Dataset Information

Crystal structure of the plexin A3 intracellular region reveals an autoinhibited conformation through active site sequestration.

ABSTRACT: Plexin cell surface receptors bind to semaphorin ligands and transduce signals for regulating neuronal axon guidance. The intracellular region of plexins is essential for signaling and contains a R-Ras/M-Ras GTPase activating protein (GAP) domain that is divided into two segments by a Rho GTPase-binding domain (RBD). The regulation mechanisms for plexin remain elusive, although it is known that activation requires both binding of semaphorin to the extracellular region and a Rho-family GTPase (Rac1 or Rnd1) to the RBD. Here we report the crystal structure of the plexin A3 intracellular region. The structure shows that the N- and C-terminal portions of the GAP homologous regions together form a GAP domain with an overall fold similar to other Ras GAPs. However, the plexin GAP domain adopts a closed conformation and cannot accommodate R-Ras/M-Ras in its substrate-binding site, providing a structural basis for the autoinhibited state of plexins. A comparison with the plexin B1 RBD/Rnd1 complex structure suggests that Rnd1 binding alone does not induce a conformational change in plexin, explaining the requirement of both semaphorin and a Rho GTPase for activation. The structure also identifies an N-terminal segment that is important for regulation. Both the N-terminal segment and the RBD make extensive interactions with the GAP domain, suggesting the presence of an allosteric network connecting these three domains that integrates semaphorin and Rho GTPase signals to activate the GAP. The importance of these interactions in plexin signaling is shown by both cell-based and in vivo axon guidance assays.

SUBMITTER: He H

PROVIDER: S-EPMC2747167 | biostudies-literature | 2009 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Crystal structure of the plexin A3 intracellular region reveals an autoinhibited conformation through active site sequestration.

He Huawei H Yang Taehong T Terman Jonathan R JR Zhang Xuewu X

Proceedings of the National Academy of Sciences of the United States of America 20090826 37

Plexin cell surface receptors bind to semaphorin ligands and transduce signals for regulating neuronal axon guidance. The intracellular region of plexins is essential for signaling and contains a R-Ras/M-Ras GTPase activating protein (GAP) domain that is divided into two segments by a Rho GTPase-binding domain (RBD). The regulation mechanisms for plexin remain elusive, although it is known that activation requires both binding of semaphorin to the extracellular region and a Rho-family GTPase (Ra ...[more]

PMID: 19717441

Similar Datasets

Project description:Encephalomyocarditis virus (EMCV) is a member of the Cardiovirus genus within the large Picornaviridae family, which includes a number of important human and animal pathogens. The RNA-dependent RNA polymerase (RdRp) 3Dpol is a key enzyme for viral genome replication. In this study, we report the X-ray structures of two different crystal forms of the EMCV RdRp determined at 2.8- and 2.15-? resolution. The in vitro elongation and VPg uridylylation activities of the purified enzyme have also been demonstrated. Although the overall structure of EMCV 3Dpol is shown to be similar to that of the known RdRps of other members of the Picornaviridae family, structural comparisons show a large reorganization of the active-site cavity in one of the crystal forms. The rearrangement affects mainly motif A, where the conserved residue Asp240, involved in ribonucleoside triphosphate (rNTP) selection, and its neighbor residue, Phe239, move about 10 ? from their expected positions within the ribose binding pocket toward the entrance of the rNTP tunnel. This altered conformation of motif A is stabilized by a cation-? interaction established between the aromatic ring of Phe239 and the side chain of Lys56 within the finger domain. Other contacts, involving Phe239 and different residues of motif F, are also observed. The movement of motif A is connected with important conformational changes in the finger region flanked by residues 54 to 63, harboring Lys56, and in the polymerase N terminus. The structures determined in this work provide essential information for studies on the cardiovirus RNA replication process and may have important implications for the development of new antivirals targeting the altered conformation of motif A.The Picornaviridae family is one of the largest virus families known, including many important human and animal pathogens. The RNA-dependent RNA polymerase (RdRp) 3Dpol is a key enzyme for picornavirus genome replication and a validated target for the development of antiviral therapies. Solving the X-ray structure of the first cardiovirus RdRp, EMCV 3Dpol, we captured an altered conformation of a conserved motif in the polymerase active site (motif A) containing the aspartic acid residue involved in rNTP selection and binding. This altered conformation of motif A, which interferes with the correct positioning of the rNTP substrate in the active site, is stabilized by a number of residues strictly conserved among picornaviruses. The rearrangements observed suggest that this motif A segment is a dynamic element that can be modulated by external effectors, either activating or inhibiting enzyme activity, and this type of modulation appears to be general to all picornaviruses.

Project description:During embryogenesis motor axons navigate to their target muscles, where individual motor axons develop complex branch morphologies. The mechanisms that control axonal branching morphogenesis have been studied intensively, yet it still remains unclear when branches begin to form or how branch locations are determined. Live cell imaging of individual zebrafish motor axons reveals that the first axonal branches are generated at the ventral extent of the myotome via bifurcation of the growth cone. Subsequent branches are generated by collateral branching restricted to their synaptic target field along the distal portion of the axon. This precisely timed and spatially restricted branching process is disrupted in turnout mutants we identified in a forward genetic screen. Molecular genetic mapping positioned the turnout mutation within a 300 kb region encompassing eight annotated genes, however sequence analysis of all eight open reading frames failed to unambiguously identify the turnout mutation. Chimeric analysis and single cell labeling reveal that turnout function is required cell non-autonomously for intraspinal motor axon guidance and peripheral branch formation. turnout mutant motor axons form the first branch on time via growth cone bifurcation, but unlike wild-type they form collateral branches precociously, when the growth cone is still navigating towards the ventral myotome. These precocious collateral branches emerge along the proximal region of the axon shaft typically devoid of branches, and they develop into stable, permanent branches. Furthermore, we find that null mutants of the guidance receptor plexin A3 display identical motor axon branching defects, and time lapse analysis reveals that precocious branch formation in turnout and plexin A3 mutants is due to increased stability of otherwise short-lived axonal protrusions. Thus, plexin A3 dependent intrinsic and turnout dependent extrinsic mechanisms suppress collateral branch morphogenesis by destabilizing membrane protrusions before the growth cone completes navigation into the synaptic target field.

Project description:BackgroundThe Escherichia coli enzyme tryptophanase (TnaA) converts tryptophan to indole, which triggers physiological changes and regulates interactions between bacteria and their mammalian hosts. Tryptophanase production is induced by external tryptophan, but the activity of TnaA is also regulated by other, more poorly understood mechanisms. For example, the enzyme accumulates as a spherical inclusion (focus) at midcell or at one pole, but how or why this localization occurs is unknown.ResultsTnaA activity is low when the protein forms foci during mid-logarithmic growth but its activity increases as the protein becomes more diffuse, suggesting that foci may represent clusters of inactive (or less active) enzyme. To determine what protein characteristics might mediate these localization effects, we constructed 42 TnaA variants: 6 truncated forms and 36 missense mutants in which different combinations of 83 surface-exposed residues were converted to alanine. A truncated TnaA protein containing only domains D1 and D3 (D1D3) localized to the pole. Mutations affecting the D1D3-to-D1D3 interface did not affect polar localization of D1D3 but did delay assembly of wild type TnaA foci. In contrast, alterations to the D1D3-to-D2 domain interface produced diffuse localization of the D1D3 variant but did not affect the wild type protein. Altering several surface-exposed residues decreased TnaA activity, implying that tetramer assembly may depend on interactions involving these sites. Interestingly, changing any of three amino acids at the base of a loop near the catalytic pocket decreased TnaA activity and caused it to form elongated ovoid foci in vivo, indicating that the alterations affect focus formation and may regulate how frequently tryptophan reaches the active site.ConclusionsThe results suggest that TnaA activity is regulated by subcellular localization and by a loop-associated occlusion of its active site. Equally important, these new TnaA variants are immediately available to the research community and should be useful for investigating how tryptophanase is localized and assembled, how substrate accesses its active site, the functional role of acetylation, and other structural and functional questions.

Dataset Information

Crystal structure of the plexin A3 intracellular region reveals an autoinhibited conformation through active site sequestration.

Publications

Crystal structure of the plexin A3 intracellular region reveals an autoinhibited conformation through active site sequestration.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets