Identification of Key Residues in the A-Raf Kinase Important for Phosphoinositide Lipid Binding Specificity

ABSTRACT Rac1 is a small guanine nucleotide binding protein that cycles between an inactive GDP-bound and active GTP-bound state to regulate cell motility and migration. Rac1 signaling is initiated from the plasma membrane (PM). Here, we used high-resolution spatial mapping and manipulation of PM lipid composition to define Rac1 nanoscale organization. We found that Rac1 proteins in the GTP- and GDP-bound states assemble into nonoverlapping nanoclusters; thus, Rac1 proteins undergo nucleotide-dependent segregation. Rac1 also selectively interacts with phosphatidic acid (PA) and phosphoinositol (3,4,5)-trisphosphate (PIP3), resulting in nanoclusters enriched in these lipids. These lipids are structurally important because depleting the PM of PA or PIP3 impairs both Rac1 PM binding and Rac1 nanoclustering. Lipid binding specificity of Rac1 is encoded in the amino acid sequence of the polybasic domain (PBD) of the C-terminal membrane anchor. Point mutations within the PBD, including arginine-to-lysine substitutions, profoundly alter Rac1 lipid binding specificity without changing the electrostatics of the protein and result in impaired macropinocytosis and decreased cell spreading. We propose that Rac1 nanoclusters act as lipid-based signaling platforms emulating the spatiotemporal organization of Ras proteins and show that the Rac1 PBD-prenyl anchor has a biological function that extends beyond simple electrostatic engagement with the PM.

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The Intermolecular Interaction between the PH Domain and the C-terminal Domain of Arabidopsis Dynamin-like 6 Determines Lipid Binding Specificity

Journal of Biological Chemistry ◽

10.1074/jbc.m204770200 ◽

2002 ◽

Vol 277 (35) ◽

pp. 31842-31849 ◽

Cited By ~ 22

Author(s):

Sung Hoon Lee ◽

Jing Bo Jin ◽

Jinhee Song ◽

Myung Ki Min ◽

Dae Sup Park ◽

...

Keyword(s):

Intermolecular Interaction ◽

Binding Specificity ◽

Lipid Binding ◽

Ph Domain ◽

Terminal Domain

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Determining the Lipid-Binding Specificity of SMP Domains: An ERMES Subunit as a Case Study

Methods in Molecular Biology - Intracellular Lipid Transport ◽

10.1007/978-1-4939-9136-5_16 ◽

2019 ◽

pp. 213-235 ◽

Cited By ~ 1

Author(s):

Andrew P. AhYoung ◽

Pascal F. Egea

Keyword(s):

Binding Specificity ◽

Lipid Binding

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A novel prenyl-polybasic domain code determines lipid-binding specificity of the K-Ras membrane anchor

Small GTPases ◽

10.1080/21541248.2017.1379583 ◽

2018 ◽

pp. 1-5 ◽

Cited By ~ 6

Author(s):

Yong Zhou ◽

John F. Hancock

Keyword(s):

Binding Specificity ◽

Lipid Binding ◽

Membrane Anchor

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Peptide binding specificity of HLA-DR4 molecules: correlation with rheumatoid arthritis association.

Journal of Experimental Medicine ◽

10.1084/jem.181.5.1847 ◽

1995 ◽

Vol 181 (5) ◽

pp. 1847-1855 ◽

Cited By ~ 216

Author(s):

J Hammer ◽

F Gallazzi ◽

E Bono ◽

R W Karr ◽

J Guenot ◽

...

Keyword(s):

Rheumatoid Arthritis ◽

Peptide Binding ◽

Binding Specificity ◽

Specific Pattern ◽

Single Amino Acid ◽

Amino Acid Exchange ◽

Peptide Binding Specificity ◽

Hla Dr ◽

Key Residues ◽

Acid Exchange

We have investigated whether sequence 67 to 74 shared by beta chains of rheumatoid arthritis (RA)-associated HLA-DR molecules imparts a specific pattern of peptide binding. The peptide binding specificity of the RA-associated molecules, DRB1*0401, DRB1*0404, and the closely related, RA nonassociated DRB1*0402 was, therefore, determined using designer peptide libraries. The effect of single key residues was tested with site-directed mutants of DRB1*0401. The results have demonstrated striking differences between RA-linked and unlinked DR allotypes in selecting the portion of peptides that interacts with the 67-74 area. Most differences were associated with a single amino acid exchange at position 71 of the DR beta chain, and affected the charge of residues potentially contacting position 71. The observed binding patterns permitted an accurate prediction of natural protein derived peptide sequences that bind selectively to RA-associated DR molecules. Thus, the 67-74 region, in particular position 71, induces changes of binding specificity that correlate with the genetic linkage of RA susceptibility. These findings should facilitate the identification of autoantigenic peptides involved in the pathogenesis of RA.

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The PX domain: a new phosphoinositide-binding module

Journal of Cell Science ◽

10.1242/jcs.115.6.1099 ◽

2002 ◽

Vol 115 (6) ◽

pp. 1099-1105 ◽

Cited By ~ 11

Author(s):

Chris D. Ellson ◽

Simon Andrews ◽

Len R. Stephens ◽

Phill T. Hawkins

Keyword(s):

Cell Survival ◽

Membrane Trafficking ◽

Critical Role ◽

Binding Specificity ◽

Structural Studies ◽

Sorting Nexins ◽

Px Domain ◽

Key Residues

The PX domain, which until recently was an orphan domain, has emerged as the latest member of the phosphoinositide-binding module superfamily. Structural studies have revealed that it has a novel fold and identified key residues that interact with the bound phosphoinositide, enabling some prediction of phosphoinositide-binding specificity. Specificity for PtdIns(3)P appears to be the most common, and several proteins containing PX domains localise to PtdIns(3)P-rich endosomal and vacuolar structures through their PX domains: these include the yeast t-SNARE Vam7p, mammalian sorting nexins (involved in membrane trafficking events) and the Ser/Thr kinase CISK, which is implicated in cell survival. Additionally,phosphoinositide binding to the PX domains of p40phox and p47phox appears to play a critical role in the active assembly of the neutrophil oxidase complex.

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Structure of Usutu virus SAAR-1776 displays fusion loop asymmetry

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2107408118 ◽

2021 ◽

Vol 118 (34) ◽

pp. e2107408118

Author(s):

Baldeep Khare ◽

Thomas Klose ◽

Qianglin Fang ◽

Michael G. Rossmann ◽

Richard J. Kuhn

Keyword(s):

Current Knowledge ◽

Lipid Binding ◽

Glycosylation Site ◽

Usutu Virus ◽

Host Interactions ◽

M Proteins ◽

Current Knowledge Base ◽

Donated Blood ◽

Key Residues ◽

Fusion Loop

Usutu virus (USUV) is an emerging arbovirus in Europe that has been increasingly identified in asymptomatic humans and donated blood samples and is a cause of increased incidents of neuroinvasive human disease. Treatment or prevention options for USUV disease are currently nonexistent, the result of a lack of understanding of the fundamental elements of USUV pathogenesis. Here, we report two structures of the mature USUV virus, determined at a resolution of 2.4 Å, using single-particle cryogenic electron microscopy. Mature USUV is an icosahedral shell of 180 copies of envelope (E) and membrane (M) proteins arranged in the classic herringbone pattern. However, unlike previous reports of flavivirus structures, we observe virus subpopulations and differences in the fusion loop disulfide bond. Presence of a second, unique E glycosylation site could elucidate host interactions, contributing to the broad USUV tissue tropism. The structures provide a basis for exploring USUV interactions with glycosaminoglycans and lectins, the role of the RGD motif as a receptor, and the inability of West Nile virus therapeutic antibody E16 to neutralize the mature USUV strain SAAR-1776. Finally, we identify three lipid binding sites and predict key residues that likely participate in virus stability and flexibility during membrane fusion. Our findings provide a framework for the development of USUV therapeutics and expand the current knowledge base of flavivirus biology.

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