Structural characterization of the oligomerization of full-length Hantaan virus polymerase into symmetric dimers and hexamers.

Hantaan virus is a dangerous human pathogen whose segmented negative-stranded RNA genome is replicated and transcribed by a virally-encoded multi-functional polymerase. Here we describe the complete cryo-electron microscopy structure of Hantaan virus polymerase in several oligomeric forms. Apo polymerase protomers can adopt two drastically different conformations, which assemble into two distinct symmetric homodimers, that can themselves gather to form hexamers.

High-Resolution Electron Diffraction of Hydrated Protein Crystals at Room Temperature.

Structural characterization is crucial to understanding protein function. Compared with X-ray diffraction methods, electron crystallography can be performed on nanometer-sized crystals and can provide additional information from the resulting Coulomb potential map. Whereas electron crystallography has successfully resolved three-dimensional structures of vitrified protein crystals, its widespread use as a structural biology tool has been limited.

High pressure freezing and cryo-sectioning can be used for protein structure determination by electron diffraction.

Electron diffraction of three-dimensional nanometer sized crystals has emerged since 2013 as an efficient technique to solve the structure of both small organic molecules and model proteins. However, the major bottleneck of the technique when applied to protein samples is to produce nano-crystals that do not exceed 200 to 300 nm in at least one dimension and to deposit them on a grid while keeping the minimum amount of solvent around them. Since the presence of amorphous solvent around the crystal, necessary to preserve its integrity, increases the amount of diffuse scattering, thus degrading the signal-to noise ratio of the diffraction signal, other sample preparation strategies have been developed.

Insights into the ligand binding specificity of SREC-II (scavenger receptor expressed by endothelial cells).

SREC-II (scavenger receptor expressed by endothelial cells II) is a membrane protein encoded by the SCARF2 gene, with high homology to class F scavenger receptor SR-F1, but no known scavenging function. We produced the extracellular domain of SREC-II in a recombinant form and investigated its capacity to interact with common scavenger receptor ligands, including acetylated low-density lipoprotein (AcLDL) and maleylated or acetylated BSA (MalBSA or AcBSA). Whereas no binding was observed for AcLDL, SREC-II ectodomain interacted strongly with MalBSA and bound with high affinity to AcBSA, a property shared with the SR-F1 ectodomain.

Statistically correcting dynamical electron scattering improves the refinement of protein nanocrystals, including charge refinement of coordinated metals.

Electron diffraction allows protein structure determination when only nanosized crystals are available. Nevertheless, multiple elastic (or dynamical) scattering, which is prominent in electron diffraction, is a concern. Current methods for modeling dynamical scattering by multi-slice or Bloch wave approaches are not suitable for protein crystals because they are not designed to cope with large molecules.

Molecular and Cellular Interactions of Scavenger Receptor SR-F1 With Complement C1q Provide Insights Into Its Role in the Clearance of Apoptotic Cells.

The scavenger receptor SR-F1 binds to and mediates the internalization of a wide range of ligands, and is involved in several immunological processes. We produced recombinant SR-F1 ectodomain and fragments deleted from the last 2 or 5 C-terminal epidermal growth factor-like modules and investigated their role in the binding of acetylated low density lipoprotein (AcLDL), complement C1q, and calreticulin (CRT). C1q measured affinity was in the 100 nM range and C1q interaction occurs its collagen-like region.

Cytosolic PCNA interacts with p47phox and controls NADPH oxidase NOX2 activation in neutrophils.

Neutrophils produce high levels of reactive oxygen species (ROS) by NADPH oxidase that are crucial for host defense but can lead to tissue injury when produced in excess. We previously described that proliferating cell nuclear antigen (PCNA), a nuclear scaffolding protein pivotal in DNA synthesis, controls neutrophil survival through its cytosolic association with procaspases. We herein showed that PCNA associated with p47phox, a key subunit of NADPH oxidase, and that this association regulated ROS production.

High concentrations of GTP induce conformational changes in the essential bacterial GTPase EngA and enhance its binding to the ribosome.

EngA is a conserved bacterial GTPase involved in ribosome biogenesis. While essential in bacteria, EngA does not have any human orthologue and can thus be an interesting target for new antibacterial compounds. EngA is the only known GTPase bearing two G domains, making unique its catalytic cycle and the induced modulation of its conformation and interaction with the ribosome.

Analysis of relationships between peptide/MHC structural features and naive T cell frequency in humans.

The structural rules governing peptide/MHC (pMHC) recognition by T cells remain unclear. To address this question, we performed a structural characterization of several HLA-A2/peptide complexes and assessed in parallel their antigenicity, by analyzing the frequency of the corresponding Ag-specific naive T cells in A2(+) and A2(-) individuals, as well as within CD4(+) and CD8(+) subsets. We were able to find a correlation between specific naive T cell frequency and peptide solvent accessibility and/or mobility for a subset of moderately prominent peptides.

Potassium acts as a GTPase-activating element on each nucleotide-binding domain of the essential Bacillus subtilis EngA.

EngA proteins form a unique family of bacterial GTPases with two GTP-binding domains in tandem, namely GD1 and GD2, followed by a KH (K-homology) domain. They have been shown to interact with the bacterial ribosome and to be involved in its biogenesis. Most prokaryotic EngA possess a high GTPase activity in contrast to eukaryotic GTPases that act mainly as molecular switches.

The C-terminal polyproline-containing region of ELMO contributes to an increase in the life-time of the ELMO-DOCK complex.

The eukaryotic Engulfment and CellMotility (ELMO) proteins form an evolutionary conserved family of key regulators which play a central role in Rho-dependent biological processes such as engulfment and cell motility/migration. ELMO proteins interact with a subset of Downstream of Crk (DOCK) family members, a new type of guanine exchange factors (GEF) for Rac and cdc42 GTPases. The physiological function of DOCK is to facilitate actin remodeling, a process which occurs only in presence of ELMO.

Crystallization and preliminary X-ray crystallographic characterization of a public CMV-specific TCR in complex with its cognate antigen.

The T-cell response to human cytomegalovirus is characterized by a dramatic reduction of clonal diversity in patients undergoing chronic inflammation or immunodepression. In order to check whether all the selected high-avidity T-cell clones recognize the immunodominant pp65 peptide antigen pp65(495-503) (NLVPMVATV) presented by the major histocompatibility complex (MHC) molecule HLA-A2 in a similar manner, several public high-affinity T-cell receptors (TCRs) specific for the pp65(495-503)-HLA-A2 complex have been investigated. Expression, purification and crystallization were performed and preliminary crystallographic data were collected to 4.

Structural bases for the affinity-driven selection of a public TCR against a dominant human cytomegalovirus epitope.

Protective T cell responses elicited along chronic human CMV (HCMV) infections are sometimes dominated by CD8 T cell clones bearing highly related or identical public TCR in unrelated individuals. To understand the principles that guide emergence of these public T cell responses, we have performed structural, biophysical, and functional analyses of an immunodominant public TCR (RA14) directed against a major HLA-A*0201-restricted HCMV Ag (pp65(495-503)) and selected in vivo from a diverse repertoire after chronic stimulations. Unlike the two immunodominant public TCRs crystallized so far, which focused on one peptide hotspot, the HCMV-specific RA14 TCR interacts with the full array of available peptide residues.

Steric hindrance and fast dissociation explain the lack of immunogenicity of the minor histocompatibility HA-1Arg Null allele.

The di-allelic HLA-A2 restricted minor histocompatibility Ag HA-1 locus codes for the highly immunogenic HA-1(His) and the nonimmunogenic HA-1(Arg) nonapeptides, differing in one amino acid. The HA-1(His) peptide is currently used for boosting the graft-vs-tumor responses after HLA matched HA-1 mismatched stem cell transplantation; usage of the HA-1(Arg) peptide would significantly enlarge the applicability for this therapy. Our studies on mechanisms causing the HA-1 unidirectional immunogenicity revealed marginal differences in proteasomal digestion, TAP translocation, and binding affinity, whereas both dissociation rates and structural analyses clearly showed marked differences in the stability of these two HLA-A2 bound alleles.

Structural insights into a new homodimeric self-activated GTPase family.

The human XAB1/MBDin GTPase and its close homologues form one of the ten phylogenetically distinct families of the SIMIBI (after signal recognition particle, MinD and BioD) class of phosphate-binding loop NTPases. The genomic context and the partners identified for the archaeal and eukaryotic homologues indicate that they are involved in genome maintenance--DNA repair or replication. The crystal structure of PAB0955 from Pyrococcus abyssi shows that, unlike other SIMIBI class G proteins, these highly conserved GTPases are homodimeric, regardless of the presence of nucleotides.

Expression, purification, crystallization and preliminary crystallographic analysis of the PAB0955 gene product.

PAB0955 from Pyrococcus abyssi is a prototype of a new Walker-type ATPase/GTPase conserved in archaea and eukaryota but not found in bacteria. PAB0955 has been expressed, purified and crystallized, and it has been shown that this thermostable protein is dimeric in reductive conditions. Crystals have been obtained either without nucleotide or in the presence of GDP or GTPgammaS.

Ab initio structure determination and refinement of a scorpion protein toxin.

The structure of toxin II from the scorpion Androctonus australis Hector has been determined ab initio by direct methods using SnB at 0.96 A resolution. For the purpose of this structure redetermination, undertaken as a test of the minimal function and the SnB program, the identity and sequence of the protein was withheld from part of the research team.

CDR3 loop flexibility contributes to the degeneracy of TCR recognition.

T cell receptor (TCR) binding degeneracy lies at the heart of several physiological and pathological phenomena, yet its structural basis is poorly understood. We determined the crystal structure of a complex involving the BM3.3 TCR and an octapeptide (VSV8) bound to the H-2K(b) major histocompatibility complex molecule at a 2.

A T cell receptor CDR3beta loop undergoes conformational changes of unprecedented magnitude upon binding to a peptide/MHC class I complex.

The elongated complementary-determining region (CDR) 3beta found in the unliganded KB5-C20 TCR protrudes from the antigen binding site and prevents its docking onto the peptide/MHC (pMHC) surface according to a canonical diagonal orientation. We now present the crystal structure of a complex involving the KB5-C20 TCR and an octapeptide bound to the allogeneic H-2K(b) MHC class I molecule. This structure reveals how a tremendously large CDR3beta conformational change allows the KB5-C20 TCR to adapt to the rather constrained pMHC surface and achieve a diagonal docking mode.

Pairing of Vbeta6 with certain Valpha2 family members prevents T cell deletion by Mtv-7 superantigen.

Superantigens (SAg) are proteins of bacterial or viral origin able to activate T cells by forming a trimolecular complex with both MHC class II molecules and the T cell receptor (TCR), leading to clonal deletion of reactive T cells in the thymus. SAg interact with the TCR through the beta chain variable region (Vbeta), but the TCR alpha chain has been shown to have an influence on the T cell reactivity. We have investigated here the role of the TCR alpha chain in the modulation of T cell reactivity to Mtv-7 SAg by comparing the peripheral usage of Valpha2 in Vbeta6(+) (SAg-reactive) and Vbeta8.

Crystal structure of a T cell receptor bound to an allogeneic MHC molecule.

Many T cell receptors (TCRs) that are selected to respond to foreign peptide antigens bound to self major histocompatibility complex (MHC) molecules are also reactive with allelic variants of self-MHC molecules. This property, termed alloreactivity, causes graft rejection and graft-versus-host disease. The structural features of alloreactivity have yet to be defined.

The uteroglobin fold.

Uteroglobin (UTG) forms a fascinating homodimeric structure that binds small- to medium-sized ligands through an internal hydrophobic cavity, located at the interface between the two monomers. Previous studies have shown that UTG fold is not limited to the UTG/CC10 family, whose sequence/structure relationships are highlighted here, but can be extended to the cap domain of Xanthobacter autotrophicus haloalkane dehalogenase. We show here that UTG fold is adopted by several other cap domains within the alpha/beta hydrolase family, making it a well-suited "geode" structure allowing it to sequester various hydrophobic molecules.

Towards the charge-density study of proteins: a room-temperature scorpion-toxin structure at 0.96 A resolution as a first test case.

The number of protein structures refined at a resolution higher than 1.0 A is continuously increasing. Subatomic structures may deserve a more sophisticated model than the spherical atomic electron density.