The plant hormone abscisic acid (ABA) has a central role in coordinating the adaptive response in situations of decreased water availability as well as the regulation of plant growth and development. Recently, a 14-member family of intracellular ABA receptors, named PYR/PYL/RCAR, has been identified. These proteins inhibit in an ABA-dependent manner the activity of a family of key negative regulators of the ABA signalling pathway: the group-A protein phosphatases type 2C (PP2Cs). Here we present the crystal structure of Arabidopsis thaliana PYR1, which consists of a dimer in which one of the subunits is bound to ABA. In the ligand-bound subunit, the loops surrounding the entry to the binding cavity fold over the ABA molecule, enclosing it inside, whereas in the empty subunit they form a channel leaving an open access to the cavity, indicating that conformational changes in these loops have a critical role in the stabilization of the hormone-receptor complex. By providing structural details on the ABA-binding pocket, this work paves the way for the development of new small molecules able to activate the plant stress response.

Nipah virus (NiV) is a highly pathogenic emergent paramyxovirus causing deadly encephalitis in humans. Its replication requires a constant supply of unassembled nucleoprotein (N(0)) in complex with its viral chaperone, the phosphoprotein (P). To elucidate the chaperone function of P, we reconstituted NiV the N(0)-P core complex and determined its crystal structure. The binding of the N-terminal region of P blocks the polymerization of N by interfering with subdomain exchange between N protomers and keeps N(0) in an open conformation, ready to grasp an RNA molecule. We found that a peptide derived from the N-binding region of P protects cells against viral infection and demonstrated by structure-based mutagenesis that this peptide acts by inhibiting N(0)-P formation. These results provide new insights about the assembly of N along genomic RNA and validate the N(0)-P complex as a target for drug development.

The genome of measles virus is encapsidated by multiple copies of the nucleoprotein (N), forming helical nucleocapsids of molecular mass approaching 150 Megadalton. The intrinsically disordered C-terminal domain of N (N TAIL ) is essential for transcription and replication of the virus via interaction with the phosphoprotein P of the viral polymerase complex. The molecular recognition element (MoRE) of N TAIL that binds P is situated 90 amino acids from the folded RNA-binding domain (N CORE ) of N, raising questions about the functional role of this disordered chain. Here we report the first in situ structural characterization of N TAIL in the context of the entire N-RNA capsid. Using nuclear magnetic resonance spectroscopy, small angle scattering, and electron microscopy, we demonstrate that N TAIL is highly flexible in intact nucleocapsids and that the MoRE is in transient interaction with N CORE . We present a model in which the first 50 disordered amino acids of N TAIL are conformationally restricted as the chain escapes to the outside of the nucleocapsid via the interstitial space between successive N CORE helical turns. The model provides a structural framework for understanding the role of N TAIL in the initiation of viral transcription and replication, placing the flexible MoRE close to the viral RNA and, thus, positioning the polymerase complex in its functional environment.is a member of the Paramyxoviridae family of the Mononegavirales order of negative sense, single stranded RNA viruses. The viral genome is encapsidated by multiple copies of the nucleoprotein (N) forming a helical nucleocapsid. Transcription and replication of the viral RNA are initiated by an interaction between N and the polymerase complex, composed of the phosphoprotein (P) and the RNAdependent RNA polymerase (1). N consists of two domains: N CORE (residues 1-400), responsible for the interaction with the viral RNA and for maintaining the nucleocapsid structure, and a long intrinsically disordered domain, N TAIL (residues 401-525) serving as the anchor point for the polymerase complex (2, 3). The molecular recognition element (MoRE) (residues 485-502) of the disordered N TAIL interacts with the C-terminal three-helix bundle domain, XD, of P (residues 459-507) (4) and thereby recruits the polymerase complex onto the nucleocapsid template (5, 6).The realization that intrinsically disordered proteins (IDPs) are functional despite a lack of structure (7-9) has revealed entirely new paradigms that appear to redefine our understanding of the role of conformational flexibility in molecular interactions (10-12). Until now most IDPs have been studied in isolation, or in the presence of a single interaction partner, although it is evident that a real physiological environment could influence the nature and relevance of apparent intrinsic disorder. In this context resolving the question of whether the protein is actually disordered in situ is of paramount importance. In this case the mechanistic role of the extensive disorder present in N TA...

In enterobacteria, the ampD gene encodes a cytosolic protein which acts as a negative regulator of beta-lactamase expression. It is shown here that the AmpD protein is a novel N-acetylmuramyl-L-alanine amidase (E.C.3.5.1.28) participating in the intracellular recycling of peptidoglycan fragments. Surprisingly, AmpD exhibits an exclusive specificity for substrates containing anhydro muramic acid. This anhydro bond is mainly found in the peptidoglycan degradation products formed by the periplasmic lytic transglycosylases and thus might behave as a 'recycling tag' allowing the enzyme to distinguish these fragments from the newly synthesized peptidoglycan precursors. The AmpD substrate (or substrates) which accumulates in the absence of the corresponding enzymatic activity acts as an intracellular positive effector for beta-lactamase expression and might represent an element of a communication network between the chromosome and the cell wall peptidoglycan.

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ALIX recruits ESCRT-III CHMP4 and is involved in membrane remodeling during endosomal receptor sorting, budding of some enveloped viruses and cytokinesis. We show that ALIX dimerizes via the middle domain (ALIX-V) in solution. Structural modeling based on small angle X-ray scattering (SAXS) data reveal an elongated crescent shaped conformation for dimeric ALIX lacking the proline rich domain (ALIXBRO1-V). Mutations at the dimerization interface prevent dimerization and induce an open elongated monomeric conformation of ALIX-V as determined by SAXS modeling. ALIX dimerizes in vivo and dimeric ALIX co-localizes with CHMP4B upon co-expression. We show further that ALIX dimerization affects HIV-1 budding. C-terminally truncated activated CHMP4B retaining the ALIX binding site forms linear, circular and helical filaments in vitro, which can be bridged by ALIX. Our data suggest that dimeric ALIX represents the active form that interacts with ESCRT-III CHMP4 polymers and functions as a scaffolding protein during membrane remodeling processes.

Replication of non-segmented negative-strand RNA viruses requires the continuous supply of the nucleoprotein (N) in the form of a complex with the phosphoprotein (P). Here, we present the structural characterization of a soluble, heterodimeric complex between a variant of vesicular stomatitis virus N lacking its 21 N-terminal residues (NΔ21) and a peptide of 60 amino acids (P60) encompassing the molecular recognition element (MoRE) of P that binds RNA-free N (N0). The complex crystallized in a decameric circular form, which was solved at 3.0 Å resolution, reveals how the MoRE folds upon binding to N and competes with RNA binding and N polymerization. Small-angle X-ray scattering experiment and NMR spectroscopy on the soluble complex confirms the binding of the MoRE and indicates that its flanking regions remain flexible in the complex. The structure of this complex also suggests a mechanism for the initiation of viral RNA synthesis.