Self-catalyzed growth of S layers via an amorphous-to-crystalline transition limited by folding kinetics.
ABSTRACT: The importance of nonclassical, multistage crystallization pathways is increasingly evident from theoretical studies on colloidal systems and experimental investigations of proteins and biomineral phases. Although theoretical predictions suggest that proteins follow these pathways as a result of fluctuations that create unstable dense-liquid states, microscopic studies indicate these states are long-lived. Using in situ atomic force microscopy to follow 2D assembly of S-layer proteins on supported lipid bilayers, we have obtained a molecular-scale picture of multistage protein crystallization that reveals the importance of conformational transformations in directing the pathway of assembly. We find that monomers with an extended conformation first form a mobile adsorbed phase, from which they condense into amorphous clusters. These clusters undergo a phase transition through S-layer folding into crystalline clusters composed of compact tetramers. Growth then proceeds by formation of new tetramers exclusively at cluster edges, implying tetramer formation is autocatalytic. Analysis of the growth kinetics leads to a quantitative model in which tetramer creation is rate limiting. However, the estimated barrier is much smaller than expected for folding of isolated S-layer proteins, suggesting an energetic rationale for this multistage pathway.
Project description:In a previous study, a surprising drying transition was observed to take place inside the nanoscale hydrophobic channel in the tetramer of the protein melittin. The goal of this paper is to determine if there are other protein complexes capable of displaying a dewetting transition during their final stage of folding. We searched the entire protein data bank (PDB) for all possible candidates, including protein tetramers, dimers, and two-domain proteins, and then performed the molecular dynamics (MD) simulations on the top candidates identified by a simple hydrophobic scoring function based on aligned hydrophobic surface areas. Our large scale MD simulations found several more proteins, including three tetramers, six dimers, and two two-domain proteins, which display a nanoscale dewetting transition in their final stage of folding. Even though the scoring function alone is not sufficient (i.e., a high score is necessary but not sufficient) in identifying the dewetting candidates, it does provide useful insights into the features of complex interfaces needed for dewetting. All top candidates have two features in common: (1) large aligned (matched) hydrophobic areas between two corresponding surfaces, and (2) large connected hydrophobic areas on the same surface. We have also studied the effect on dewetting of different water models and different treatments of the long-range electrostatic interactions (cutoff vs PME), and found the dewetting phenomena is fairly robust. This work presents a few proteins other than melittin tetramer for further experimental studies of the role of dewetting in the end stages of protein folding.
Project description:All organisms have evolved specialized DNA repair mechanisms in order to protect their genome against detrimental lesions such as DNA double-strand breaks. In plant organelles, these damages are repaired either through recombination or through a microhomology-mediated break-induced replication pathway. Whirly proteins are modulators of this second pathway in both chloroplasts and mitochondria. In this precise pathway, tetrameric Whirly proteins are believed to bind single-stranded DNA and prevent spurious annealing of resected DNA molecules with other regions in the genome. In this study, we add a new layer of complexity to this model by showing through atomic force microscopy that tetramers of the potato Whirly protein WHY2 further assemble into hexamers of tetramers, or 24-mers, upon binding long DNA molecules. This process depends on tetramer-tetramer interactions mediated by K67, a highly conserved residue among plant Whirly proteins. Mutation of this residue abolishes the formation of 24-mers without affecting the protein structure or the binding to short DNA molecules. Importantly, we show that an Arabidopsis Whirly protein mutated for this lysine is unable to rescue the sensitivity of a Whirly-less mutant plant to a DNA double-strand break inducing agent.
Project description:The contribution of almost each amino acid side chain to the thermodynamic stability of the tetramerization domain (residues 326-353) of human p53 has been quantitated using 25 mutants with single-residue truncations to alanine (or glycine). Truncation of either Leu344 or Leu348 buried at the tetramer interface, but not of any other residue, led to the formation of dimers of moderate stability (8-9 kcal/mol of dimer) instead of tetramers. One-third of the substitutions were moderately destabilizing (<3.9 kcal/mol of tetramer). Truncations of Arg333, Asn345 or Glu349 involved in intermonomer hydrogen bonds, Ala347 at the tetramer interface or Thr329 were more destabilizing (4.1-5.7 kcal/mol). Strongly destabilizing (8.8- 11.7 kcal/mol) substitutions included those of Met340 at the tetramer interface and Phe328, Arg337 and Phe338 involved peripherally in the hydrophobic core. Truncation of any of the three residues involved centrally in the hydrophobic core of each primary dimer either prevented folding (Ile332) or allowed folding only at high protein concentration or low temperature (Leu330 and Phe341). Nine hydrophobic residues per monomer constitute critical determinants for the stability and oligomerization status of this p53 domain.
Project description:Members of the p53 tumor-suppressor family are expressed as multiple isoforms. Isoforms with an N-terminal transactivation domain are transcriptionally active, while those ones lacking this domain often inhibit the transcriptional activity of other family members. In squamous cell carcinomas, the high expression level of ?Np63? inhibits the tumor-suppressor function of TAp73?. This can in principle be due to blocking of the promoter or by direct interaction between both proteins. p63 and p73 can hetero-oligomerize through their tetramerization domains and a hetero-tetramer consisting of two p63 and two p73 molecules is thermodynamically more stable than both homo-tetramers. Here we show that cells expressing both p63 and p73 exist in mouse epidermis and hair follicle and that hetero-tetramer complexes can be detected by immunoprecipitation in differentiating keratinocytes. Through structure determination of the hetero-tetramer, we reveal why this hetero-tetramer is the thermodynamically preferred species. We have created mutants that exclusively form either hetero-tetramers or homo-tetramers, allowing to investigate the function of these p63/p73 hetero-tetramers. Using these tools, we show that inhibition of TAp73? in squamous cell carcinomas is due to promoter squelching and not direct interaction.
Project description:The biotin-binding tetrameric proteins, streptavidin from Streptomyces avidinii and chicken egg white avidin, are excellent models for the study of subunit-subunit interactions of a multimeric protein. Efforts are thus being made to prepare mutated forms of streptavidin and avidin, which would form monomers or dimers, in order to examine their effect on quaternary structure and assembly. In the present communication, we compared the crystal structures of binding site W-->K mutations in streptavidin and avidin. In solution, both mutant proteins are known to form dimers, but upon crystallization, both formed tetramers with the same parameters as the native proteins. All of the intersubunit bonds were conserved, except for the hydrophobic interaction between biotin and the tryptophan that was replaced by lysine. In the crystal structure, the binding site of the mutated apo-avidin contains 3 molecules of structured water instead of the 5 contained in the native protein. The lysine side chain extends in a direction opposite that of the native tryptophan, the void being partially filled by an adjacent lysine residue. Nevertheless, the binding-site conformation observed for the mutant tetramer is an artificial consequence of crystal packing that would not be maintained in the solution-phase dimer. It appears that the dimer-tetramer transition may be concentration dependent, and the interaction among subunits obeys the law of mass action.
Project description:Interleukin-15 (IL-15) is essential for the development and maintenance of natural killer (NK) cells. IL-15 activates STAT5 proteins, which can form dimers or tetramers. We previously found that NK cell numbers are decreased in Stat5a-Stat5b tetramer-deficient double knockin (DKI) mice, but the mechanism was not investigated. Here we show that STAT5 dimers are sufficient for NK cell development, whereas STAT5 tetramers mediate NK cell maturation and the expression of maturation-associated genes. Unlike the defective proliferation of Stat5 DKI CD8+ T cells, Stat5 DKI NK cells have normal proliferation to IL-15 but are susceptible to death upon cytokine withdrawal, with lower Bcl2 and increased active caspases. These findings underscore the importance of STAT5 tetramers in maintaining NK cell homeostasis. Moreover, defective STAT5 tetramer formation could represent a cause of NK cell immunodeficiency, and interrupting STAT5 tetramer formation might serve to control NK leukaemia. Overall design: ChIP-Seq analyses using purified splenic NK cells from both WT and Stat5 DKI, with rabbit IgG or anti-STAT5B. RNA-Seq analyses using total RNA from bone marrow NK cells and splenic NK subpopulations from both WT and Stat5 DKI mice.
Project description:Interleukin-15 (IL-15) is essential for the development and maintenance of natural killer (NK) cells. IL-15 activates STAT5 proteins, which can form dimers or tetramers. We previously found that NK cell numbers are decreased in Stat5a-Stat5b tetramer-deficient double knockin (DKI) mice, but the mechanism was not investigated. Here we show that STAT5 dimers are sufficient for NK cell development, whereas STAT5 tetramers mediate NK cell maturation and the expression of maturation-associated genes. Unlike the defective proliferation of Stat5 DKI CD8+ T cells, Stat5 DKI NK cells have normal proliferation to IL-15 but are susceptible to death upon cytokine withdrawal, with lower Bcl2 and increased active caspases. These findings underscore the importance of STAT5 tetramers in maintaining NK cell homoeostasis. Moreover, defective STAT5 tetramer formation could represent a cause of NK cell immunodeficiency, and interrupting STAT5 tetramer formation might serve to control NK leukaemia.
Project description:This study is to find the cellular and molecular mechanisms by which a naturally-occurring deltaNp53 isoform causes accelerated aging in humans. The biological function of deltaNp53, which lacks only 40 N-terminal amino acids, represents an example of p53 as a regulator of mammalian aging. When expressed together with WTp53 in mice, ΔNp53 causes an aging phenotype such as shorter life span, reduced body mass, organ atrophy and osteoporosis. Because p53 must form a tetramer to regulate transcription, we generated p53 clones (based upon the structure of the native p53 tetramer) containing one ΔNp53 linked with one WTp53 to form a functional ΔNp53:WTp53 tetramer with 1:1 stoichiometry. Thus, our strategy ensured each p53 tetramer contained 2 ΔNp53 and 2 WTp53 proteins. Importantly, ΔNp53:WTp53 form stable tetramers, based upon gel filtration chromatography and structural analysis using electron microscopy. Furthermore, the ΔNp53:WTp53 tetramer activates transcription equally well compared with WTp53 tetramers in an in vitro reconstituted transcription system. Having verified the stoichiometry, stability, structure, and activity of these ΔNp53:WTp53 tetramers, here we used microarray analysis to compare global gene expression patterns in p53-null H1299 cells expressing either WTp53 or ΔNp53:WTp53. As expected, global gene expression was largely similar, since the differences between ΔNp53:WTp53 tetramers and WTp53 tetramers are slight: only 2 of 4 p53 proteins will be different in the ΔNp53:WTp53 tetramer. Among only several dozen genes that were selectively up- or down-regulated 2-fold or greater, many genes known to regulate mammalian aging were altered in cells expressing ΔNp53:WTp53, including insulin signaling pathway members (IRS1, INPP5D, PLK3, MAP3K1, FGF5) and regulators of glucose metabolism (SLC2A2, CRYAB, LRCH1). Expression of other key metabolic genes were also altered in cells expressing ΔNp53:WTp53 tetramers, suggesting that global me tabolic changes might contribute to ΔNp53:WTp53 pathology. In collaboration with Metabolon (Durham, NC), we identified approximately one hundred metabolites that were significantly up- or down-regulated in H1299 cells expressing ΔNp53:WTp53. The metabolome analysis was a powerful complement to the gene expression data, and further suggested that the mTOR pathway (e.g. across-the-board up-regulation of amino acid levels) and mitochondrial function (e.g. up-regulation of carnitine, important for a-oxidation of fatty acids) was altered in cells expressing ΔNp53:WTp53. These findings were subsequently validated using biochemical and cell-based approaches. Furthermore, whereas equal expression of ΔNp53 and WTp53 cause accelerated aging in mammals, due to alternative splicing and translation initiation ΔNp53 is a naturally-occurring isoform whose expression levels can change throughout the lifetime. Thus, the cellular and molecular mechanisms identified from this work will likely reflect changes common to normal, physiological aging. Comparison of global gene expression profiles in WTp53 or ΔNp53:WTp53 expressed H1299 RNA from FACS sorted GFP positive hybrid to Affymatrix Gene The exp 0, exp 4 and exp 5 are independent replicate/transfections/biological experiments. The exp d, exp e and exp f are control experiments.
Project description:STAT proteins bind DNA as dimers and tetramers to control cellular development, differentiation, survival, and expansion. The tetramer binding sites are comprised of two dimer-binding sites repeated in tandem. The genome-wide distribution of the spacings between the dimer binding sites shows a distinctive, non-random pattern. Here, we report on estimating the feasibility of building possible molecular models of STAT5A tetramers bound to a DNA double helix with all possible spacings between the dimer binding sites. We found that the calculated feasibility estimates correlated well with the experimentally measured frequency of tetramer-binding sites. This suggests that the feasibility of forming the tetramer complex was a major factor in the evolution of this DNA sequence variation.
Project description:Both ?-galactosidase (GAL) and ?-glucuronidase (GUS) are tetrameric enzymes used widely as reporter proteins. However, little is known about the folding and assembly of these enzymes. Although the refolding kinetics of GAL from a denatured enzyme have been reported, it is not known how the kinetics differ when coupled with a protein translation reaction. Elucidating the assembly kinetics of GAL and GUS when coupled with protein translation will illustrate the differences between these two reporter proteins and also the assembly process under conditions more relevant to those in vivo. In this study, we used an in vitro translation/transcription system to synthesize GAL and GUS, measured the time development of the activity and oligomerization state of these enzymes, and determined the rate constants of the monomer to tetramer assembly process. We found that at similar concentrations, GAL assembles into tetramers faster than GUS. The rate constant of monomer to dimer assembly of GAL was 50-fold faster when coupled with protein translation than that of refolding from the denatured state. Furthermore, GAL synthesis was found to lack the rate-limiting step in the assembly process, whereas GUS has two rate-limiting steps: monomer to dimer assembly and dimer to tetramer assembly. The consequence of these differences when used as reporter proteins is discussed.