scholarly journals Conserved charged residues in the leucine-rich repeat domain of the Ran GTPase activating protein are required for Ran binding and GTPase activation

1999 ◽  
Vol 343 (3) ◽  
pp. 653-662 ◽  
Author(s):  
Jörg HABERLAND ◽  
Volker GERKE
Keyword(s):  
Nucleocytoplasmic Transport ◽  
Small Gtpases ◽  
Structural Basis ◽  
Ran Gtpase ◽  
Protein Protein Interaction ◽  
Charged Residues ◽  
Gtpase Activation ◽  
Gtpase Cycle ◽  
Leucine Rich Repeats ◽  
Lrr Domain

GTPase activating proteins (GAPs) for Ran, a Ras-related GTPase participating in nucleocytoplasmic transport, have been identified in different species ranging from yeast to man. All RanGAPs are characterized by a conserved domain consisting of eight leucine-rich repeats (LRRs) interrupted at two positions by so-called separating regions, the latter being unique for RanGAPs within the family of LRR proteins. The cytosolic RanGAP activity is essential for the Ran GTPase cycle which in turn provides directionality in nucleocytoplasmic transport, but the structural basis for the interaction between Ran and its GAP has not been elucidated. In order to gain a better understanding of this interaction we generated a number of mutant RanGAPs carrying amino acid substitutions in the LRR domain and analysed their complex formation with Ran as well as their ability to stimulate the intrinsic GTPase activity of the G protein. We show that conserved charged residues present in the separating regions of the LRR domain are indispensable for efficient Ran binding and GAP activity. These separating regions contain three conserved arginines which could possibly serve as catalytic residues similar to the arginine fingers identified in GAPs for other small GTPases. However, mutations in two of these arginines do not affect the GAP activity and replacement of the third conserved arginine (Arg91 in human RanGAP) severely interferes not only with GAP activity but also with Ran binding. This indicates that RanGAP-stimulated GTP hydrolysis on Ran does not involve a catalytic arginine residue but requires certain charged residues of the LRR domain of the GAP for mediating the protein-protein interaction.

scholarly journals Crystal structure of the substrate-recognition domain of theShigellaE3 ligase IpaH9.8

2016 ◽  
Vol 72 (4) ◽  
pp. 269-275 ◽  
Author(s):  
Kenji Takagi ◽  
Minsoo Kim ◽  
Chihiro Sasakawa ◽  
Tsunehiro Mizushima
Keyword(s):  
Crystal Structure ◽  
Pathogenic Bacteria ◽  
Inflammatory Responses ◽  
Substrate Recognition ◽  
Structural Features ◽  
Host Cells ◽  
Structural Basis ◽  
Global Public Health ◽  
Leucine Rich Repeats ◽  
Lrr Domain

Infectious diseases caused by bacteria have significant impacts on global public health. During infection, pathogenic bacteria deliver a variety of virulence factors, called effectors, into host cells. TheShigellaeffector IpaH9.8 functions as an ubiquitin ligase, ubiquitinating the NF-κB essential modulator (NEMO)/IKK-γ to inhibit host inflammatory responses. IpaH9.8 contains leucine-rich repeats (LRRs) involved in substrate recognition and an E3 ligase domain. To elucidate the structural basis of the function of IpaH9.8, the crystal structure of the LRR domain ofShigellaIpaH9.8 was determined and this structure was compared with the known structures of other IpaH family members. This model provides insights into the structural features involved in substrate specificity.

Reading the phosphorylation code: binding of the 14-3-3 protein to multivalent client phosphoproteins

2020 ◽  
Vol 477 (7) ◽  
pp. 1219-1225 ◽  
Author(s):  
Nikolai N. Sluchanko
Keyword(s):  
Protein Function ◽  
Intrinsically Disordered Protein ◽  
Structural Basis ◽  
Major Protein ◽  
Post Translational Modifications ◽  
Protein Protein Interaction ◽  
Intrinsically Disordered ◽  
Biochemical Journal ◽  
Multiple Binding ◽  
And Control

Many major protein–protein interaction networks are maintained by ‘hub’ proteins with multiple binding partners, where interactions are often facilitated by intrinsically disordered protein regions that undergo post-translational modifications, such as phosphorylation. Phosphorylation can directly affect protein function and control recognition by proteins that ‘read’ the phosphorylation code, re-wiring the interactome. The eukaryotic 14-3-3 proteins recognizing multiple phosphoproteins nicely exemplify these concepts. Although recent studies established the biochemical and structural basis for the interaction of the 14-3-3 dimers with several phosphorylated clients, understanding their assembly with partners phosphorylated at multiple sites represents a challenge. Suboptimal sequence context around the phosphorylated residue may reduce binding affinity, resulting in quantitative differences for distinct phosphorylation sites, making hierarchy and priority in their binding rather uncertain. Recently, Stevers et al. [Biochemical Journal (2017) 474: 1273–1287] undertook a remarkable attempt to untangle the mechanism of 14-3-3 dimer binding to leucine-rich repeat kinase 2 (LRRK2) that contains multiple candidate 14-3-3-binding sites and is mutated in Parkinson's disease. By using the protein-peptide binding approach, the authors systematically analyzed affinities for a set of LRRK2 phosphopeptides, alone or in combination, to a 14-3-3 protein and determined crystal structures for 14-3-3 complexes with selected phosphopeptides. This study addresses a long-standing question in the 14-3-3 biology, unearthing a range of important details that are relevant for understanding binding mechanisms of other polyvalent proteins.

scholarly journals Structural basis for activation of trimeric Gi proteins by multiple growth factor receptors via GIV/Girdin

2014 ◽  
Vol 25 (22) ◽  
pp. 3654-3671 ◽  
Author(s):  
Changsheng Lin ◽  
Jason Ear ◽  
Krishna Midde ◽  
Inmaculada Lopez-Sanchez ◽  
Nicolas Aznar ◽  
...  
Keyword(s):  
Growth Factor ◽  
G Proteins ◽  
Protein Interaction ◽  
Cross Talk ◽  
Tyrosine Kinases ◽  
Molecular Mechanisms ◽  
Growth Factor Receptors ◽  
Structural Basis ◽  
Nucleotide Exchange ◽  
Protein Protein Interaction

A long-standing issue in the field of signal transduction is to understand the cross-talk between receptor tyrosine kinases (RTKs) and heterotrimeric G proteins, two major and distinct signaling hubs that control eukaryotic cell behavior. Although stimulation of many RTKs leads to activation of trimeric G proteins, the molecular mechanisms behind this phenomenon remain elusive. We discovered a unifying mechanism that allows GIV/Girdin, a bona fide metastasis-related protein and a guanine-nucleotide exchange factor (GEF) for Gαi, to serve as a direct platform for multiple RTKs to activate Gαi proteins. Using a combination of homology modeling, protein–protein interaction, and kinase assays, we demonstrate that a stretch of ∼110 amino acids within GIV C-terminus displays structural plasticity that allows folding into a SH2-like domain in the presence of phosphotyrosine ligands. Using protein–protein interaction assays, we demonstrated that both SH2 and GEF domains of GIV are required for the formation of a ligand-activated ternary complex between GIV, Gαi, and growth factor receptors and for activation of Gαi after growth factor stimulation. Expression of a SH2-deficient GIV mutant (Arg 1745→Leu) that cannot bind RTKs impaired all previously demonstrated functions of GIV—Akt enhancement, actin remodeling, and cell migration. The mechanistic and structural insights gained here shed light on the long-standing questions surrounding RTK/G protein cross-talk, set a novel paradigm, and characterize a unique pharmacological target for uncoupling GIV-dependent signaling downstream of multiple oncogenic RTKs.

Deregulated expression of the RanBP1 gene alters cell cycle progression in murine fibroblasts

1997 ◽  
Vol 110 (19) ◽  
pp. 2345-2357 ◽  
Author(s):  
A. Battistoni ◽  
G. Guarguaglini ◽  
F. Degrassi ◽  
C. Pittoggi ◽  
A. Palena ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Cell Cycle Progression ◽  
Protein Import ◽  
Proliferating Cells ◽  
Cycle Progression ◽  
Ran Gtpase ◽  
Genes Encoding ◽  
Active Transcription ◽  
Gtpase Cycle

RanBP1 is a molecular partner of the Ran GTPase, which is implicated in the control of several processes, including DNA replication, mitotic entry and exit, cell cycle progression, nuclear structure, protein import and RNA export. While most genes encoding Ran-interacting partners are constitutively active, transcription of the RanBP1 mRNA is repressed in non proliferating cells, is activated at the G1/S transition in cycling cells and peaks during S phase. We report here that forced expression of the RanBP1 gene disrupts the orderly execution of the cell division cycle at several stages, causing inhibition of DNA replication, defective mitotic exit and failure of chromatin decondensation during the telophase-to-interphase transition in cells that achieve nuclear duplication and chromosome segregation. These results suggest that deregulated RanBP1 activity interferes with the Ran GTPase cycle and prevents the functioning of the Ran signalling system during the cell cycle.

scholarly journals Ran GTPase Cycle and Importins α and β Are Essential for Spindle Formation and Nuclear Envelope Assembly in LivingCaenorhabditis elegans Embryos

2002 ◽  
Vol 13 (12) ◽  
pp. 4355-4370 ◽  
Author(s):  
Peter Askjaer ◽  
Vincent Galy ◽  
Eva Hannak ◽  
Iain W. Mattaj
Keyword(s):  
Nuclear Envelope ◽  
Cell Function ◽  
Small Gtpase ◽  
Spindle Formation ◽  
Ran Gtpase ◽  
Early Embryos ◽  
Nuclear Envelope Assembly ◽  
Importin Α ◽  
Gtpase Cycle

The small GTPase Ran has been found to play pivotal roles in several aspects of cell function. We have investigated the role of the Ran GTPase cycle in spindle formation and nuclear envelope assembly in dividing Caenorhabditis elegans embryos in real time. We found that Ran and its cofactors RanBP2, RanGAP, and RCC1 are all essential for reformation of the nuclear envelope after cell division. Reducing the expression of any of these components of the Ran GTPase cycle by RNAi leads to strong extranuclear clustering of integral nuclear envelope proteins and nucleoporins. Ran, RanBP2, and RanGAP are also required for building a mitotic spindle, whereas astral microtubules are normal in the absence of these proteins. RCC1(RNAi) embryos have similar abnormalities in the initial phase of spindle formation but eventually recover to form a bipolar spindle. Irregular chromatin structures and chromatin bridges due to spindle failure were frequently observed in embryos where the Ran cycle was perturbed. In addition, connection between the centrosomes and the male pronucleus, and thus centrosome positioning, depends upon the Ran cycle components. Finally, we have demonstrated that both IMA-2 and IMB-1, the homologues of vertebrate importin α and β, are essential for both spindle assembly and nuclear formation in early embryos.

scholarly journals In silico analyses of CD14 molecule reveals significant evolutionary diversity potentially associated with speciation and variable immune response in mammals

2019 ◽  
Author(s):  
Olanrewaju B Morenikeji ◽  
Bolaji N Thomas
Keyword(s):  
Immune Response ◽  
Complex Disease ◽  
Mammalian Species ◽  
Coding Region ◽  
Protein Protein Interaction ◽  
Species Response ◽  
Gene Coding ◽  
Protein Interactome ◽  
Mutational Hotspots ◽  
Leucine Rich Repeats

Cluster differentiation gene (CD14) is a family of monocyte differentiating genes that works in conjunction with lipopolysaccharide binding protein (LBP), forming a complex with TLR4 or LY96 to mediate innate immune response to pathogens. In this paper, we used different computational methods to elucidate the evolution of CD14 gene coding region in 14 mammalian species. Our analyses identified leucine rich repeats (LRRs) as the only significant domain across the CD14 protein of the 14 species, presenting with frequencies ranging from 1-4. Importantly, we found signal peptides located at mutational hotspots demonstrating this gene is conserved across these species. Out of the 10 selected variants analyzed in this study, only 6 were predicted to possess significant deleterious effect. Our predicted protein interactome showed a significant varying protein-protein interaction with CD14 protein across the species. This may be important for drug target and therapeutic manipulation for the treatment of many diseases. We conclude that these results contribute to our understanding of the CD14 molecular evolution, which underlays varying species response to complex disease traits.

scholarly journals Crystal structure of yeast Sis1 peptide-binding fragment and Hsp70 Ssa1 C-terminal complex

2006 ◽  
Vol 398 (3) ◽  
pp. 353-360 ◽  
Author(s):  
Jingzhi Li ◽  
Yunkun Wu ◽  
Xinguo Qian ◽  
Bingdong Sha
Keyword(s):  
Crystal Structure ◽  
Close Contact ◽  
Critical Role ◽  
Peptide Binding ◽  
Structural Basis ◽  
Cellular Processes ◽  
Terminal Form ◽  
Charged Residues ◽  
Domain I

Heat shock protein (Hsp) 40 facilitates the critical role of Hsp70 in a number of cellular processes such as protein folding, assembly, degradation and translocation in vivo. Hsp40 and Hsp70 stay in close contact to achieve these diverse functions. The conserved C-terminal EEVD motif in Hsp70 has been shown to regulate Hsp40–Hsp70 interaction by an unknown mechanism. Here, we provide a structural basis for this regulation by determining the crystal structure of yeast Hsp40 Sis1 peptide-binding fragment complexed with the Hsp70 Ssa1 C-terminal. The Ssa1 extreme C-terminal eight residues, G634PTVEEVD641, form a β-strand with the domain I of Sis1 peptide-binding fragment. Surprisingly, the Ssa1 C-terminal binds Sis1 at the site where Sis1 interacts with the non-native polypeptides. The negatively charged residues within the EEVD motif in Ssa1 C-terminal form extensive charge–charge interactions with the positively charged residues in Sis1. The structure-based mutagenesis data support the structural observations.

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