scholarly journals Structure of a designed, right-handed coiled-coil tetramer containing all biological amino acids

2007 ◽  
Vol 16 (10) ◽  
pp. 2224-2232 ◽  
Author(s):  
Mark Sales ◽  
Joseph J. Plecs ◽  
James M. Holton ◽  
Tom Alber
Keyword(s):  
Amino Acids ◽  
Coiled Coil

scholarly journals Involvement of FtsE ATPase and FtsX Extracellular Loops 1 and 2 in FtsEX-PcsB Complex Function in Cell Division of Streptococcus pneumoniae D39

mBio ◽  
2013 ◽  
Vol 4 (4) ◽  
Author(s):  
Lok-To Sham ◽  
Katelyn R. Jensen ◽  
Kevin E. Bruce ◽  
Malcolm E. Winkler
Keyword(s):  
Signal Transduction ◽  
Amino Acids ◽  
Cell Division ◽  
Streptococcus Pneumoniae ◽  
Amino Acid ◽  
Stationary Phase ◽  
Coiled Coil ◽  
Extracellular Loop ◽  
Coiled Coil Domain ◽  
Loop Domain

ABSTRACT The FtsEX protein complex has recently been proposed to play a major role in coordinating peptidoglycan (PG) remodeling by hydrolases with the division of bacterial cells. According to this model, cytoplasmic FtsE ATPase interacts with the FtsZ divisome and FtsX integral membrane protein and powers allosteric activation of an extracellular hydrolase interacting with FtsX. In the major human respiratory pathogen Streptococcus pneumoniae (pneumococcus), a large extracellular-loop domain of FtsX (ECL1FtsX) is thought to interact with the coiled-coil domain of the PcsB protein, which likely functions as a PG amidase or endopeptidase required for normal cell division. This paper provides evidence for two key tenets of this model. First, we show that FtsE protein is essential, that depletion of FtsE phenocopies cell defects caused by depletion of FtsX or PcsB, and that changes of conserved amino acids in the FtsE ATPase active site are not tolerated. Second, we show that temperature-sensitive (Ts) pcsB mutations resulting in amino acid changes in the PcsB coiled-coil domain (CCPcsB) are suppressed by ftsX mutations resulting in amino acid changes in the distal part of ECL1FtsX or in a second, small extracellular-loop domain (ECL2FtsX). Some FtsX suppressors are allele specific for changes in CCPcsB, and no FtsX suppressors were found for amino acid changes in the catalytic PcsB CHAP domain (CHAPPcsB). These results strongly support roles for both ECL1FtsX and ECL2FtsX in signal transduction to the coiled-coil domain of PcsB. Finally, we found that pcsB CC(Ts) mutants (Ts mutants carrying mutations in the region of pcsB corresponding to the coiled-coil domain) unexpectedly exhibit delayed stationary-phase autolysis at a permissive growth temperature. IMPORTANCE Little is known about how FtsX interacts with cognate PG hydrolases in any bacterium, besides that ECL1FtsX domains somehow interact with coiled-coil domains. This work used powerful genetic approaches to implicate a specific region of pneumococcal ECL1FtsX and the small ECL2FtsX in the interaction with CCPcsB. These findings identify amino acids important for in vivo signal transduction between FtsX and PcsB for the first time. This paper also supports the central hypothesis that signal transduction between pneumococcal FtsX and PcsB is linked to ATP hydrolysis by essential FtsE, which couples PG hydrolysis to cell division. The classical genetic approaches used here can be applied to dissect interactions of other integral membrane proteins involved in PG biosynthesis. Finally, delayed autolysis of the pcsB CC(Ts) mutants suggests that the FtsEX-PcsB PG hydrolase may generate a signal in the PG necessary for activation of the major LytA autolysin as pneumococcal cells enter stationary phase.

scholarly journals The inv(16) Fusion Protein Associates with Corepressors via a Smooth Muscle Myosin Heavy-Chain Domain

2003 ◽  
Vol 23 (2) ◽  
pp. 607-619 ◽  
Author(s):  
Kristie L. Durst ◽  
Bart Lutterbach ◽  
Tanawan Kummalue ◽  
Alan D. Friedman ◽  
Scott W. Hiebert
Keyword(s):  
Amino Acids ◽  
Smooth Muscle ◽  
Fusion Protein ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Transcriptional Repression ◽  
Coiled Coil ◽  
Smooth Muscle Myosin ◽  
Muscle Myosin ◽  
Repression Domain

ABSTRACT Inversion(16) is one of the most frequent chromosomal translocations found in acute myeloid leukemia (AML), occurring in over 8% of AML cases. This translocation results in a protein product that fuses the first 165 amino acids of core binding factor β to the coiled-coil region of a smooth muscle myosin heavy chain (CBFβ/SMMHC). CBFβ interacts with AML1 to form a heterodimer that binds DNA; this interaction increases the affinity of AML1 for DNA. The CBFβ/SMMHC fusion protein cooperates with AML1 to repress the transcription of AML1-regulated genes. We show that CBFβ/SMMHC contains a repression domain in the C-terminal 163 amino acids of the SMMHC region that is required for inv(16)-mediated transcriptional repression. This minimal repression domain is sufficient for the association of CBFβ/SMMHC with the mSin3A corepressor. In addition, the inv(16) fusion protein specifically associates with histone deacetylase 8 (HDAC8). inv(16)-mediated repression is sensitive to HDAC inhibitors. We propose a model whereby the inv(16) fusion protein associates with AML1 to convert AML1 into a constitutive transcriptional repressor.

scholarly journals Sec2 protein contains a coiled-coil domain essential for vesicular transport and a dispensable carboxy terminal domain.

1990 ◽  
Vol 110 (6) ◽  
pp. 1897-1909 ◽  
Author(s):  
J Nair ◽  
H Müller ◽  
M Peterson ◽  
P Novick
Keyword(s):  
Amino Acids ◽  
Secretory Pathway ◽  
Synthetic Lethality ◽  
Coiled Coil ◽  
Protein Product ◽  
Temperature Sensitive ◽  
Vesicular Traffic ◽  
Amino Terminal ◽  
Native Molecular Mass ◽  
Coiled Coil Domain

SEC2 function is required at the post-Golgi apparatus stage of the yeast secretory pathway. The SEC2 sequence encodes a protein product of 759 amino acids containing an amino terminal region that is predicted to be in an alpha-helical, coiled-coil conformation. Two temperature-sensitive alleles, sec2-41 and sec2-59, encode proteins truncated by opal stop codons and are suppressible by an opal tRNA suppressor. Deletion analysis indicates that removal of the carboxyl terminal 251 amino acids has no apparent phenotype, while truncation of 368 amino acids causes temperature sensitivity. The amino terminal half of the protein, containing the putative coiled-coil domain, is essential at all temperatures. Sec2 protein is found predominantly in the soluble fraction and displays a native molecular mass of greater than 500 kD. All phenotypes of the temperature-sensitive sec2 alleles are partially suppressed by duplication of the SEC4 gene, but the lethality of a sec2 disruption is not suppressed. The sec2-41 mutation exhibits synthetic lethality with the same subset of the late acting sec mutants as does sec4-8 and sec15-1. The Sec2 protein may function in conjunction with the Sec4 and Sec15 proteins to control vesicular traffic.

scholarly journals Characterization of kinectin, a kinesin-binding protein: primary sequence and N-terminal topogenic signal analysis.

1995 ◽  
Vol 6 (2) ◽  
pp. 171-183 ◽  
Author(s):  
H Yu ◽  
C V Nicchitta ◽  
J Kumar ◽  
M Becker ◽  
I Toyoshima ◽  
...  
Keyword(s):  
Amino Acids ◽  
Transmembrane Domain ◽  
Binding Protein ◽  
Coiled Coil ◽  
Integral Membrane Protein ◽  
In Vitro Translation ◽  
Predicted Amino Acid Sequence ◽  
Hydrophobic Region ◽  
Reading Frame

Kinectin is a kinesin-binding protein (Toyoshima et al., 1992) that is required for kinesin-based motility (Kumar et al., 1995). A kinectin cDNA clone containing a 4.7-kilobase insert was isolated from an embryonic chick brain cDNA library by immunoscreening with a panel of monoclonal antibodies. The cDNA contained an open reading frame of 1364 amino acids encoding a protein of 156 kDa. A bacterially expressed product of the full length cDNA bound purified kinesin. Transient expression in CV-1 cells gave an endoplasmic reticulum distribution that depended upon the N-terminal domain. Analysis of the predicted amino acid sequence indicated a highly hydrophobic near N-terminal stretch of 28 amino acids and a large portion (326-1248) of predicted alpha helical coiled coils. The 30-kDa fragment containing the N-terminal hydrophobic region was produced by cell-free in vitro translation and found to assemble with canine pancreas rough microsomes. Cleavage of the N terminus was not observed confirming its role as a potential transmembrane domain. Thus, the kinectin cDNA encodes a cytoplasmic-oriented integral membrane protein that binds kinesin and is likely to be a coiled-coil dimer.

scholarly journals Mutations in the Drosophila hook gene inhibit endocytosis of the boss transmembrane ligand into multivesicular bodies.

1996 ◽  
Vol 133 (6) ◽  
pp. 1205-1215 ◽  
Author(s):  
H Krämer ◽  
M Phistry
Keyword(s):  
Amino Acids ◽  
Transmembrane Protein ◽  
Coiled Coil ◽  
Specific Pattern ◽  
Multivesicular Bodies ◽  
Endocytic Compartment ◽  
Coiled Coil Domain ◽  
Type Pattern ◽  
Cell Type Specific ◽  
Endocytic Vesicles

Transmembrane ligands can be internalized across cell boundaries into receptor-expressing cells. In the developing Drosophila eye imaginal disc, the bride of sevenless transmembrane protein (boss) is expressed on the surface of R8 cells. After internalization into neighboring R7 cells, the boss protein accumulates in multivesicular bodies. In a search for genes that affect this cell-type-specific pattern of boss endocytosis, we found that mutations in the hook gene inhibit the accumulation of boss in multivesicular bodies of R7 cells. In addition, hook flies exhibit pleiotropic phenotypes including abnormal bristle morphology and eye degeneration. The wild-type-pattern of boss endocytosis was restored in hook mutants by a genomic rescue fragment containing the hook gene or by a hook cDNA expressed in R7 cells under control of a sevenless (sev) enhancer. The hook gene encodes a novel cytoplasmic protein of 679 amino acids with a central coiled-coil domain of some 200 amino acids. Truncated, epitope-tagged hook proteins coimmunoprecipitated the full-length protein, indicating dimerization mediated by the coiled-coil domain. The hook protein localizes to vesicular structures that are part of the endocytic compartment. The requirement of the hook protein in R7 cells for the accumulation of boss protein in multivesicular bodies, and the localization of the hook protein to endocytic vesicles indicate that the hook gene encodes a novel component of the endocytic compartment that plays an important role in the endocytosis of transmembrane ligands or their transport to multivesicular bodies.

scholarly journals Nonideal polar residues in the core of the coiled coil of FtsLB are critical for modulating its stability and activation

2021 ◽  
Author(s):  
Samuel J Craven ◽  
Samson G.F. Condon ◽  
Gladys Diaz-Vazquez ◽  
Qiang Cui ◽  
Alessandro Senes
Keyword(s):  
Amino Acids ◽  
Cell Wall ◽  
Cell Division ◽  
Conformational Changes ◽  
Structural Model ◽  
Coiled Coil ◽  
Fine Tuning ◽  
Marginal Stability ◽  
Structural Element ◽  
The Core

The FtsLB complex is a critical regulator of bacterial cell division, acting as a switch that modulates cell wall reconstruction. Evidence indicates that FtsLB exists in either an off or on state which supports the corresponding activation state of the peptidoglycan synthase complex FtsWI. In Escherichia coli, residues within FtsLB that are critical for this activation are located in a region near the C-terminal end of the periplasmic coiled coil, raising questions about the precise role of this conserved domain in the mechanism. Here, we investigate an unusual cluster of polar amino acids occurring within the core of the coiled coil. These amino acids likely reduce the structural stability of the domain and thus may be important for governing conformational changes. We found that mutating these positions to hydrophobic residues increased the thermal stability of FtsLB but caused cell division defects, suggesting that the coiled-coil domain is an intentionally "detuned" structural element. In addition, suppressor mutations were identified within the polar cluster, indicating that the precise identity of the polar amino acids is important for fine-tuning the structural balance between the off and on states. Based on energetic and sequence propensity considerations, we propose a revised structural model of the tetrameric FtsLB (named the "Y-model") in which the periplasmic domain splits into a pair of coiled-coil branches. In this configuration, the polar amino acids participate in packing within the core, but their hydrophilic terminal moieties remain more favorably exposed to water than in the original four-helix bundle model ("I-model"). The Y-model remains well structured during molecular dynamics simulations, unlike the I-model, and satisfies all known experimental constraints. For this reason, we propose the Y-model as the configuration of the coiled coil of FtsLB and that a shift in this architecture, dependent on its marginal stability, is involved in activating the complex during the process that triggers septal cell wall reconstruction.

scholarly journals The Vaccinia Virus 14-Kilodalton (A27L) Fusion Protein Forms a Triple Coiled-Coil Structure and Interacts with the 21-Kilodalton (A17L) Virus Membrane Protein through a C-Terminal α-Helix

1998 ◽  
Vol 72 (12) ◽  
pp. 10126-10137 ◽  
Author(s):  
María-Isabel Vázquez ◽  
German Rivas ◽  
David Cregut ◽  
Luis Serrano ◽  
Mariano Esteban
Keyword(s):  
Amino Acids ◽  
Vaccinia Virus ◽  
Structural Model ◽  
Analytical Ultracentrifugation ◽  
Transmembrane Domain ◽  
Coiled Coil ◽  
C Terminus ◽  
Α Helix ◽  
Mutant Forms ◽  
Helical Region

ABSTRACT The vaccinia virus 14-kDa protein (encoded by the A27L gene) plays an important role in the biology of the virus, acting in virus-to-cell and cell-to-cell fusions. The protein is located on the surface of the intracellular mature virus form and is essential for both the release of extracellular enveloped virus from the cells and virus spread. Sequence analysis predicts the existence of four regions in this protein: a structureless region from amino acids 1 to 28, a helical region from residues 29 to 37, a triple coiled-coil helical region from residues 44 to 72, and a Leu zipper motif at the C terminus. Circular dichroism spectroscopy, analytical ultracentrifugation, and chemical cross-linking studies of the purified wild-type protein and several mutant forms, lacking one or more of the above regions or with point mutations, support the above-described structural division of the 14-kDa protein. The two contiguous cysteine residues at positions 71 and 72 are not responsible for the formation of 14-kDa protein trimers. The location of hydrophobic residues at the a and d positions on a helical wheel and of charged amino acids in adjacent positions, e and g, suggests that the hydrophobic and ionic interactions in the triple coiled-coil helical region are involved in oligomer formation. This conjecture was supported by the construction of a three-helix bundle model and molecular dynamics. Binding assays with purified proteins expressed in Escherichia coli and cytoplasmic extracts from cells infected with a virus that does not produce the 14-kDa protein during infection (VVindA27L) show that the 21-kDa protein (encoded by the A17L gene) is the specific viral binding partner and identify the putative Leu zipper, the predicted third α-helix on the C terminus of the 14-kDa protein, as the region involved in protein binding. These findings were confirmed in vivo, following transfection of animal cells with plasmid vectors expressing mutant forms of the 14-kDa protein and infected with VVindA27L. We find the structural organization of 14kDa to be similar to that of other fusion proteins, such as hemagglutinin of influenza virus and gp41 of human immunodeficiency virus, except for the presence of a protein-anchoring domain instead of a transmembrane domain. Based on our observations, we have established a structural model of the 14-kDa protein.

Cloning and characterization of the mouse polyamine-modulatedfactor-1 (mPMF-1) gene: an alternatively spliced homologue of the human transcription factor

2001 ◽  
Vol 359 (2) ◽  
pp. 387-392 ◽  
Author(s):  
Yanlin WANG ◽  
Wendy DEVEREUX ◽  
Patrick M. WOSTER ◽  
Robert A. CASERO
Keyword(s):  
Amino Acids ◽  
Transcription Factor ◽  
Transcriptional Activation ◽  
Splice Variants ◽  
Coiled Coil ◽  
Human Protein ◽  
Related Factor ◽  
Polyamine Analogues ◽  
Coiled Coil Region ◽  
Alternatively Spliced

The natural polyamines and their analogues have been implicated in transcriptional regulation of specific genes. Human polyamine-modulated factor-1 (hPMF-1) was the first polyamine-responsive transcription factor identified. Here the mouse homologue of the hPMF-1 gene is described. Interestingly, the mouse gene (mPMF-1) codes for two alternatively spliced mRNAs. Both of the mouse splice variants, mPMF-1S and mPMF-1L, possess C-terminal coiled-coil domains nearly identical to that found in hPMF-1 and are highly homologous with the human protein. The C-terminal coiled-coil structure is necessary for transcriptional activation. However, the shorter protein, mPMF-1S, does not contain an N-terminal coiled-coil region as do both hPMF-1 and the longer mPMF-1L. mPMF-1L mRNA codes for a protein of 202 amino acids, 37 amino acids longer than the human protein. By contrast, mPMF-1S codes for only 133 amino acids, as a result of two exons being omitted compared with mPMF-1L. Both mouse transcription factors can interact with Nrf-2 (nuclear factor-E2-related factor 2), the normal partner of hPMF-1, substantiating the importance of the C-terminal coiled-coil region responsible for this interaction. Finally, the expression of mPMF-1 is induced when mouse M1 myeloid leukaemia cells are exposed to polyamine analogues, suggesting control similar to that observed for the hPMF-1.

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