Giant protein kinases: domain interactions and structural basis of autoregulation.

The myosin-associated giant protein kinases twitchin and titin are composed predominantly of fibronectin- and immunoglobulin-like modules. We report the crystal structures of two autoinhibited twitchin kinase fragments, one from Aplysia and a larger fragment from Caenorhabditis elegans containing an additional C-terminal immunoglobulin-like domain. The structure of the longer fragment shows that the immunoglobulin domain contacts the protein kinase domain on the opposite side from the catalytic cleft, laterally exposing potential myosin binding residues. Together, the structures reveal the cooperative interactions between the autoregulatory region and the residues from the catalytic domain involved in protein substrate binding, ATP binding, catalysis and the activation loop, and explain the differences between the observed autoinhibitory mechanism and the one found in the structure of calmodulin-dependent kinase I.     

Interaction of the mutants sx,tra and dsx in Drosophila melanogaster. Chaetotaxy of the basitarsus of the forelegs in males and females

The basitarsal bristle pattern of the mutants sx (sexcombless), tra (transformer), and dsx (doublesex), and of the combinations sx-dsx and tra-dsx is described. Epistasis of dsx over both sx and tra for many of the chaetotaxal characteristics was found. The various effects of interaction observed, in individuals of male as well as female chromosomal constitution, are discussed in the light of the levels of action of the mutant genes in modifying the development of sex. It is suggested that intersexes induced by dsx are a class by itself, and that the action of dsx might be at a primary level of sex determination.     

Cohabitation of juvenile females with mature males promotes sexual cannibalism in fishing spiders

Precopulatory sexual cannibalism, or female predation of a potentialmate before mating, illustrates an extreme intersexual conflict.Unlike sexual cannibalism occurring after mating, precopulatorysexual cannibalism cannot be construed as a male strategy. Thus,research on the adaptive significance of this phenomenon hasfocused on female benefits. In the present study, I test theidea that precopulatory sexual cannibalism represents an adaptivefemale trade-off between the material costs and benefits ofmating with a male (forgoing food, securing sperm) and preyingon a male (forgoing sperm, securing food). I pay particularattention to the rarely tested prediction that precopulatorysexual cannibalism by virgin females should increase as eachfemale's expectation of future mating opportunities increases.I use the phenomenon of cohabitation between adult males andjuvenile females nearing sexual maturity as a means to manipulatefemale expectation of future mate availability. Results indicatethat feeding on a male has significant positive effects on severalmeasures of female fecundity. However, the likelihood of precopulatoryattacks was not explained by a female's recent feeding history.Finally, as predicted, juvenile female cohabitation with maturemales (expectation of future mating opportunities) heightensthe prevalence of precopulatory attacks by virgin females, suggestingthat juvenile experience can alter a female's propensity forsexual cannibalism. This is the first study to suggest thatjuvenile experience can alter a female's propensity for sexualcannibalism. This finding emphasizes the point that studiesof sexual selection and mating systems need to consider theeffects of juvenile experience on adult behavior.     

Differences in rat kidney morphology between males, females and testosterone-treated females.

Kidneys of normal female and male Wistar-Kyoto rats were studied by standard morphological techniques and morphometry in order to evaluate possible differences in the overall kidney morphology between both sexes. Furthermore, we investigated the role of testosterone (DHT) on kidney morphology by treating females with daily DHT injections. Kidney weight and volume in relation to body weight were not significantly different between males and females and were not affected by DHT. Differences were found in the volume distribution among the kidney zones. The cortex was larger in males than in females, whereas the medulla was conspicuously larger in females than in males. The greater volume of the cortex in males was mainly due to a more extensive development of proximal tubules. DHT treatment in females increased the volume of their proximal tubules. Glomerular volume was similar among the three groups. Within the medulla, the difference was most prominent in the inner stripe (14.9% of the total kidney volume in females vs. 8.9% in males) and was also important in the inner medulla (7.0 vs. 4.8%). The absolute epithelial volume of thick ascending limbs in this zone was larger in females than in males. This difference was more pronounced in short loops (approximately 20%) than in long loops (approximately 10%). The values of the DHT-treated females ranged in between. In spite of the greater development of medulla and thick ascending limbs in females, urine concentration was higher in males than in females and maximum urinary concentrating ability after 48 h dehydration was not different between both sexes.     

The N-terminal DNA-binding domain of Rad52 promotes RAD51-independent recombination in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the Rad52 protein plays a role in both RAD51-dependent and RAD51-independent recombination pathways. We characterized a rad52 mutant, rad52-329, which lacks the C-terminal Rad51-interacting domain, and studied its role in RAD51-independent recombination. The rad52-329 mutant is completely defective in mating-type switching, but partially proficient in recombination between inverted repeats. We also analyzed the effect of the rad52-329 mutant on telomere recombination. Yeast cells lacking telomerase maintain telomere length by recombination. The rad52-329 mutant is deficient in RAD51-dependent telomere recombination, but is proficient in RAD51-independent telomere recombination. In addition, we examined the roles of other recombination genes in the telomere recombination. The RAD51-independent recombination in the rad52-329 mutant is promoted by a paralogue of Rad52, Rad59. All components of the Rad50-Mre11-Xrs2 complex are also important, but not essential, for RAD51-independent telomere recombination. Interestingly, RAD51 inhibits the RAD51-independent, RAD52-dependent telomere recombination. These findings indicate that Rad52 itself, and more precisely its N-terminal DNA-binding domain, promote an essential reaction in recombination in the absence of RAD51.     

The N-terminal domain of RfaH plays an active role in protein fold-switching

The bacterial elongation factor RfaH promotes the expression of virulence factors by specifically binding to RNA polymerases (RNAP) paused at a DNA signal. This behavior is unlike that of its paralog NusG, the major representative of the protein family to which RfaH belongs. Both proteins have an N-terminal domain (NTD) bearing an RNAP binding site, yet NusG C-terminal domain (CTD) is folded as a β-barrel while RfaH CTD is forming an α-hairpin blocking such site. Upon recognition of the specific DNA exposed by RNAP, RfaH is activated via interdomain dissociation and complete CTD structural rearrangement into a β-barrel structurally identical to NusG CTD. Although RfaH transformation has been extensively characterized computationally, little attention has been given to the role of the NTD in the fold-switching process, as its structure remains unchanged. Here, we used Associative Water-mediated Structure and Energy Model (AWSEM) molecular dynamics to characterize the transformation of RfaH, spotlighting the sequence-dependent effects of NTD on CTD fold stabilization. Umbrella sampling simulations guided by native contacts recapitulate the thermodynamic equilibrium experimentally observed for RfaH and its isolated CTD. Temperature refolding simulations of full-length RfaH show a high success towards α-folded CTD, whereas the NTD interferes with βCTD folding, becoming trapped in a β-barrel intermediate. Meanwhile, NusG CTD refolding is unaffected by the presence of RfaH NTD, showing that these NTD-CTD interactions are encoded in RfaH sequence. Altogether, these results suggest that the NTD of RfaH favors the α-folded RfaH by specifically orienting the αCTD upon interdomain binding and by favoring β-barrel rupture into an intermediate from which fold-switching proceeds.     

Regulation of phosducin-like protein by casein kinase 2 and N-terminal splicing

Phosducin-like protein (PhLP) is a member of the phosducin family of G-protein betagamma-regulators and exists in two splice variants. The long isoform PhLP(L) and the short isoform PhLP(S) differ by the presence or absence of an 83-amino acid N terminus. In isolated biochemical assay systems, PhLP(L) is the more potent Gbetagamma-inhibitor, whereas the functional role of PhLP(S) is still unclear. We now report that in intact HEK 293 cells, PhLP(S) inhibited Gbetagamma-induced inositol phosphate generation with approximately 20-fold greater potency than PhLP(L). Radiolabeling of transfected HEK 293 cells with [(32)P] revealed that PhLP(L) is constitutively phosphorylated, whereas PhLP(S) is not. Because PhLP(L) has several consensus sites for the constitutively active kinase casein kinase 2 (CK2) in its N terminus, we tested the phosphorylation of the recombinant proteins by either HEK cell cytosol in the presence or absence of kinase inhibitors or by purified CK2. PhLP(L) was a good CK2 substrate, whereas PhLP(S) and phosducin were not. Progressive truncation and serine/threonine to alanine mutations of the PhLP(L) N terminus identified a serine/threonine cluster (Ser-18/Thr-19/Ser-20) within a small N-terminal region of PhLP(L) (amino acids 5-28) as the site in which PhLP(L) function was modified in HEK 293 cells. In native tissue, PhLP(L) also seems to be regulated by phosphorylation because phosphorylated and non-phosphorylated forms of PhLP(L) were detected in mouse brain and adrenal gland. Moreover, the alternatively spliced isoform PhLP(S) was also found in adrenal tissue. Therefore, the physiological control of G-protein regulation by PhLP seems to involve phosphorylation by CK2 and alternative splicing of the regulator.     

The N-terminal domain influences the structure and property of protein phosphatase 1

Molecular and Cellular Biochemistry - The protein phosphatase 1 has conserved cores in PPP gene family flanked by non-conserved N-terminal domains. PP1 with residues 1–8 deleted or...     

The expanded octarepeat domain selectively binds prions and disrupts homomeric prion protein interactions

Insertion of additional octarepeats into the prion protein gene has been genetically linked to familial Creutzfeldt Jakob disease and hence to de novo generation of infectious prions. The pivotal event during prion formation is the conversion of the normal prion protein (PrPC) into the pathogenic conformer PrPSc, which subsequently induces further conversion in an autocatalytic manner. Apparently, an expanded octarepeat domain directs folding of PrP toward the PrPSc conformation and initiates a self-replicating conversion process. Here, based on three main observations, we have provided a model on how altered molecular interactions between wild-type and mutant PrP set the stage for familial Creutzfeldt Jakob disease with octarepeat insertions. First, we showed that wild-type octarepeat domains interact in a copper-dependent and reversible manner, a "copper switch." This interaction becomes irreversible upon domain expansion, possibly reflecting a loss of function. Second, expanded octarepeat domains of increasing length gradually form homogenous globular multimers of 11-21 nm in the absence of copper ions when expressed as soluble glutathione S-transferase fusion proteins. Third, octarepeat domain expansion causes a gain of function with at least 10 repeats selectively binding PrPSc in a denaturant-resistant complex in the absence of copper ions. Thus, the combination of both a loss and gain of function profoundly influences homomeric interaction behavior of PrP with an expanded octarepeat domain. A multimeric cluster of prion proteins carrying expanded octarepeat domains may therefore capture and incorporate spontaneously arising short-lived PrPSc-like conformers, thereby providing a matrix for their conversion.     

The N-terminal domain of a rab protein is involved in membrane-membrane recognition and/or fusion.

Proteins of the YPT1/SEC4/rab family are well documented to be involved in the regulation of membrane transport. We have previously reported that rab5 regulates endosome-endosome recognition and/or fusion in vitro. Here, we show that this process depends on the rab5 N-terminal domain. Treatment of early endosomal membranes at a low trypsin concentration essentially abolished fusion and cleaved rab5 to a 1 kDa smaller polypeptide. Two-dimensional gel analysis suggested that rab5 is one of the few, if not the only, polypeptides cleaved by trypsin under these conditions. Whereas endosome fusion could be stimulated by cytosol prepared from cells overexpressing rab5 (and thus containing high amounts of the protein), this stimulation was abolished by trypsin-treatment of the cytosol. Trypsin-treated cytosol prepared from mock-transfected cells, which contains very low amounts of rab5, showed no inhibitory activity indicating that rab5 is the target of trypsin in these experiments. Purified rab5 prepared after expression in Escherichia coli was treated with trypsin, which cleaved the protein at the N-terminus. A synthetic peptide of rab5 N-terminal domain inhibited endosome fusion in our cell-free assay. A version of the same peptide truncated at the N-terminus or a peptide of rab3 N-terminal domain were without effects. Altogether, these observations suggest that the N-terminal domain of rab5 is involved in the process of early endosome recognition and/or fusion, presumably because it interacts with another component of the transport machinery.     

The coevolution of sexual imprinting by males and females

Sexual imprinting is the learning of a mate preference by direct observation of the phenotype of another member of the population. Sexual imprinting can be paternal, maternal, or oblique if individuals learn to prefer the phenotypes of their fathers, mothers, or other members of the population, respectively. Which phenotypes are learned can affect trait evolution and speciation rates. “Good genes” models of polygynous systems predict that females should evolve to imprint on their fathers, because paternal imprinting helps females to choose mates that will produce offspring that are both viable and sexy. Sexual imprinting by males has been observed in nature, but a theory for the evolution of sexual imprinting by males does not exist. We developed a good genes model to study the conditions under which sexual imprinting by males or by both sexes can evolve and to ask which sexual imprinting strategies maximize the fitness of the choosy sex. We found that when only males imprint, maternal imprinting is the most advantageous strategy. When both sexes imprint, it is most advantageous for both sexes to use paternal imprinting. Previous theory suggests that, in a given population, either males or females but not both will evolve choosiness in mating. We show how environmental change can lead to the evolution of sexual imprinting behavior by both sexes in the same population.     

Biotin protein ligase from Saccharomyces cerevisiae. The N-terminal domain is required for complete activity.

Catalytically active biotin protein ligase from Saccharomyces cerevisiae (EC 6.3.4.15) was overexpressed in Escherichia coli and purified to near homogeneity in three steps. Kinetic analysis demonstrated that the substrates ATP, biotin, and the biotin-accepting protein bind in an ordered manner in the reaction mechanism. Treatment with any of three proteases of differing specificity in vitro revealed that the sequence between residues 240 and 260 was extremely sensitive to proteolysis, suggesting that it forms an exposed linker between an N-terminal 27-kDa domain and the C-terminal 50-kDa domain containing the active site. The protease susceptibility of this linker region was considerably reduced in the presence of ATP and biotin. A second protease-sensitive sequence, located in the presumptive catalytic site, was protected against digestion by the substrates. Expression of N-terminally truncated variants of the yeast enzyme failed to complement E. coli strains defective in biotin protein ligase activity. In vitro assays performed with purified N-terminally truncated enzyme revealed that removal of the N-terminal domain reduced BPL activity by greater than 3500-fold. Our data indicate that both the N-terminal domain and the C-terminal domain containing the active site are necessary for complete catalytic function.     

The N-terminal A domain of fibronectin-binding proteins A and B promotes adhesion of Staphylococcus aureus to elastin

The ability of Staphylococcus aureus to adhere to components of the extracellular matrix is an important mechanism for colonization of host tissues during infection. We have previously shown that S. aureus binds elastin, a major component of the extracellular matrix. The integral membrane protein, elastin-binding protein (EbpS), binds soluble elastin peptides and tropoelastin via its surface-exposed N-terminal domain. In this study, we demonstrate that some strains of S. aureus adhere strongly to immobilized human elastin and that this interaction is independent of EbpS but instead is mediated by the fibronectin-binding proteins, FnBPA and FnBPB. Our results show that EbpS mutant cells adhere to elastin-coated plates, whereas the cells negative for FnBPA and FnBPB do not adhere to the plates. Furthermore, only wild-type cells from the exponential phase of growth adhered when FnBPs were expressed maximally. We show that adherence to elastin promoted by FnBPA was not affected by soluble fibronectin, suggesting that the elastin binding domain is distinct from the fibronectin binding regions. Recombinant FnBPA(37-544) (rFnBPA(37-544)) protein corresponding to the A region of FnBPA and anti-FnBPA(37-544) antibodies inhibited FnBPA-mediated bacterial adherence to immobilized elastin. Finally, recombinant A domain proteins, rFnBPA(37-544) and rFnBPB(37-540), bound immobilized elastin dose-dependently and saturably. This interaction was inhibited by soluble elastin peptides, suggesting a specific receptor-ligand interaction.     

Alternative splicing of human synexin mRNA in brain, cardiac, and skeletal muscle alters the unique N-terminal domain.

Several synexin (annexin VII) mRNAs have been identified by screening a human fibroblast cDNA library. One type of message contained an alternatively spliced cassette exon, predicting two isoforms of synexin differing in the N-terminal domain. Polymerase chain reaction analysis of synexin mRNA from various fetal and adult tissues, from human and monkey, revealed that the alternative splicing event is tissue-regulated; synexin mRNA containing the cassette exon is prevalent in brain, heart, and skeletal muscle. This is supported by Western blot analysis showing that muscle synexin (annexin VIIb) is larger than synexin from lung (annexin VIIa). The muscle and lung isoforms have the same molecular mass as the recombinant synexins expressed in Escherichia coli using cDNAs containing or lacking the cassette exon, respectively. The difference in size is consistent with the molecular masses predicted from the proteins encoded by the alternatively spliced synexin mRNAs. Another type of synexin mRNA contained a longer 3'-noncoding region generated by the selection of an alternate poly(A) signal. Northern analysis of human fibroblast RNA showed the presence of two bands (2.0- and 2.4-kilobase) when hybridized to a cDNA fragment of the coding region of synexin, but only the 2.4-kilobase band hybridized to a probe made from the longer 3' end.     

The adaptive significance of polymorphism in the rotifer Asplanchna. Humps in males and females

Summary Experiments show that humps or body wall outgrowths protect males and cruciform females of Asplanchna sieboldi from predation by conspecific, cannibal females. Humped prey were eaten by large cannibals to a significantly lesser extent than similarly-sized or even larger, non-humped prey.Since males and male-producing females, which are typically cruciform, co-occur with the largest, most cannibalistic morphotypes, their protection by humps may greatly increase the efficiency of sexual reproduction.An analysis of female diets and the occurrence of humped males and females in the different species of Asplanchna shows that these humped forms are found only in those species in which the females can eat comparatively large prey. This relationship provides strong indirect evidence for the theory that humps in both males and polymorphic females specifically evolved as a defense mechanism against predation by conspecific, cannibal females.Supported by National Science Foundation Grant GB-32182.     

The protein splicing domain of the homing endonuclease PI-sceI is responsible for specific DNA binding.

The homing endonuclease PI- Sce I consists of a protein splicing domain (I) and an endonucleolytic domain (II). To characterize the two domains with respect to their contribution to DNA recognition we cloned, purified and characterized the isolated domains. Both domains have no detectable endonucleolytic activity. Domain I binds specifically to the PI- Sce I recognition sequence, whereas domain II displays only weak non-specific DNA binding. In the specific complex with domain I the DNA is bent to a similar extent as observed with the initial complex formed between PI- Sce I and DNA. Our results indicate that protein splicing domain I is also involved in recognition of the DNA substrate.