Determining selectivity of phosphoinositide-binding domains

Narayan, K A; Lemmon, Mark A.

doi:10.1016/j.ymeth.2006.05.006

Cited by 119 publications

(131 citation statements)

References 73 publications

(114 reference statements)

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“…The latter can be explained by the fact that the RFLR motif is present and may contribute to PtdIns ( for PtdIns(3)P to be 2.3 µM. This value is within the range of other PtdIns(3)P-binding protein domains such as the EEA1 FYVE and the Vam7p PX domains (131,132), but is 3-10-fold higher to that estimated for Avr1b and AvrL567 using effector binding to cells and liposome binding assays (11); this higher affinity value may be explained by the presence of additional tags in the proteins tested in these assays that may contribute to the binding (134). Nonetheless, a modest affinity of a protein to phosphoinositides, such as Avh5, may be required for its further release from intracellular membranes to target other subcellular compartments (135).…”

Section: Discussionmentioning

confidence: 53%

Structural Basis for Interactions of thePhytophthora sojaeRxLR Effector Avh5 with Phosphatidylinositol 3-Phosphate and for Host Cell Entry

Sun¹,

Kale²,

Azurmendi³

et al. 2013

MPMI

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Oomycetes, such as Phytophthora sojae, are plant pathogens that employ protein effectors that enter host cells to facilitate infection. Plants may overcome infection by recognizing pathogen effectors via intracellular receptors (R proteins) that form part of their defense system. Entry of some effector proteins into plant cells is mediated by conserved RxLR motifs in the effectors and phosphoinositides (PIPs) resident in the host plasma membrane such as phosphatidylinositol 3-phosphate (PtdIns(3)P). Recent reports differ regarding the regions on RxLR effector proteins involved in PIP recognition. To clarify these differences, I have structurally and functionally characterized the P. sojae effector, avirulence homolog-5 (Avh5).

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Section: Discussionmentioning

confidence: 53%

Structural Basis for Interactions of thePhytophthora sojaeRxLR Effector Avh5 with Phosphatidylinositol 3-Phosphate and for Host Cell Entry

Sun¹,

Kale²,

Azurmendi³

et al. 2013

MPMI

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“…We used surface plasmon resonance (SPR) to measure the interaction of F2F3 with phospholipid bilayers immobilized on a hydrophobically modified Biacore L1 SPR chip (30). All measurements were made relative to a surface containing 100% phosphatidylcholine (PC).…”

Section: Resultsmentioning

confidence: 99%

Affinity of talin-1 for the β3-integrin cytosolic domain is modulated by its phospholipid bilayer environment

Moore

Nygren

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Binding of the talin-1 FERM (4.1/ezrin/radixin/moesin) domain to the β3 cytosolic tail causes activation of the integrin αIIbβ3. The FERM domain also binds to acidic phospholipids. Although much is known about the interaction of talin-1 with integrins and lipids, the relative contribution of each interaction to integrin regulation and possible synergy between them remain to be clarified. Here, we examined the thermodynamic interplay between FERM domain binding to phospholipid bilayers and to its binding sites in the β3 tail. We found that although both the F0F1 and F2F3 subdomains of the talin-1 FERM domain bind acidic bilayers, the full-length FERM domain binds with an affinity similar to F2F3, indicating that F0F1 contributes little to the overall interaction. When free in solution, the β3 tail has weak affinity for the FERM domain. However, appending the tail to acidic phospholipids increased its affinity for the FERM domain by three orders of magnitude. Nonetheless, the affinity of the FERM for the appended tail was similar to its affinity for binding to bilayers alone. Thus, talin-1 binding to the β3 tail is a ternary interaction dominated by a favorable surface interaction with phospholipid bilayers and set by lipid composition. Nonetheless, interactions between the FERM domain, the β3 tail, and lipid bilayers are not optimized for a high-affinity synergistic interaction, even at the membrane surface. Instead, the interactions appear to be tuned in such a way that the equilibrium between inactive and active integrin conformations can be readily regulated.cytoskeleton | plasma membrane | platelet aggregation T he integrin αIIbβ3 resides on the platelet surface in equilibrium between resting and active conformations (1-5). On circulating platelets, αIIbβ3 is constrained in its inactive conformation to prevent spontaneous platelet aggregation. Similarly, the cytoskeletal protein talin-1, whose binding to the β3 cytosolic tail (CT) stabilizes the active conformation of αIIbβ3 (6-8), is sequestered away from the β3 CT (9). Besides interacting with integrins, talin-1 interacts with negatively charged phospholipids (10-12) and phosphoinositides (13)(14)(15)(16). Stimuli generated at sites of vascular damage recruit talin-1 to the platelet plasma membrane, thereby promoting αIIbβ3 activation (17-19). Much is known about the interaction of talin-1 with integrins and lipids, but the relative contribution of each interaction to integrin regulation and possible synergy between them remain to be clarified. Elucidating these interactions is important for understanding events leading to and controlling the formation of integrin complexes and for potential pharmacologic modulation of integrin signaling.Talin-1 is a 250-kD protein containing a 45-kD N-terminal head domain attached via a flexible linker to a 200-kD C-terminal rod domain (7). Because the head domain is packed against the rod domain in its inactive state (20), talin-1 recruitment to the membrane and to integrin β CTs requires disruption of this interaction (1...

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“…The proteins were then tested for their ability to bind to lipids using the well-established lipid vesicle sedimentation (LVS) assay (Narayan and Lemmon 2006). This method involves the quantitative pelleting of liposomal vesicles from a lipid/ protein mixture.…”

Section: Lipid Binding Assaysmentioning

confidence: 99%

“…The assay requires that the protein does not precipitate, and its precipitation is not promoted by the presence of the lipid due to nonspecific binding. Therefore, we used proteins that do not precipitate and several control-lipids (Narayan and Lemmon 2006). Control lipids are lipids that have similar or identical hydrophobic tails to lipids that Hsp70s bind but have different head groups and do not bind or show minimal binding to HspA1A (for example PC or PE are control lipids for PS).…”

Section: Lipid Binding Assaysmentioning

confidence: 99%

Biochemical characterization of the interaction between HspA1A and phospholipids

McCallister¹,

Kdeiss²,

Nikolaidis³

2016

Cell Stress and Chaperones

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Seventy-kilodalton heat shock proteins (Hsp70s) are molecular chaperones essential for maintaining cellular homeostasis. Apart from their indispensable roles in protein homeostasis, specific Hsp70s localize at the plasma membrane and bind to specific lipids. The interaction of Hsp70s with lipids has direct physiological outcomes including lysosomal rescue, microautophagy, and promotion of cell apoptosis. Despite these essential functions, the Hsp70-lipid interactions remain largely uncharacterized. In this study, we characterized the interaction of HspA1A, an inducible Hsp70, with five phospholipids. We first used high concentrations of potassium and established that HspA1A embeds in membranes when bound to all anionic lipids tested. Furthermore, we found that protein insertion is enhanced by increasing the saturation level of the lipids. Next, we determined that the nucleotide-binding domain (NBD) of the protein binds to lipids quantitatively more than the substrate-binding domain (SBD). However, for all lipids tested, the full-length protein is necessary for embedding. We also used calcium and reaction buffers equilibrated at different pH values and determined that electrostatic interactions alone may not fully explain the association of HspA1A with lipids. We then determined that lipid binding is inhibited by nucleotide-binding, but it is unaffected by protein-substrate binding. These results suggest that the HspA1A lipid-association is specific, depends on the physicochemical properties of the lipid, and is mediated by multiple molecular forces. These mechanistic details of the Hsp70-lipid interactions establish a framework of possible physiological functions as they relate to chaperone regulation and localization.

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Determining selectivity of phosphoinositide-binding domains

Cited by 119 publications

References 73 publications

Structural Basis for Interactions of thePhytophthora sojaeRxLR Effector Avh5 with Phosphatidylinositol 3-Phosphate and for Host Cell Entry

Structural Basis for Interactions of thePhytophthora sojaeRxLR Effector Avh5 with Phosphatidylinositol 3-Phosphate and for Host Cell Entry

Affinity of talin-1 for the β3-integrin cytosolic domain is modulated by its phospholipid bilayer environment

Biochemical characterization of the interaction between HspA1A and phospholipids

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