Joan Pous

The Birnavirus Crystal Structure Reveals Structural Relationships Among Icosahedral Viruses.

Double-stranded RNA virions are transcriptionally competent icosahedral particles that must translocate across a lipid bilayer to function within the cytoplasm of the target cell. Birnaviruses are unique among dsRNA viruses as they have a single T = 13 icosahedral shell, lacking the characteristic inner capsid observed in the others. We determined the crystal structures of the T = 1 subviral particle (260 angstroms in diameter) and of the T = 13 intact virus particle (700 angstroms in diameter) of an avian birnavirus to 3 angstroms and 7 angstroms resolution, respectively. Our results show that VP2, the only component of the virus icosahedral capsid, is homologous both to the capsid protein of positive-strand RNA viruses, like the T = 3 nodaviruses, and to the T = 13 capsid protein of members of the Reoviridae family of dsRNA viruses. Together, these results provide important insights into the multiple functions of the birnavirus capsid and reveal unexpected structural relationships among icosahedral viruses.

The Structure of an Engineered Domain-Swapped Ribonuclease Dimer and Its Implications for the Evolution of Proteins toward Oligomerization

BACKGROUND Domain swapping has been proposed as a mechanism that explains the evolution from monomeric to oligomeric proteins. Bovine and human pancreatic ribonucleases are monomers with no biological properties other than their RNA cleavage ability. In contrast, the closely related bovine seminal ribonuclease is a natural domain-swapped dimer that has special biological properties, such as cytotoxicity to tumour cells. Several recombinant ribonuclease variants are domain-swapped dimers, but a structure of this kind has not yet been reported for the human enzyme. RESULTS The crystal structure at 2 A resolution of an engineered ribonuclease variant called PM8 reveals a new kind of domain-swapped dimer, based on the change of N-terminal domains between the two subunits. The swapping is fastened at both hinge peptides by the newly introduced Gln101, involved in two intermolecular hydrogen bonds and in a stacking interaction between residues of different chains. Two antiparallel salt bridges and water-mediated hydrogen bonds complete a new interface between subunits, while the hinge loop becomes organized in a 3(10) helix structure. CONCLUSIONS Proteins capable of domain swapping may quickly evolve toward an oligomeric form. As shown in the present structure, a single residue substitution reinforces the quaternary structure by forming an open interface. An evolutionary advantage derived from the new oligomeric state will fix the mutation and favour others, leading to a more extended complementary dimerization surface, until domain swapping is no longer necessary for dimer formation. The newly engineered swapped dimer reported here follows this hypothetical pathway for the rapid evolution of proteins.

Structural Peptides of a Nonenveloped Virus Are Involved in Assembly and Membrane Translocation

ABSTRACT The capsid of infectious bursal disease virus (IBDV), a nonenveloped virus of the family Birnaviridae, has a T=13l icosahedral shell constituted by a single protein, VP2, and several disordered peptides, all derived from the precursor pVP2. In this study, we show that two of the peptides, pep11 and pep46, control virus assembly and cell entry. Deletion of pep11 or even simple substitution of most of its residues blocks the capsid morphogenesis. Removal of pep46 also prevents capsid assembly but leads to the formation of subviral particles formed by unprocessed VP2 species. Fitting with the VP2 atomic model into three-dimensional reconstructions of these particles demonstrates that the presence of uncleaved pep46 causes a steric hindrance at the vertices, blocking fivefold axis formation. Mutagenesis of the pVP2 maturation sites confirms that C terminus processing is necessary for VP2 to acquire the correct icosahedral architecture. All peptides present on virions are accessible to proteases or biochemical labeling. One of them, pep46, is shown to induce large structural rearrangements in liposomes and to destabilize target membranes, demonstrating its implication in cell entry.

Crystal structure of a complex of DNA with one AT-hook of HMGA1.

We present here for the first time the crystal structure of an AT-hook domain. We show the structure of an AT-hook of the ubiquitous nuclear protein HMGA1, combined with the oligonucleotide d(CGAATTAATTCG)2, which has two potential AATT interacting groups. Interaction with only one of them is found. The structure presents analogies and significant differences with previous NMR studies: the AT-hook forms hydrogen bonds between main-chain NH groups and thymines in the minor groove, DNA is bent and the minor groove is widened.

The interaction of manganese ions with DNA

We present the structure of the duplex formed by a fragment of auto-complementary DNA with the sequence d(CGTTAATTAACG). The structure was determined by X-ray crystallography. Up to date it is the first structure presenting the interaction between a DNA oligonucleotide and manganese ions. The presence of Mn2+ creates bonds among the N7 atom of guanines and phosphates. These bonds stabilize and determine the crystallographic network in a P3(2) space group, unusual in oligonucleotide crystals. The crystal structure observed is compared with those found in the presence of Mg2+, Ca2+ and Ni2+, which show different kinds of interactions. The double helices show end-to-end interactions, in a manner that the terminal guanines interact with the minor groove of the neighboring duplex, while the terminal cytosines are disordered. We have chosen this sequence since it contains a TTAA repeat. Such repeats are very rare in all genomes. We suggest that this sequence may be very susceptible to the formation of closely spaced thymine dimers.

Structural insights into the Ca2+ and PI(4,5)P2 binding modes of the C2 domains of rabphilin 3A and synaptotagmin 1

Significance Vesicle fusion is an important event in neuronal transmission and endocrine cell secretion. A myriad of proteins containing double C2 domains are involved in this complex process; however, how Ca2+ and the different types of membrane lipids regulate their function is still not well understood. In this work, we provide structural insights to explain the ability of different C2 domains to interact with Ca2+ and PI(4,5)P2 and demonstrate the existence of a specific PI(4,5)P2-binding motif that provides these domains with specific properties to interact with the membrane and initiate vesicle fusion. We also demonstrate a unique molecular mechanism conferring their specificity for the different phosphoinositides, which resides in additional amino acidic residues surrounding the key interacting lysines. Proteins containing C2 domains are the sensors for Ca2+ and PI(4,5)P2 in a myriad of secretory pathways. Here, the use of a free-mounting system has enabled us to capture an intermediate state of Ca2+ binding to the C2A domain of rabphilin 3A that suggests a different mechanism of ion interaction. We have also determined the structure of this domain in complex with PI(4,5)P2 and IP3 at resolutions of 1.75 and 1.9 Å, respectively, unveiling that the polybasic cluster formed by strands β3–β4 is involved in the interaction with the phosphoinositides. A comparative study demonstrates that the C2A domain is highly specific for PI(4,5)P2/PI(3,4,5)P3, whereas the C2B domain cannot discriminate among any of the diphosphorylated forms. Structural comparisons between C2A domains of rabphilin 3A and synaptotagmin 1 indicated the presence of a key glutamic residue in the polybasic cluster of synaptotagmin 1 that abolishes the interaction with PI(4,5)P2. Together, these results provide a structural explanation for the ability of different C2 domains to pull plasma and vesicle membranes close together in a Ca2+-dependent manner and reveal how this family of proteins can use subtle structural changes to modulate their sensitivity and specificity to various cellular signals.

Structure of the Triatoma virus capsid

The crystallographic structure of TrV shows specific morphological and functional features that clearly distinguish it from the type species of the Cripavirus genus, CrPV.

Three-dimensional structure of human RNase 1ΔN7 at 1.9 Å resolution

Human pancreatic ribonuclease 1 (RNase 1) is considered to be the human counterpart of bovine pancreatic RNase A. Truncation of seven amino-acid residues from the amino-terminal sequence resulted in RNase 1ΔN7, which has a reduced ribonucleolytic activity and a lower affinity for the human placental RNase inhibitor (PRI). This RNase 1 variant has been cloned, heterologously overexpressed, purified and crystallized. Its crystal structure has been determined and refined using data to 1.9 A resolution. The molecule displays the α + β folding topology typical of members of the RNase A superfamily. The main distinct features found in RNase 1ΔN7 are basically located in three loops affecting the fitting of the enzyme to the active site of subtilisin and the shape of the B2 subsite. These changes, taken with the lack of the catalytically active residue Lys7, may explain the reduced affinity of RNase 1ΔN7 for PRI and the low ribonucleolytic activity of the protein when compared with the native enzyme.

Minor group human rhinovirus-receptor interactions: Geometry of multimodular attachment and basis of recognition

X‐ray structures of human rhinovirus 2 (HRV2) in complex with soluble very‐low‐density lipoprotein receptors encompassing modules 1, 2, and 3 (V123) and five V3 modules arranged in tandem (V33333) demonstrates multi‐modular binding around the virions five‐fold axes. Occupancy was 60% for V123 and 100% for V33333 explaining the high‐avidity of the interaction. Surface potentials of 3D‐models of all minor group HRVs and K‐type major group HRVs were compared; hydrophobic interactions between a conserved lysine in the viruses and a tryptophan in the receptor modules together with coulombic attraction via diffuse opposite surface potentials determine minor group HRV receptor specificity.

Purification, crystallization and preliminary X‐ray diffraction studies of the bacteriophage φ29 connector particle

The connector or portal particle from double‐stranded DNA bacteriophage φ29 has been crystallized. This structure, which connects the head of the virus with the tail and plays a central role in prohead assembly and DNA packaging and translocation, is formed by 12 subunits of the p10 protein and has a molecular weight of 430 kDa. The connector structure was proteolysed with endoproteinase Glu‐C from Staphylococcus aureus V8, which removes 13 and 18 amino acids from the amino‐ and carboxy‐terminal regions of the p10 protein, respectively. Two crystal forms were grown from drops containing an alcohol solution and paraffin oil. Crystals of form I are monoclinic, space group C2 with cell dimensions a=416.86 Å, b=227.62 Å, c=236.68 Å and β=96.3° and contain four connector particles per asymmetric unit. Crystals of form II are tetragonal, space group P42212 with cell dimensions a=b=170.2 Å, c=156.9 Å and contain half a particle per asymmetric unit. X‐ray diffraction data from both native crystal forms have been collected to 6.0 and 3.2 Å respectively, using synchrotron radiation. Crystals of form II are likely to have the same packing arrangement as the two‐dimensional crystals analyzed previously by electron microscopy.