Structure, function, and regulation of myosin 1C.

Myosin 1C, the first mammalian single-headed myosin to be purified, cloned, and sequenced, has been implicated in the translocation of plasma membrane channels and transporters. Like other forms of myosin I (of which eight exist in humans) myosin 1C consists of motor, neck, and tail domains. The neck domain binds calmodulins more tightly in the absence than in the presence of Ca(2+). Release of calmodulins exposes binding sites for anionic lipids, particularly phosphoinositides. The tail domain, which has an isoelectic point of 10.5, interacts with anionic lipid headgroups. When both neck and tail lipid binding sites are engaged, the myosin associates essentially irreversibly with membranes. Despite this tight membrane binding, it is widely believed that myosin 1C docking proteins are necessary for targeting the enzyme to specific subcellular location. The search for these putative myosin 1C receptors is an active area of research.

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Identification, characterization and cloning of myr 1, a mammalian myosin-I.

The Journal of Cell Biology ◽

10.1083/jcb.120.6.1393 ◽

1993 ◽

Vol 120 (6) ◽

pp. 1393-1403 ◽

Cited By ~ 71

Author(s):

C Ruppert ◽

R Kroschewski ◽

M Bähler

Keyword(s):

Amino Acid ◽

Light Chain ◽

Brush Border ◽

Binding Sites ◽

Neuronal Development ◽

Amino Acid Sequences ◽

Dependent Manner ◽

Tail Domain ◽

Myosin I ◽

Splice Forms

We have identified, characterized and cloned a novel mammalian myosin-I motor-molecule, called myr 1 (myosin-I from rat). Myr 1 exists in three alternative splice forms: myr 1a, myr 1b, and myr 1c. These splice forms differ in their numbers of putative calmodulin/light chain binding sites. Myr 1a-c were selectively released by ATP, bound in a nucleotide-dependent manner to F-actin and exhibited amino acid sequences characteristic of myosin-I motor domains. In addition to the motor domain, they contained a regulatory domain with up to six putative calmodulin/light chain binding sites and a tail domain. The tail domain exhibited 47% amino acid sequence identity to the brush border myosin-I tail domain, demonstrating that myr 1 is related to the only other mammalian myosin-I motor molecule that has been characterized so far. In contrast to brush border myosin-I which is expressed in mature enterocytes, myr 1 splice forms were differentially expressed in all tested tissues. Therefore, myr 1 is the first mammalian myosin-I motor molecule with a widespread tissue distribution in neonatal and adult tissues. The myr 1a splice form was preferentially expressed in neuronal tissues. Its expression was developmentally regulated during rat forebrain ontogeny and subcellular fractionation revealed an enrichment in purified growth cone particles, data consistent with a role for myr 1a in neuronal development.

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Multivalent lipid targeting by the calcium-independent C2A domain of Slp-4/granuphilin

10.1101/2020.05.29.123596 ◽

2020 ◽

Author(s):

Aml A Alnaas ◽

Abena Watson-Siriboe ◽

Sherleen Tran ◽

Mikias Negussie ◽

Jack A. Henderson ◽

...

Keyword(s):

Plasma Membrane ◽

Lipid Vesicles ◽

Lipid Binding ◽

Snare Complex ◽

Rab Proteins ◽

Secretory Vesicles ◽

High Affinity ◽

Anionic Lipids ◽

Dynamics Simulations ◽

C2a Domain

ABSTRACTSynaptotagmin-like protein 4 (Slp-4), also known as granuphilin, is a Rab effector responsible for docking secretory vesicles to the plasma membrane before exocytosis. Slp-4 binds vesicular Rab proteins via an N-terminal Slp homology (SHD) domain, interacts with plasma membrane SNARE complex proteins via a central linker region, and contains tandem C-terminal C2 domains (C2A and C2B) with affinity for phosphatidylinositol-(4,5)-bisphosphate (PIP2). The Slp-4 C2A domain binds with low nanomolar apparent affinity to PIP2 in lipid vesicles that also contain background anionic lipids such as phosphatidylserine (PS), but much weaker when either the background anionic lipids or PIP2 are removed. Through computational and experimental approaches, we show that this high affinity membrane interaction arises from concerted interaction at multiple sites on the C2A domain. In addition to a conserved PIP2-selective lysine cluster, there exists a larger cationic surface surrounding the cluster which contributes substantially to the affinity for physiologically relevant lipid compositions. While mutations at the PIP2-selective site decrease affinity for PIP2, multiple mutations are needed to decrease binding to physiologically relevant lipid compositions. Docking and molecular dynamics simulations indicate several conformationally flexible loops that contribute to the nonspecific cationic surface. We also identify and characterize a covalently modified variant in the bacterially expressed protein, which arises through reactivity of the PIP2-binding lysine cluster with endogenous bacterial compounds and has a low membrane affinity. Overall, multivalent lipid binding by the Slp-4 C2A domain provides selective recognition and high affinity docking of large dense-core secretory vesicles to the plasma membrane.

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Identification and assessment of cardiolipin interactions with E. coli inner membrane proteins

Science Advances ◽

10.1126/sciadv.abh2217 ◽

2021 ◽

Vol 7 (34) ◽

pp. eabh2217

Author(s):

Robin A. Corey ◽

Wanling Song ◽

Anna L. Duncan ◽

T. Bertie Ansell ◽

Mark S. P. Sansom ◽

...

Keyword(s):

Membrane Proteins ◽

Binding Site ◽

Binding Sites ◽

Lipid Binding ◽

Inner Membrane ◽

Ample Evidence ◽

Anionic Lipid ◽

E Coli ◽

Bacterial Proteins ◽

Dynamics Simulations

Integral membrane proteins are localized and/or regulated by lipids present in the surrounding bilayer. While bacteria have relatively simple membranes, there is ample evidence that many bacterial proteins bind to specific lipids, especially the anionic lipid cardiolipin. Here, we apply molecular dynamics simulations to assess lipid binding to 42 different Escherichia coli inner membrane proteins. Our data reveal an asymmetry between the membrane leaflets, with increased anionic lipid binding to the inner leaflet regions of the proteins, particularly for cardiolipin. From our simulations, we identify >700 independent cardiolipin binding sites, allowing us to identify the molecular basis of a prototypical cardiolipin binding site, which we validate against structures of bacterial proteins bound to cardiolipin. This allows us to construct a set of metrics for defining a high-affinity cardiolipin binding site on bacterial membrane proteins, paving the way for a heuristic approach to defining other protein-lipid interactions.

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Multivalent lipid targeting by the calcium-independent C2A domain of synaptotagmin-like protein 4/ granuphilin

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.014618 ◽

2020 ◽

pp. jbc.RA120.014618

Author(s):

Aml A Alnaas ◽

Abena Watson-Siriboe ◽

Sherleen Tran ◽

Mikias Negussie ◽

Jack A. Henderson ◽

...

Keyword(s):

Plasma Membrane ◽

Lipid Vesicles ◽

Lipid Binding ◽

Snare Complex ◽

Protein Domain ◽

Rab Proteins ◽

Secretory Vesicles ◽

High Affinity ◽

Anionic Lipids ◽

C2a Domain

Synaptotagmin-like protein 4 (Slp-4), also known as granuphilin, is a Rab effector responsible for docking secretory vesicles to the plasma membrane before exocytosis. Slp-4 binds vesicular Rab proteins via an N-terminal Slp homology domain, interacts with plasma membrane SNARE complex proteins via a central linker region, and contains tandem C-terminal C2 domains (C2A and C2B) with affinity for phosphatidylinositol-(4,5)-bisphosphate (PIP2). The Slp-4 C2A domain binds with low nanomolar apparent affinity to PIP2 in lipid vesicles that also contain background anionic lipids such as phosphatidylserine (PS), but much weaker when either the background anionic lipids or PIP2 are removed. Through computational and experimental approaches, we show that this high affinity membrane binding arises from concerted interaction at multiple sites on the C2A domain. In addition to a conserved PIP2-selective lysine cluster, a larger cationic surface surrounding the cluster contributes substantially to the affinity for physiologically relevant lipid compositions. Although the K398A mutation in the lysine cluster blocks PIP2 binding, this mutated protein domain retains the ability to bind physiological membranes in both a liposome binding assay and MIN6 cells. Molecular dynamics simulations indicate several conformationally flexible loops that contribute to the nonspecific cationic surface. We also identify and characterize a covalently modified variant that arises through reactivity of the PIP2-binding lysine cluster with endogenous bacterial compounds and binds weakly to membranes. Overall, multivalent lipid binding by the Slp-4 C2A domain provides selective recognition and high affinity docking of large dense-core secretory vesicles to the plasma membrane.

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Two distinct attachment sites for vimentin along the plasma membrane and the nuclear envelope in avian erythrocytes: a basis for a vectorial assembly of intermediate filaments.

The Journal of Cell Biology ◽

10.1083/jcb.105.1.105 ◽

1987 ◽

Vol 105 (1) ◽

pp. 105-115 ◽

Cited By ~ 128

Author(s):

S D Georgatos ◽

G Blobel

Keyword(s):

Plasma Membrane ◽

Nuclear Envelope ◽

Binding Sites ◽

Nuclear Surface ◽

Tail Domain ◽

Binding Studies ◽

Amino Terminal ◽

Attachment Sites ◽

10 Nm Filaments

In vitro binding studies with isolated bovine lens vimentin and avian erythrocyte membranes reveal the existence of two functionally distinct sets of intermediate filament attachment sites. One population of such receptors is located along the nuclear envelope and comprises polypeptides recognizing the carboxy-terminal tail domain of vimentin. Vimentin associates with these nuclear surface receptors in a cooperative manner and forms extensive 10-nm filaments in a concentration-dependent fashion. Conversely, the plasma membrane contains binding sites that interact in a noncooperative, saturable fashion with vimentin, recognizing its amino-terminal head domain. The functional dichotomy of the vimentin-binding sites under in vitro conditions may reflect a vectorial assembly process whereby 10-nm filaments, although structurally apolar, acquire polar features brought about by the differential attachment to specific receptors arranged along the plasma membrane and the nuclear envelope.

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Localization and specificity of the phospholipid and actin binding sites on the tail of Acanthamoeba myosin IC

The Journal of Cell Biology ◽

10.1083/jcb.117.6.1241 ◽

1992 ◽

Vol 117 (6) ◽

pp. 1241-1249 ◽

Cited By ~ 86

Author(s):

SK Doberstein ◽

TD Pollard

Keyword(s):

Binding Sites ◽

Binding Assay ◽

Lipid Binding ◽

Actin Binding ◽

Acidic Phospholipid ◽

Myosin I ◽

Head Groups ◽

Acidic Phospholipids ◽

Beta Galactosidase ◽

Actin Binding Site

We used bacterially expressed beta-galactosidase fusion proteins to localize the phospholipid binding domain of Acanthamoeba myosin IC to the region between amino acids 701 and 888 in the NH2-terminal half of the tail. Using a novel immobilized ligand lipid binding assay, we determined that myosin I can bind to several different acidic phospholipids, and that binding requires a minimum of 5 mol% acidic phospholipid in a neutral lipid background. The presence of di- and triglycerides and sterols in the lipid bilayer do not contribute to the affinity of myosin I for membranes. We confirm that the ATP-insensitive actin binding site is contained in the COOH-terminal 30 kD of the tail as previously shown for Acanthamoeba myosin IA. We conclude that the association of the myosin IC tail with acidic phospholipid head groups supplies much of the energy for binding myosin I to biological membranes, but probably not specificity for targeting myosin I isoforms to different cellular locations.

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Rat myr 4 defines a novel subclass of myosin I: identification, distribution, localization, and mapping of calmodulin-binding sites with differential calcium sensitivity.

The Journal of Cell Biology ◽

10.1083/jcb.126.2.375 ◽

1994 ◽

Vol 126 (2) ◽

pp. 375-389 ◽

Cited By ~ 69

Author(s):

M Bähler ◽

R Kroschewski ◽

H E Stöffler ◽

T Behrmann

Keyword(s):

Sequence Analysis ◽

Light Chain ◽

Binding Sites ◽

Adult Brain ◽

Regulatory Domain ◽

Tail Domain ◽

Iq Motif ◽

Calmodulin Binding ◽

Myosin I ◽

Punctate Staining

We report the identification and characterization of myr 4 (myosin from rat), the first mammalian myosin I that is not closely related to brush border myosin I. Myr 4 contains a myosin head (motor) domain, a regulatory domain with light chain binding sites and a tail domain. Sequence analysis of myosin I head (motor) domains suggested that myr 4 defines a novel subclass of myosin I's. This subclass is clearly different from the vertebrate brush border myosin I subclass (which includes myr 1) and the myosin I subclass(es) identified from Acanthamoeba castellanii and Dictyostelium discoideum. In accordance with this notion, a detailed sequence analysis of all myosin I tail domains revealed that the myr 4 tail is unique, except for a newly identified myosin I tail homology motif detected in all myosin I tail sequences. The Ca(2+)-binding protein calmodulin was demonstrated to be associated with myr 4. Calmodulin binding activity of myr 4 was mapped by gel overlay assays to the two consecutive light chain binding motifs (IQ motifs) present in the regulatory domain. These two binding sites differed in their Ca2+ requirements for optimal calmodulin binding. The NH2-terminal IQ motif bound calmodulin in the absence of free Ca2+, whereas the COOH-terminal IQ motif bound calmodulin in the presence of free Ca2+. A further Ca(2+)-dependent calmodulin binding site was mapped to amino acids 776-874 in the myr 4 tail domain. These results demonstrate a differential Ca2+ sensitivity for calmodulin binding by IQ motifs, and they suggest that myr 4 activity might be regulated by Ca2+/calmodulin. Myr 4 was demonstrated to be expressed in many cell lines and rat tissues with the highest level of expression in adult brain tissue. Its expression was developmentally regulated during rat brain ontogeny, rising 2-3 wk postnatally, and being maximal in adult brain. Immunofluorescence localization demonstrated that myr 4 is expressed in subpopulations of neurons. In these neurons, prominent punctate staining was detected in cell bodies and apical dendrites. A punctate staining that did not obviously colocalize with the bulk of F-actin was also observed in C6 rat glioma cells. The observed punctate staining for myr 4 is reminiscent of a membranous localization.

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Specific cardiolipin-SecY interactions are required for proton-motive-force stimulation of protein secretion

10.1101/202184 ◽

2017 ◽

Author(s):

Robin A. Corey ◽

Euan Pyle ◽

William J. Allen ◽

Marina Casiraghi ◽

Bruno Miroux ◽

...

Keyword(s):

Plasma Membrane ◽

Protein Secretion ◽

Binding Sites ◽

Protein Transport ◽

Proton Motive Force ◽

Lipid Binding ◽

Proton Exchange ◽

Motive Force ◽

Coarse Grained ◽

Stimulation Of

AbstractThe transport of proteins across or into membranes is a vital biological process, achieved in every cell by the conserved Sec machinery. In bacteria, SecYEG combines with the SecA motor protein for secretion of pre-proteins across the plasma membrane, powered by ATP hydrolysis and the trans-membrane proton-motive-force (PMF). The activities of SecYEG and SecA are modulated by membrane lipids, particularly by cardiolipin, a specialised phospholipid known to associate with a range of energy-transducing machines. Here, we identify two specific cardiolipin binding sites on the Thermotoga maritima SecA-SecYEG complex, through application of coarse-grained molecular dynamics simulations. We validate the computational data and demonstrate the conserved nature of the binding sites using in vitro mutagenesis, native mass spectrometry and biochemical analysis of Escherichia coli SecYEG. The results show that the two sites account for the preponderance of functional cardiolipin binding to SecYEG, and mediate its roles in ATPase and protein transport activity. In addition, we demonstrate an important role for cardiolipin in the conferral of PMF-stimulation of protein transport. The apparent transient nature of the CL interaction might facilitate proton exchange with the Sec machinery and thereby stimulate protein transport, by an as yet unknown mechanism. This study demonstrates the power of coupling the high predictive ability of coarse-grained simulation with experimental analyses, towards investigation of both the nature and functional implications of protein-lipid interactions.Significance StatementMany proteins are located in lipid membranes surrounding cells and cellular organelles. The membrane can impart important structural and functional effects on the protein, making understanding of this interaction critical. Here, we apply computational simulation to the identification of conserved lipid binding sites on an important highly conserved bacterial membrane protein, the Sec translocase (SecA-SecYEG), which uses ATP and the proton motive force (PMF) to secrete proteins across the bacterial plasma membrane. We experimentally validate and reveal the conserved nature of these binding sites, and use functional analyses to investigate the biological significance of this interaction. We demonstrate that these interactions are specific, transient, and critical for both ATP- and PMF- driven protein secretion.

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