scholarly journals Structural and immunological characterization of the myosin-like 110-kD subunit of the intestinal microvillar 110K-calmodulin complex: evidence for discrete myosin head and calmodulin-binding domains.

1988 ◽  
Vol 107 (5) ◽  
pp. 1749-1757 ◽  
Author(s):  
J M Carboni ◽  
K A Conzelman ◽  
R A Adams ◽  
D A Kaiser ◽  
T D Pollard ◽  
...  
Keyword(s):  
Binding Site ◽  
Brush Border ◽  
Myosin Head ◽  
Myosin Ii ◽  
Structural Similarity ◽  
Actin Binding ◽  
Atp Binding Site ◽  
Calmodulin Binding ◽  
Tryptic Fragment ◽  
Binding Domains

The actin bundle within each microvillus of the intestinal brush border is tethered laterally to the membrane by spirally arranged bridges. These bridges are thought to be composed of a protein complex consisting of a 110-kD subunit and multiple molecules of bound calmodulin (CM). Recent studies indicate that this complex, termed 110K-CM, is myosin-like with respect to its actin binding and ATPase properties. In this study, possible structural similarity between the 110-kD subunit and myosin was examined using two sets of mAbs; one was generated against Acanthamoeba myosin II and the other against the 110-kD subunit of avian 110K-CM. The myosin II mAbs had been shown previously to be cross-reactive with skeletal muscle myosin, with the epitope(s) localized to the 50-kD tryptic fragment of the subfragment-1 (S1) domain. The 110K mAbs (CX 1-5) reacted with the 110-kD subunit as well as with the heavy chain of skeletal but not with that of smooth or brush border myosin. All five of these 110K mAbs reacted with the 25-kD, NH2-terminal tryptic fragment of chicken skeletal S1, which contains the ATP-binding site of myosin. Similar tryptic digestion of 110K-CM revealed that these five mAbs all reacted with a 36-kD fragment of 110K (as well as larger 90- and 54-kD fragments) which by photoaffinity labeling was shown to contain the ATP-binding site(s) of the 110K subunit. CM binding to these same tryptic digests of 110K-CM revealed that only the 90-kD fragment retained both ATP- and CM-binding domains. CM binding was observed to several tryptic fragments of 60, 40, 29, and 18 kD, none of which contain the myosin head epitopes. These results suggest structural similarity between the 110K and myosin S1, including those domains involved in ATP- and actin binding, and provide additional evidence that 110K-CM is a myosin. These studies also support the results of Coluccio and Bretscher (1988. J. Cell Biol. 106:367-373) that the calmodulin-binding site(s) and the myosin head region of the 110-kD subunit lie in discrete functional domains of the molecule.

scholarly journals A Variable Domain near the ATP-Binding Site in Drosophila Muscle Myosin Is Part of the Communication Pathway between the Nucleotide and Actin-binding Sites

2007 ◽  
Vol 368 (4) ◽  
pp. 1051-1066 ◽  
Author(s):  
Becky M. Miller ◽  
Marieke J. Bloemink ◽  
Miklós Nyitrai ◽  
Sanford I. Bernstein ◽  
Michael A. Geeves
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Variable Domain ◽  
Actin Binding ◽  
Atp Binding ◽  
Atp Binding Site ◽  
Communication Pathway ◽  
Muscle Myosin

Calmodulin binding domains: characterization of a phosphorylation and calmodulin binding site from myosin light chain kinase

Biochemistry ◽  
1986 ◽  
Vol 25 (6) ◽  
pp. 1458-1464 ◽  
Author(s):  
Thomas J. Lukas ◽  
Wilson H. Burgess ◽  
Franklyn G. Prendergast ◽  
Wai Lau ◽  
D. Martin Watterson
Keyword(s):  
Binding Site ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Calmodulin Binding ◽  
Binding Domains

scholarly journals Biochemical and immunological characterization of p190-calmodulin complex from vertebrate brain: a novel calmodulin-binding myosin.

1992 ◽  
Vol 118 (2) ◽  
pp. 359-368 ◽  
Author(s):  
F S Espindola ◽  
E M Espreafico ◽  
M V Coelho ◽  
A R Martins ◽  
F R Costa ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Brush Border ◽  
Myosin Ii ◽  
Gel Filtration ◽  
Cross Reactivity ◽  
Mammalian Brain ◽  
Rat Tissues ◽  
Tail Region ◽  
Calmodulin Binding ◽  
Myosin I

We have recently identified a novel 190-kD calmodulin-binding protein (p190) associated with the actin-based cytoskeleton from mammalian brain (Larson, R. E., D. E. Pitta, and J. A. Ferro. 1988. Braz. J. Med. Biol. Res. 21:213-217; Larson, R. E., F. S. Espindola, and E. M. Espreafico. 1990. J. Neurochem. 54:1288-1294). These studies indicated that p190 is a phosphoprotein substrate for calmodulin-dependent kinase II and has calcium- and calmodulin-stimulated MgATPase activity. We now have biochemical and immunological evidence that this protein is a novel calmodulin-binding myosin whose properties include (a) Ca2+ dependent action activation of its Mg-ATPase activity, which seems to be mediated by Ca2+ binding directly to calmodulin(s) associated with p190 (maximal activation by actin requires the presence of Ca2+ and is further augmented by addition of exogenous calmodulin); (b) ATP-sensitive cross-linking of skeletal muscle F-actin, as demonstrated by the low-speed actin sedimentation assay; and (c) cross-reactivity with mAbs specific for epitopes in the head of brush border myosin I. We also show that p190 has properties distinct from conventional brain myosin II and brush border myosin I, including (a) separation of p190 from brain myosin II by gel filtration on a Sephacryl S-500 column; (b) lack by p190 of K(+)-stimulated EDTA ATPase activity characteristic of most myosins; (c) lack of immunological cross-reactivity of polyclonal antibodies which recognize p190 and brain myosin II, respectively; (d) lack of immunological recognition of p190 by mAbs against an epitope in the tail region of brush border myosin I; and (e) distinctive proteolytic susceptibility to calpain. A survey of rat tissues by immunoblotting indicated that p190 is expressed predominantly in the adult forebrain and cerebellum, and could be detected in embryos 11 d post coitus. Immunocytochemical studies showed p190 to be present in the perikarya and dendritic extensions of Purkinje cells of the cerebellum.

scholarly journals Exploring conformational equilibria of a heterodimeric ABC transporter

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
M Hadi Timachi ◽  
Cedric AJ Hutter ◽  
Michael Hohl ◽  
Tufa Assafa ◽  
Simon Böhm ◽  
...  
Keyword(s):  
Binding Site ◽  
High Temperatures ◽  
Abc Transporter ◽  
Thermotoga Maritima ◽  
Nucleotide Binding ◽  
Atp Binding ◽  
Molecular Feature ◽  
Atp Binding Site ◽  
Binding Domains ◽  
Conformational Equilibria

ABC exporters pump substrates across the membrane by coupling ATP-driven movements of nucleotide binding domains (NBDs) to the transmembrane domains (TMDs), which switch between inward- and outward-facing (IF, OF) orientations. DEER measurements on the heterodimeric ABC exporter TM287/288 from Thermotoga maritima, which contains a non-canonical ATP binding site, revealed that in the presence of nucleotides the transporter exists in an IF/OF equilibrium. While ATP binding was sufficient to partially populate the OF state, nucleotide trapping in the pre- or post-hydrolytic state was required for a pronounced conformational shift. At physiologically high temperatures and in the absence of nucleotides, the NBDs disengage asymmetrically while the conformation of the TMDs remains unchanged. Nucleotide binding at the degenerate ATP site prevents complete NBD separation, a molecular feature differentiating heterodimeric from homodimeric ABC exporters. Our data suggest hydrolysis-independent closure of the NBD dimer, which is further stabilized as the consensus site nucleotide is committed to hydrolysis.

Multiple unconventional myosin domains of the intestinal brush border cytoskeleton

1994 ◽  
Vol 107 (12) ◽  
pp. 3535-3543 ◽  
Author(s):  
M.B. Heintzelman ◽  
T. Hasson ◽  
M.S. Mooseker
Keyword(s):  
Brush Border ◽  
Myosin Ii ◽  
Actin Binding ◽  
Myosin Vi ◽  
Myosin V ◽  
Terminal Web ◽  
Intestinal Brush Border ◽  
Myosin I ◽  
Biochemical Fractionation

Representatives of class V and class VI unconventional myosins are identified as components of the intestinal brush border cytoskeleton. With brush border myosin-I and myosin-II, this brings to four the number of myosin classes associated with this one subcellular domain and represents the first characterization of four classes of myosins expressed in a single metazoan cell type. The distribution and cytoskeletal association of each myosin is distinct as assessed by both biochemical fractionation and immunofluorescence localization. Myosin-VI exists in both the microvillus and terminal web although the terminal web is the predominant site of concentration. Myosin-V is present in the terminal web and, most notably, at the distal ends of the microvilli, thus becoming the first actin-binding protein to be localized to this domain as assessed by both immunohistochemical and biochemical methods. In the undifferentiated enterocytes of the intestinal crypts, myosin-VI is expressed but not yet localized to the brush border, in contrast to myosin-V, which does demonstrate an apical distribution in these cells. An assessment of myosin abundance indicates that while myosin-II is the most abundant in the cell and in the brush border, brush border myosin-I is only slightly less abundant in contrast to myosins-V and -VI, both of which are two orders of magnitude less abundant than the others. Extraction studies indicate that of these four myosins, myosin-V is the most tightly associated with the brush border membrane, as detergent, in addition to ATP, is required for efficient solubilization.

Functional Characterization of the Secondary Actin Binding Site of Myosin II†

Biochemistry ◽  
1999 ◽  
Vol 38 (46) ◽  
pp. 15078-15085 ◽  
Author(s):  
Juliette Van Dijk ◽  
Marcus Furch ◽  
Chrystel Lafont ◽  
Dietmar J. Manstein ◽  
Patrick Chaussepied
Keyword(s):  
Binding Site ◽  
Myosin Ii ◽  
Functional Characterization ◽  
Actin Binding ◽  
Actin Binding Site

scholarly journals The effect of berbamine derivatives on activated Ca2+-stimulated Mg2+-dependent ATPase in erythrocyte membranes

1987 ◽  
Vol 248 (3) ◽  
pp. 985-988 ◽  
Author(s):  
Y Xu ◽  
J Liu ◽  
S Zhang ◽  
L Liu
Keyword(s):  
Oleic Acid ◽  
Binding Site ◽  
Enzyme Inhibition ◽  
Structural Similarity ◽  
Erythrocyte Membranes ◽  
Atp Binding ◽  
Atp Binding Site ◽  
Dependent Atpase ◽  
Naturally Occurring ◽  
Derivatives Of

Ca2+-stimulated Mg2+-dependent ATPase (Ca2+ + Mg2+-ATPase) stimulated by calmodulin, by partial proteolysis or by oleic acid in erythrocyte membranes was inhibited by various derivatives of the naturally occurring alkaloid berbamine. The ability of these derivatives to inhibit trypsin-activated Ca2+ + Mg2+-ATPase correlated well with their ability to inhibit the calmodulin-stimulated enzyme. Inhibition of the trypsin-activated Ca2+ + Mg2+-ATPase by O-4-(ethoxybutyl)berbamine (EBB) was competitive with respect to ATP. The Ki for inhibition was about 8 microM. These results suggest that the binding site of EBB on the activated Ca2+ + Mg2+-ATPase may bear structural similarity to that on calmodulin, and may be closely related to the ATP-binding site on the enzyme.

scholarly journals Structural and functional variations in skeletal-muscle and scallop muscle actins

1988 ◽  
Vol 256 (3) ◽  
pp. 853-859 ◽  
Author(s):  
H K Hue ◽  
J P Labbé ◽  
M C Harricane ◽  
J C Cavadore ◽  
Y Benyamin ◽  
...  
Keyword(s):  
Binding Site ◽  
Striated Muscle ◽  
Myosin Head ◽  
Smooth Muscle Actin ◽  
Actin Binding ◽  
Conformational Epitopes ◽  
Structural And Functional Properties ◽  
Actin Isoform ◽  
Antigenic Reactivity ◽  
Actin Binding Site

Structural and functional properties in two striated-muscle actins, one from a vertebrate, the other from an invertebrate (scallop), were compared in relation to a smooth-muscle actin isoform (aortic actin). In spite of differences in the variable N-terminal region, the two striated-muscle isoactins showed, in contrast with aortic actin, a large structural homology revealed by proteinase-susceptibility and interaction with the myosin head. Thus the myosin head may bind to the two striated-muscle actins in constant parts of the 18-113 sequence. In contrast, antigenic reactivity of conformational epitopes of these actins strongly differentiated scallop actin from the two others. The behaviour of the scallop actin appears to be related to several amino acid substitutions located near or at functional domains such as monomer-monomer binding site, DNAase-I-dependent actin-actin binding site and actin-severing domain, which modified the polypeptide chain exposure.

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