transcription factor (HSF) DNA-binding domain is required for full activity

The yeast heat shock transcription factor (HSF) belongs to the winged helix family of proteins. HSF binds DNA as a trimer, and additional trimers can bind DNA co-operatively. Unlike other winged helix–turn–helix proteins, HSF’s wing does not appear to contact DNA, as based on a previously solved crystal structure. Instead, the structure implies that the wing is involved in protein–protein interactions, possibly within a trimer or between adjacent trimers. To understand the function of the wing in the HSF DNA-binding domain, a Saccharomyces cerevisiae strain was created that expresses a wingless HSF protein. This strain grows normally at 30°C, but shows a decrease in reporter gene expression during constitutive and heat-shocked conditions. Removal of the wing does not affect the stability or trimeric nature of a protein fragment containing the DNA-binding and trimerization domains. Removal of the wing does result in a decrease in DNA-binding affinity. This defect was mainly observed in the ability to form the first trimer-bound complex, as the formation of larger complexes is unaffected by the deletion. Our results suggest that the wing is not involved in the highly co-operative nature of HSF binding, but may be important in stabilizing the first trimer bound to DNA.

Cofilin Phosphorylation by Protein Kinase Testicular Protein Kinase 1 and Its Role in Integrin-mediated Actin Reorganization and Focal Adhesion Formation

Testicular protein kinase 1 (TESK1) is a serine/threonine kinase with a structure composed of a kinase domain related to those of LIM-kinases and a unique C-terminal proline-rich domain. Like LIM-kinases, TESK1 phosphorylated cofilin specifically at Ser-3, both in vitro and in vivo. When expressed in HeLa cells, TESK1 stimulated the formation of actin stress fibers and focal adhesions. In contrast to LIM-kinases, the kinase activity of TESK1 was not enhanced by Rho-associated kinase (ROCK) or p21-activated kinase, indicating that TESK1 is not their downstream effector. Both the kinase activity of TESK1 and the level of cofilin phosphorylation increased by plating cells on fibronectin. Y-27632, a specific inhibitor of ROCK, inhibited LIM-kinase-induced cofilin phosphorylation but did not affect fibronectin-induced or TESK1-induced cofilin phosphorylation in HeLa cells. Expression of a kinase-negative TESK1 suppressed cofilin phosphorylation and formation of stress fibers and focal adhesions induced in cells plated on fibronectin. These results suggest that TESK1 functions downstream of integrins and plays a key role in integrin-mediated actin reorganization, presumably through phosphorylating and inactivating cofilin. We propose that TESK1 and LIM-kinases commonly phosphorylate cofilin but are regulated in different ways and play distinct roles in actin reorganization in living cells.

The Cia5 gene controls formation of the carbon concentrating mechanism in Chlamydomonas reinhardtii

Wild-type Chlamydomonas reinhardtii cells shifted from high concentrations (5%) of CO2 to low, ambient levels (0.03%) rapidly increase transcription of mRNAs from several CO2-responsive genes. Simultaneously, they develop a functional carbon concentrating mechanism that allows the cells to greatly increase internal levels of CO2 and HCO\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document}. The cia5 mutant is defective in all of these phenotypes. A newly isolated gene, designated Cia5, restores transformed cia5 cells to the phenotype of wild-type cells. The 6,481-bp gene produces a 5.1-kb mRNA that is present constitutively in light in high and low CO2 both in wild-type cells and the cia5 mutant. It encodes a protein that has features of a putative transcription factor and that, likewise, is present constitutively in low and high CO2 conditions. Complementation of cia5 can be achieved with a truncated Cia5 gene that is missing the coding information for 54 C-terminal amino acids. Unlike wild-type cells or cia5 mutants transformed with an intact Cia5 gene, cia5 mutants complemented with the truncated gene exhibit constitutive synthesis of mRNAs from CO2-responsive genes in light under both high and low CO2 conditions. These discoveries suggest that posttranslational changes to the C-terminal domain control the ability of CIA5 to act as an inducer and directly or indirectly control transcription of CO2-responsive genes. Thus, CIA5 appears to be a master regulator of the carbon concentrating mechanism and is intimately involved in the signal transduction mechanism that senses and allows immediate responses to fluctuations in environmental CO2 and HCO\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document} concentrations.

Three-dimensional domain swapping in p13suc1 occurs in the unfolded state and is controlled by conserved proline residues

p13suc1 has two native states, a monomer and a domain-swapped dimer. We show that their folding pathways are connected by the denatured state, which introduces a kinetic barrier between monomer and dimer under native conditions. The barrier is lowered under conditions that speed up unfolding, thereby allowing, to our knowledge for the first time, a quantitative dissection of the energetics of domain swapping. The monomer–dimer equilibrium is controlled by two conserved prolines in the hinge loop that connects the exchanging domains. These two residues exploit backbone strain to specifically direct dimer formation while preventing higher-order oligomerization. Thus, the loop acts as a loaded molecular spring that releases tension in the monomer by adopting its alternative conformation in the dimer. There is an excellent correlation between domain swapping and aggregation, suggesting they share a common mechanism. These insights have allowed us to redesign the domain-swapping propensity of suc1 from a fully monomeric to a fully dimeric protein.