Structural basis of phosphodiesterase 6 inhibition by the C-terminal region of the γ-subunit

ABSTRACT Macrophagetropic R5 human immunodeficiency virus type 1 (HIV-1) isolates often evolve into dualtropic R5X4 variants during disease progression. The structural basis for CCR5 coreceptor function has been studied in a limited number of prototype strains and suggests that R5 and R5X4 Envs interact differently with CCR5. However, differences between unrelated viruses may reflect strain-specific factors and do not necessarily represent changes resulting from R5 to R5X4 evolution of a virus in vivo. Here we addressed CCR5 domains involved in fusion for a large set of closely related yet functionally distinct variants within a primary isolate swarm, employing R5 and R5X4 Envs derived from the HIV-1 89.6PI quasispecies. R5 variants of 89.6PI could fuse using either N-terminal or extracellular loop CCR5 sequences in the context of CCR5/CXCR2 chimeras, similar to the unrelated R5 strain JRFL, but R5X4 variants of 89.6PI were highly dependent on the CCR5 N terminus. Similarly, R5 89.6PI variants and isolate JRFL tolerated N-terminal CCR5 deletions, but fusion by most R5X4 variants was markedly impaired. R5 89.6PI Envs also tolerated multiple extracellular domain substitutions, while R5X4 variants did not. In contrast to CCR5 use, fusion by R5X4 variants of 89.6PI was largely independent of the CXCR4 N-terminal region. Thus, R5 and R5X4 species from a single swarm differ in how they interact with CCR5. These results suggest that R5 Envs possess a highly plastic capacity to interact with multiple CCR5 regions and support the concept that viral evolution in vivo results from the emergence of R5X4 variants with the capacity to use the CXCR4 extracellular loops but demonstrate less-flexible interactions with CCR5 that are strongly dependent on the N-terminal region.

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Orexin-A is composed of a highly conservedC-terminal and a specific, hydrophilicN-terminal region, revealing the structural basis of specific recognition by the orexin-1 receptor

Journal of Peptide Science ◽

10.1002/psc.747 ◽

2006 ◽

Vol 12 (7) ◽

pp. 443-454 ◽

Cited By ~ 20

Author(s):

Tomoyo Takai ◽

Takao Takaya ◽

Mutsuko Nakano ◽

Hideo Akutsu ◽

Atsushi Nakagawa ◽

...

Keyword(s):

Terminal Region ◽

Structural Basis ◽

Orexin A ◽

Specific Recognition

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Interaction sites of the C-terminal region of the cGMP phosphodiesterase inhibitory subunit with the GDP-bound transducin α-subunit

Biochemical Journal ◽

10.1042/bj3370281 ◽

1999 ◽

Vol 337 (2) ◽

pp. 281-288 ◽

Cited By ~ 2

Author(s):

Yu LIU ◽

Vadim Y. ARSHAVSKY ◽

Arnold E. RUOHO

Keyword(s):

Bound States ◽

Present Report ◽

Active Form ◽

Terminal Region ◽

Functional Difference ◽

Α Subunit ◽

Cgmp Phosphodiesterase ◽

Interaction Sites ◽

Γ Subunit ◽

Inactive Form

In the present report, the region of interaction between the GDP-bound α-subunit of transducin (αt.GTP) and the cGMP phosphodiesterase inhibitory γ-subunit (Pγ) has been studied. It is widely accepted that the αt.GTP is the active form of transducin and that the GDP-bound transducin α-subunit (αt.GDP) is the inactive form. We have reported previously that the binding region of the C-terminal of Pγ on αt.GTP is in a region between the exposed face of the α3 and α4 helices of αt.GTP [Liu, Arshavsky and Ruoho (1996) J. Biol. Chem. 271, 26900–26907]. We now report that N-[(3-[125I]iodo-4-azidophenylpropionamido-S-(2-thiopyridyl)]cysteine ([125I]ACTP)-derivatized Pγ (at Cys-68) reversibly undergoes a unique disulphide exchange of the radioiodinated moiety N-(3-[125I]iodo-4-azidophenylpropionamido)cysteine ([125I]APC) from Cys-68 of Pγ to αt.GDP but not to the guanosine 5´-(γ-thio)-triphosphate (GTP[S])-bound transducin α-subunit (αt-GTP[S]). The specificity of the interaction was demonstrated by the fact that exchange was protected by the functionally active Cys-68 → Ala Pγ mutant, and by pretreatment of the αt.GDP with the βγ-subunit of transducin. Chemical cleavage and amino acid sequencing demonstrated that the [125I]ACTP-derived Pγ specifically transferred the [125I]APC group to Cys-250 and Cys-210 of αt.GDP. These data indicate that the C-terminal region (especially Cys-68–Trp-70) of Pγ interacts with αt.GDP on the exposed interface between α2/β4 and α3/β5 of the α-subunit of transducin. Disulphide exchange was also observed with the α-subunit of holotransducin but this was only approx. 60% of that of pure αt.GDP. The variation in the binding pattern between αt.GDP and αt.GTP with the C-terminal region of Pγ may contribute to the functional difference between the GDP- and GTP-bound states.

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Structural basis for distinctive recognition of fibrinogen γC peptide by the platelet integrin αIIbβ3

The Journal of Cell Biology ◽

10.1083/jcb.200801146 ◽

2008 ◽

Vol 182 (4) ◽

pp. 791-800 ◽

Cited By ~ 143

Author(s):

Timothy A. Springer ◽

Jianghai Zhu ◽

Tsan Xiao

Keyword(s):

Metal Ion ◽

Structural Data ◽

Structural Basis ◽

Side Chain ◽

Α Subunit ◽

Integrin Αiibβ3 ◽

Rgd Motif ◽

Γ Subunit ◽

Fibrinogen Binding ◽

C Terminus

Hemostasis and thrombosis (blood clotting) involve fibrinogen binding to integrin αIIbβ3 on platelets, resulting in platelet aggregation. αvβ3 binds fibrinogen via an Arg-Asp-Gly (RGD) motif in fibrinogen's α subunit. αIIbβ3 also binds to fibrinogen; however, it does so via an unstructured RGD-lacking C-terminal region of the γ subunit (γC peptide). These distinct modes of fibrinogen binding enable αIIbβ3 and αvβ3 to function cooperatively in hemostasis. In this study, crystal structures reveal the integrin αIIbβ3–γC peptide interface, and, for comparison, integrin αIIbβ3 bound to a lamprey γC primordial RGD motif. Compared with RGD, the GAKQAGDV motif in γC adopts a different backbone configuration and binds over a more extended region. The integrin metal ion–dependent adhesion site (MIDAS) Mg2+ ion binds the γC Asp side chain. The adjacent to MIDAS (ADMIDAS) Ca2+ ion binds the γC C terminus, revealing a contribution for ADMIDAS in ligand binding. Structural data from this natively disordered γC peptide enhances our understanding of the involvement of γC peptide and integrin αIIbβ3 in hemostasis and thrombosis.

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Faculty Opinions recommendation of Structural basis of receptor sulfotyrosine recognition by a CC chemokine: the N-terminal region of CCR3 bound to CCL11/eotaxin-1.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.725363658.793504332 ◽

2015 ◽

Author(s):

Dimitrios Morikis

Keyword(s):

Terminal Region ◽

Structural Basis ◽

Cc Chemokine

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H,K-ATPase α subunit C-terminal membrane topology: epitope tags in the insect cell expression system

Biochemical Journal ◽

10.1042/bj3400601 ◽

1999 ◽

Vol 340 (3) ◽

pp. 601-611

Author(s):

Adam J. SMOLKA ◽

Kellie A. LARSEN ◽

Clifford W. SCHWEINFEST ◽

Charles E. HAMMOND

Keyword(s):

Expression System ◽

Recombinant Baculovirus ◽

Transmembrane Domains ◽

Terminal Region ◽

Structural Basis ◽

Sf9 Cells ◽

Β Subunit ◽

Α Subunit ◽

Baculovirus Infection ◽

P Type

The H,K-ATPase responsible for gastric acidification is a heterodimeric (α and β subunit) P-type ATPase, an integral protein of parietal cell apical membranes, which promotes the electroneutral exchange of K+ for protons, is stimulated by K+ and is inhibited by 2-methyl-8-(phenylmethoxy)imidazo[1,2-α]pyridine-3-acetonitrile (SCH 28080). Hydropathy analysis of the catalytic α subunit has been interpreted in terms of four N-terminal transmembrane domains, a cytoplasmically oriented segment containing ATP binding and phosphorylation sites, and a C-terminal region with four or six putative transmembrane domains. Several lines of evidence implicate the C-terminal region of P-type ATPases in cation-binding and occlusion, conformational changes, and interactions with the β subunit (HKβ), making the definition of topology a prerequisite for understanding the structural basis of these functions. Influenza haemagglutinin epitopes (YPYDVPDYA; flu tag) were inserted in predicted hydrophilic segments of the α subunit (HKα) to establish the membrane orientation of two amino acids with different predicted topologies in the C-terminal four- and six-transmembrane models. Wild-type and mutated HKα and HKβ cDNA species were expressed in insect cells (Sf9) via recombinant baculovirus infection, and expression of H,K-ATPase was verified by immunoblotting with HKα- and HKβ-specific and flu-tag-specific antibodies. Functional assays showed K+-stimulated, SCH 28080-sensitive ATPase activity, confirming neo-native topology in H,K-ATPase heterodimers expressed in Sf9 cells. The topology of flu tags was determined by microsomal protease protection assays in Sf9 cells and immunolabelling of HKα and HKβ in intact and permeabilized Sf9 cells. In addition, MS of native H,K-ATPase tryptic peptides identified cytoplasmically oriented HKα residues. The results indicated cytoplasmic exposure of Leu844 and Phe996, and luminal exposure of Pro898, leading to a revised secondary structure model of the C-terminal third of HKα.

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