Secondary Structure of the Novel Myosin Binding Domain WYR and Implications within Myosin Structure

Structural changes in the myosin II light meromyosin (LMM) that influence thick filament mechanical properties and muscle function are modulated by LMM-binding proteins. Flightin is an LMM-binding protein indispensable for the function of Drosophila indirect flight muscle (IFM). Flightin has a three-domain structure that includes WYR, a novel 52 aa domain conserved throughout Pancrustacea. In this study, we (i) test the hypothesis that WYR binds the LMM, (ii) characterize the secondary structure of WYR, and (iii) examine the structural impact WYR has on the LMM. Circular dichroism at 260–190 nm reveals a structural profile for WYR and supports an interaction between WYR and LMM. A WYR–LMM interaction is supported by co-sedimentation with a stoichiometry of ~2.4:1. The WYR–LMM interaction results in an overall increased coiled-coil content, while curtailing ɑ helical content. WYR is found to be composed of 15% turns, 31% antiparallel β, and 48% ‘other’ content. We propose a structural model of WYR consisting of an antiparallel β hairpin between Q92-K114 centered on an ASX or β turn around N102, with a G1 bulge at G117. The Drosophila LMM segment used, V1346-I1941, encompassing conserved skip residues 2-4, is found to possess a traditional helical profile but is interpreted as having <30% helical content by multiple methods of deconvolution. This low helicity may be affiliated with the dynamic behavior of the structure in solution or the inclusion of a known non-helical region in the C-terminus. Our results support the hypothesis that WYR binds the LMM and that this interaction brings about structural changes in the coiled-coil. These studies implicate flightin, via the WYR domain, for distinct shifts in LMM secondary structure that could influence the structural properties and stabilization of the thick filament, scaling to modulation of whole muscle function.

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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

Journal of Virology ◽

10.1128/jvi.72.12.10126-10137.1998 ◽

1998 ◽

Vol 72 (12) ◽

pp. 10126-10137 ◽

Cited By ~ 44

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.

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A Structural Model for Phosphorylation Control of Dictyostelium Myosin II Thick Filament Assembly

The Journal of Cell Biology ◽

10.1083/jcb.147.5.1039 ◽

1999 ◽

Vol 147 (5) ◽

pp. 1039-1048 ◽

Cited By ~ 30

Author(s):

Wenchuan Liang ◽

Hans M. Warrick ◽

James A. Spudich

Keyword(s):

Structural Model ◽

Myosin Ii ◽

Coiled Coil ◽

Thick Filament ◽

Tail Region ◽

Filament Assembly ◽

Dictyostelium Myosin ◽

Thick Filaments ◽

Coil Structure

Myosin II thick filament assembly in Dictyostelium is regulated by phosphorylation at three threonines in the tail region of the molecule. Converting these three threonines to aspartates (3×Asp myosin II), which mimics the phosphorylated state, inhibits filament assembly in vitro, and 3×Asp myosin II fails to rescue myosin II–null phenotypes. Here we report a suppressor screen of Dictyostelium myosin II–null cells containing 3×Asp myosin II, which reveals a 21-kD region in the tail that is critical for the phosphorylation control. These data, combined with new structural evidence from electron microscopy and sequence analyses, provide evidence that thick filament assembly control involves the folding of myosin II into a bent monomer, which is unable to incorporate into thick filaments. The data are consistent with a structural model for the bent monomer in which two specific regions of the tail interact to form an antiparallel tetrameric coiled–coil structure.

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Differential Requirement for the Nonhelical Tailpiece and the C Terminus of the Myosin Rod in Caenorhabditis elegansMuscle

Molecular Biology of the Cell ◽

10.1091/mbc.e02-11-0728 ◽

2003 ◽

Vol 14 (4) ◽

pp. 1677-1690 ◽

Cited By ~ 10

Author(s):

Pamela E. Hoppe ◽

Rebecca C. Andrews ◽

Payal D. Parikh

Keyword(s):

Muscle Development ◽

Striated Muscle ◽

Contractile Function ◽

Protein Localization ◽

Coiled Coil ◽

Thick Filament ◽

Normal Morphology ◽

Antibody Staining ◽

C Terminus

Myosin heavy chain (MHC) is a large, multidomain protein important for both cellular structure and contraction. To examine the functional role of two C-terminal domains, the end of the coiled-coil rod and the nonhelical tailpiece, we have generated constructs in which residues within these domains are removed or mutated, and examined their behavior in Caenorhabditis elegans striated muscle. Genetic tests demonstrate that MHC lacking only tailpiece residues is competent to support the timely onset of embryonic contractions, and therefore viability, in animals lacking full-length MHC. Antibody staining experiments show that this truncated molecule localizes as wild type in early stages of development, but may be defective in processes important for thick filament organization later in embryogenesis. Ultrastructural analysis reveals thick filaments of normal morphology in disorganized arrangement, as well as occasional abnormal assemblages. In contrast, molecules in which the four terminal residues of the coiled coil are absent or mutated fail to rescue animals lacking endogenous MHC. Loss of these four residues is associated with delayed protein localization and delayed contractile function during early embryogenesis. Our results suggest that these two MHC domains, the rod and the tailpiece, are required for distinct steps during muscle development.

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The myosin II coiled-coil domain atomic structure in its native environment

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2024151118 ◽

2021 ◽

Vol 118 (14) ◽

pp. e2024151118

Author(s):

Hamidreza Rahmani ◽

Wen Ma ◽

Zhongjun Hu ◽

Nadia Daneshparvar ◽

Dianne W. Taylor ◽

...

Keyword(s):

Atomic Structure ◽

Coiled Coil ◽

Thick Filament ◽

Terminal Segment ◽

The Other ◽

Cardiac Myosin ◽

Thick Filaments ◽

C Terminus ◽

Α Helix ◽

Lethocerus Indicus

The atomic structure of the complete myosin tail within thick filaments isolated from Lethocerus indicus flight muscle is described and compared to crystal structures of recombinant, human cardiac myosin tail segments. Overall, the agreement is good with three exceptions: the proximal S2, in which the filament has heads attached but the crystal structure doesn’t, and skip regions 2 and 4. At the head–tail junction, the tail α-helices are asymmetrically structured encompassing well-defined unfolding of 12 residues for one myosin tail, ∼4 residues of the other, and different degrees of α-helix unwinding for both tail α-helices, thereby providing an atomic resolution description of coiled-coil “uncoiling” at the head–tail junction. Asymmetry is observed in the nonhelical C termini; one C-terminal segment is intercalated between ribbons of myosin tails, the other apparently terminating at Skip 4 of another myosin tail. Between skip residues, crystal and filament structures agree well. Skips 1 and 3 also agree well and show the expected α-helix unwinding and coiled-coil untwisting in response to skip residue insertion. Skips 2 and 4 are different. Skip 2 is accommodated in an unusual manner through an increase in α-helix radius and corresponding reduction in rise/residue. Skip 4 remains helical in one chain, with the other chain unfolded, apparently influenced by the acidic myosin C terminus. The atomic model may shed some light on thick filament mechanosensing and is a step in understanding the complex roles that thick filaments of all species undergo during muscle contraction.

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Auto-regulation of Rab5 GEF activity in Rabex5 by allosteric structural changes, catalytic core dynamics and ubiquitin binding

eLife ◽

10.7554/elife.46302 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 3

Author(s):

Janelle Lauer ◽

Sandra Segeletz ◽

Alice Cezanne ◽

Giambattista Guaitoli ◽

Francesco Raimondi ◽

...

Keyword(s):

Mass Spectrometry ◽

Structural Changes ◽

Structural Model ◽

Regulation Mechanism ◽

Rab Gtpases ◽

Nucleotide Exchange ◽

Catalytic Core ◽

Exchange Mass Spectrometry ◽

C Terminus ◽

Ubiquitin Binding

Intracellular trafficking depends on the function of Rab GTPases, whose activation is regulated by guanine exchange factors (GEFs). The Rab5 GEF, Rabex5, was previously proposed to be auto-inhibited by its C-terminus. Here, we studied full-length Rabex5 and Rabaptin5 proteins as well as domain deletion Rabex5 mutants using hydrogen deuterium exchange mass spectrometry. We generated a structural model of Rabex5, using chemical cross-linking mass spectrometry and integrative modeling techniques. By correlating structural changes with nucleotide exchange activity for each construct, we uncovered new auto-regulatory roles for the ubiquitin binding domains and the Linker connecting those domains to the catalytic core of Rabex5. We further provide evidence that enhanced dynamics in the catalytic core are linked to catalysis. Our results suggest a more complex auto-regulation mechanism than previously thought and imply that ubiquitin binding serves not only to position Rabex5 but to also control its Rab5 GEF activity through allosteric structural alterations.

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Secondary Structural Model of MALAT1 Becomes Unstructured in Chronic Myeloid Leukemia and Undergoes Structural Rearrangement in Cervical Cancer

Non-Coding RNA ◽

10.3390/ncrna7010006 ◽

2021 ◽

Vol 7 (1) ◽

pp. 6

Author(s):

Matthew C. Wang ◽

Phillip J. McCown ◽

Grace E. Schiefelbein ◽

Jessica A. Brown

Keyword(s):

Cervical Cancer ◽

Chronic Myeloid Leukemia ◽

Hela Cells ◽

Binding Sites ◽

Myeloid Leukemia ◽

Structural Changes ◽

Structural Model ◽

Dimethyl Sulfate ◽

K562 Cells ◽

Cancer Types

Long noncoding RNAs (lncRNAs) influence cellular function through binding events that often depend on the lncRNA secondary structure. One such lncRNA, metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), is upregulated in many cancer types and has a myriad of protein- and miRNA-binding sites. Recently, a secondary structural model of MALAT1 in noncancerous cells was proposed to form 194 hairpins and 13 pseudoknots. That study postulated that, in cancer cells, the MALAT1 structure likely varies, thereby influencing cancer progression. This work analyzes how that structural model is expected to change in K562 cells, which originated from a patient with chronic myeloid leukemia (CML), and in HeLa cells, which originated from a patient with cervical cancer. Dimethyl sulfate-sequencing (DMS-Seq) data from K562 cells and psoralen analysis of RNA interactions and structure (PARIS) data from HeLa cells were compared to the working structural model of MALAT1 in noncancerous cells to identify sites that likely undergo structural alterations. MALAT1 in K562 cells is predicted to become more unstructured, with almost 60% of examined hairpins in noncancerous cells losing at least half of their base pairings. Conversely, MALAT1 in HeLa cells is predicted to largely maintain its structure, undergoing 18 novel structural rearrangements. Moreover, 50 validated miRNA-binding sites are affected by putative secondary structural changes in both cancer types, such as miR-217 in K562 cells and miR-20a in HeLa cells. Structural changes unique to K562 cells and HeLa cells provide new mechanistic leads into how the structure of MALAT1 may mediate cancer in a cell-type specific manner.

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The SPI2-encoded SseA chaperone has discrete domains required for SseB stabilization and export, and binds within the C-terminus of SseB and SseD

Microbiology ◽

10.1099/mic.0.26997-0 ◽

2004 ◽

Vol 150 (7) ◽

pp. 2055-2068 ◽

Cited By ~ 10

Author(s):

Daniel V. Zurawski ◽

Murry A. Stein

Keyword(s):

Type Iii Secretion ◽

Coiled Coil ◽

Amphipathic Helix ◽

Yeast Two Hybrid ◽

C Terminus ◽

N Terminus ◽

Domain Mapping ◽

Self Association ◽

Discrete Domains ◽

Two Hybrid

SseA, a key Salmonella virulence determinant, is a small, basic pI protein encoded within the Salmonella pathogenicity island 2 and serves as a type III secretion system chaperone for SseB and SseD. Both SseA partners are subunits of the surface-localized translocon module that delivers effectors into the host cell; SseB is predicted to compose the translocon sheath and SseD is a putative translocon pore subunit. In this study, SseA molecular interactions with its partners were characterized further. Yeast two-hybrid screens indicate that SseA binding requires a C-terminal domain within both partners. An additional central domain within SseD was found to influence binding. The SseA-binding region within SseB was found to encompass a predicted amphipathic helix of a type participating in coiled-coil interactions that are implicated in the assembly of translocon sheaths. Deletions that impinge upon this putative coiled-coiled domain prevent SseA binding, suggesting that SseA occupies a portion of the coiled-coil. SseA occupancy of this motif is envisioned to be sufficient to prevent premature SseB self-association inside bacteria. Domain mapping on the chaperone was also performed. A deletion of the SseA N-terminus, or site-directed mutations within this region, allowed stabilization of SseB, but its export was disrupted. Therefore, the N-terminus of SseA provides a function that is essential for SseB export, but dispensable for partner binding and stabilization.

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Phosphorylation of KLC1 modifies interaction with JIP1 and abolishes the enhanced fast velocity of APP transport by kinesin-1

Molecular Biology of the Cell ◽

10.1091/mbc.e17-05-0303 ◽

2017 ◽

Vol 28 (26) ◽

pp. 3857-3869 ◽

Cited By ~ 4

Author(s):

Kyoko Chiba ◽

Ko-yi Chien ◽

Yuriko Sobu ◽

Saori Hata ◽

Shun Kato ◽

...

Keyword(s):

Coiled Coil ◽

Amyloid Β ◽

Tetratricopeptide Repeat ◽

Amyloid Β Protein ◽

The Novel ◽

Anterograde Transport ◽

Interacting Protein ◽

Protein Precursor ◽

Kinesin Light Chain ◽

Β Protein

In neurons, amyloid β-protein precursor (APP) is transported by binding to kinesin-1, mediated by JNK-interacting protein 1b (JIP1b), which generates the enhanced fast velocity (EFV) and efficient high frequency (EHF) of APP anterograde transport. Previously, we showed that EFV requires conventional interaction between the JIP1b C-terminal region and the kinesin light chain 1 (KLC1) tetratricopeptide repeat, whereas EHF requires a novel interaction between the central region of JIP1b and the coiled-coil domain of KLC1. We found that phosphorylatable Thr466 of KLC1 regulates the conventional interaction with JIP1b. Substitution of Glu for Thr466 abolished this interaction and EFV, but did not impair the novel interaction responsible for EHF. Phosphorylation of KLC1 at Thr466 increased in aged brains, and JIP1 binding to kinesin-1 decreased, suggesting that APP transport is impaired by aging. We conclude that phosphorylation of KLC1 at Thr466 regulates the velocity of transport of APP by kinesin-1 by modulating its interaction with JIP1b.

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Reggie/flotillin proteins are organized into stable tetramers in membrane microdomains

Biochemical Journal ◽

10.1042/bj20061686 ◽

2007 ◽

Vol 403 (2) ◽

pp. 313-322 ◽

Cited By ~ 118

Author(s):

Gonzalo P. Solis ◽

Maja Hoegg ◽

Christina Munderloh ◽

Yvonne Schrock ◽

Edward Malaga-Trillo ◽

...

Keyword(s):

T Cell Activation ◽

Cell Activation ◽

Coiled Coil ◽

Cellular Prion Protein ◽

Membrane Microdomains ◽

Protein Assemblies ◽

Cellular Processes ◽

C Terminus ◽

Functional Relevance ◽

Interfering Rna

Reggie-1 and -2 proteins (flotillin-2 and -1 respectively) form their own type of non-caveolar membrane microdomains, which are involved in important cellular processes such as T-cell activation, phagocytosis and signalling mediated by the cellular prion protein and insulin; this is consistent with the notion that reggie microdomains promote protein assemblies and signalling. While it is generally known that membrane microdomains contain large multiprotein assemblies, the exact organization of reggie microdomains remains elusive. Using chemical cross-linking approaches, we have demonstrated that reggie complexes are composed of homo- and hetero-tetramers of reggie-1 and -2. Moreover, native reggie oligomers are indeed quite stable, since non-cross-linked tetramers are resistant to 8 M urea treatment. We also show that oligomerization requires the C-terminal but not the N-terminal halves of reggie-1 and -2. Using deletion constructs, we analysed the functional relevance of the three predicted coiled-coil stretches present in the C-terminus of reggie-1. We confirmed experimentally that reggie-1 tetramerization is dependent on the presence of coiled-coil 2 and, partially, of coiled-coil 1. Furthermore, since depletion of reggie-1 by siRNA (small interfering RNA) silencing induces proteasomal degradation of reggie-2, we conclude that the protein stability of reggie-2 depends on the presence of reggie-1. Our data indicate that the basic structural units of reggie microdomains are reggie homo- and hetero-tetramers, which are dependent on the presence of reggie-1.

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Secondary structure and biophysical activity of synthetic analogues of the pulmonary surfactant polypeptide SP-C

Biochemical Journal ◽

10.1042/bj3070535 ◽

1995 ◽

Vol 307 (2) ◽

pp. 535-541 ◽

Cited By ~ 65

Author(s):

J Johansson ◽

G Nilsson ◽

R Strömberg ◽

B Robertson ◽

H Jörnvall ◽

...

Keyword(s):

Secondary Structure ◽

Pulmonary Surfactant ◽

Transmembrane Helix ◽

Helical Structure ◽

Helical Conformation ◽

Synthetic Analogues ◽

Charged Amino Acids ◽

Phospholipid Micelles ◽

Helical Content ◽

Biophysical Activity

Native pulmonary-surfactant-associated lipopolypeptide SP-C, its chemically depalmitoylated form and several synthetic analogues lacking the palmitoylcysteine residues were analysed for secondary structure in phospholipid micelles and for biophysical activity in 1,2-dipalmitoyl-sn-glycero-3- phosphocholine/phosphatidylglycerol/palmitic acid (68:22:9, by wt.). Compared with the native molecule, with the entire poly-valyl part in a known alpha-helical conformation, depalmitoylated SP-C was found to be still mainly alpha-helical, but with an approx. 20% decrease in the helical content. A synthetic hybrid polypeptide where the entire poly-valyl alpha-helical part of native SP-C had been replaced with the amino acid sequence of a transmembrane helix of bacteriorhodopsin is also predominantly alpha-helical. In contrast, synthetic SP-C analogues lacking only the palmitoyl groups, by replacement of the palmitoylcysteine residues with cysteine, phenylalanine or serine, or lacking the positively charged amino acids by replacement with alanine, are considerably less alpha-helical than both native and depalmitoylated SP-C. The data indicate that the SP-C palmitoyl groups are important for maintenance of the alpha-helical conformation in parts of the polypeptide, and that the poly-valyl alpha-helical conformation is not fully formed in synthetic SP-C polypeptides. Furthermore, the helical structure of both native and depalmitoylated SP-C in dodecylphosphocholine micelles is very resistant to thermal denaturation, exhibiting ordered structure at 90 degrees C. The alpha-helical content grossly parallels the peptide-induced acceleration of the spreading of phospholipids at an air/water interface and the increase of surface pressure. The data suggest that the alpha-helical conformation itself, rather than just the covalent structure, is of prime importance for the biological function of synthetic pulmonary-surfactant peptides.

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