N.Verdaguer and S.Corbalan-Garcia contributed equally to this workThe C2 domain acts as a membrane-targeting module in a diverse group of proteins including classical protein kinase Cs (PKCs), where it plays an essential role in activation via calcium-dependent interactions with phosphatidylserine. The three-dimensional structures of the Ca 2⍣ -bound forms of the PKCα-C2 domain both in the absence and presence of 1,2-dicaproyl-snphosphatidyl-L-serine have now been determined by X-ray crystallography at 2.4 and 2.6 Å resolution, respectively. In the structure of the C2 ternary complex, the glycerophosphoserine moiety of the phospholipid adopts a quasi-cyclic conformation, with the phosphoryl group directly coordinated to one of the Ca 2⍣ ions. Specific recognition of the phosphatidylserine is reinforced by additional hydrogen bonds and hydrophobic interactions with protein residues in the vicinity of the Ca 2ϩ binding region. The central feature of the PKCα-C2 domain structure is an eight-stranded, antiparallel β-barrel with a molecular topology and organization of the Ca 2⍣ binding region closely related to that found in PKCβ-C2, although only two Ca 2⍣ ions have been located bound to the PKCα-C2 domain. The structural information provided by these results suggests a membrane binding mechanism of the PKCα-C2 domain in which calcium ions directly mediate the phosphatidylserine recognition while the calcium binding region 3 might penetrate into the phospholipid bilayer.

Different mutants of Cowpea Mosaic Virus (CPMV) have been used as scaffolds to bind 2 and 5 nm gold nanoparticles through gold−sulfur bond formation at specific locations on the virus to produce patterns of specific interparticle distances. TEM images confirm that the bound gold particles produce patterns of gold nanoparticles that correlate well with models built from the known locations of the inserted cysteine groups on the capsid. These results demonstrate that it is possible to use CPMV mutants as nanoscale scaffolds to place gold nanoparticles at fixed interparticle distances.

The C2 domain of protein kinase Calpha (PKCalpha) corresponds to the regulatory sequence motif, found in a large variety of membrane trafficking and signal transduction proteins, that mediates the recruitment of proteins by phospholipid membranes. In the PKCalpha isoenzyme, the Ca2+-dependent binding to membranes is highly specific to 1,2-sn-phosphatidyl-l-serine. Intrinsic Ca2+ binding tends to be of low affinity and non-cooperative, while phospholipid membranes enhance the overall affinity of Ca2+ and convert it into cooperative binding. The crystal structure of a ternary complex of the PKCalpha-C2 domain showed the binding of two calcium ions and of one 1,2-dicaproyl-sn-phosphatidyl-l-serine (DCPS) molecule that was coordinated directly to one of the calcium ions. The structures of the C2 domain of PKCalpha crystallised in the presence of Ca2+ with either 1,2-diacetyl-sn-phosphatidyl-l-serine (DAPS) or 1,2-dicaproyl-sn-phosphatidic acid (DCPA) have now been determined and refined at 1.9 A and at 2.0 A, respectively. DAPS, a phospholipid with short hydrocarbon chains, was expected to facilitate the accommodation of the phospholipid ligand inside the Ca2+-binding pocket. DCPA, with a phosphatidic acid (PA) head group, was used to investigate the preference for phospholipids with phosphatidyl-l-serine (PS) head groups. The two structures determined show the presence of an additional binding site for anionic phospholipids in the vicinity of the conserved lysine-rich cluster. Site-directed mutagenesis, on the lysine residues from this cluster that interact directly with the phospholipid, revealed a substantial decrease in C2 domain binding to vesicles when concentrations of either PS or PA were increased in the absence of Ca2+. In the complex of the C2 domain with DAPS a third Ca2+, which binds an extra phosphate group, was identified in the calcium-binding regions (CBRs). The interplay between calcium ions and phosphate groups or phospholipid molecules in the C2 domain of PKCalpha is supported by the specificity and spatial organisation of the binding sites in the domain and by the variable occupancies of ligands found in the different crystal structures. Implications for PKCalpha activity of these structural results, in particular at the level of the binding affinity of the C2 domain to membranes, are discussed.

For most dsRNA viruses, the genome-enclosing capsid comprises 120 copies of a single capsid protein (CP) organized into 60 icosahedrally equivalent dimers, generally identified as 2 nonsymmetrically interacting CP molecules with extensive lateral contacts. The crystal structure of a partitivirus, Penicillium stoloniferum virus F (PsV-F), reveals a different organization, in which the CP dimer is related by almost-perfect local 2-fold symmetry, forms prominent surface arches, and includes extensive structure swapping between the 2 subunits. An electron cryomicroscopy map of PsV-F shows that the disordered N terminus of each CP molecule interacts with the dsRNA genome and probably participates in its packaging or transcription. Intact PsV-F particles mediate semiconservative transcription, and transcripts are likely to exit through negatively charged channels at the icosahedral 5-fold axes. Other findings suggest that the PsV-F capsid is assembled from dimers of CP dimers, with an arrangement similar to flavivirus E glycoproteins.capsid assembly ͉ mycovirus ͉ Partitiviridae ͉ partitivirus

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Cowpea mosaic virus (CPMV) is a robust, icosahedrally symmetric platform successfully used for attaching a variety of molecular substrates including proteins, fluorescent labels, and metals. The symmetric distribution and high local concentration of the attached molecules generates novel properties for the 30 nm particles. We report new CPMV reagent particles generated by systematic replacement of surface lysines with arginine residues. The relative reactivity of each lysine on the native particle was determined, and the two most reactive lysine residues were then created as single attachment sites by replacing all other lysines with arginine residues. Structural analysis of gold derivatization not only corroborated the specific reactivity of these unique lysine residues but also demonstrated their dramatically different presentation environment. Combined with site-directed cystine mutations, it is now possible to uniquely double label CPMV, expanding its use as an addressable nanoblock.

Infectious myonecrosis virus (IMNV) is an emerging pathogen of penaeid shrimp in global aquaculture. Tentatively assigned to familyTotiviridae, it has a nonsegmented dsRNA genome of 7,560 bp and an isometric capsid of the 901-aa major capsid protein. We used electron cryomicroscopy and 3D image reconstruction to examine the IMNV virion at 8.0-Å resolution. Results reveal a totivirus-like, 120-subunit T ‫؍‬ 1 capsid, 450 Å in diameter, but with fiber complexes protruding a further 80 Å at the fivefold axes. These protrusions likely mediate roles in the extracellular transmission and pathogenesis of IMNV, capabilities not shared by most other totiviruses. The IMNV structure is also notable in that the genome is centrally organized in five or six concentric shells. Within each of these shells, the densities alternate between a dodecahedral frame that connects the threefold axes vs. concentration around the fivefold axes, implying certain regularities in the RNA packing scheme.dsRNA virus ͉ electron cryomicroscopy ͉ nonenveloped virus ͉ penaeid shrimp ͉ Totiviridae I nfectious myonecrosis virus (IMNV) was first isolated from whiteleg shrimp, Litopenaeus vannamei, from aquaculture farms in northeast Brazil (1, 2). The associated disease was characterized by skeletal muscle necrosis, most markedly in distal abdomen and tail, with mortality over the harvest cycle nearing 70%. Purified IMNV virions reproduce this disease in pathogen-free L. vannamei (2). Diagnostics are available using reverse transcription and PCR to distinguish IMNV from other RNA viruses of shrimp (3, 4), and a survey from Pernambuco, Brazil, detected IMNV in 9 of 11 farms (5). IMNV has also been detected in L. vannamei from Indonesian farms, probably after transfer of aquaculture stocks (4).The genome sequence and many features of IMNV have been reported (2). Negatively stained virions exhibit an isometric capsid with a diameter of Ϸ400 Å. The virions contain a major capsid protein (MCP) of relative molecular weight (M r ) 106,000 and an N terminus of sequence IVSMENQSEID as shown by Edman degradation. The genome, a single molecule of 7,560-bp dsRNA, contains two extended ORFs in different frames of the plus strand: ORF1 in frame 1 (nucleotides 136-4953) and ORF2 in frame 3 (nucleotides 5241-7451). ORF1 encodes a 1,605-aa protein that includes the N-terminal sequence of the MCP starting at amino acid 705. The protein spanning amino acids 705-1605 have a sequencepredicted mass of 99 kDa, consistent with the M r of the MCP. The MCP thus seems to be cleaved from a larger precursor. A 60-aa region at the N terminus of ORF1 shares sequence similarities with dsRNA-binding proteins. ORF2 encodes a 736-aa protein that contains typical motifs of an RNA-dependent RNA polymerase (RdRp). Proteins representing ORF2 and the first 704 aa of ORF1 have yet to be identified, although candidate minor proteins have been seen in denaturing gels of IMNV virions. Phylogenetic analyses link IMNV to members of the family Totiviridae of nonsegmented dsRNA viruses with isomet...

Genetic economy leads to symmetric distributions of chemically identical subunits in icosaherdal and helical viruses. Modification of the subunit genes of a variety of viruses has permitted the display of polypeptides on both the infectious virions and virus particles made in expression systems. Icosahedral chimeric particles of this type often display novel properties resulting in high local concentrations of the insert. Here we report an extension of this concept in which entire proteins were chemically cross-linked to lysine and cysteine residues genetically engineered on the coat protein of icosahedral Cowpea mosaic virus particles. Three exogenous proteins, the LRR domain of internalin B, the T4 lysozyme, and the Intron 8 gene product of the of the HER2 tyrosine kinase receptor were derivatized with appropriate bifunctional cross-linkers and conjugated to the virus capsid. Characterization of these particles demonstrated that (1) virtually 100% occupancy of the 60 sites was achieved; (2) biological activity (either enzyme or binding specificity) of the attached protein was preserved; (3) in one case (LRR-internalin B) the attached protein conformed with the icosahedral symmetry to the extent that a reconstruction of the derivatized particles displayed added density with a shape consistent with the X-ray structure of the attached protein. Strategies demonstrated here allow virus particle targeting to specific cell types and the use of an icosahedral virus as a platform for structure determination of small proteins at moderate resolution.