The Structure and Regulation of Human Muscle α-Actinin

SummaryThe spectrin superfamily of proteins plays key roles in assembling the actin cytoskeleton in various cell types, crosslinks actin filaments, and acts as scaffolds for the assembly of large protein complexes involved in structural integrity and mechanosensation, as well as cell signaling. α-actinins in particular are the major actin crosslinkers in muscle Z-disks, focal adhesions, and actin stress fibers. We report a complete high-resolution structure of the 200 kDa α-actinin-2 dimer from striated muscle and explore its functional implications on the biochemical and cellular level. The structure provides insight into the phosphoinositide-based mechanism controlling its interaction with sarcomeric proteins such as titin, lays a foundation for studying the impact of pathogenic mutations at molecular resolution, and is likely to be broadly relevant for the regulation of spectrin-like proteins

Structure of the Vesicular Stomatitis Virus N0-P Complex

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

Structure of the C-Terminal Domain of Lettuce Necrotic Yellows Virus Phosphoprotein

International audienceLettuce necrotic yellows virus (LNYV) is a prototype of the plant-adapted cytorhabdoviruses. Through a meta-prediction of disorder, we localized a folded C-terminal domain in the amino acid sequence of its phosphoprotein. This domain consists of an autonomous folding unit that is monomeric in solution. Its structure, solved by X-ray crystallography, reveals a lollipop-shaped structure comprising five helices. The structure is different from that of the corresponding domains of other Rhabdoviridae, Filoviridae, and Paramyxovirinae; only the overall topology of the polypeptide chain seems to be conserved, suggesting that this domain evolved under weak selective pressure and varied in size by the acquisition or loss of functional modules

Self-organization of the vesicular stomatitis virus nucleocapsid into a bullet shape.

International audienceThe typical bullet shape of Rhabdoviruses is thought to rely on the matrix protein for stabilizing the nucleocapsid coil. Here we scrutinize the morphology of purified and recombinant nucleocapsids of vesicular stomatitis virus in vitro. We elucidate pH and ionic strength conditions for their folding into conical tips and further growth into whole bullets, and provide cryo-electron microscopy reconstructions of the bullet tip and the helical trunk. We address conformational variability of the reconstituted nucleocapsids and the issue of constraints imposed by the binding of matrix protein. Our findings bridge the gap between the isolated nucleoprotein-RNA string in its form of an undulating ribbon, and the tight bullet-shaped virion skeleton

CC2D1A is a regulator of ESCRT-III CHMP4B.

International audienceEndosomal sorting complexes required for transport (ESCRTs) regulate diverse processes ranging from receptor sorting at endosomes to distinct steps in cell division and budding of some enveloped viruses. Common to all processes is the membrane recruitment of ESCRT-III that leads to membrane fission. Here, we show that CC2D1A is a novel regulator of ESCRT-III CHMP4B function. We demonstrate that CHMP4B interacts directly with CC2D1A and CC2D1B with nanomolar affinity by forming a 1:1 complex. Deletion mapping revealed a minimal CC2D1A-CHMP4B binding construct, which includes a short linear sequence within the third DM14 domain of CC2D1A. The CC2D1A binding site on CHMP4B was mapped to the N-terminal helical hairpin. Based on a crystal structure of the CHMP4B helical hairpin, two surface patches were identified that interfere with CC2D1A interaction as determined by surface plasmon resonance. Introducing these mutations into a C-terminal truncation of CHMP4B that exerts a potent dominant negative effect on human immunodeficiency virus type 1 budding revealed that one of the mutants lost this effect completely. This suggests that the identified CC2D1A binding surface might be required for CHMP4B polymerization, which is consistent with the finding that CC2D1A binding to CHMP4B prevents CHMP4B polymerization in vitro. Thus, CC2D1A might act as a negative regulator of CHMP4B function

Solution structure of the C-terminal nucleoprotein-RNA binding domain of the vesicular stomatitis virus phosphoprotein

International audienceBeyond common features in their genome organization and replication mechanisms, the evolutionary relationships among viruses of the Rhabdoviridae family are difficult too decipher because of the great variability in the amino acid sequence of their proteins. The phosphoprotein (P) of vesicular stomatitis virus (VSV) is an essential component of the RNA transcription and replication machinery; in particular, it contains binding sites for the RNA-dependent RNA polymerase and for the nucleoprotein. Here, we devised a new method for defining boundaries of structured domains from multiple disorder prediction algorithms, and we identified an autonomous folding C-terminal domain in VSV P(P-CTD). We show that, like the C-terminal domain of rabies virus (RV) P, VSV P-CTD binds to the viral nucleocapsid (nucleoprotein-RNA complex). We solved the three-dimensional structure of VSV P-CTD by NMR spectroscopy and found that the topology of its polypeptide chain resembles that of RV P-CTD. The common part of both proteins could be superimposed with a backbone RMSD from mean atomic coordinates of 2.6 angstrom. VSV P-CTD has a shorter N-terminal helix (alpha(1)) than RV P-CTD; it lacks two alpha-helices (helices alpha(3) and alpha(6) of RV P), and the loop between strands beta(1) and beta(2) is longer than that in RV. Dynamical properties measured by NMR relaxation revealed the presence of fast motions (below the nanosecond timescale) in loop regions (amino acids 209-214) and shower conformational exchange in the N- and C-terminal helices. Characterization of a longer construct indicated that P-CTD is preceded by a flexible linker. The results presented here support a modular organization of VSV P, with independent folded domains separated by flexible linkers, which is conserved among different genera of Rhabdoviridae and is similar to that proposed for the P proteins of the Paramyxoviridae

Ensemble Structure of the Highly Flexible Complex Formed between Vesicular Stomatitis Virus Unassembled Nucleoprotein and its Phosphoprotein Chaperone.

International audienceNucleocapsid assembly is an essential process in the replication of the non-segmented, negative-sense RNA viruses (NNVs). Unassembled nucleoprotein (N(0)) is maintained in an RNA-free and monomeric form by its viral chaperone, the phosphoprotein (P), forming the N(0)-P complex. Our earlier work solved the structure of vesicular stomatitis virus complex formed between an N-terminally truncated N (NΔ21) and a peptide of P (P60) encompassing the N(0)-binding site, but how the full-length P interacts with N(0) remained unknown. Here, we combine several experimental biophysical methods including size exclusion chromatography with detection by light scattering and refractometry, small-angle X-ray and neutron scattering and nuclear magnetic resonance spectroscopy with molecular dynamics simulation and computational modeling to characterize the NΔ21(0)-PFL complex formed with dimeric full-length P. We show that for multi-molecular complexes, simultaneous multiple-curve fitting using small-angle neutron scattering data collected at varying contrast levels provides additional information and can help refine structural ensembles. We demonstrate that (a) vesicular stomatitis virus PFL conserves its high flexibility within the NΔ21(0)-PFL complex and interacts with NΔ21(0) only through its N-terminal extremity; (b) each protomer of P can chaperone one N(0) client protein, leading to the formation of complexes with stoichiometries 1N:P2 and 2N:P2; and (c) phosphorylation of residues Ser60, Thr62 and Ser64 provides no additional interactions with N(0) but creates a metal binding site in PNTR. A comparison with the structures of Nipah virus and Ebola virus N(0)-P core complex suggests a mechanism for the control of nucleocapsid assembly that is common to all NNVs

Calcium modulates the domain flexibility and function of an α\alpha-actinin similar to the ancestral α-actinin

The actin cytoskeleton, a dynamic network of actin filaments and associated F-actin–binding proteins, is fundamentally important in eukaryotes. α-Actinins are major F-actin bundlers that are inhibited by Ca$^{2+}$ in nonmuscle cells. Here we report the mechanism of Ca$^{2+}$-mediated regulation of Entamoeba histolytica α-actinin-2 (EhActn2) with features expected for the common ancestor of Entamoeba and higher eukaryotic α-actinins. Crystal structures of Ca$^{2+}$-free and Ca$^{2+}$-bound EhActn2 reveal a calmodulin-like domain (CaMD) uniquely inserted within the rod domain. Integrative studies reveal an exceptionally high affinity of the EhActn2 CaMD for Ca$^{2+}$, binding of which can only be regulated in the presence of physiological concentrations of Mg$^{2+}$. C$^{2+}$ binding triggers an increase in protein multidomain rigidity, reducing conformational flexibility of F-actin–binding domains via interdomain cross-talk and consequently inhibiting F-actin bundling. In vivo studies uncover that EhActn2 plays an important role in phagocytic cup formation and might constitute a new drug target for amoebic dysentery