Rotavirus NSP4 is a multifunctional endoplasmic reticulum (ER)-resident nonstructural protein with the N terminus anchored in the ER and about 131 amino acids (aa) of the C-terminal tail (CT) oriented in the cytoplasm. Previous studies showed a peptide spanning aa 114 to 135 to induce diarrhea in newborn mouse pups with the 50% diarrheal dose approximately 100-fold higher than that for the full-length protein, suggesting a role for other regions in the protein in potentiating its diarrhea-inducing ability. In this report, employing a large number of methods and deletion and amino acid substitution mutants, we provide evidence for the cooperation between the extreme C terminus and a putative amphipathic ␣-helix located between aa 73 and 85 (AAH [73][74][75][76][77][78][79][80][81][82][83][84][85] ) at the N terminus of ⌬N72, a mutant that lacked the N-terminal 72 aa of nonstructural protein 4 (NSP4) from Hg18 and SA11. Cooperation between the two termini appears to generate a unique conformational state, specifically recognized by thioflavin T, that promoted efficient multimerization of the oligomer into high-molecular-mass soluble complexes and dramatically enhanced resistance against trypsin digestion, enterotoxin activity of the diarrhea-inducing region (DIR), and double-layered particle-binding activity of the protein. Mutations in either the C terminus, AAH 73-85 , or the DIR resulted in severely compromised biological functions, suggesting that the properties of NSP4 are subject to modulation by a single and/or overlapping highly sensitive conformational domain that appears to encompass the entire CT. Our results provide for the first time, in the absence of a three-dimensional structure, a unique conformation-dependent mechanism for understanding the NSP4-mediated pleiotropic properties including virus virulence and morphogenesis.Rotavirus is the most common cause of life-threatening, severe dehydrating diarrhea in children and animals (50). Rotavirus infection can be either symptomatic or asymptomatic. But the genetic/molecular basis for rotavirus virulence is not yet clearly understood. The recent identification of the nonstructural protein 4 (NSP4) as the first viral enterotoxin has attracted considerable attention toward understanding its structure and function. But analysis of NSP4 sequences from more than 175 strains failed to reveal any sequence motifs or amino acids that segregated with the virulence phenotype of the virus. Furthermore, a peptide spanning amino acids (aa) 114 to 135 was reported to induce diarrhea at an approximately 100-fold molar excess compared to the full-length protein (6). This suggested that other regions in the protein might influence its diarrhea-inducing potential. Also, the extreme C terminus, including the terminal methionine, was shown to be important for double-layered particle (DLP)-binding activity. NSP4 is 175 aa in length, with the N-terminal region anchored in the endoplasmic reticulum (ER) and approximately 131 aa of the C terminus oriented in the cytoplasm. The...
Arabidopsis Toc33 (atToc33) is a GTPase and a member of the Toc (translocon at the outer-envelope membrane of chloroplasts) complex that associates with precursor proteins during protein import into chloroplasts. By inference from the crystal structure of psToc34, a homologue in pea, the arginine at residue 130 (Arg 130 ) has been implicated in the formation of the atToc33 dimer and in intermolecular GTPase activation within the dimer. Here we report the crystal structure at 3.2-Å resolution of an atToc33 mutant, atToc33(R130A), in which Arg 130 was mutated to alanine. Both in solution and in crystals, atToc33(R130A) was present in its monomeric form. In contrast, both wild-type atToc33 and another pea Toc GTPase homologue, pea Toc159 (psToc159), were able to form dimers in solution. Dimeric atToc33 and psToc159 had significantly higher GTPase activity than monomeric atToc33, psToc159, and atToc33(R130A). Molecular modeling using the structures of psToc34 and atToc33(R130A) suggests that, in an architectural dimer of atToc33, Arg 130 from one monomer interacts with the -phosphate of GDP and several other amino acids of the other monomer. These results indicate that Arg 130 is critical for dimer formation, which is itself important for GTPase activity. Activation of GTPase activity by dimer formation is likely to be a critical regulatory step in protein import into chloroplasts.Chloroplast biogenesis relies on the import from the cytosol of the majority of chloroplast proteins, which are synthesized as precursors with N-terminal targeting signals. Import of precursor proteins into chloroplasts is mediated by a protein translocon complex, which is composed of the Toc 2 (translocon at the outer envelope membrane of chloroplasts) and the Tic (translocon at the inner envelope membrane of chloroplasts) proteins (for reviews, see Refs. 1 and 2). Three Toc proteins, Toc159, Toc75, and Toc34, form the core of the Toc complex (3). In Arabidopsis, Toc34 is encoded by two genes, atToc33 and atToc34, which share 59 and 64% amino acid identity with pea Toc34 (psToc34), respectively (4, 5). atToc33 is the major functional form in leaf chloroplasts (6, 7).Toc75 forms a protein-conducting channel across the outer membrane (8, 9). Toc34 has a cytosol-exposed GTPase domain followed by a C-terminal membrane anchor (10). Toc159 consists of an acidic N-terminal domain, followed by a GTPase domain homologous to that of Toc34, and a membrane-protected C-terminal domain (11). Toc159 and Toc34 function as the initial receptors for incoming precursor molecules. Nonhydrolyzable GTP analogues severely inhibit precursor binding to chloroplasts (12, 13). It has also been shown in vitro that the sequential transfer of precursors between these two receptors is regulated by GTP hydrolysis (14). Targeting of the two receptors themselves to chloroplasts is also dependent on GTP (15-19). Therefore activation and regulation of the GTPase activity of these two receptors are critical for protein import into chloroplasts.Previously we have reported th...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.